Annual Report 2007-08 Health Hazards in Ship Breaking Workers of Alang RR Tiwari, SK Dave, SR Tripathi, NG Sathwara, PK Majumdar, BD Patel, RA Rathod and JB Vyas Abstract The ship breaking workers are exposed to various hazards such as accidents and injuries, exposure to asbestos, heavy metals like lead and polychlorinated biphenyls and noise exposure. The present cross sectional study was carried out to find the pattern and prevalence of health morbidities among ship breaking workers and their associated epidemiological factors. The study included 405 ship-breaking workers. Data collection on pre-designed proforma was followed by complete clinical examination, pulmonary function testing and chest radiography (PA View) of each worker. Blood sample of gas cutters was collected for lead estimation. The study also included audiometry of 187 workers and workplace monitoring for air lead levels. The radiological evaluation showed 12(2.96%) subjects with 1/1 and higher opacities whereas the spirometric evaluation revealed 65 (16%) subjects with obstructive, 6 (1.5%) with restrictive and 5 (1.2%) with combined type of impairment. The workplace air lead level monitoring revealed normal lead values while 3 23(18.4%) subjects showed blood lead levels >30g/m . The audiometric evaluation of workers suggested normal results for all the workers. The workers in ship breaking are exposed to dust and lead. Keywords: Ship-breaking, lead, asbestos, pulmonary function test Introduction Materials and Methods Ship breaking is the process of dismantling an obsolete vessel's structure for scrapping or disposal. The Alang-Sosiya Ship-Breaking Yard (ASSBY) located in the Gulf of Cambay in the Bhavnagar District of Gujarat State in India is the biggest ship-breaking yard, which employ some 15,000 to 25,000 workers directly for ship-breaking activities1. The complete dismantling of an obsolete ship is further divided into sub processes which include removal of asbestos containing materials by workers known as Gudadiwale in local language, pulling the ship with the help of wires to the beaching point, sorting of the materials according to different metals of which they are made of, cutting the iron parts of the ship with the help of gas cutter and other miscellaneous work like housekeeping of the plot on which these activities are carried out, loading and unloading of the heavy parts on and off the trolley for marketing purpose, driving the trolleys, vehicles and cranes, etc. All these processes expose workers to a wide range of hazards or workplace activities or conditions likely to cause injury or illness. These include exposure of asbestos fibres, heavy and toxic metals such as lead, mercury, cadmium, zinc and copper, PCB in paint, PVC and welding fumes2,3. Thus, the present cross sectional study was carried out to find the pattern and prevalence of health morbidities among ship breaking workers and their associated epidemiological factors. The present study was carried out among 405 ship breaking workers. Interview technique was used as tool for data collection, which was later on recorded on a pre-designed proforma. This was followed by complete clinical examination, pulmonary function testing and chest radiography (PA View) of each worker. 5ml blood sample was drawn from cubital vein in a heparinised vacutainers in aseptic condition from those workers working in the process of gas cutting to estimate the blood lead levels as they were directly exposed to fumes generated during the cutting process. Also the audiometry of about 187 workers was carried out. The workplace monitoring was carried out for air lead levels. The environmental monitoring for asbestos fibre levels could not be done as during the study period in none of the ship the process of removal of asbestos containing material was going on. For lead estimation, blood samples were digested using HNO3 and H2O2 mixture in Microwave digester (ETHOS 1600). The digested samples were analyzed using AAS, Perkin Elmer, U.S.A. model no 3100. Air samples were collected using personal sampler at the rate of 2-3 LPM for 2-3 hours and respirable high volume sampler (Envirotech Ltd, New Delhi) at the rate of 0.92 to 1.1 m3/min for 8 hrs. Collected air samples were digested using HNO3 and HClO4 (2:1) mixture. 7 Annual Report 2007-08 Table 1: Distribution of pulmonary function status according to duration of exposure Exposure N Pulmonary Function Status duration Normal Obstructive Restrictive Combined <5 101 87 (86.1) 11(10.9) 1 (1.0) 2(2.0) 5-10 135 104 (77) 25(18.4) 3 (2.2) 3 (2.2) 10-15 119 100 (84) 18 (15.1) 1 (0.8) 1(2.3) ³15 50 38 (76) 11 (22) 1 (2) Total 405 329 (81.3) 65 (16) 6 (1.5) 5 (1.2) Table 2: Findings on chest radiography in ship breaking workers Opacities No % Normal 357 88.2 Pleural thickening 6 1.5 Old healed tuberculosis 2 0.5 Emphysema 2 0.5 Cardiomegaly 4 1.0 1/0 small regular opacities 3 0.7 1/0 small irregular opacities 19 4.7 1/1 small regular opacities 1 0.2 1/1 small irregular opacities 7 1.7 1/2 small irregular opacities 1 0.2 Small regular and irregular opacities 2 0.5 Large opacities 1 0.2 Numbers in parenthesis indicate the percentage The digested samples were analyzed using Atomic Absorption Spectrophotometer (Perkin Elmer double beam model no. 3100) at the wavelength of 283.3 nm. Calibration of the instrument and the samples were compared with the lead standard of National Bureau of Standards (NBS), USA. Data analysis was carried out using Epi-Info5 software (WHO). when radiographs were classified according to the ILO Classification of Pneumoconiosis3, 12 (2.96%) were found to have findings suggestive of interstitial lung fibrosis. This can be attributed to lung parenchymal pathologies such as asbestosis. However, when the radiological abnormalities were analyzed according to different processes, it was found that such cases were evenly distributed among all processes. This is because each workers works in multiple processes. Results and Discussion The demographic characteristics of study subjects showed that the majority of the workers were less than 35 years of age with mean age being 30.86±7.79 years. All the subjects were male. This is due to the fact that the work demands heavy labour. About 81% of the workers were married while remaining were single. Though half of the workers were educated up to middle and secondary levels, about one-third were illiterate also. 57.4% of the workers were working in this occupation for more than 9 years. The mean duration of exposure was 9.67±0.28 years. 125 (41%) workers were working in gas cutting followed by 49(16.1%) in maal paani and 31(10.2%) in Jodi. Only 10 (3.2%) were working in miscellaneous process like as cook, removing the cabins in the ships and machine repairer. The respiratory complaints such as cough and breathlessness were present only in 3.2% and 3.9% workers respectively. Other complaints included anorexia in 28 (6.9%), bleeding from gums in 21(5.5%) and frequent burning in micturition in 19 (5%) workers. The musculoskeletal complaints like low back pain and joint pains were more common being present in 21.1% and 10.5% respectively. This could be due to heavy lifting5,6 and continuous working in squatting posture7. Any type of injury from trivial abrasions to major injury resulted during work in ship breaking processes in last one year period was present in 17.1% subjects. Though some of the workers were using personal protective devices like helmets and gumboots but the quality and maintenance of such PPEs were found to be sub-standard. Table 3 shows the mean lead levels according to the duration of exposure among the gas cutters. Though an increase in mean blood lead levels is observed with increasing duration of exposure, the difference was found to be statistically non-significant. Also the mean . levels were below the permissible levels of 30mg/dL The reason being the process of gas cutting is carried out in open space, the lead Table 1 shows the distribution of pulmonary function abnormalities according to the duration of exposure. Table 2 shows the findings observed on the chest radiography of study subjects. 359 (88.6%) had normal chest radiographs. In remaining subjects small irregular opacities of category 1/0 of ILO classification of Pneumoconiosis4 were present in 19 (4.7%), 6 (1.5%) had pleural thickening while other abnormalities included old tuberculosis in 2(0.5%), emphysema in 2 (0.5%) and cardiomegaly in 4 (1%) subjects. In all 48 (11.4%) had radiological abnormalities. The spirometric parameters revealed that only 6 (1.5%) had restrictive while 5 (1.2%) had combined restrictive and obstructive type of abnormality. However Table 3: Mean blood lead levels according to exposure duration Exposure group N Blood Pb levels (mg/dL) <5 14 13.32 ± 9.79 5-10 41 20.33 ± 9.93 10-15 51 19.21 ± 9.65 ³15 19 24.61 ± 9.04 Total 125 19.74 ± 9.99 8 Annual Report 2007-08 Right ear Left ear Figure 2: Average hearing level (dB) according to duration of exposure Figure 1: Average hearing level (dB) according to duration of exposure liberated in the fumes gets diluted by the ambient air. However as the workers work without any respiratory protective device, individual blood lead levels were more than 30mg/dL in 23 (18.4%) workers. Similar results were reported in earlier study8. Acknowledgment: The investigators are grateful to AR Shah, MH Vakharia, MR Varia, RC Patel, Raksha Agarwal, Divija Patel, MI Sheikh and Shaswat Dodia for the technical help rendered during this study. References Table 4 shows the air lead levels at the worksite and away from the worksite. The air lead levels were also below the normal levels 3 prescribed. The normal values for open area is 1.5mg/m while that for workplace area is 50mg/m3. The workplace and ambient lead levels at working plots were within normal level. 1. Rousmaniere P and Raj N. Ship breaking in the developing world: Problems and Prospects. Int J Occup Environ Hlth 2007; 13:359-368.2. International Labour Organization. Safety and Health in Shipbreaking: Guidelines for Asian Countries and Turkey,2004.http://www.ilo.org/public/english/standards/rel m/gb/docs/gb289/pdf/meshs-1.pdf. Accessed February 25,09. 3. Mattorano D, Harney J, Cook C, Roegner K. Metal exposure during ship repair and ship breaking procedures. Appl Occup Environ Hyg. 2001; 16:339-349. 4. International Labour Organization. Encyclopaedia of Occupational Health and Safety, volume II.3rd revised edition, ILO, Geneva, 1983: 2102-2104. 5. Miranda H, Viikari-Juntura E, Punnett L, Riihimäki H. Occupational loading, health behavior and sleep disturbance as predictors of low-back pain Scand J Work Environ Health. 2008; 34:411-419. 6. van der Burg JC, van Dieën JH, Toussaint HM. Lifting an unexpectedly heavy object: the effects on low-back loading and balance loss. Clin Biomech. 2000; 15:469-477 7. Van Vuuren BJ, Becker PJ, van Heerden HJ, Zinzen E, Meeusen R. Lower back problems and occupational risk factors in a South African steel industry. Am J Ind Med. 2005; 47:451-457. 8. Nosal RM, Wilhelm WJ. Lead toxicity in the ship breaking industry: the Ontario experience. Can J Public Health. 1990; 81:259-262. The result of hearing assessment for right and left ear is shown in Figures 1 & 2 respectively. Mean threshold of hearing of exposed group was just above the normal level in the right ear but the left ear showed mild hearing loss. The 20-25 years exposure group had little higher levels at frequencies 4000-8000Hz. Audiometric evaluations showed that the hearing acuity of all the workers was within normal limits. Though the workers are exposed to noise, it is intermittent in nature and this might not affect the hearing much. Thus to conclude, the ship breaking workers are exposed to asbestos dust, heavy metals like lead and intermittent noise. Table 4: Air lead levels at and away from the worksite (mg/m3) Place Plot No. Open area Worksite Sosiya 3 0.772 4.42 127-128 1.19 14.65 132 0.07 0.76 Alang 73 0.19 2.56 7 1.68 7.08 9 Annual Report 2007-08 Musculoskeletal Pain and Discomfort and Work Stressors Among Women in Fish Processing Activities A Nag, PK Nag, H Vyas and P Shah Abstract The objective of the study is to describe workers with musculoskeletal pain and discomfort (MSDs), to identify work aspects associated with pain and discomfort and to correlate the stress symptoms to pain among women engaged in fish processing activities. A descriptive study was conducted among women workers (N=450) selected at random from fish processing units of the coastal regions of Gujarat in Western part of India. The major activities performed by the workers were grading, sorting, peeling, and packing. About 72% of workers reported pain and discomforts chiefly in upper back, knee and shoulders. Relative risk estimate suggests that workers suffering from fatigue and those reported socio domestic disruption were at a higher risk to pain and discomforts. Analysis of correlation coefficient between work aspects (work stressors) and MSD suggest that physical work environment, physical activity, manual material handling, cold work environment and poor task clarity were correlated. In fish processing industry work involves repetitive movements and may be associated with a considerable risk of musculo-skeletal disorders. Physical and psychosocial risk factors at work may potentially interact to further increase the risk of symptoms of MSDs. Strategies aimed at the prevention of these work related musculo-skeletal disorders should reduce exposure to physical and psychosocial workplace risk factors. Keywords: Musculoskeletal discomfort, fish processing, work stressors Introduction Materials and Methods It has already been mentioned in our previous Annual Reports that due to the nature of the job, women in fish processing activities are exposed to cold environment, use improper hand tools and implements and adopt awkward working postures to perform occupational activities. Many studies in literature establish relationship between work factors and MSDs. Personal, psychosocial job stress and work organizational factors in sewing machine operators1, work tasks and psychosocial job stress associated to MSD in forestry2. Physically strenuous works, carrying heavy weights and 3 environmental factors and mental stress of German workforce have been associated to pain and discomfort. Such correlation studies help to down line the key causative factors that are associated to pain and discomforts and thereby help to draw plans that intervene to make the work and work factors less stressful. The objective of the study is to find out the prevalence of musculo-skeletal pain and discomfort among fish processing workers, to identify occupational aspects associated with pain and discomfort and to correlate the psychosocial stress symptoms with the pain and discomforts prevailing among women engaged in fish processing activities. The prevalence of musculo-skeletal pain and discomfort among the women fish processing workers were evaluated using NIOSH 4 checklist , including checkpoints on the severity of the pain, the workday lost due to pain, the remedial measures adopted by the workers to minimize the pain and the worker's perception on the causative factors for pain. To identify the work aspects and stressors, the workers were individually interviewed by using a multi-method ergonomic checklist5. The physical health and well-being were examined using the standard questionnaire. The checklist entries were responded by a single digit on a five-point common severity agreement/ disagreement scale - strong disagreement (1) to strong agreement (5), suggesting that the low value is the positive indicator of the perception of absence of the stress. The score for each section of the checkpoints were arrived by adding the individual scores obtained for each item and then dividing it by maximum cumulative score possible under that section. In other words, for each of the work stressors, the outcome value would range within 0 to 1. Required ethical clearance for the study was obtained from the ethical committee of the Institute. 11 Annual Report 2007-08 Results and Discussion Annual Report 2007-08 Table 2: Responses of women to variables related to MSD (N=450) Age (Mean ± SD) 23 ± 6.4 75.3 Work experience (Mean ± SD) 2.8 ± 2.0 <3years (%) 47.6 Education -- Literate (%) 72 Marital status -- Unmarried (%) 69 Work shift (hr) 12 Tasks (Women performs more than one task) (%) Grading 39 Sorting 15 Packing 50 Mixed tasks 15 Peeling 27 Ring cutting 16 BMI (Mean ± SD) 21.4 ± 4.1 12 0.8 50 0.7 0.6 Relative loading 40 30 20 16 20 44 0.5 0.4 0.3 0.2 20 0.1 Machinery Machinery safety Work organization MMH Tools Seating arrangement Task clarity Training Task situation Thigh Skill requirement Finger Job feed back Ankle Mental overload Calf Auxillary support Elbow Work posture Hand Job autonomy Lower Shoulder Neck back Physical activity Knee Work place design IIIumination Uppper back Temperature 0 Job speicalization 0 10 Figure 1. Prevalence of MSD in fish processing workers Work stressors and MSD: The descriptive values obtained gave the response of the workers to the work aspect as a stressor. The response of the workers to these work stressors is shown in Figure 2. Illumination, low temperature, dust, constrained work posture, high physical activity, psychosocial aspects as work scheduling, poor job autonomy and poor job feedback had more weighttage among the work stressors as perceived. The Pearson's correlation matrix showed that high physical activity, job specialization, cold environment, poor job feedbacks and strenuous work postures were associated to pain and discomfort in the lower back. Psychosocial aspects as poor job autonomy and mental overload had correlation to discomfort in knee. Pain in hand was associated to work environment, absence of auxiliary support and manual material handling. High physical activities, job specialization, task environment, cold environment, poor job feedbacks and poor job autonomy and task clarity, strenuous work postures and high mental overload were associated to pain and discomfort in lower back. Physical activity and poor task clarity associated to discomfort in upper back. Pain in hand was associated to work environment (cold), absence of auxiliary support, manual handling and poor task clarity. Discomforts in hand and finger associated to cold environment, high physical activity, manual material handling. The double fold risk of development of MSD among younger group of women workers, who were less than 25 years of age, attracts special attention regarding implementation of ergonomic intervention in the fish in processing units with an immediate effect. processors (N=450) <25 years (%) 23 35 6 27 9 2 treatment for prevention of pain and only 16% of the women consulted the doctors to minimize the pain. Table 1: Physical and occupational characteristics of women fish 0.9 60 52 35 13 Prevalence of MSD (%) Severity of pain (%) Mild Moderate Severe Worker's perception to the cause of pain (%) Posture Work environment Work method Work load Exhaustion Others (Non occupational) Worker's selection of remedial measures (%) Self chosen pain killer Seek advice from doctors Apply ointment None The workgroup (N=450) was relatively young with a mean age and job involvement of 23 ± 6.4 and 2.8 ± 2.0 years respectively (Table 1). Work activities primarily performed by the workers in units were grading, peeling, ring cutting, sorting and packing. More than 70% of workers were literate and approximately 70% were unmarried. About 72% of workers reported pain and discomfort (MSD) in different regions of the body and there was high prevalence of pain and discomfort in upper back (54%) followed by lower back (33 %), knee (35 %), shoulder (27 %), hand (17%), finger (9%) and calf (13%) shown in Figure 1. The women workers less than 25 yrs of age (OR 2.6, 95% CI 1.3-5.2) showed higher risk of development of MSD in lower back than the women who were more than 25 yrs of age. Risk estimates suggest that workers performing grading (OR 2.4, CI 1.34.2) and peeling (OR 2.5, 95% CI 1.3-4.8) were at relatively higher risk to develop pain in upper back and hands which is associated to repetitive hand movements involved in the processing activities. Risk to overall pain and discomfort was found among women engaged in ring cutting (OR 20.7, CI 9.8-43.8) and mixed tasks (OR 4.5, CI 1.9-10.5). Table 2 shows that severe pain was experienced by 13% of the women whose pain persisted, even in the night, long after the cessation of work. Women had mixed responses when asked about their perception to the cause of pain and discomfort. As perceived by the workers, working posture, work environment and workload were the factors contributed to the development of pain and discomfort. The selection of remedial measures differed among women and nearly 44% of the women were indifferent towards adopting Figure 2. Relative weightage of work stressors Workers' chosen perception to the cause of MSD like constrained posture (23%), cold environment (35%) and work load (27%) were found to have correlation with the pain in lower back, upper arm ect., signify the necessity of the involvement of the workers representation while taking into account the intervention program to minimize the MSD among these women. Repeated handling of unergonomically designed tools as knives and scissors, impose biomechanical strain and increase their risk to MSD and need to be replaced by properly designed hand tools. Also women perform processing activities in cold environment without any personal protective garment, might be one of the causative factors for the development of MSD in different body regions. High prevalence of MSD among the women fish processing workers suggest that they are at risk and the intervention strategy must be developed and implemented immediately. MSD among fish processors is a major health problem. Fish processing activities share many similarities that are attributed to the poor work conditions, which pose varieties of health hazards including MSD. Women can face significant risks from the processing activities they perform, underlining the importance of adequate risk-assessment and prevention measures for them. Musculo-skeletal pain and discomfort are the results of interaction of the factors associated with the job and work environment, environmental factors and characteristics of the individual factors. As observed in the study, working conditions and methods, unergonomically designed work station and tools, cold work environment, constant standing posture, long working hours etc., were the predominant factors contributing to the development of MSD. Perceptions of intensified workload, monotonous work, limited job control, low job clarity, and low social support were also associated with various work-related MSD. Conclusion to the health hazard can be mitigated by suitable intervention and make the work safe and less strenuous. Fish processing workers are at high risk to pain and discomfort in different body regions, due to varied nature of activities in inclement working conditions. The behavioral symptoms, job specialization, skill requirements, physical activity, psychosocial aspects, work environment and postural constraints have association with the development of MSD. The study points out the importance of suitable work designs and ergonomics intervention to prevent musculo-skeletal problems among the women. References 1. Wang, PC, Rampel, DM, Harrison, RJ, Chan, J., Ritz, BR 2007. Work organizational, and personal factors associated with upper body musculoskeletal disorders among sewing machine operators. Occu. Enviro. Med. 64(12): 806-813. 2. Hagen, K, Magnus, P, & Vetlesen, K, 1998. Neck /shoulder and lowback disorder in the forestry industry: Relationship to work tasks and perceived psychosocial job stress. Ergonomics. 41-15101518. 3. Schneider S., Lipinski S, Schiltenwolf M., 2006 Occupations associated with a high risk of self-reported back pain: representative outcomes of a back pain prevalence study in the Federal Republic of Germany. Eur Spine J. 15,821-833. 4. Cohen, AL, Gjessing, CC, Fine, LJ, Bernard, BP, McGlothlin, JD, 1997. Elements of Ergonomics Program-A primer based on workplace evaluation of musculo-skeletal disorders. DHHS (NIOSH) Publ. No. 97-117. 5. Nag PK. 1998. Work systems-checklists. In: ILO Encyclopedia of Occupational health and Safety, Fourth ed. Geneva, ILO Ergonomics pp. 29.14-29.24 (Chapter 29). Work related pain and discomfort lead to sickness absence and to add to the burden of disease. The components at work that might attribute 13 Split contour & Porous backrest Annual Report 2007-08 Arm rest Angle & Height Adjustment Vibrators Waterfall seatpan Chair Height Adjustment Stabilometric Signal Analysis of Women Sitting Postures Tilt mechanisam of seatpan A Nag, PK Nag, S Pal and H Vyas Foldable Footrest with Castor Abstract The study examines the utility of the force platform measurements- Centre of pressure (CoP) displacement parameters and force exertion in order to explore the influence of sitting modes governed by sitting behavior and seat components. A test rig of two piezoelectric force platform, one platform was placed to serve as a chair seat pan (P2) and second platform (P1) was placed on ground to serve as a footrest. Eleven women participated and adopted eleven sitting modes arms right angled with upright back, arms on lap with upright back, arms on lap with slouched back, one leg crossed over the other with upright back, back supported with arms on lap, upright unsupported and supported back (95 recline) with variations in armrest height which was adjusted at 17, 20 and 23 cm. Force exertion and CoP displacements parameters derived from force platform were included in the study. In addition to descriptive, in order to understand the influence of sitting modes on CoP displacement parameters, one-way (sitting modes) repeated measures analysis of variance (ANOVA) was done with time blocks as covariate. The combined inclusion of back support and armrest in the seat system reduced the load at seat pan; this is suggestive of increased dissipation of load from seat pan to armrest, back support and marginally to feet. ANOVA results indicated that changes in the sitting modes had discernable effects on the medio-lateral and anterio-posterior CoP displacement. The typical characteristics of stabilometric dimension brings an insight into the influence of seat components and sitting behavior on the postural balance characteristics therefore is important in human seat interface design. Keywords: Stabilometric dimension, sitting postures, CoP displacement Introduction three orthogonal axes Fx, Fy, and Fz, and the three moments around the three axes Mx, My, and Mz. These signals were analyzed using SMART analyzer software (BTS Bioengineering, Italy) to derive force components and platform signals from P2 (seat pan) were used to obtain the x and y coordinates of the CoP displacement corresponding to medio-lateral (ML) and anterio-posterior (AP) time series respectively with SWAY software (BTS Bioengineering, Italy). The statistical analysis was accomplished by the software SPSS 16.0 package. In order to understand the influence of postures on CoP displacement parameters, one-way (sitting modes) repeated measures analysis of variance (ANOVA) was used with time blocks as covariate. The least significant difference test was applied to produce post hoc multiple comparison of the test measures, with respect to variation in sitting modes, arms and back support. The sitting habits and behavior vary considerably with the designs of chair-desk complex. Since prolonged sitting work is a potential risk to the spinal, paraspinal and other musculo-skeletal structures, researches have been focused on the postural load at the intervertebral discs. This contribution describes the orthogonal force components recorded from the ground reaction force and analysis of body CoP displacements due to chair sitting modes and the components of seat. Materials and Methods F Tz Force Platform signals Dynamic change in Kg Force Force Componants Eleven healthy, young, sedentary women (age - 31.7 ± 6.9 years, body height - 150.4 ± 5.1 cms, body weight - 48.3 ± 5.7 kg, body mass index: 21.6 ± 3.2, spine length - 44.7 ± 4.7 cms) volunteered in the study. A test rig of two piezoelectric force platform (sized 40 x 60 cm, Kistler, Switzerland, model 9268AA), one being placed to serve as a chair seat pan (P2), stabled and vertically adjusted by a heavy duty mechanical jacking mechanism and the second platform placed at ground (P1) as footrest (Figure 1). The sitting on a force platform recorded three counteracting forces due to the body weight along the Results Force distribution (kgf) at seat pan and feet that was derived in eleven chair-sitting modes is given in Table 1. The force distributed at the seat pan (38.9 kgf) was recorded highest in Mode C (slouched back), and the force at feet was 8.9 kgf (i.e., the combined force at P1 and P2 was 47.8 kgf, which was 99% of the body weight measured in 15 Annual Report 2007-08 standing posture). With the back supported, the load at the seat pan reduced by about 8%, as compared between Mode B (unsupported back) and Mode E (supported back at 950 recline). With unsupported back and increase in the height of armrest from 17 to 23 cm, the force distributed at the seat pan reduced from 35.9 to 34.2 kgf. The combined inclusion of back support and armrest in the seat system for Modes F, G and H (arm rest height 17, 20 and 23 respectively) reduced the load at seat pan to 32.9, 32.6 and 32.3 kgf respectively; this is suggestive of increased dissipation of load from seat pan to armrest, back support and marginally to feet. Directional ML and AP CoP displacements of eleven sitting modes are shown in Figure 2. Repeated measures of ANOVA indicated that the ML CoP displacement varied across the sitting modes (p<0.01). The unsupported upright sitting modes (Modes A and B) had highest AP CoP displacement (194 mm), however ML CoP displacement (p<0.001) was significantly less in Mode A compared to Mode B. Among the sitting modes, Mode E (supported back) caused highest ML CoP displacement, when back was supported at 950 recline. The post hoc analysis showed that the legs being crossed one over the other (Mode D) caused relatively larger ML CoP displacement (p<0.001), with reference to sitting mode B. The unsupported upright sitting (Mode B) resulted in significantly greater AP CoP displacement (p<0.001), compared to those in slouch sitting (Mode C). The supported back (Mode E) caused reduction in AP CoP displacement (p<0.001) and increased ML CoP displacement (p<0.001) compared to those in upright unsupported sitting (Mode B). For the pooled data, the scatter of the ML and AP CoP displacements was negatively correlated (r = - 0.283, p<0.001). The linear regression equation indicated that the unit increase in the ML CoP displacement had a corresponding decrease in the AP CoP displacement. Discussion The present study emphasizes that seat components and sitting modes implicate a change in body passive acceleration, CoP displacement and CoP oscillation pattern2-4 The armrest and backsupport led to reduction in 16.3% of the net load exerted at the seat pan. The armrest took about 8% of the net force exerted at the seat pan. The extent of load dissipated by the components of seat indicated likely mitigation of the compressive and shear stresses on the spinal and other paraspinal structures. The present observation emphasizes that the armrest, as a seat component, had greater contribution to reduce load at seat. Load distribution outlined the overall reduction and transfer of the load from seat pan to armrest, back support and feet. The study defines amplitude of ML and AP CoP displacement to range narrowly within 6-14 mm and 160 to 195 mm respectively in chair-seated modes. In chair seating modes oscillation patterns found to be predominant in anterior-posterior and medio-lateral direction. Annual Report 2007-08 Table 1. Force distribution (Kgf) at seat pan and feet in different chair sitting modes Modes Posture configuration A Upright unsupported back, legs right-angled at knee and armsright-angled at elbow B Upright sitting unsupported back, legs right-angled at knee and arms rested on lap C Slouched back, legs right-angled at knee and arms rested on lap D Upright unsupported back, legs crossed one over the other, and arms rested on lap E Supported back at a recline of 95o with arms rested on lap F Supported back, armrest at the height of 17 cm G Supported back, armrest at the height of 20 cm H Supported back, armrest at the height of 23 cm I Upright unsupported back, armrest at the height of 17 cm J Upright unsupported back, armrest at the height of 20 cm K Upright unsupported back, armrest at the height of 23 cm (P2- Seat pan) Kgf 38.8±5.4 38.6±5.6 38.9±5.7 38.5±4.5 36.0±5.3 32.9±5.0 32.6±5.8 32.3±5.3 35.9±6.0 34.4±5.6 34.2±5.7 (P1- feet) Kgf 8.9±1.2 9.0±1.3 8.9±1.3 9.0±1.0 9.6±1.2 10.3±1.1 10.3±1.3 10.4±1.2 9.6±1.4 9.9±1.3 10.0±1.3 Values are mean ± SD References Platform (P2) seat pan Height adjustment Platform(P1) foot rest Armrest heights caused significant change in the ML (p<0.001) and AP CoP (p<0.001) displacements when the back was upright. The ML CoP displacement in Mode J (armrest height 20 cm) was relatively less compared to Mode I (armrest height 17 cm) (p<0.01) and Mode K (armrest height 23 cm) and higher than Mode B (arms on lap) (p<0.0001). Mode I (arm rest height 17 cm) varied from Mode K (armrest height 23 cm) in AP CoP displacement (p<0.001). With back unsupported, the increase in height of armrest from 20 cm (Mode J) to 23 cm (Mode K) significantly influenced the AP CoP displacement (p<0.001). Mode G had considerably higher ML CoP displacement (p<0.05) than Mode H. The study includes analysis of nearly eleven hundred platform signals of women recorded for different sitting modes and thereby identified seven distinct CoP patterns (Figure 3). The anterior-posterior (Pattern 3) CoP displacement was predominant in Mode A (upright sitting, arms at right angle), Mode C (slouched back), Mode D (crossed legs) and Mode E (supported back). The medio- lateral (Pattern 1) CoP displacement was predominant in Mode B (upright sitting arms on lap) i.e. about 42% of the total distribution of patterns. Figure 1.. Force platform for stabilometric study Men and women have subtle postural orientation due to circumstances of sitting as well as seat used. Analysis of seat dynamics bears significance to assess design and consequences for sitter. The present stabilometric study quantifies relative share of body weight distributed at seat pan and feet and low amplitude body oscillation characteristics influenced by back unsupported or supported or slouched sitting, back supported sitting, position of arms (on lap, at right angle from elbow and on armrest) and position of leg. The study finding strongly indicated that the backrest and armrest shifted weight from the seat pan. The armrest influenced the directional CoP displacements. With the backrest, medio-lateral CoP displacement increased in comparison to back unsupported sitting. The typical characteristics of stabilometric dimensions bring an AP CoP displaced (mm) = 187.7 1.02 ML CoP displaced (mm) 16 Figure 2: CoP displacement in different chair seated modes Figure 3 17 1. SWAY software manual, release 1.4.4, BTS Engineering, Milano, Italy. 2. Kanaya, T., Gresty, M.A., Bronstein, A.M., Buckwell, D., Day, B., 1995. Control of the head in response to tilt of the body in normal and labyrinthine-defective human subjects. Journal of Physiology, 489, 895-910. 3. Keshner, E.A., 2000. Modulating active stiffness affects head stabilizing strategies in young and elderly adults during trunk rotations in the vertical plane. Gait and Posture, 11, 1-11. 4. Fenety, P.A., Putnam, C., Walker, J.M., 2000. Inchair movement: validity, reliability and implications for measuring sitting discomfort. Applied Ergonomics, 31, 383-393. Annual Report 2007-08 Assessment of Feasibility of Dust Control Devices for Small Silica Flour Milling Units in India LJ Bhagia, MI Shaikh, SL Dodia and P Sekhar Abstract Exposure to crystalline silica produces silicosis and silico-tuberculosis. There are two type of silica mills viz. hammer type (product mesh size <100) and ball mill type (product mesh size 100-500). At Godhra all mills (17) are hammer type whereas at Beawar most of the mills (about 300) are ball type. However, there are some hammer types of mills at Beawar also. In ball mills, exposure occurs during feeding and bagging operations. Exposure to silica dust in ball mills is reported. Total dust concentrations ranged from 9.88 to 13.14 and 11.78 to 25.36 mg/m3 in feeding and bagging operations respectively. The respirable dust varied from 2.19 to 2.48 3 in feeding and 2.89 to 4.39 mg/m bagging operations. Keywords: Silica milling, quartz, dust control system Introduction Silicosis is the most ancient and common of all occupational diseases. Even today, it continues to be among the most serious occupational diseases. The crystalline free silica, the agent responsible for the causation of silicosis, is one of the most powerful fibrogenic matter found in nature. Silicosis occurs in occupations like silica milling1, agate industry2-4, slate pencil industry5,6, ceramic and pottery7, foundries, glass, mining of stones containing free silica, etc. Ambient silica dust exposure in vicinity of agate and slate-pencil industry has 8,9 also been reported. There is no cure for silicosis at present but the disease is preventable. For the prevention, exposure of workers to crystalline silica needs to be reduced. Engineering control is the most powerful tool for the reduction of dust exposure. This study was conducted with the objectives to monitor dust levels in silica mills, design a dust control system and test the efficacy of dust control system. Materials and Methods Type of mills: There are two type of mills viz. hammer type (mesh size <100) and ball mill type (mesh size 100-500). At Godhra all mills (17) are hammer type whereas at Beawar most of the mills (about 300) are ball type. About 25 hammer types of mills are also at Beawar. Manufacturing process (Ball mills): The process includes feeding of raw materials to ball mill. Different size of particles is separated by mesh separator (100 - 500 mesh size). Normally 200 19 mesh size product is manufactured. Over size particles are fed back to ball mill. Product is collected manually in bags. Exposure occurs at feeding and bagging operations Exposure assessment: Five ball mills were selected for monitoring dust levels at Beawar. House keeping in general was very poor in all the mills. The quality of bags used for product was also very poor. The exposure of workers to silica dust occurs at feeding and bagging operations. Workers are exposed continuously for 8 hours. Total and respirable dust concentrations were evaluated at feeding and bagging sites. Dust concentrations were evaluated by using SKC samplers. Dust was collected on membrane filters. Results and Discussion Total and respirable dust concentrations are shown in Table 1. Total dust concentrations ranged from 9.88 to 13.14 and 11.78 to 25.36 3 mg/m in feeding and bagging operations respectively. The respirable 3 dust varied from 2.19 to 2.48 and 2.89 to 4.39 mg/m in feeding and bagging operations respectively. Dust levels need to be reduced by proper engineering control. At a glance Table 1 show that dust concentrations are higher than the permissible level of 0.1 mg/m3 for respirable and 0.3 mg/m3 for total dust. Dust levels in bagging are more than the same in feeding of raw materials. Hence more suction is required at bagging site. Keeping this in view dust control system is designed. Annual Report 2007-08 Annual Report 2007-08 Environmental cum Epidemiological Survey in Asbestos Jointing Material Manufacturing Industry Table1: Dust concentrations (mg/m3) in ball mills Mill A B C D E Feeding Total dust 10.74±6.42 (5) 9.88±2.68 (4) 10.03±2.97 (6) 12.88±2.88 (5) 13.14±4.29 (4) Bagging Respirable dust 2.45±0.71 (6) 2.37±0.69 (6) 2.31±1.76 (6) 2.19±1.62 (6) 2.48±1.57 (6) Total dust 12.90±3.32 (5) 11.78±1.02 (4) 17.65±2.04 (6) 15.82±3.36 (5) 25.36±8.22 (4) Respirable dust 3.70±1.26(6) 3.44±0.97 (6) 3.51±0.40 (6) 2.89±0.64 (6) 4.39±1.92 (6) Numbers in parenthesis indicate number of samples Design for the dust control system: Dust control system is designed by NIOH using ACGIH ventilation guidelines10. Dust control system involves hood (one each at feeding and bagging points) ducting, centrifugal fan and bag filters with reverse pulse jet system. The reverse pulse jet systems are most commonly used. The pulse jet cleaning mechanism uses a high pressure jet of air to remove dust from the bag. Bags in the bag house compartment are supported internally by rings or cages. Bags are held firmly in place at the top by clasps and have an enclosed bottom. Dust laden-air is filtered through the bag, depositing dust on the outside surface of the bag. The dust cake is removed from the bag by blast of compressed air injected into the top of the bag tube. The blast of high pressure air stops the normal flow of the air through the filter. The air blast develops into a standing wave that causes the bag to expand as the bubble travels down the bag tube. As the bag flexes, the cake fractures and deposited particles are discharged from the bag. Total flow of the system is 1200 Cubic feet per minute (CFM). Horse-Power of the motor is 5 hp (1440 RPM). Speed of the fan is increased to 2300 RPM by using pulleys. Owners were encouraged to install dust control system for feeding and bagging operations. One of the owners has agreed to install the system on experimental basis. MoU has been signed with the mill at Beawar for installation of dust control system. The work is in progress for testing the efficacy of dust control system. References 1. Bhagia LJ, Tiwari RR, Sharma YK, Saiyed HN. Silicosis and its control in small-scale silica mills in India. WHO newsletter Gohnet (Global Occupational Health Network), 12; 12-15, 2007. 2. NIOH Report. Prevention, control and treatment of silicosis and silico tuberculosis in agate industry- report submitted by NIOH to the Ministry of Health and family welfare, Govt. of India, 2004. 3. Bhagia LJ, Parikh DJ, Pandya GL, Vyas JB and Saiyed HN. Assessment and control of silica dust exposure in agate grinding units, Khambhat, India. Indian journal of Occupational Hygiene and Safety. 1;2: 20-27, 2007. 4. Sadhu HG, Parikh DJ, Sharma YK, et al. A follow up study of health status of Small-scale Agate Industry Workers. Indian J. of Ind. Med. 41;3: 101-105,1995. 5. Saiyed HN, Parikh, DJ, Ghodasara, NB, et al: Silicosis in slate pencil workers. An environmental/medical study. Am. J. Ind. Med. 8: 127-133,1985. 6. Saiyed HN and Chatterjee, BB: Rapid progression of silicosis in slate pencil workers: A follow up study. Am. J. Ind. Med., 1985; 8: 135-142. 7. Saiyed HN, Ghodasara NB, Sathwara NG, Patel GC, Parikh DJ, Kashyap SK. Dustiness, Silicosis and Tuberculosis in Small Scale Pottery Workers. Indian J Med Res.; 102: 138-142,1995. 8. Bhagia LJ. Non-Occupational exposure to silica dust in vicinity of slate pencil industry, India Published Online in Environmental Monitoring and assessment (Springer publication), April 2008. 9. Bhagia LJ, Parikh DJ, Saiyed HN. Ambient silica monitoring in vicinity of agate industry, Khambhat, India. Indian journal of Occupational Hygiene and Safety, 1; 6-10, 2007. 10. ACGIH: Industrial ventilation, A manual of recommended practice, 25th edition, Signature Publications, 2004. A Saha, LJ Bhagia, YK Sharma, PK Majumdar and JB Vyas Abstract A cross sectional study was carried out among the workers of asbestos jointing material industry with the aim of evaluating the working environment as well as the workers. Seventy workers participated in this study. Every individual subject was interviewed with a pre-designed questionnaire to collect information in relation to personal, occupational and morbidity details. Lung function test was carried out in all subjects. All the study subjects were evaluated by radiological examination as per ILO guidelines. Environmental monitoring of asbestos fibers was also carried out. Pulmonary function test revealed that 11.4% workers were having obstructive and 2.9% were having restrictive abnormality. Fiber level in all the workplaces were below national and international standards. No worker was found to have radiographic features suggestive of interstitial lung fibrosis. Keywords: Asbestos, jointing material, pulmonary function Introduction Asbestos is the generic name given to a group of fibrous mineral silicates found in nature. Exposures to asbestos fibres occur during mining, milling, processing, and transportation of asbestos and also during manufacturing of asbestos products. Known major health hazards of asbestos include cancer of lung, mesothelioma of pleura and peritoneum and a specific fibrous disease of lung known as asbestosis. All types of asbestos fibres are responsible for human mortality and morbidity. Chrysotile is thought to be less harmful than other fibre types but it is difficult to substantiate after standardization for exposure levels, type of exposure, duration of exposure, etc. Harmful effects of asbestos also depend on several factors like size and type of fibres, duration of exposure, use of personal protective devices, etc. Chrysotile asbestos, the most common variety in use, has been consumed in roofing, insulation, pipe and other moulded goods, gaskets, friction materials (brake linings, clutch facings) etc.1,2 . Asbestos-cement industries manufacturing corrugated and flat sheets, moulded goods, pipes etc. are the major consumers of asbestos worldwide, accounting for about 85% of all use. These products contain 10-15% of asbestos3. Asbestos fibers when breathed in may get trapped in the lungs and may remain there for a long time. Over time, these fibers can 20 accumulate and cause scarring and inflammation, which can affect breathing and lead to serious health problems4. Asbestos has been classified as a known human carcinogen (a substance that causes cancer) by the U.S. Department of Health and Human Services, the EPA, and the International Agency for Research on Cancer5-8. The occurrence of health effects of asbestos exposure also depends upon the type of asbestos used. While the occurrence of asbestosis, lung cancer and mesothelioma is widely reported with the use of amphibole variety the target cells10. Murlidhar and Kanhere11 reported 23% asbestosis in a survey of 181workers of an asbestos industry of India. The mean age of the workers was 54 years and all had a minimum of 20 years of exposure to asbestos. Lung function test revealed that 62% of workers had FVC values less than 80% of predicted standard. Radiological findings revealed that 92% and 17% of the subjects were having parenchymal fibrosis and pleural abnormalities respectively11. Such studies conducted in India are insufficient and considerably old. Hence, need of such studies is desperately felt to assess the present day asbestos handling workplaces and workers. The Company manufactures Compressed Asbestos Fibre Jointing Sheets as one of its major products. Asbestos fibre is mixed with binders solution and fillers and then passed through the Calendar Machine to form Compressed Asbestos Fibre Jointing Sheets. These sheets are cut, as par required size, graphited, if required printed, bundled and packed. 21 Annual Report 2007-08 Materials and Methods Fiber concentrations in the workplaces were evaluated using SKC personal samplers with a flow rate of 1-2 ltr./min. The membrane filter method using phase contrast microscopy was used. Samples were collected on cellulose acetate membrane filters (diameter 25mm, pore size 0.8mm) using personal samplers with flow rates of 1-2 LPM. Samples were shifted to the laboratory and slides were prepared using acetone-triacetin method. Fibres (length >5 m, width <3 m and aspect ratio ³3:1) were counted using Walton-Beckett graticule at magnification of 400X. Fibre levels were then compared with the permissible levels mentioned in the Indian Factories Act, 1948 (1 fibre/ml) and the recommended exposure levels of OSHA (0.1 fibre/ml), NIOSH (0.1 fibre/ml). In India the chief use of asbestos is in roofing, pipeline and fireproofing. Asbestos cement product manufacturing industries producing AC sheets and pipes are the major user of asbestos in our country. Apart from this a portion of asbestos use in our country is committed by manufacturers of other asbestos containing materials (e.g. friction materials, jointing materials etc.). This study was carried out among the workers of asbestos jointing material industry with the aim of evaluating the working environment as well as the workers. Seventy workers participated in this study. The aim and purpose of the study was explained to the participants and consent was obtained accordingly. Every individual subject was interviewed with a predesigned questionnaire to collect information in relation to personal, occupational and morbidity details of the workers. Lung function test was carried out in all subjects. Forced vital capacity (FVC) was recorded by Spirovit-sp-10 (Schiller Health Care Ltd, Switzerland). Three successive recording were made in standing posture and the nose clip was used. The best of the three performances was considered for calculation purpose. Other different parameters like FEV1, FEF25-75 were also calculated from the same tracings. All volumes obtained were expressed in body temperature on atmospheric pressure of air saturated with water vapour (BTPS). Body height and body weight were measured in bare feet on a standard scale. Pulmonary function test values were predicted from the standard prediction equation. The instrument was calibrated every day before starting the experiment. FVC and FEV1 were expressed in litres, FEF25-75 in litres/sec and FEV1% was presented as the ratio of FEV1 and FVC. Results and Discussion Mean age of the study subjects was 37.8±9.02 years (range 22-60 years). Majority of the workers were more than 30 years of age. Twenty-four workers (39.3%) were of more than forty years age. Thirty workers (42.9%) were in 31-40 years age group. Sixteen workers (22.9%) were up to thirty years of age. Among the total number of workers 63 (90%) were married and the rest were not. Only 27.1% workers had more than secondary level education and about 14.3 % workers illiterate. Mean body mass index of the workers was 20.6±4.4. Majority (57.1%) had normal body mass index values (18.5-24.99) whereas 30% and 12.9% workers had values lower and higher than normal respectively. Among the total number of workers, 10 (14.3%) were smokers and 28 (40%) workers were chewers. Majority of the workers (51.4%) were from Process Division followed by Maintenance (24.3%) and Packing/Loading (8.6%). Other workers participated in the study were from Stores and other departments including persons employed for cleaning (15.7%). Mean job duration of the workers was 11.77 ± 7.71 years. Minimum experience was 1 year and maximum experience was 30 years. Majority of workers (45.7%) had experience of more than 10 years, whereas 25.7% All the study subjects were evaluated by radiological examination as per ILO guidelines. Chest X rays (PA view) were obtained with the subject in inspiration. These X rays were examined using ILO Classification of Pneumoconioses Radiographs. Data were analyzed using Epi Info 5 and SPSS software. Table 1: Distribution of pulmonary function test parameters according to age and experience Age Group (yrs.) FEV1 FVC Up to 30 31-40 41-60 Significance (One way ANOVA) Experience Group (yrs.) Upto 5 6-10 11 and above Significance (One way ANOVA) FEF 25%- 75% 3.57 ± 0.62 3.29 ± 0.55 2.87±0.75 F=5.98; p<0.01 4.07 ± 0.75 3.76 ± 0.65 3.43 ± 0.97 F=3.19; p<0.05 4.22 ± 1.36 3.94 ± 1.18 3.15 ± 1.23 F=4.34; p<0.05 3.19 ± 0.61 3.33 ± 0.76 3.16 ± 0.7 NS 3.65 ± 0.71 3.85 ± 0.84 3.69 ± 0.89 NS 3.98 ± 1.67 3.67 ± 1.46 3.61 ± 1.30 NS 23 Annual Report 2007-08 workers had duration of work of 6-10 years. Almost twenty nine percent of workers were having experience of up to 5 years of work. 2. HSDB. 2003. Hazardous Substances Database. National Library of Medicine. http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen/HSDB. So far as morbidity is concerned, 5.7 % of the workers were suffering from frequent cough during work, 2.9% from backache and 2.9% from joint pain. Headache (5.7%), eye irritation (2.9%) and nasal irritation (2.9%) during or immediately after work were the other complaints of the workers. Pulmonary function test revealed that 11.4% workers were having obstructive disorder, 2.9% were having restrictive abnormality and rests were normal. Significant effect of age (Table 1) on pulmonary function test parameters' values was observed but no such significant effect was observed in relation to work experience. Radiological findings suggestive of Koch's infection (Right Upper Zone) were observed in two workers. One of them had rightsided pleural effusion also. 3. Pigg BJ (1994). The uses of chrysotile. Ann Occup Hyg 38: 453458. 4. Agency for Toxic Substances and Disease Registry (June 2006). Asbestos:Health Effects http://www.atsdr.cdc.gov/asbestos/ Environmental monitoring was carried out at four sampling locations. Fiber concentrations are shown in Table 2. Permissible Exposure level (PEL) in India is 1 fiber/ml whereas other agencies like NIOSH, ACGIH and OSHA recommend 0.1 fiber/ml. It can be seen from table 2 that fiber concentrations are lower than PELs. Table 2: Mean Fiber Concentration (fibers/ml) at different locations Location No. of Fiber levels Samples (Mean±SD) Fiber Feeding 12 0.0382 ± 0.0224 Calendermachine 12 0.0135 ± 0.0128 Cutting/finishing 6 0.0107 ± 0.0071 Near godown 6 0.0217 ± 0.0114 asbestos/health_effects/index.html. 5. http://www.atsdr.cdc.gov/toxprofiles/phs61.html. 6. National Toxicology Program. Report on Carcinogens. Eleventh Edition. U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program, 2005. 7. U.S. Environmental Protection Agency. Health Effects Assessment for Asbestos. U.S. Environmental Protection Agency, 1984. EPA/540/1-86/049 (NTIS PB86134608). 8. International Agency for Research on Cancer. Asbestos. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 14. Lyon, France. http://monographs.iarc.fr/ENG/Monographs/vol14/volume14.pdf. 9. Conclusion Fiber level in all the workplaces were below the national and international standards. Only three percent workers were found to have restrictive type of pulmonary function impairment. No worker was found to have radiographic features suggestive of interstitial lung fibrosis. Harvey G, Page M, Dumas L. Binding of environmental carcinogens to asbestos and mineral fibres. Br J Ind Med 1984, 41: 396-400. 10. Gerde P & Scholander P (1989). A model for the influence of inhaled mineral fibers on the pulmonary uptake of polycyclic aromatic hydrocarbons (PAH) from cigarette smoke. In: Wehner AP & Felton DV ed. Biological interaction of inhaled fibers and cigarette smoke. Richland, Washington, Battelle Pacific, pp 97120. 11. Murlidhar V, Kanhere V. Asbestosis in a composite mill at Mumbai: a prevalence study. Environmental Health: A Global Access Science Source 2005; 4:24. Acknowledgment: We are thankful to Idrish Shaikh, S Dodia and M Joshi for the help rendered during the study. References 1. Agency for Toxic Substances and Disease Registry (September 2001). Public Health Statement for Asbestos. ATSDR. 2001. Toxicological Profile for Asbestos. NTIS Accession No. PB/2001/109101. Atlanta, GA: Agency for Toxic Substances and Disease Registry. 146 pp. 24 Annual Report 2007-08 Respiratory Response to Tobacco Dust Exposure in the Bidi Binders BP Chattopadhyay, PK Gangopadhyay, AK Mukherjee, DS Munda, S Das and SM Hossain Abstract Bidi industry is one of the country's oldest home-based industries in the unorganised sector. Bidi making is a skilled job. The occupational stress is associated with long hours of work in sitting posture, and exposure to tobacco dusts. In the present study total 198 bidi binders (Male-109 and Female-89) were investigated. A detailed history of illness was collected and clinical examination was performed. The pulmonary function status was made by spirometric method. Slow Vital capacity (SVC) and forced vital capacity (FVC) was recorded spirometrically and peak expiratory flow rates by Wright's peak flow meter. All other lung volumes (SVC, FVC, FEV1) and flow rates (FEF0.2-1.2ml, FEF25-75% and FEF75-85%were calculated from the same tracings. Blood samples were taken from 169 bidi binders' for IgE and total and differential count of leucocytes. Total dust concentrations of the bidi manufacturing process are between 0.49 to 1.09 mg/m3, and respirable dust was between 0.26 - 0.68 mg/m3, which are below the ACGIH TLV of nuisance dust. About 53.7% of the workers complained of cough; chronic bronchitis in 41% and breathlessness by 39% binders. The lung volumes and flow rates of the male bidi binders are significantly higher compared to the females. The PFT values are gradually reduced as the duration of work exposure increased. The mean eosinophil count and IgE level were found higher than normal range in both sexes. As a whole 25.7% male and 11.2% female bidi binders had respiratory function impairments. The occurrence rate is much higher among the males compared to females. The respiratory impairments among the bidi binders may be due to their exposure to tobacco dust during the work. Keywords: Bidi binders, Tendu leaf, Pulmonary function tests, Spirometry breathlessness were reported symptoms of the women bidi workers . Introduction 3 Bidi industry is one of the country's oldest home based industries employing over 3 million workers consisting of males, females and children. There are different processes involved in bidi making. About 85% of World's bidi production is coming from 290,000 bidi making 1 units in India . Bidi is manufactured in different states like West Bengal, Kerala, Karnataka, Andhra Pradesh and Madhya Pradesh. On an average, each worker can roll from 400 to 1000 bidis per day depending on individual skill and time. The ventilatory capacities of tobacco factory workers in Libya decreased in comparison with age matched unexposed workers4. The respiratory symptoms and lung function impairment in workers in a 5 tobacco processing plant was evidenced in Croatia The health status of the workers exposed to tobacco dust showed a fairly high frequency 6 of respiratory diseases . In bidi binders the occupational stress is associated with long hours of work in floor sitting posture, and exposure to tobacco dusts 7 and poor ill ventilated working conditions. Gopal reported the main symptoms among the female bidi workers are backaches, neck aches, headaches, burning senses on of eyes, pain in the legs and numbness of the fingers, cough, giddiness, breathlessness, urinary burning sensation, white discharge, joint pains and swelling etc. Keeping in view the hazards of tobacco dust, the present study was under taken to investigate respiratory health status among the bidi binders. The air borne tobacco dust and microbes generated during bidi processing is inhaled by the workers. These are the probable risk factors of causing respiratory disorders. Studies carried out on tobacco processing workers reported symptoms like headache, 2 nausea, giddiness, vomiting, tiredness, loss of appetite etc . It revealed that the rate of urinary excretion of nicotine and cotinine was high in exposed workers. Backache, headache, pain in the neck, pain in the hands & legs, cough with or without expectoration, 25 Annual Report 2007-08 Materials and Methods Bidi making is a skilled job. Bidi is an indigenous cigarette in which tobacco is rolled in a processed tendu leaf and tied with a cotton thread. The rolled bidis are bundled and then dried by using a Chula for 10 - 15 minutes. The tendu leaves are moistened and then cut into piece of required size and shape by the workers with the help of scissor. Processed tobacco is mixed up by hand and then it is wrapped and rolled in the cut piece of tendu leaf in such a way that one end is broader and the other end is narrower and that end is tied with thread so as to maintain shape and tobacco should not come out. Environmental Study: Work place environmental monitoring in five bidi manufacturing units was carried out at Mograhat areas of West Bengal. Assessment of personal exposure to respirable dust carried out by attaching personal sampler with respirable sampling head to the workers and sampling for 2-3 hours at a rate of 2 litres per minute (LPM) during the work. Work place area was also monitored for total dust levels by placing a high volume dust sampler in the processing and bidi binding area with a 4.7 mm sampling head on the Glass fibre filter paper at a rate of 28.36 LPM for 2-3 hours consecutively for two days. During the course of sampling, thermal parameters like dry bulb (DB), wet bulb (WB), natural wet bulb (WNB), globe temperature (GT) were recorded and wet bulb globe temperature (WBGT) in the work areas were calculated. Medical Study: In the present study 198 bidi binders (Male-109 and Female-89) were investigated. Mograhat area was chosen considering the approachability and assurance of participation of the subjects voluntarily. A questionnaire, tested and validated in field condition, was administered to each participant. Prior to administering questionnaire written consent was obtained from each subject for his or her voluntary participation that was required for clinical examination. The data were analysed using Epi Info software (WHO). surface area (BSA) was calculated from the Du-Bois and Du-Bois 11 formula . Pulmonary function test values were predicted from the standard prediction equation of the Kolkata normal subjects12. The criteria followed for categorization of the severity of restrictive impairments is based upon the ratios between predicted and observed values of SVC and obstructive impairments based on the observed 12 FEV1% . Immunological Study and Leucocyte Count: Blood samples were taken from 169 bidi binders for immunological study that is for IgE and total and differential count of leucocytes. Immunoglobulin E (IgE) enzyme immunoassay was done by enzyme linked Immunosorbent assay (ELISA) method13 by taking the serum from the blood sample and using the test kit of Biocheck Inc. Burlingame. Leucocytes count was made by standard method using Eosin and Haematoxylin stain. Results and Discussion The samples collected during area monitoring within the bidimanufacturing units (Table-1) showed total dust concentrations are 3 3 0.49-1.09 mg/m , which are below the ACGIH TLV of 10 mg/m , the TLV of total nuisance dust as there is no separate TLV for the tobacco dust. The personal exposure of the workers engaged in the bidi processing units to respirable dust was between 0.26 - 0.68 mg/m3. The exposure level of respirable dust was also found below the permissible standard of nuisance dust. In the manufacturing process there are heating operations for drying and baking of the rolled bidis which release pungent smell and fumes in the work areas. Pulmonary Function Tests: The pulmonary function status of the bidi binders was assessed by spirometric method. Slow Vital capacity (SVC) and forced vital capacity (FVC) was recorded spirometrically (Chest Graph-801, Tokyo, Japan) and peak expiratory flow rates by Wright's peak flow meter (Clement & Clark, UK). Other lung volumes and flow rates were measured from the same tracing. The smoking history including frequency of smoking of the subjects were recorded. Bidi binders are divided into non-smokers, smokers and left ex8 smokers . The personal history, present duration of work along with their past and present work history was recorded9. The PFT measurements were made in a comfortable standing position and body height and body weight was measured by a standard scale without footwear. All measured lung volumes obtained were expressed in body temperature pressure saturated with water vapour (BTPS) 10. Body 27 Table 1: Static air and personal exposure of workers to respirable dust in the bidi binding units Bidi binding unit 1 2 3 4 5 Static air concentration Personal exposure to of total dust (mg/m3) † respirable dust (mg/m3) # 1.09 0.26 ± 0.19, (0.08 - 0.56) (n=4) 0.80 0.39 ± 0.13, (0.20 - 0.49) (n=5) 0.63 0.52 ± 0.69, (0.05 - 1.89) (n=5) 0.62 0.33 ± 0.11, (0.24 - 0.48) (n=5) 0.49 0.68 ± 0.30, (0.32 - 1.13) (n=5) †: High Volume Sampling. #: Low Volume Sampling, Figures in the parenthesis are ranges, n = number of sampling Table 4: Distribution of musculo-skeletal symptoms as reported by the subjects Bidi Binders Symptoms Male Female Total (n=109) (n=92) (n=201) Headache 19 (17.4) 18 (19.6) 37 (18.4) Pain in Neck 72 (66.1) 72 (78.3) 144 (71.6) Joint Pain 67 (61.5) 47 (51.1) 114 (56.7) Low Back Pain 80 (73.4) 72 (78.3) 152 (75.6) Pain in Hands and Legs 19 (17.4) 7 (7.6) 26 (12.9) Neuropathy 87 (79.8) 58 (63.0) 145 (72.1) Table 2: Meteorological parameters recorded in the different bidi-binding units Bidi binding No. of DB (ºC) % RH WBGT (ºC) 23.7 ± 0.2 20.8 ± 7.2 20.8 ± 7.1 20.6 ± 7.4 25.2 ± 3.4 51.3 ± 14.6 48.9 ± 18.7 48.4 ± 19.0 48.8 ± 20.2 48.8 ± 8.5 21.7 ± 0.7 19.5 ± 6.5 19.5 ± 6.5 19.3 ± 6.8 23.4 ± 2.6 Table 2 shows the measurements of thermal parameters in these manufacturing units. The maximum value of WBGT temperature obtained was 23.4 ºC in the manufacturing unit 5 and the minimum was 19.3 ºC in the manufacturing unit 3. The WBGT temperatures in all these manufacturing units were below the WBGT TLV of 30 ºC for light nature of continuous job as recommended by ACGIH. The dust levels measured in the bidi processing units are within the limit for the nuisance dust. Among the total workers studied the average age were 48.25 ± 12.82 years. The mean height and weight were 155.87 ± 7.81 cms and 48.00 ± 8.95 Kgs respectively. Majority of them were literate more in case of males. The general symptoms of the subjects are presented in Table 3. Weakness, chronic diarrhoea and dyspepsia were more prevalent in male, whereas giddiness showed higher prevalence in females. About 53.7% workers complained of cough and chronic bronchitis was noted in 40.8% subjects. Breathlessness was complained by substantial number of subjects (39.3%). Rhonchi were detected in 10.4% of the subjects. These symptoms and signs were present in higher frequencies in male subjects. Incidentally a large number of male workers were smoker. Inhalation of tobacco dust and smoking might be contributing in developing these complaints. The symptoms related to musculoskeletal system is summarised in Table 4. Pain in neck, low back pain and joint pain were highly prevalent. Typical posture of bidi rolling might be a probable cause of these ailments. Presence of headache, Burning and itching of eye, conjunctival redness and dimness of vision were the other complaints of the workers. Table 3: Distribution of general symptoms as reported by the subjects Symptoms Bidi Binders Male (n= 109) Female (n = 92) Weakness 64 (58.7) 49 (53.3) Giddiness 40 (36.7) 41 (45.1) Chronic Diarrhoea 45 (41.3) 29 (31.5) Dyspepsia 60 (55.0) 46 (50.0) Values in the parenthesis indicate percentage Total (n= 201) 113 (56.2) 81 (40.5) 74 (36.8) 106 (52.7) BMI range (kg/m2 ) Values in the parenthesis indicate percentage Nutritional deficiency disorders like anaemia (17.9%), angular stomatitis (57.2%) and glossitis (33.3%) were also prevalent. Glossitis and angular stomatitis prevalence was more in males whereas anaemia more in females. Other symptoms like loss of appetite, feeling of cramps, etc. were also present among the bidi binders. About 5.2% workers were hypertensive (>140/90 mm of Hg; male 6.4%, female 4.3%). Most of the symptoms and signs may be sequelae of handling tobacco, awkward posture and nutritional deficiency. The lung volumes (SVC, FVC, FEV1, FEV1%) and flow rates (FEF0.21.21ml, FEF25-75%, FEF75-85%, PEFR) of the male bidi binders were significantly higher compared to the females. The pulmonary function test (PFT) values were low in females compared to male bidi binders. In male bidi binders there was a gradual decrement of PFT values as the age increases. Female bidi binders in the age range 20 yrs had low lung volumes and the flow rates compared to the age range (21-30 and 3140yrs). Male bidi binders had higher PFT values, compared to females. The PFT values of the bidi binders (Table 5) were divided according to their BMI into three categories, that is <18.5, 18.5 to <25 and ³ 25. The PFT values of the male bidi binders are less in <18.5 category, compared to other two categories. Only male bidi binders are smokers, who were divided into nonsmokers, smokers and ex-smokers, according to their smoking habit (Table 6). The mean values of all pulmonary function parameters were significantly higher in non-smokers followed by smokers and exsmokers. The work exposure of the bidi binders of both sexes were divided into <10 yrs, 11-20 yrs, 21-30 yrs, 31-40 yrs, 41-50 yrs and ³ 50 years. No female subjects had working history more than 31-40 years and above. The PFT values are gradually reduced as the duration of work exposure increased from 11-20 years onwards. The PFT values of 28 F e m a le (n=89) observation 3 11 5 5 12 Table 5: Pulmonary function test values of the male and female bidi binders according to BMI range (Mean ± SD) FEV 1 (l) FVC (l) SVC (l) FEV 1% FEF.2 -1.21 (l/sec) FEF25 -75% (l/sec) FEF 75-85% (l/sec) PEFR (l/min) <18.5 (n=46) 2.70 ±0.66 2.67 ±0.67 1.17 ±0.71 80 ±10.96 4.07 ±1.84 2.38 ±1.31 0.83 ±0.50 350 ±105.89 18.5 to <25 (n=58) 3.02* ±0.65 2.91 ±0.71 2.42*** ±0.66 83 ±8.21 5.06** ±1.88 2.74 ±1.32 0.89 ±0.48 393* ±91.01 >=25 (n=5) 3.18 ±0.36 3.13 ±0.38 2.63 ±0.18 ### 84 ±6.03 6.46 ±1.23 3.02 ±0.74 0.85 ±0.20 446 ±49.30 <18.5 (n=26) 2.40 ±0.41 2.35 ±0.41 2.14 ±0.41 91 ±9.12 3.70 ±1.02 2.91 ±0.78 1.33 ±0.64 324* ±63.57 18.5 to <25 (n=57) 2.29 ±0.37 2.21 ±0.37 1.93* ±0.40 87 ±10.82 3.21 ±1.13 2.49* ±0.89 1.04* ±0.51 329 ±56.36 >=25 (n=6) 2.39 ±0.65 2.19 ±0.86 1.81 ±0.78 87 ±11.77 3.22 ±1.97 2.39 ±1.07 1.04 ±0.43 303 ±51.64 *p<0.05, **p<0.01, ***p<0.001 when compared with corresponding earlier BMI range ## ## <0.01, ###p<0.001BMI <18.5 Vs >=25 Table 6: Pulmonary function test values of the male control subject and bidi binders according to smoking habits (Mean ± SD) SVC (l) FVC (l) Non -Sm (n=19) 3.13 ±0.60 3.10 ±0.65 2.70 ±0.64 87 ±7.97 Sm (n=78) 2.89 ±0.64 2.82 ±0.64 2.31 * ±0.62 81 ** ±8.18 Ex -Sm (n=12) 2.56 ±0.80 # 2.35 ±0.85 Smoking category M a le (n=109) unit 1 2 3 4 5 Annual Report 2007-08 M a le (n=109) Annual Report 2007-08 FEV1 (l) @## FEV1% @ ## # 1.83 ±0.82 77 ±15.11 FEF.2 -1.21 (l/sec) FEF 25 -75% (l/sec) FEF 75 - 85% (l/sec) PEFR (l/min) 5.65 ±1.38 3.43 ±1.11 1. 19 ±0.63 429 ±66.07 4.61 * ±1.88 2.48 ** ±1.27 0.82 ** ±0.42 376 * ±96.40 ## 315 ±120.93 # 3.76 ±2.55 ## 2.10 ±1.34 0.64 ±0.35 ## Level of significance* p<0.05, **p<0.01 Non-smoker compared with the smokers. @P<0.05 @P<0.01 Smoker compared with the # ## Ex-smokers p<0.05, p<0.01 Non-smoker compared with the Ex-smokers the male bidi binders were less compared to the 11-20 years exposure group, but in females, no such trend was observed. The Hb, total RBC and WBC count of the male and female bidi binders were within normal range. The eosinophil count was higher than the normal range in male and female bidi binders. The serum IgE level was found to be higher in both male and female bidi binders compared to the normal range (0-200 IU/ml). The bidi binders are divided into the three groups according to their serum IgE level, 0-200 IU/ml, 200-400 IU/ml and >400 IU/ml. The maximum number of bidi binders had >400 IU/ml of serum IgE level. The PFT values in male bidi binders were found to be higher in the low IgE level compared to the high IgE level but in females, higher IgE level group had high PFT values. The pulmonary function impairments are of restrictive (7.34%), obstructive (11.01%) and combined (restrictive and 29 obstructive) type (7.34%) among the male bidi binders. The corresponding values in females are 3.37% and 7.87% but there was no combined type of impairment. As a whole, 25.7% male bidi binders and 11.24% female bidi binders had respiratory function impairments. The pulmonary function impairments are shown in Figure 1. Among the bidi binders, 5.26% of the non-smokers had restrictive and obstructive type of impairments (Table 7). Among the smokers, the impairment was 24.4% and in ex-smokers 58.3%, but in non-smokers it was 10.5%. Mustajbegovic et.al18 reported that the ventilator capacity in tobacco workers showed a reduction in FEV1, FEF50% and FEF25% in relation to predicted values. In the present study reduction of lung volumes i.e., SVC, FVC, FEV1, FEV1%, flow rates were noted in bidi Annual Report 2007-08 Annual Report 2007-08 were higher among the male bidi binders (25.7%) and the impairments were comparatively low among the female bidi binders (11.24%). 11. Du-Bois D, Du-Bois EF. Clinical calorimetry. A formula to estimate the approximate surface area if height and weight be known. Arch. Intern. Med 1916; 17:868-871. 12. Chatterjee S, Saha D, Chattopadhayay BP. Pulmonary function studies in healthy non-smoking men of Calcutta. Annals of Human Biology. 1988; 15: 865-874. Acknowledgment: The investigators thankfully acknowledge cooperation of the bidi binders and the technical assistance rendered by SK Bhattacharya, J Alam, SK Roy, M Chatterjee, D Chakraborty, and T Dasgupta. This project was partially supported by the Department of Science and Technology, Govt. of West Bengal. The authors thankfully acknowledge the co-operation rendered by Mondal of Mograhat gram Panchyet, Kalo Gazi, Jugal Baidya and other members of the gram panchyet. 30 25 13. Engvall E, Perlmann P. Quantitation of specific antibodies by enzyme linked anti immunoglobulin in antigen-coated tubes. J Immnol 1973,109: 129-135. rd 14. Morgan WKC, Seaton A. Occupational Lung Diseases. 3 Edition, Philadelphia, London, Toronto, Montreal, Sydney, Tokyo, W.B. Saunders Company 1975 p79. References Bidis more lethal than cigarettes: Health Ministry, The Statesman, Kolkata edition, 14th May 2008. 15. Ignacak A, Guizik TJ, Gorski L, Czerniawska-Mysik G, AdamekGuzik T: Influence of tobacco dust on the respiratory system and selected immunological parameters. Przegl lek. 2002; 59(10): 789-792. 2. National Institute of Occupational Health, Ahmedabad, Occupational Health survey of Women Workers engaged in Bidi making (rolling). Annual Report 1980. 16. Mahimkar MB, Bhisey RA: Occupational exposure to bidi tobacco increases chromosomal aberrations in tobacco processors. Mutat Res. 1995; 334(2): 139-144. 3. National Institute of Occupational Health, Ahmedabad, Occupational Health survey of Women Workers engaged in Bidi making (rolling), pp 78-86. Annual Report 1983-84. 17. Bagwe AN, Bhisey RA: Occupational exposure to tobacco and resultant genotoxicity in bidi industry workers. Mutat Res. 1993; 299(2): 103-109. 4. Mengesha YA and Bekele A: Relative chronic effects of occupational dust on respiratory indices and health of workers in three Ethiopian factories. Am J Ind Med 1998; 34: 373-80. 18. Mustajbegovic J, Zuskin E, Schachter EN, Kern J, Luburic- Milas M, Pucarin J.: Respiratory findings in tobacco workers. Chest; 123 (5): 1740- 1748. 5. Imbus HR: Clinical aspects of occupational medicine. In: Occupational medicine. Edited by Carl Zenz, O. Bruce. Dickerson, Edward, P. Horvath, JR, Third edition, Mosby, London, 1994; 3. 19. Osim EE, Musabayane CT, Mujamda J. Lung function of Zimbabwean farm workers exposed to flue caring and stacking of tobacco leaves. S Afr Med J. 1998; 88(9): 1127-1133. 6. Umadevi B, Swarna M, Padmavathi P Jyothi A, Reddy PP. Cytogenetic effect in workers occupationally exposed to tobacco dust; Mutat. Res. 2003; 535(2): 147-154. 20. Valic F, Beritic D, Butkovic D. Respiratory response to tobacco dust exposure. Am Rev Respir Dis 1976; 113(6): 751-755. 1. Figure 2 : Respiratory impariments of male and female bidi binders Table 7: Respiratory impairments of male bidi binders according to smoking habit Smoking Habit Restrictive Obstructive Male Non-smoker (n=19) 1 (5.26) 1 (5.26) Bidi Smoker (n=78) 5 (6.41) 8 (10.26) binders (n=109) Ex-smoker (n=12) 2 (16.67) 3 (25.00) Combined _ 6 (7.69) 2 (16.67) Gross Total 2 (10.52) 19 (24.36) 7 (58.33) Values in parenthesis are percentages tobacco dust might have negative health effects in female workers. The respiratory symptoms reported by the present female bidi binders are more compared to male, which corroborates the study of Viegi22. There was a gradual decrement of pulmonary functions as their duration of work exposure increased. The PFT values are found higher in the low IgE level bidi binders compared to the high IgE level. binders. Smoking bidi or cigarettes got a profound effect on PFT values and smokers had much less PFT values, compared to the nonsmokers8. Osim et.al19 evidenced that the FEV1% of the tobacco farm workers was different from that of the control. The present study showed that as the duration of exposure increases there was a decrement of lung volumes and flow rates in male and female bidi binders. The respiratory symptoms were more in female bidi binders compared to males. The low prevalence of respiratory symptoms was found in non-exposed persons20. Kjaergaard et.al21 stated that higher prevalence of cough and shortness of breath was present among the tobacco workers. Their results highly corroborated with the results of the present study. Viegi 22 established that prolonged exposure to 23 Mukhtar et.al. stated that the small airways are affected by exposure to tobacco dust. Huuskonen et.al24 reported a tendency towards restriction and obstruction especially in small airways of tobacco industry workers and the diffusion capacity also decreased in 18% of the workers. In the present study the respiratory impairments 30 7. Gopal. M. Health of women workers in the bidi industry. Occu India (2000) Medico Friends Circle Bulletin, Jan-Feb 2000. 21. Kjaergaard SK, Pederson OF, Frydenberg M, Schanheyder H, Anderson P, Bande GJ. Respiratory disease and lung function in a tobacco industry. Arch Environ Health. 1989; 44(3): 164-170. Chattopadhayay BP, Alam J. Spirometric function of ventilatory function of non-smokers and different graded of smokers of Calcutta. Ind J Environ Protec. 1996 14: 274-279. 22. Viegi,G, Paggiaro PL, Begliomini E, Vaghetti E, Paoletti P, Giuntini, C. Respiratory effects of occupational exposure to tobacco dust. Br J Ind Med. 1986; 43(12): 802-808. 9. Chattopadhayay BP, Saiyed HN, Alam J, Roy SK, Thakur S, Dasgupta TK. Injury into occurrence of Byssinosis in Jute mill workers. J Occup Hlth 1999; 41: 225-231. 23. Mukhtar, MS, Rao GM, Gamra NS, Afan AM, Zendah MI. Respiratory effects of occupational exposure to tobacco dust. Respiration; 1991; 58(5-6): 271-276. 10. Cotes JE. Lung function assessment and application in Medicine3rd Edition, Oxford, Blackwell Scientific Publication, 1976. 24. Huuskonen, MS, Husman K, Janvisalo J, et al. Extrinsic allergic alveolitis in the tobacco industry. Br J Ind Med. 1984; 41(1): 77-83. 8. 31 Annual Report 2007-08 Studies on Hydrocarbon Chain (C6-C9) Characteristics of Benzene and Alteration of Heme-Metabolism in Lymphocytes of Human Exposed to Benzene and Substituted Benzene AK Mukherjee, SK Bhattacharya and SK Roy Abstract The study was undertaken for exposure assessment of eight C6 - C9 aromatic hydrocarbons in and around the petrol pumps, biological monitoring of urinary metabolites among the exposed and to correlate the exposure of benzene and substituted benzene with the early health effects. Environmental levels of aromatic hydrocarbons were collected in charcoal sorbent tubes and analysed by GC, FID technique. The urinary metabolites were estimated by reverse phase HPLC. The personal exposure to PM10 observed in the range, 174-960 µg/m3 and the levels at the immediate outdoor air around petrol pumps were between, 176-401 3 µg/m . The PM10 around the petrol pumps having particles, 18.3 to 53.3 % of < 4.7 µm size and 1.4 to 20.1% particles, below 1.1 µm size. The exposures to toluene were highest (AM, 735.7 µg/m3; (GM, 500.2 µg/m3) among all the hydrocarbons; followed by benzene (AM, 199.1 µg/m3 & GM, 145.7 µg/m3), ethyl benzene (AM, 152.7 µg/m3 & GM, 119.9 µg/m3), o-xylene (AM, 112.3 µg/m3 & GM, 109.6 µg/m3), m-, p-xylenes (AM, 122.1 3 3 3 3 µg/m & GM, 85.1 µg/m ) combined, propyl benzene (AM, 73.5 µg/m ; GM, 62.1 µg/m ) and mesitylene (AM, 51.2 µg/m3; GM, 44.1 µg/m3). The outdoor air hydrocarbons also followed the similar trend but the levels were lower. The pre-shift and post-shift urinary tt-MA were 0.24 and 0.91 mg/g creatinine and for SPMA were 0.90 and 1.33 mg/g creatinine respectively. The urinary MA, HA and MHA in pre- and post-shift samples were 1.69 and 2.89 mg/g creatinine, 5.67 and 10.01 mg/g creatinine and 0.45 and 0.66 mg/g creatinine respectively. Air benzene levels, both in personal exposure and immediate outdoors were observed to be higher than the recommended levels of exposure. Air benzene level correlated significantly with pre- and post-shift urinary tt-MA and SPMA and the air ethylbenzene, toluene and xylene also correlated significantly with pre- and post-shift urinary MA, HA and MHA. The post-shift urinary metabolites were significantly higher than the pre-shift values in case of the tt-MA and MA among the petrol pump workers. A better correlation was observed between the personal exposure to benzene and relative excretion SPMA than tt-MA. A decreasing order in case of urinary excretion of the metabolites, HA, MA and MHA was noticed similarly with the sequence of environmental levels of hydrocarbons, toluene, ethylbenzene and xylene The post-shift urinary metabolites of workers were significantly higher than that of the occupationally unexposed population, particularly for tt-MA and HA. Keywords: Aromatic hydrocarbon, benzene, human exposure,particulate matter, tt-MA Introduction Petrol pumps are one of the major sources of VOCs pollution in ambient air of the urban localities. The petrol pump workers are exposed to these compounds, which might enter into the body by inhalation and skin absorption. The inhaled compounds (about 20%) are excreted through exhale air and more than 80% of the absorbed VOCs are metabolised in the body and excreted as urinary metabolites. Benzene produces a variety of urinary products like phenolic compounds, ttmuconic acid (tt-MA) and S-phenyl mercapturic acid (SPMA), which are 33 used as exposure marker of benzene at different levels. tt-MA and SPMA have been proposed to be the two urinary metabolites for biological monitoring of benzene exposure at levels below 5 ppm. SPMA has been proved to be a more reliable biomarker at level below 1 1 ppm than tt-MA due to its longer elimination half-life . Exposure to toluene occurs simultaneously with benzene and hippuric acid (HA) is well known indicator of toluene exposure2. Moreover, hippuric acid production by human showed significant inter- and intra-individual variation influenced by factors like diet, Annual Report 2007-08 intake of medicines, alcohol consumption etc.3,4. Xylene with its three isomeric forms (-o, -m and -p) is one of the major constituent of VOCs. It is reported that about 95% of absorbed xylene is metabolised in liver to methyl hippuric acid (MHA)5. Mandelic acid is a recognized biomarker of ethyl benzene6. Jang et al.7, reported significant correlation between urinary metabolites like mandelic acid and methyl hippuric acid for exposure to ethyl benzene and xylene respectively, along with the environmental concentrations of ethyl benzene and xylene. At low exposure both methyl hippuric acid and mandelic acid can be used as a indicator for commercial xylene exposure7. Muzyka et al8, observed the alteration of heme metabolism in lymphocytes of workers exposed to diesel fuel. Both ALA and PP levels have significantly increased among the exposed workers with respect to lymphocytes compared to the control. ALA synthase activity was 2.5 fold higher in lymphocyte of workers exposed whereas ferrochelatase activities have been found decreased, and protoporphyrin level elevated8. 9 Studies are limited in Indian cities. Mohan Rao et al , in their study of non-methane hydrocarbons in industrial locations of Bombay, observed benzene concentrations above 5 ppb at four of the five chosen sites and its concentration was more than that of toluene9. Samanta et. al10., reported the levels of VOCs in road side air of Kolkata over a period of three years from 1992 to 1994 during winter10. A similar study in Delhi11 reported total ambient non methane VOC in 13 sites and the levels were correlated with traffic volume. The diurnal and 12 seasonal variations were also observed. Mukherjee et al conducted the exposure of drivers and conductors to benzene, toluene and xylene (BTX) in state transport buses of Kolkata12. Studies have been reported on behavioral pattern, pulmonary function status and biological monitoring among the petrol pump workers in India13 -16. In the light of the literature review the present report incorporates the environmental assessment of C6 - C9 hydrocarbons around the petrol pumps, personal exposure of the workers to C6 - C9 hydrocarbons (e.g. benzene, toluene, ethyl benzene, o-, m-, -p-xylene and n-propyl benzene) at work and biological monitoring of these compounds in urine of the workers. The urinary metabolites, namely, tt-muconic acid, S-phenylmercapturic acid (SPMA), mandelic acid (MA), hippuric Acid and methyl hippuric acid were studied. Materials and Methods Ten petrol pumps, located in North and Central Kolkata were selected for personal exposure assessment of workers in the petrol pumps with respect to respirable dust (PM10) and mono aromatic C6 - C9 hydrocarbons namely, benzene, toluene, ethylbenzene, o-, m- and pxylenes, n-propanyl benzene, isopropanyl benzene. Environmental 34 monitoring of these components at the immediate air around the petrol pumps was undertaken. All the petrol pumps are located within the busy traffic zones and are situated by the side of main roads. Biological monitoring of the C6 - C9 hydrocarbons in urine were carried out among the petrol pump workers. The urinary metabolites, trans, trans- muconic acid (tt-MA), Sphenylmercapturic acid (SPMA), hippuric acid (HA), methyl hippuric acid (MHA) and mandelic acid (MA) were estimated. Environmental study: Static air sampling: Air sampling for static air PM10 was undertaken in the outdoor air at a height of 7 to 10 feet from the ground level by a 8-stage cascade impactor (1 ACFM) for a period of 6 8 hours at rate of 1 Cubic Feet per Minute (CFM). Three consecutive days samples were collected on the same filter paper in each site. The gravimetric weight differences of the 8-impactor stages (9.0, 5.8, 4.7, 3.3, 2.1, 1.1, 0.7 and 0.4 µm.) were used for estimation of dust concentration and particle size distribution. Monitoring of the hydrocarbons in static air around were carried out at the outdoor static air by using a Air Check low volume sampler, model 224-PCXR4, SKC Inc. Air samples were collected in sorbent tubes (size, o.d. 6mm x length 70 mm.), packed with activated charcoal (60 80 mesh) in two compartments (100 / 50 mg) and kept in holder connected to a constant flow pump, by drawing air at a rate of 0.1 LPM for a maximum period of 3 - 4 hours within the period of 9 AM to 5 PM. The flow rate of air sampling pump was calibrated regularly before and after sampling by using a primary airflow calibrator (Ultra Flow, SKC, USA). Personal Exposure: The study included both the, filler group and non-filler group of workers for exposure monitoring in petrol pumps. The filler group is engaged to fill the fuel (diesel / petrol) into the vehicles and the non-filler categories in the petrol pumps donot fill fuels into vehicles, e.g., manager, cashier and other supervisory staff. Personal exposure monitoring of 38 petrol pump workers were done for the aromatic hydrocarbons by attaching a personal sampler (Air Check low volume sampler, model 224-PCXR4, SKC Inc.) to the lapel of the worker fixed with an activated charcoal sorbent tube under similar sampling conditions. Gas chromatographic analysis of VOCs: The VOC samples collected in activated charcoal tubes were desorbed in carbon disulfide (CS2) for 1 - 2 hour and analysed for benzene, toluene, ethylbenzene, o-,m- and p-xylene, n-propanyl benzene, isopropanyl benzene. Estimations were done by using a Gas Chromatograph, model, Auto System XL GC (Perkin Elmer) attached with a flame ionization detector (FID) and a capillary column, length 30 m, i.d, 0.53 mm., PE 624 (Perkin Elmer). Annual Report 2007-08 extraction (SPE). The tt-MA, SPMA, MA, HA calibration curves showed the value of co-efficient of determination (R2), 0.995 or more except SPMA (R2 = 0.919). The percentage recovery of each metabolite was studied in the spiked normal urine samples of occupationally nonexposed group. The quantification of individual hydrocarbons in the samples was done by using a seven point calibration curve of different concentrations of the standard compounds prepared in CS2. Fluorobenzene was used as internal standard to avoid injection error and trace amount of benzene contamination in the solvent. The retention times (RT) obtained for benzene, fluoro benzene, toluene, ethylbenzene, m- & p-xylene, o-xylene, n-propanyl benzene and mesitylene were 7.16, 8.32, 13.46, 18.52, 19.31, 20.75, 23.78 and 24.50 minutes respectively. All the calibration curves showed the value of co-efficient of determination (r2), 0.995 or more. Results and Discussion The petrol pump workers included in the study are mostly come from the outskirts of Calcutta and belong to lower socio-economic status and low educational background. This occupation was classified as low-skilled but with high risk of benzene exposure along with co-exposure to other aromatic volatiles25. Out of the total 31 exposed subjects, 9 subjects (25.8 %) were reported to be smoker in the category, 1 - 10 cigarettes per day, 1 subject (3.2%) in the category, >11 - 24 cigarettes per day and 22 subjects (71%) were non-smokers. Most of the workers are non-vegetarian. The mean age of these workers was 32.45 years, (range:17-71 years) The years of exposure, < 5 years, 20 subjects, > 5 - 10 years, 4 subjects and > 10 years 7 subjects. The mean body weight of the 31 subjects was 54.7 kg. (range:45-66 kg). Non-occupationally exposed group of 22 persons were selected as positive control. 20 subjects were males and only 2 were females. All the subjects were non-vegetarian with only 4 smokers. The average age of 22 subjects was 40.5 years (range:22-58 years). The mean weight of these subjects was 63.3 Kg. (range:48-84 kg). Biological monitoring of VOCs: Study subjects: The biological monitoring included an exposed population of 31 subjects from the petrol pumps. Besides, 22 subjects were included who belong to occupations involved in official and other jobs mainly, not exposed to the nature of conditions as the petrol pump workers. The details of the occupational history, age, weight, smoking habit, food habit, and year of exposure in the job were recorded. Spot urine samples were collected from the exposed workers in the morning hours before starting the work (Pre-shift) and also at the end of the shift (Post-shift). Spot urine samples of occupationally unexposed population were also collected. Analysis of urinary metabolites: The collected urine samples were undergone sample pre-treatment using the strong anion exchanger (SAX) column (500mg/3ml., Whatman) for solid phase extraction (SPE), attached with Vac-Elute vacuum elution system. Prior to extraction, the SAX cartridge was conditioned by washing with methanol (2 to 3 times), followed by washing with phosphate buffer (pH 6.4) and the elution rate was adjusted to 2 to 3 ml. 10ml urine-phosphate buffer mixture (1:1) was passed through SAX column adding 1ml. each time. The SAX column was cleaned by washing with water, then with phosphate buffer and 1.0% aqueous acetic acid. The column was dried. Finally, the analyte was eluted by 10% aqueous acetic acid to a volume 4 - 5 ml.6 The analysis was performed by a HPLC (Shimadzu, Japan) consisted of an HPLC system controller (Model : SLC-10 A, VP-Series), connected with an automated liquid sampler (Model: LC- 10AT, VP series), spectrophotometer (Model: SPD M 10A, Diode Array Detector), and column oven. The column used was ODS-2 Hypersil, 250mm x 4.6mm., particle size 5 mm. (Thermo). The mobile phase used was 0.1% Trifluoro Acetic Acid (TFA) in acetonitrile and 0.1% Trifluoro Acetic Acid (TFA) in water with a programmable time range, 0-50 min. at a flow rate of 1 liter per minute. Each metabolite was estimated by scanning the chromatogram at specific λmax. The tt-MA was measured at λmax, 263 nm, SPMA at 205 nm, MA at 202 nm, HA at 228nm and MHA at 202 nm. Table 1 shows the respirable dust (PM10) exposure of petrol pump workers and the static air concentration at immediate outdoor around the petrol pump. The mean PM10 level of the workers found to be very high (481.1 mg/m3) and the concentrations depended on factors like the location of the pump with respect to the traffic junction, incoming of vehicles to the pump for re-fuelling and meteorological conditions. The highest value of personal exposure (960 mg/m3) found to be alarmingly high. The mean PM10 level in the ambient air around the petrol pump was 248 mg/m3. The PM10 around the petrol pumps consists 18.3 to 53.3 % particles, < 4.7 mm size and 1.4 to 20.1% particles, below 1.1 mm. The particle size distribution found to vary from pump to pump depending on the size of the pump, its location and traffic volume near the pump. Table 1 : Personal exposures of the workers to PM10 and static air concentration of PM10 around the petrol pump The reverse phase high performance liquid chromatographic (HPLC) technique was used for estimation of the metabolites (e.g. ttMA, SPMA, MA, HA and MHA). A control urine-phosphate buffer mixture (1:1) was spiked with 6.25 mg/ml, 12.5 mg/ml and 18.75 mg/ml of standard mixture respectively and the each spiked urine was injected to the HPLC after the clean up procedure by solid phase * Parameters PM10 Concentrations (µg/m3) Personal exposure pump workers* (n) 12 Static air around petrol pump† (n) 15 of petrol 481.1 ± 237.8 (174.5 - 960.4) 247.7 ± 90.9 (176.0 - 401.2) † Personal sampling technique., Static air sampling technique, Values are mean ± SD, (range), n=number 35 Annual Report 2007-08 Table 2: Personal exposure and static air concentrations of Volatile organic compounds (VOCs) in and around different petrol pumps Type of samples Benzene µg/m3 Toluene µg/m3 Et. Benzene µg/m3 m-,p-Xylene µg/m3 o-Xylene µg/m3 Prop. benzene Mesytylene µg/m3 Personal Exposure 199.1±181.4 35 - 728 735.7±702.7 85 -2965 145.7* 122.1±110.1 26 - 403 85.1* 112.3±107.0 24 - 489 109.6* 73.5±60.8 30-345 62.1* 51.2±30.1 22-147 44.1* 34# 30# 28# 92.7±77.6 23-241 72.6±54.4 23-187 66.3* 69.7* 57.6* 11# 11# 10# Static air around petrol pump 500.2* 152.7±119.0 33 - 590 119.9* 37# 38# 34# 29# 110.9±60.1 40-238 443.7±304.0 86-980 120.3±70.3 22-268 137.0±128.1 22-419 96.5* 336.4* 98.5* 89.7* 11# 11# 11# 11# Values are expressed as AM±SD, Range; *GM = Geometric mean; number of samples 78.2±44.6 28-153 # 3 Table 2 shows the personal exposure of workers and static air concentrations of VOCs around petrol pumps. The arithmetic and geometric mean outdoor air (static air) concentrations found to follow more or less the similar trend but with values lower than that of personal exposure due to the aerial dilution. As the petrol pump workers perform their job in the open air, their exposure levels was expected to be much lower than the prescribed workplace ACGIH TLVs 3 for benzene (1.6 mg/m or 0.5 ppm), toluene (50 ppm, 188.14 mg/m ), 3 ethylbenzene (100 ppm, 433.54 mg/m ), xylene (100 ppm, 433.54 mg/m3) 17. But when personal exposure to benzene within the petrol pump region is compared to the Air Quality Standards prescribed in different countries, the value is about 5 to 40 times higher. The air benzene levels at the immediate(50 ppm, 188.14 mg/m3), ethylbenzene (100 ppm, 433.54 mg/m3), xylene (100 ppm, 433.54 Table 3: Levels of urinary metabolites of different aromatic hydrocarbons among the workers exposed in the petrol pumps Pre - shift Metabolites tt-MA Urine (mg/ L ) 0.41 ± 0.58 (0.04 – 1.84) (n = 20) Post - shift Urine -Creatinine (mg/g creatinine) 0.24 ± 0.30 (0.02 – 1.57) (n = 20) Urine (mg/ L ) 0.95 ± 0.39 * * (0.05 – 1.68) (n = 31) Urine - Creatinine (mg/g creatinine) 0.91 ± 0.51# # (0.03– 2.56) (n = 31) 1 10 ± 1.05 (0.09 – 3.37) (n = 26) 0.90 ± 0.92 (0.09 – 3.34) (n = 26) 1.29 ± 1.03 (0.11 – 4.14) (n = 33) 1.33 ± 1.58 (0.11 – 8.16) (n = 33) MA 1.85 ± 1.82 (0.16 – 6.99) (n = 21) 1.69 ± 1.86 (0.10 – 6.72) (n = 21) 2.98 ± 1.43 * (0.22 – 15.92) (n = 30) 2.89 ± 1.77 (0.28 – 12.55) (n = 30) HA 8.08 ± 7.15 (0.11 – 25.72) (n = 21) 5.67 ± 5.80 (0.17 – 20.30) (n = 21) 12.44 ± 10.68 (0.67 – 44.07) (n = 26) 10.01 ± 8.76 (0.72 – 35.35) (n = 26) MHA 0.52 ± 0 .49 (0.09 – 2.02) (n=18) 0.45 ± 0.43 (0.06 – 1.56) (n=18) 0.77 ± 0.67 (0.12 – 2.58) (n=20) 0.66 ± 0.65 (0.16 – 2.96) (n=20) SPMA Values are expressed as mean±SD; Range and number of samples; tt-MA = tt- Muconic Acid SPMA = S-Phenylmercapturic acid; MA = Mandelic Acid; HA = Hippuric Acid and MHA = Methyl Hippuric Acid. * p<0.05; ** p < 0.001; (When compared with urine metabolite results of Pre-& Post-shifts values of urine). # p< 0.05; # #p<0.001 (compared with urine metabolite results of Pre-& Post-shifts values with respect to creatinine) 36 # Annual Report 2007-08 mg/m3) 17. But when personal exposure to benzene within the petrol pump region is compared to the Air Quality Standards prescribed in different countries, the value is about 5 to 40 times higher. The air benzene levels at the immediate outdoor static air around petrol pump was more than 22 times higher than the prescribed European 3 Commission limit value for benzene (5 μg/m , annual average) and also found several times higher than the recommended Air Quality Standard of 5 ppb (16.25 μg/m3) as annual mean (Air Quality Standard, 2007) by the UK expert panel for benzene in air with a target to reduce to 1 ppb 18. The benzene levels at the immediate outdoor static air around petrol pump in Kolkata when compared to the reported values in different urban environment like residential area, traffic intersections, petrol pumps of Delhi, the values were comparable and particularly to that of the petrol pumps. The highest value obtained in case of the personal exposure of petrol pump workers of Kolkata, is much higher than the air benzene level reported at petrol pump of Delhi. The values obtained in this study are higher than the air benzene levels reported from the major cities of the world. Besides, the values of VOCs (e.g, benzene, toluene, Etbenzene, m-, p- xylene, and o- xylene) obtained in the petrol pump areas of Kolkata city are much higher than that of different areas in Delhi. The toluene levels were reported highest among all the VOCs in Delhi air as observed in the present study19. Table 3 presents the levels of urinary metabolites of the different aromatic hydrocarbons among the workers engaged in the petrol pumps. The values of the metabolites are expressed, both in terms of milligram per liter of urine and milligram per gram of creatinine. Several studies on estimation of urinary metabolites, like tt-MA, SPMA, MA, HA Table 4: Levels of urinary metabolites of different aromatic hydrocarbons among non-occupationally exposed population in and around Kolkata city Metabolites tt-MA (n = 20) SPMA (n = 21) MA (n = 20) HA (n = 14 ) MHA (n=17) Levels of urinary metabolites of non - occupationally exposed group. Urine (mg/ L ) Urine creatinine (mg/g creatinine) 0.31± 0.18 0.36 ± 0.20 (0.02 - 1.71) (0.03 - 2.06) 0.73 ± 0.56 0.73 ± 0.52 (0.05 - 5.16) (0.10 - 6.22) 1.58 ± 1.34 1.95 ± 1.67 (0.11 - 6.36) (0.11 - 6.36) 1.86 ± 1.91 3.00 ± 3.00 (0.12 - 4.39) (0.11 - 8.36) 0.70 ± 0.69 0.69 ± 0.65 (0.08 - 2.85) (0.07 - 2.37) and MHA reported as individual metabolite or combined with one or two. In the present study all the five metabolites are estimated together by same method after the solid phase extraction (SPE) using strong anion exchange (SAX) cartridge, followed by elution in minimum volume of 10% acetic acid 20. It was observed that the postshift values are significantly higher than the pre-shift values for the metabolites, tt-MA (p <0.001) and MA (p <0.05) among the petrol pump workers. The estimations of metabolites in the normal urine of occupationally unexposed subjects and the same urine spiked with standard metabolites (6.25 ppm) showed the recovery ranges, 82.0 Figure 1: Correlation between personal exposure to benzene (µg/m3) and relative excretion of tt-MA. Figure 2: Correlaton between personal exposure to benzene (µg/m3) and relative excretion of SPMA tt-MA = tt- muconic acid; SPMA = s-phenylmercapturic acid; MA = Mandelic Acid; HA = Hippuric Acid and MHA = Methyl Hippuric Acid 37 Annual Report 2007-08 98.4%, 81.3 104.5%, 91.8 100.8%, 80.5 104.5%, 80.5 96.4%, 81.4 92.8% respectively for tt-MA, SPMA, MA, HA and MHA. Urinary benzene metabolites, tt-MA and SPMA are well-established biomarkers of benzene exposure at level below 5 ppm. It is also reported that at exposure below 1 ppm (3.2 mg/m3) SPMA is very specific biomarker of benzene than tt-MA20. Estimation of these metabolites, tt-MA and SPMA simultaneously in this study revealed a better picture of benzene exposure among the petrol pump workers in the range, <1ppm to 5 ppm. The benzene levels both, personal exposure and static outdoor air, at the petrol pumps showed values much below 1 ppm. The lower values of tt-MA compared to SPMA may be accounted due to the fact that SPMA is comparatively more sensitive at levels < 1ppm (Table 3). It may also be observed in Figures 1 and 2 showing a correlation between personal exposure to benzene (g/m3) and relative excretion of tt-MA and SPMA respectively. Table 4 shows the urinary metabolites of aromatic hydrocarbons among the occupationally unexposed population. On comparing the levels of urinary metabolites in post-shift samples of exposed worker, engaged in petrol pumps to that of the occupationally unexposed population, it was observed that the levels of tt-MA (p< 0.001), SPMA (p< 0.05), MA (p< 0.001) and HA (p< 0.001), expressed in mg/L urine among exposed group were significantly higher. However, in case of the values of urinary metabolites expressed in mg/g creatinine, the levels of tt-MA (p< 0.001) and HA (p< 0.01) were found to be significantly higher. References 1. Boogaard PJ and Sittert NJV. Suitability of S-phenyl mercapturic acid and trans trans-muconic acid as biomarkers for exposure to low concentrations of Benzene. Env. Health Perspectives, 1996;104: Suppl. 6. 2. Duydu Y, Suzen S, Erdem N, Uysal H, Vural N. Validation of hippuric acid as a biomarker of toluene exposure. Bulletin of Environmental Contamination and Toxicology 1999; 63: 1, 1- 8. 3. Angerer, J. “Occupational chronic exposure to organic solvents.” XII. o-cresol excretion after toluene exposure”. Int. Archives of Occupational and Environmental Health 1985; 56 (4):, 323- 328. 4. J. Angerer, A. Kramer, Occupational chronic exposure to organic solvents XVI. Ambient and biological monitoring of workers exposed to toluene. International Achieves of occupational and environmental health, 1997; 69: 91-96. 5. Jacobson GA, and McLean S. Biological monitoring of low level occupational xylene exposure and the role of recent exposure. Ann. Of Occupational Hyg, 2003; 47: 4, 331- 336. 6. S J Shahtaheri, M Abdollahi, F Golbabaei, A Rahimi-Froushani, F Ghamari. Optimization of SPE for Analysis of Mandelic Acid as a Biomarker of Exposure to Ethyl Benzene. Iranian J Env Health Sci Eng, 2004, Vol.1, No.2, Vppo.l7.10,-N80. 7. J. Y. Jang, P.O. Droz, S. Kim, Biological monitoring of workers exposed to ethylbenzene and co-exposed to xylene. International Achieves Occup. Environ. Health, 2001, 74:31-37. 38 8. V. Muzyka, S. Bogovski, A. Viitak, T. Veidebaum, Alteration of heme metabolism in lymphocytes and metal content in blood plasma as markers of diesel fuels effects on human organism. The Science of the Total Environment, 2002; 286: 73-81. 9. AM Mohon Rao, GG Pandit, P Sain, S Sharma, T M Krishnamoorthy and KSV Nambi. Non-methane hydrocarbons in industrial locations of Bombay. Atmospheric Environ. 1997; 31: 7, 1077-1085. 10. Gautam Samanta, Gautam Chattopadhyay, Badal K Mondal, Tarit Roy chowdhuri, Partha P. Chowdhury, Chitta R. Chanda, Prabal Banarjee, Dilip Lodh, Dipankar Das and dipankar Chakraborty, Air pollution in Calcutta during winter A three year study; Current science , 1998; vol. 75 no. 2. 11. PK Padhy, C.K. Varshney, Total non-methane volatile organic compounds (TNMVOC) in the atmosphere of Delhi. Atmospheric Environment 34 (2000) 577-584. 12. A K Mukherjee, SK Bhattacharya, S Ahmed, SK Roy, Roychowdhury, A, S Sen “Occupational exposure of drivers and conductors to noise, heat dust and Volatile Organic Compounds in the state transport special buses of Kolkata City”. Transportation Research Part D Elsevier Science Ltd, 2003; 8: 11-19. 13. P Kumar, BN Gupta, KP Pandya, SH Clerk. Behavioral studies in petrol pump workers. Int. Archives of Occupational and Environmental Health, 1988; 61: 1-2. 14. M Singhal, F Khaliq, S Singhal and O.P. Tandon. Pulmonary functions in petrol pump workers: A preliminary study. Indian J. Physiology Pharmacology, 2007; 51: 3, 244-248. 15. C. Kesavachandra, S. K. Rastogi, M. Anand, N. Mathur, A. Dhawan. Lung function abnormalities among petrol pump workers of Lucknow, North India. Scientific Correspondence, Current Sci, 2006;90: 9, 1177-1178 16. Y Verma and SVS Rana. Biological monitoring of exposure to benzene in petrol pump workers and dry cleaners. Industrial Health, 2001; 39: 4, 330-333. 17. American Conference of Governmental Industrial Hygienists (ACGIH). 1999 TLVs and BEIs. Threshold Limit Values for Chemical substances and Physical Agents. Biological Exposure Indices. Cincinnati OH. 1999. 18. J W S Longhurst, S J Lindley, A F R Watson, D E Conlan. The introduction of local air quality management in the United Kingdom: A review and theoretical framework. Atmospheric Environment, 1996; 30: 23, 3975-3985. 19. Parivesh, a news letter from ENVIS Centre Central Pollution Control Board, (2002). 20. Barbieri A, Sabatini L, Accorsi A, Roda A and Violante FS. Simultaneous determination of t,tmuconic acid, Sphenylmercapturic and S-benzylmercapturic acids in urine by a rapid sensitive liquid chromatography / eletrospray tandem mass spectrometry method. Rapid Communications in Mass Spectroscopy, 2004; 18, 1983-1988. Annual Report 2007-08 Biomonitoring of Exposure to Urban Air pollutants in Traffic Policemen HR Rajmohan and B Ravichandran Abstract The urinary excretion of 8-hydroxy deoxyguanosine has been used as a biomarker of oxidative DNA damage in both the clinical and occupational setting. The urinary 8-hydroxydeoxyguanosine in traffic policemen posted at busy traffic junctions were estimated along with the control population away from the busy traffic junctions those doing administrative job. A total of 100 urinary samples (56 samples of traffic policemen and 44 samples of control population were collected for estimation of 8-OHdG and analysed using enzyme linked immunosorbent assay (ELISA). The mean 8-OHdG significantly higher (14.24 ± 12.53 mg/g creatinine) than level of those of control group (8.34 ± 6.24 mg/g creatinine) (p<0.01). The comparison made between the study and control groups taking into consideration over all experience the results showed 13.88 ± 12.35 mg/g creatinine 8-OHdG in traffic policemen and 8.34 ± 6.24 mg/g creatinine 8-OHdG in control subjects which was statistically significant (p<0.01). The 8-OHdG levels were significantly higher in non smokers of study group compared to the control group (p<0.01). The study showed that urinary 8-OHdG is also associated with occupational and other lifestyle factors. Keywords: Urinary 8-hydroxy deoxyguanosine, traffic policemen, ELISA Introduction benzene concentration was in the range of 123.20 - 4048.00 mg/g creatinine in traffic policemen and 30.30 - 2462.90 m/g creatinine in petrol filling station workers. It is recognized that these population are at high risk. Hence, biomonitoring of urinary 8-OHdG, an indicator of oxidative DNA damage might indicate the likely extent of harmful effect that may occur on these groups. Urinary 8-OHdG is easy and non-invasive to collecting compared to collect tissue or leukocyte samples. Urban air contains a diversity of chemical compounds, some of which are genotoxins. The urinary excretion of 8-hydroxy deoxyguanosine has been used as a biomarker in both the clinical and occupational setting.1-6. Exposure to various environmental pollutants such as polyaromatic hydrocarbons, fly ash and fine particulate matter 2.5 containing metal fumes of vanadium, chromium, manganese, nickel, copper and lead increased the level of urinary 8-OHdG 7-9. Occupational exposure to benzene is a known carcinogen. A dose response relationship was found between personal exposure to benzene and urinary 8-OHdG concentrations3. In order to expound the health effects arising out of occupational exposure, monitoring of biomarkers of early genetic effect will be a useful tool. Materials and Methods The urinary 8-hydroxydeoxyguanosine in traffic policemen posted at busy traffic junctions were estimated along with the control population away from the busy traffic junctions those doing administrative job. A total of 100 urinary samples (56 samples of traffic policemen and 44 samples of control population) were collected for estimation of 8 OHdG and analysed using enzyme linked immunosorbent assay (ELISA). The collected urine samples were centrifuged at 2000 rpm for 10 minutes and the supernatants were used for the determination of 8 OHdG. Urinary 8 - OHdG levels and subsequently adjusted by urinary creatinine levels. Studies were carried out on air pollution revealed that urban population is exposed to various inorganic and organic pollutants. An increased risk of cancer has been reported in occupations with heavy exposure to traffic related pollution10. Recently study on health risk assessment of rural and urban population due to indoor / ambient air pollution of Bangalore city showed that the concentration of benzene, toluene and m- xylene varied from 4.35-55.10, 8.73-103.61 and 1.53-135.67 mg/m3 respectively in traffic policemen and 1.200444.6, 0.5 - 130.8 and 0.50-195.6 mg/m3 in petrol filling station workers. The trans, trans-muconic acid a known biomarker of The data pertaining to personal habits like smoking, alcohol consumption, nutritional data and other potential confounding factors were collected using a questionnaire survey. 39 Annual Report 2007-08 Results and Discussion The levels of 8-OHdG among the study and control subjects according to their age groups are given in Table 1. It was observed that the traffic policemen below the age group of <30 showed 10.06 ± 4.37 mg/g creatinine levels of 8-OHdG compared to the same age group of people in control subjects (6.45 ± 2.11 mg/g creatinine). In the 31-40 age group of traffic policemen the levels were 18.50 ± 16.15 mg/g creatinine where as in the control group it was 10.42 ± 8.71 mg/g creatinine. Similarly in the higher age group traffic policemen the levels were 11.18 ± 9.13 mg/g creatinine and 12.16 ± 9.39 mg/g creatinine in 41-50 & ³51 age groups respectively. The 8-OHdG levels in the control subjects of the same group were 9.02 ± 6.96 mg/g creatinine and 7.22 ± 4.97 mg/g creatinine respectively. The mean of total study population the 8OhdG levels were significantly higher (14.24 ± 12.53 mg/g creatinine) than the control group (8.34 ± 6.24 mg/g creatinine of 8-OHdG) (p<0.01). The distribution of participants according to duration of exposure is shown in Table 2. The 8-OHdG levels according to their years of experience is given in Table-3. The traffic policemen those working less than 10 years were shown the levels of 8-OHdG 17.61 ± 17.10 mg/g creatinine and those working more than 11 years and below 20 years were shown 11.47 ± 7.56 mg/g creatinine of 8-OHdG levels. These values when compared to the control subjects not working in the traffic zones were shown 9.94 ± 7.61 mg/g creatinine among those below 10 years of experience. Even when we look into the control group working between 11-20 and 21-30 years of experience the levels were 9.73 ± 5.06 mg/g creatinine, and 7.20 ± 6.41 mg/g creatinine respectively. The levels were significantly (p<0.05) higher than the control group in 21-30 years of experienced traffic policemen. Those working more than 31 years the levels were 8.06 ± 6.35 mg/g creatinine in traffic policemen and 7.30 ± 6.83 mg/g creatinine in control subjects. The comparison made between the study and control groups taking into consideration over all experience the results showed 13.88 ± 12.35 mg/g creatinine 8-OHdG in traffic policemen and 8.34 ± 6.24 mg/g creatinine 8-OHdG in control subjects which were statistically significant (p<0.01). Table 1: Levels of 8-hydroxy -2 deoxyguanosine (µg/g creatinine) in study and control groups according to age group Age group (yrs) Study Control <30 31-40 10.06±4.37 (7) 6.45±2.11 (3) 18.50±16.15 (23) 10.42±8.71 (6) 41-50 11.18±9.13 (15) 9.02±6.96 (18) ³51 12.16±9.39 (11) 7.22±4.97 (17) Total 14.24±12.53** (56) 8.34±6.24 (44) Mean±SD ; (Number) ; **p<0.01 Studies on urinary 8-OHdG as a biomarker of oxidative DNA damage in workers exposed to fine particulates was carried out by Jee Young Kim et al9 and concluded that urinary 8-OHdG increased after occupational exposure to fine particulate matter. Chum-Yu Chaung et al.,10 studied the urinary 8- hydroxydeoxyguanosine (8-OHdG) on taxi drivers from traffic exhaust and/or smoking in exposed and nonexposed individuals, and found the level significantly higher in drivers than in community men (13.4 ± 4.7 vs. 11.5 ± 4.7 mg/g creatinine). Table 2: Distribution of study and control subjects according to work duration of exposure Duration of Exposure (yrs) <10 11-20 21-30 ³31 Total Study Control 20 (36.4) 22 (38.2) 10 (18.2) 4 (7.3) 56 (100.0) 7 (15.9) 12 (27.3) 19 (43.2) 6 (13.6) 44 (100.0) Table 3: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to work experience Experience (yrs) Study Control <10 17.61±17.10 (20) 9.94±7.61 (7) 11-20 11.47±7.56 (22) 9.73±5.06 (12) 21-30 13.48±9.93* (10) 7.20±6.41 (19) ³31 8.06±6.35 (4) 7.30±6.83 (6) Total 13.88±12.35** (56) 8.34±6.24 (44) *p<0.05 ; **p<0.01 ; Mean ± SD The 8-OHdG levels among study and control groups according to their smoking habits is shown in Table-4. The 8-OHdG levels compared to the smokers of traffic policemen and control group were 7.61 ± 4.72 mg/g creatinine and 7.10 ± 4.83 mg/g creatinine, respectively. The data analysis among the smokers and non-smokers of the present study contradicts the earlier studies, as regard to excretion of 8-OHdG. The earlier studies showed that the smoking habit had effect on 8-OHdG excretion. Several studies showed an increase in urinary 8-OHdG concentrations in smokers compared with 12,4 nonsmokers . Smoking can have a considerable effect on the 7 concentration of 8-OHdG. However, studies by Nilsson et al failed to show the effect of smoking on the urinary excretion of 8-OHdG. Whereas studies by Jee Young Kim et al.9 reported that mean baseline 8-OHdG concentration were not significantly different between smokers and nonsmokers. Previous occupational studies, in which workers were exposed to various carcinogens, including PAHs and asbestos, found that smoking 41 Annual Report 2007-08 Table 4: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and smoking habits Study Control Age group (yrs) ³ 30 Smokers 9.03 (2) 10.47± 2.79 (5) 8.44 (1) 5.46 (2) 31-40 10.02±6.50 (4) 18.13±15.59 (19) 10.62 (2) 10.31±11.24 (4) 41-50 6.44±2.82 (4) 12.90±10.11 (11) 1.41 (2) 9.98±6.80 (16) ³51 5.81±3.14 (3) 14.54±9.97 (8) 7.86±5.55 (4) 7.03±5.01 (13) Total 7.61±4.72 (13) 15.23±12.40 (43) 7.10±4.83 (9) 8.66±6.58 (35) Non-Smokers Smokers Non-Smokers Annual Report 2007-08 Table 7: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and alcohol consumption ≤30 31-40 41-50 *p<0.05 ; **p<0.01 ; Numbers in parenthesis indicate sample size Table 5: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and smoking habits Study Age group (yrs) Chewers Control Non Chewers Chewers Non chewers ≤30 - 9.78±5.08 (7) - 6.45±2.11 (3) 31-40 12.19 (2) 17.89±15.84 (21) - 10.42±8.71 (6) 41-50 33.98 (2) 9.03±5.31 (13) - 8.88±7.15 (18) ≥51 6.13 (2) 13.47±10.56 (9) 12.35 (1) 6.90±4.95 (16) Total 14.61±13.37 (6) 13.86±12.28 (50) 12.35 (1) 8.17±6.34 (43) ≥51 Total Yes No Yes No ≤30 9.40±4.39 (6) 14.01 (2) 6.34 (2) 6.68 (1) 31-40 18.50±16.15 (23) - 12.32±8.23 (5) 0.90 (1) 41-50 11.18±9.13 (15) - 9.85±6.92 (16) 2.39 (2) ≥51 12.72±9.70 (10) - 7.20±5.14 (16) 7.59 (1) Total 14.39±12.72* (54) 14.01 (2) 8.90±6.34 (39) 3.99±3.21 (5) 42 smokers. They concluded that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system. Studies by Yun-Chul Hong et al.15 concluded that cigarette smoke contains numerous carcinogens. It has been shown to generate reactive oxygen species and to induce oxidative damage in isolated DNA's as well as to produce 8-OHdG in cell cultures. It has been reported that smokers Yes No 6.68 (1) 8.65±6.10 (3) 6.84±5.46 (8) 6.34 (2) 12.18±11.97 (3) 10.77±7.79 (10) 12.30±10.95 (8) 15.64±10.40** (27) 11.79±4.57 (3) 11.60±9.89 (29) 10.25±3.67 (5) 7.88±4.92 (17) 6.57±5.08 (12) 8.58±6.89 (27) Study Control Vegetarian Mixed Vegetarian Mixed ≤30 5.60 (2) 11.84±3.24 (5) 7.56 (2) 4.23 (1) 31-40 26.88 (2) 10.65±6.29 (6) 7.98±6.44 (3) 11.75±8.57* (13) 17.71±16.71 (21) 11.53±10.99 (9) 13.73±10.19 (8) 14.99±13.49 (43) - 12.32±8.23 (6) 9.51±7.89 (12) 9.15±5.94 (8) 9.74±7.12 (27) ≥51 Total 8.05±5.12 (6) 5.51±3.41 (9) 6.65±3.96 (17) Mean ± S.D; *p<0.05; Numbers in parenthesis indicate sample size Table 9: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and exercise habit Age group Study Control Exercise No exercise Exercise ≤30 6.59±3.37 (3) 12.21±3.61 (4) 7.56 (2) 4.23 (1) 31-40 10.03±5.58 (10) 21.12±14.03 (13) 10.32±9.74 (5) 10.90 (1) 41-50 6.95±3.09 (4) 12.72±10.21 (11) 7.25±4.89 (10) 11.25±8.76 (8) ≥51 12.26±10.94 (8) 11.90±4.64 (3) 6.99±5.47 (12) 7.77±4.03 (5) Total 9.83±7.38 (25) 16.06±11.65* (31) 7.69±5.88 (29) 9.60±6.92 (15) *p<0.05; Numbers in parenthesis indicate sample size status was not a significant predictor of urinary 8-OHdG levels 5, 6, 13. 14 However, Besarati Nia et al ., reported that 8-OHdG was consistently increased among smokers. Heavy exposure to air pollution in occupational settings in terms of diesel exhaust, polyaromatic hydrocarbons, and benzene has been associated with increased 8OHdG excretion, whereas non-occupational exposure to ambient air pollution was not significantly lower in smokers as compared with non- No 9.77±5.08 (5) 14.70±13.85 (11) 8.75±6.92 (10) Age Group 41-50 Control Coffee / Tea Yes 10.77 (2) 18.62±10.54 (12) 16.95±11.36 * (5) Table 8: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and food habit Table 6: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and chewing habits Study Coffee / Tea Control Alcohol consumption *p<0.05; **p<0.01; Numbers in parenthesis indicate sample size *p<0.05 ; **p<0.01 ; Numbers in parenthesis indicate sample size Age Group Study Alcohol consumption Age group (yrs) *p<0.05; Numbers in parenthesis indicate sample size 43 No exercise Annual Report 2007-08 5. had a 50% higher concentration or urinary 8-OHdG than non-smokers, which correlates with the study by Yun Chul, which showed that smokers who smoke less that 20 cigarettes a day showed a 48% increase in the excretion of 8-OHdG. The 8-OHdG levels among study and control groups, according to their exercise habit is shown in Table 9. The excretion of 8-OHdG levels were high in traffic policemen those not doing exercise when compared to the administrative staff. Among study group the levels were significantly higher (p< 0.05) in those not doing exercise compared to those having habit of doing moderate exercise. 15 Hiroshi et al studied the effects of exercise, working conditions, meat intake, body mass index and smoking habit and other life styles on 8OHdG excretion. It was found that moderate physical exercise, and high body mass index reduced the 8-OHdG excretion, while physical labour, smoking and low meat intake (less than once/week) increased the excretion levels. 6. 7. 8. Conclusion The study showed that the level of urinary 8-OHdG is associated with occupational and other lifestyle factors. Differences between the results of the present study and other investigation might explain variations in sample size, sample composition, and methods of measurement of urinary 8-OHdG. Inclusion of more number of participation from traffic policemen might highlight the association of 8-OHdG excretion and their exposure to air pollutants. 9. 10. Acknowledgment: Technical assistance rendered by A Mala, Vijayendra, MN Prakash, V Sehar, S Raghavendra Rao, N S Marula Siddiah, N Thara, MR Subbamma, HC Rangaswamy, D Krishnamurthy, R Muniyappa, J Revathy, N Hema are acknowledged. Assistance rendered by Police Department, Bangalore City is gratefully acknowledged. 11. 12. References 1. 2. 3. 4. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H et al (1997): Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy 2'-deoxygunosine excretion with radiotherapy, chemotherapy and response to treatment. FEBS Lett 409: 287-291 Honda M, Yamada Y, Tomonaga M, Ichinose H, Kamihira S (2000): Correlation of urinary 8-Hydroxy-2'-Deoxyguanosine (8-OHdG) a biomarker of oxidative DNA damage and clinical features of hematological disorders: a pilot study. Leuk Res 24: 461-468. Lagorio S, Tagesson C, Forastiere F, Iavarone I, Axelson O, Carere A (1994): Exposure to benzene and urinary concentration of 8Hydroxy-2'-Deoxyguanosine, a biological marker of oxidative damage to DNA. Occup Environ Med 51: 739-743. Pilger A, Germadnik D, Schaffer A, Theiler A, Pils P, Sluka F at al. (2000): 8-Hydroxydeoxyguanosine in leukocyte DNA and urine of quartz exposed workers and patients with silicosis. Int Arch Occ. Environ Health 73:305-310. 13. 14. 15. 16. 44 Tagesson C, Chabiuk D, Axelson O, Baranski B, Palus J, Wyszynska K (1993): Increased urinary excretion of the oxidative DNA adduct, 8-Hydroxydeoxyguanosine as a possible early indicator of occupational cancer hazards in the asbestos, rubber and azo-dye industries. Pol J Occ Med Environ Health 6: 357-368 Toraason M, Hayden C, Mariow D, Rinehart R, Mathias P, Werren D et al (2001): DNA strand breaks, oxidative damage, and 1-OH pyrene in roofers with coal-tar pitch dust and/or asphalt fume exposure. Int Arch Occup Environ Health 74:396-404. Nilsson R, Nordlinder R, Moen BE, Ovrebo S, Bleie K, Skorve AH, Hollund BE, Tagesson C (2004): Increased urinary excretion of 8hydroxy deoxyguanasine in engine room personnel exposed to polycyclic aromatic hydrocarbons. Occup Environ Med 61, 692-6. Yoshida R, Ogawa Y, Mori I, Nakata A, Wang R, Ueno S, Shioji I, Hisanaga N (2003): Association between oxidative stress levels and total duration of engagement in jobs with exposure to fly ash among workers at municipal solid waste incinerators. Mutagenesis 18, 533-537. Jee Young Kim, Sutapa Mukherjee, Long Ngo and David C Christiani (2004): Urinary 8-Hydroxy-2'-Deoxyguanosine as a Biomarker of Oxidative DNA Damage in Workers Exposed to Fine Particulates. Environmental Health Perspectives 112, 6, 666-671. Chun-Yu Chuang, Chen-Chen Lee, Yu-Kang Chang and FungChang Sung (2003). Oxidative DNA damage estimated by urinary 8 hydroxydeoxyguanosine: influence of taxi driving, smoking and areca chewing.Chemosphere. 52(7):1163-1171. Burgaz S, Demircigil GC, Karahalil B, Karakaya AE (2002): Chromosomal damage in peripheral blood lymphocytes of traffic policemen and taxi drivers exposed to urban air pollution. Chemosphere Apr, 47 (1): 57-64. Loft S., Vistisen K, Ewertz M, et al. (1999) Oxidative DNA damage estimated by 8 hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis. 13: 2241-2247. Toraason M (1999) 8-hydroxyguanosine as a biomarker of workplace exposure. Biomarkers 4:3-26. A.Besarati Nia, F.J.Van Schooten, P.A.E.L.Schilderman, T.M.C.M.De Kok, G.R.Haenen, M.H.M.Van Herwijnen, E.Van Agen,D.Pachen,J.C.S.Kleinjans (2001). A Multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechansisms Carcinogenesis 21( 2):395-401. Yun-Chul Hong, Hye-sook and Eun-Hee Ha 2000 influence of genetic susceptibility on the urinary excretion of 8-hydroxydeoxyguanosine of firefighters, Occup. Environ. Med, 57:370-375. Hiroshi Kasai, Noako Iwamoto-Tanaka, Toshiaki Miyamoto, Kiyoshi Kawanami et al, (2001). Life style and urinary 8hydroxydeoxygaunosine, a marker of oxidative DNA damage, effect of exercise, working conditions, meat intake, body mass index, and smoking, Cancer Res, 92:9-15. Annual Report 2007-08 Occupational Noise and Hearing Assessment of Workers in a Refurbishment Unit SR Tripathi and R Vishwakarma* Abstract A total of 38 workers, age (21-34 yrs. with normal ears, duration of employment 1-8 yrs), working in a refurbishment unit, were subjected to hearing assessment. The data for each subject was obtained twice i.e. once (Day 1) at the pre- exposure level and on the second occasion (Day 3) at the post exposure level. The result showed that the mean threshold of the workers were well within normal threshold of hearing on low frequencies as well as on high frequencies, before work. However, after work the threshold of hearing increased a little which was also within the normal level of hearing. The sound pressure level (SPL) has been also carried out near the workers' ear level, which was found to be in the range of 85-116 dBA at different locations. Keywords: Hearing assessment, threshold limit, frequency, sound pressure level Introduction The adverse health effects of noise exposure can be prevented, or at least minimized, by reducing noise exposure to safe levels. The best method of noise reduction is to implement engineering controls such as modifications to the noise source and/or to the workplace environment. Where technology cannot adequately control the problem, personal hearing protection such as ear muffs or plugs are used. The use of personal protection is only an interim measure while engineering controls for permanently reducing noise levels are being explored and implemented. Workers in certain industries experience high exposure to potentially hazardous noise levels. These industries include agriculture, mining, construction, manufacturing and utilities, transportation, machinery, aircraft, weapons, and industrial operations contribute to a potentially noise hazardous environment. Man-made technology advances have led to the installation of broad spectrum of machines and as a result the work environment as well as the community environment is now threatened with undesired sounds resulting in noise pollution. Its effects are insidious and often go undetected1. Problems arise when sound damages the human hearing. Assessment of the magnitude of the noise problem, the levels and quality of noise generated vis-à-vis the risk to hearing impairment of workers were assessed in a gas turbine with refurbishment facility. It was also intended to identify the noisy equipment that could assault on the health of the workforce at their place of works. The main occupational activity of the workers was grinding operation. Each worker was working in a separate small cabin as a result they were working very close to the machine. With this background it was decided that for the promotion of well being of the work force following future course of action should be taken. Intensity of noise is not the only factor to determine the magnitude of hearing loss, there are other variables which is required to understand the spectrum of noise characteristics, duration of exposure, physical dimension of workroom, and other personal factor such as age, sex, temperament, susceptibility, personality etc2 Longterm occupational exposure to high level of noise carries an increased 3-5 risk of perceptive hearing loss . A number of studies have reported that noise causes serious hazards to hearing6-10 cognitive11, neuromotor functions12 and psychological disturbances such as lower level of work performance 1 3 increased annoyance 1 4 - 1 6 , hypertension17,18, hearing interference and speech intelligibility19,20, industrial accident 21. This study was conducted with the objective to quantify level of exposure to noise, particularly at high frequencies (3, 4, 6 & 8 KHz), to determine the hearing ability through audiometric performance and to recommend a hearing conservation programme to protect personnel from noise higher than permissible levels. *Prof. ENT, Civil Hospital, Ahmedabad 45 Annual Report 2007-08 Zyglo Facility F Gangway Fixture Storage Area VL The criteria for acceptance as normal ears considered were: (a) Tympanic membrane intact, (b) Rinne test positive, (c) No history of congenital or acquired conditions associated with sensory neural hearing loss. (d) Audiogram compatible with clinical findings Weber test, nerve defects, absolute non- conduction, polypus, etc. were also taken in to account for categorizing ears as normal. 8 9 10 11121314 1516 17 NDT C E Supervisor Cabin CNC G 1 2 2 3 4 5 6 7 B Table 1: Sound pressure level (SPL) at different locations of noise survey area Noise Levels Monitoring Frequency Levels Unit SLM 63 125 250 500 1K 2K 4K 8K 85 A Fr-6 Segment Abrasive wheel Abrasive cone dBA 96 60 62 67 71 75 81 82 B Fr-9 2nd Stage Mounted point dBA 92 58 70 75 81 82 82 85 89 B Fr-9 2nd Stage Abrasive cone dBA 99 62 65 72 83 97 100 105 107 Component C Fr-6 TP Abrasive cone dBA 115 62 69 79 87 92 104 113 114 D Gangway Gangway dBA 85 65 62 62 67 82 83 85 85 E Gangway Gangway dBA F Fr-9 1 Stage Abrasive cone dBA 95 57 62 65 69 72 82 87 88 G Fr-6 TP CL Abrasive cone dBA 116 65 65 70 81 89 95 105 110 dBA 85 70 72 70 68 69 74 77 78 st Grit Blasting machine Materials and Methods Following criteria were maintained for non-inclusion of workers in audiometry: (a) The subjects' present noise level was not amenable to quantitative description. (b) Not served in the armed forces, or had been exposed to gunfire. (c) Whose past noise exposure was different from that of their present occupation. (d) Who were known to have existing or previous ear disease or abnormality. (e) Head injury with history of unconsciousness or skull fracture. Subjects: A total of 38 workers with normal hearing ability (as assessed by questionnaire) included in the study. They were in the age range of 21-34 yrs. with the duration of employment 1-8 yrs and working in the noisy work environment for 8 hours daily. Instruments used for the study are sound level meter:- 2218, fitted with condenser microphone (Bruel & Kjer, Denmark), Octave frequency analyzer1616, (Bruel & Kjer, Denmark), Audiometer: Model 700MK IV (M/s Arphi India Ltd.).Tunning Fork: Vibrates at 512Hz. 46 Procedure: As testing threshold of audibility with speech signals involves problems that have not as yet been completely solved or identified, pure tone audiometry was carried out for the present investigation. In the individual experiment, in a quiet room (less than 10 dB) subjects were briefed about the nature and purpose of the study. The hearing assessment was carried out (tone lasting for 2 sec. pulsing of the tone at 0.5/ sec.) The intensity of the tone was raised by 5 dB until the threshold of hearing was determined at each test frequency viz 125 Hz, 250Hz, 500Hz, 1KHz, 1.5KHz, 2KHz, 3KHz, 4KHz, 6KHz.and 8KHz. Care was taken to ensure reliable reporting of the subjects' 'just audible sound'. Misses (error of omission) and false alarms (error of commission) were avoided. Design of the study: Pure tone air conduction hearing threshold was obtained in a quiet room. Threshold of hearing is defined as the minimum decibel level (dB) at which the subject responds at least two times on ascending trial. The data for each subject was obtained twice (i) at the pre- exposure level before the work shift (Day1) and (ii) at the post exposure level at the end of 8 hr. work shift (Day 3). Half of the subjects were tested in the sequence before- after work shift, while for the rest of the subjects the testing was in the reverse sequence of afterbefore work shift, so as to eliminate the effects of practice on the performance of the test. Pure tone threshold was obtained using descending - ascending threshold crossing technique. The data was Figure 1. Schematic diagram of the noise survey area Location analyzed for each ear of the subjects for all test frequencies. The schematic diagram of the noise survey area has been shown in Fig. 1. Criteria for hearing impairment: Hearing impairment is considered to occur when the average of the hearing threshold levels at audiometric frequencies of both ears exceed 25 dBA. D A Annual Report 2007-08 Results The sound pressure level (SPL) has been carried out near the workers' ear level, which was found to be in the range of 85-116 dBA at different locations (Table 1) and octave band analysis was also carried out at all locations (Figure 2 and 3). The noise level at the places Figure 2: Frequency spectrum of noise originating from equipment Figure 3: Frequency spectrum of noise originating from equipment Figure 4: Mean hearing level (dB) for the right ear of exposed group Figure 5: Mean hearing level (dB) for the left ear of exposed group 47 Annual Report 2007-08 Fr-6 Segment, Fr-9 2nd Stage, Fr-6 TP, Fr-9 1st Stage and Fr-6 TP CL was 96, 99, 115, 95 and 116 respectively, which is very high than the recommended safe limit value. The noise level at the places Fr-6 Segment, Fr-9 2nd Stage, Fr-6 TP, Fr-9 1st stage and Fr-6 TP CL was 96, 99, 115, 95 and 116 respectively, which is very high than the recommended safe limit value. 5. 6. The work force were subjected to hearing assessment, before entering the noisy area (pre exposure) and followed by 8 hours exposure (post exposure). The data for the workers who were found to be free from ear pathology were analyzed. The geometrical mean threshold of hearing for the right ear and left ear of the exposed group was calculated. Hence, the mean of the value of both the ears are given by duration of exposure (Figure 3 & 4). It may be noted that the mean threshold of the workers were well within normal threshold of hearing on low as well as on high frequencies, before work. However, after the work threshold of hearing increased a little which was also within the normal level of hearing. 7. 8. 9. 10. Discussion 11. The sound pressure level shows that the noise level at many places was higher but hearing impairment was not found because they were using earmuffs during their work. But it is not a permanent solution. Also the exposure is not for 8 hours daily and sound pressure level is not always at high level. It may be concluded through the problem of noise with regard to hearing impairment was not observed but the management is interested to reduce the noise levels in working area and hence consulted the engineers to prepare a sound proof-working chamber for permanent solution. 13. Conclusion 14. 12. The sound pressure level (SPL) ranged from 85-116 dBA. Octave Band Analyses revealed that noise was high frequency characteristics, which was at some places more than recommended values. Hearing assessment shows that no noise induced hearing loss was found among the workers because they are using ear protectors. These ear protectors are temporary arrangement so management is trying to make a sound proof-working chamber for permanent solution. 17. References 18. 15. 16. 1. Harris CM. Handbook of noise control. Second edition. McGraw Hill Book Company, USA, Chapters 9 &15, 1997. 2. Webb JD. Noise control in industry, Sound Research Laboratories Ltd., England, 19, 1976. 3. Bauer P, Korpert K, Neuberger M, et al. (1991) Risk factors for hearing loss at different frequencies in a population of noiseexposed workers. J Acoust Soc Am; 90:3086-98. 4. Prince MM, Stayner LT, Smith RJ, et al.(1997) A re-examination of 19. 20. 21. 48 risk estimates from the NIOSH Occupational Noise and Hearing Survey (ONHS). J Acoust Soc Am; 101:950-63. Ahmed HO, Dennis JH, Badran O, et al. Occupational noise exposure and hearing loss of workers in two plants in eastern Saudi Arabia. Ann Occup Hyg 2001; 45:371-80. Holme RH, Steel KP. J Assoc Res Otolaryngol. 2003 Dec 5 [Epub ahead of print]. Progressive Hearing Loss and Increased Susceptibility to Noise-Induced Hearing Loss in Mice Carrying a Cdh23 but not a Myo7a Mutation. Tambs K. Hearing loss caused by noise, otitis and head injuries. Tidsskr Nor Laegeforen. 2003 Nov 6; 123(21): 3047-49. Moshi NH, Riwa P (2003) Noise induced hearing loss among industrial workers in Dar es Salaam. East Afr Med. J; 80 (6) : 298 - 02. Adera T, Gaydos JC. Identifying comparison groups for evaluating occupational hearing loss: a statistical assessment of 22 industrial populations. Am J Ind Med. 1997 Feb; 31(2): 243-9. Maisarah SZ, Said H. The noise exposed factory workers the prevalence of sensori-neural hearing loss and their use of personal hearing protection devices. Med J Malaysia. 1993 Sep; 48 (3): 280-85. Bhattacharya SK, Tripathi SR and Chatterjee BB (1986) Influence of exposures to low intensities of sound at different frequencies on the cognitive abilities and sensori-motor coordination. Ind J Med Res 84,430-436. Bhattacharya, SK, Tripathi SR and Kashyap SK (1989) The combined effects of noise and illumination on the performances efficiency of visual search and neuromotor task components, J. Human Ergol, Tokyo, 18,41-43. Cohen A (1973). Extra-auditory effects of occupational noise. Part II. Effects on work performance. Natl Saf News 108 (3):68-76. Schultz TJ (1978). Synthesis of Social Surveys on Noise Annoyance. Journal of the Acoustical Society of America, Vol. 64, No. 2, , pp. 377-405. Taylor SM (1984). A path model of aircraft noise annoyance. Sound and Vibration 96(2): 243-260. Öhrström E, Björkman M, Rylander R (1988). Noise annoyance with regard to neuro-physiological sensitivity, subjective noise sensitivity and personality variables. Psychol Med 18:605-613. Wu TN, Ko YC, Chang PY (1987). Study of noise exposure and high blood pressure in shipyard workers. Am J Ind Med 12:431-438. Talbott E, Findlay R, Kuller L, et al (1990). Noise-induced hearing loss: a possible marker for high blood pressure in older noiseexposed populations. J Occup Med 32 (8): 685-689. Lindeman HE (1976). Speech intelligibility and the use of hearing protectors. Audiology, 15 348-356. Webster JC. Effects of Noise on Speech Intelligibility. In Proceedings of the Conference Noise as a Public Health Hazard, (1969), p. 49-73. Moll van Charante AW, Mulder PGH (1990). Perceptual acuity and the risk of industrial accidents. Am J Epidemiol 131(4): 652-663. Annual Report 2007-08 Pesticide Exposure Amongst Formulators and Health Effects VK Bhatnagar, AB Patel, SSA Zaidi, SR Tripathi, SN Sinha, VK Shivgotra Abstract The study was conducted in a unit engaged in production and formulation of organophosphate insecticides (Triazophos and Acephate). The findings indicated that the workers involved in production and formulation of these insecticides had significantly low level of RBCs cholinesterase activity. Serum level of IgM reflected significant increase, indicating impairment of immune system of subjects. The levels of thyroid functional tests (T3, T4 and TSH) were within normal range. The findings pertaining to neurobehavioral test battery indicated that maintenance and exposed group workers showed significant elevation in finger dexterity error test. Further, a significant decrease in neurobehavioral tests like forward and back memory, total digit symbol test, and increase in hand steadiness test was observed in maintenance group workers. Keywords: Pesticide formulators, triaziphos, acephate, cholinesterase, neurobehavioural test Introduction general physical (anthropometric data, blood pressure, conjuctiva and sclera) and systemic examination of respiratory, cardiovascular and gastrointestinal systems; allergic respiratory, allergic skin and chronic symptoms to peripheral neuropathy were noted. An electrocardiogram (ECG) of each worker was also done. Pesticides are toxic chemicals and pose risks to the user groups. These are important factors in agricultural development and protection of public health in our country, since the tropical climate is very conducive for pest breeding. Their production and formulation in industrial settings may involve exposure risk to the workers. Blood samples of the subjects from cubital vein were obtained with the help of vacutainer (BD Vacutainer, Plymouth, PL6 7BP, UK). Serum was obtained by centrifuging blood sample at 5,000 rpm for 5 0 min and stored at 20 C till the time of analysis. The activities of serum glutamate oxaloacetate transaminase (SGOT, EC 2.6.1.1), serum glutamate pyruvate transaminase (SGPT, EC 2.6.1.2) were estimated following the procedure of Bergmeyer et al 1. The activities of alkaline phosphatase (AlP, EC 3.1.3.1) and lactate dehydrogenase (LDH, EC 1.1.2.27) were estimated according to the method of Empfehlungen2 and Mathieu3. The absorbance of these parameters was recorded on RA-50 Chemistry Analyzer. Plasma Cholinesterase (ChE, EC 3.1.1.8) 4 activity was estimated according to method of Ellman et al and that of RBCs cholinesterase (ChE, EC 3.1.1.7) was estimated following the 5 procedure of Worek et al . The absorbance of plasma and RBCs ChE activities were measured on Varian Cary-100 Spectrophotometer. In a subset of samples, the immunological profile IgG, IgM and IgA) and thyroid functional tests (T3, T4 and TSH) were also estimated. Neurobehavioral test battery involving (Tweezer Dexterity, Finger Dexterity, Hand Steadiness, Card Sorting, Mental Control, Memory test, and Digit Symbol) was administered to this study. The existing database on health of workers involved in the production and formulation of pesticides in the country is scanty, therefore, the study is designed to address the magnitude of health risk among pesticide formulators with an objective to evaluate the magnitude of health risk with special reference to neurobehavioural changes. Materials and Methods The unit belongs to organized sector and engaged in production and formulation of organophosphate insecticides (Triazophos and Acephate). A total of 161 workers were included as subjects in this study. These were divided into three groups; i) Control Group (n=40) comprised of employees of administration department e.g. managers, executives, clerks, typists, stenographers, peon, orderlies etc ii) Maintenance Group (n=50) comprised of mechanics, electricians, engineers, fitters and helpers etc and iii) Exposed Group (n=71) comprised of workers, supervisors involved in the production and formulation of organophosphate insecticides. There was specific work schedule (8 hr shift duty) for exposed and maintenance group workers whereas the control group subjects had 8 hr general duties without any shift. Each subject was interviewed for their demographics, smoking habits, alcohol intake, medical history, past and present illness, work conditions and exposure to other chemicals. Their detailed occupational history (past and present) was recorded on pre-coded proforma. They underwent detailed clinical examination including Data presented as Mean ± SD. All statistical analysis was performed using SPSS software. Analysis of variance (ANOVA) was used for comparison among groups followed by group wise comparisons by Dunnett's T3 test. Linear regression was used for studying various 49 Annual Report 2007-08 Table 4: Neuro-behavioural test performance of study population Table 1: Distribution of subjects according to the duration of their Job Group N Age (Years) Mean SD Duration of Exposure 1-2 2- 5 <1 Control 40 35.029.98 4 15 10 11 50 31.389.08 18 5 15 12 Exposed 71 28.456.81 29 10 16 16 N= number of subjects Parameter RBCs ChE Data on distribution of subjects in relation to their duration of job have been mentioned in Table 1. There were 40 subjects in control group (aged: 35.02 ± 9.98 years), 50 subjects in maintenance group (aged: 31.38 ± 9.08 years), and 71 subjects in exposed group (aged: 28.45 6.81 years), The levels of various biochemical indices estimated in the blood samples of study population are given in Table 2. The serum levels of GOT, GPT, alkaline phosphatase and lactate dehydrogenase were within normal limits in the maintenance and exposed groups subjects. Control (n=40) Maintenance (n=50) Exposed (n=71) Coefficient 0.282 0.459 0.402 p value 0.078 0.001 0.001 Data pertaining to neurobehavioural test battery e.g. tweezer dexterity, finger dexterity, hand steadiness, card sorting (design and face value), hand dynamometer, mental control, memory (forward and backward) and digit symbol test have been given in Table 4. While analyzing the data, three factors (total score, error and percentage of accuracy) have been considered to evaluate the effect of pesticides on the neurobehavioral test performance. The result indicated that exposed group workers have shown significantly lower performance in digit symbol test (% of accuracy) compared to control group. Exposed workers did not reflect any deficit on memory test, mental control and also in their motor performance test (tweezer dexterity, finger dexterity and hand steadiness). Similarly no significant deficit was found on card sorting and hand dynamometer performance of maintenance and exposed group subjects. Quite a few studies of OP exposed workers showed no change in their performance in some of 8 the neurobehavioural test . In one of the studies, tremor was related to 9 10,11 exposure to combination of pesticides but not to OPs . Available data indicated that grip strength was not related to the exposure of 12 13 14 subjects to fumigants , DDT , or multiple pesticides . Table 2: Levels of various biochemical indices in the study population Exposed (n=71) 13.78 1.55 13.64 1.36 14.20 1.35 ALKPase (U/l) 136.57 4.29 141.56 3.38 136.59 2.68 LDH (U/l) 327.72 9.52 312.83 8.67 320.62 7.70 RBCs ChE (mU/umol Hb) 492.35 7.56 488.67 9.09 3421.3 1.25 3326.64 1.27 3144.47 1.28 Hb (gm/dl) The maintenance group of workers showed poor performance on accuracy score of finger dexterity (% accuracy) and card sorting. Though there is no significant difference on their total score and error score. Similarly the memory test, (both forward and backward) has shown deficiency in their performance scores than the control group subjects. Similar results have been shown in workers exposed to OPs and applicators in field conditions15,16. Card sorting (face value) and mental control (accuracy 30 seconds time) have also shown poor accuracy. 464.6 7.97* # Plasma ChE (U/l) # SGPT (U/l) 22.72 1.39 22.26 1.52 23.48 1.50 # SGOT (U/l) 26.11 1.25 26.1 1.24 27.57 1.27 Finger Dexterity # Data expressed as mean ± SE; GM ± GSD; *p < 0.05 50 2.78 0.378 2.04 0.240 2.87 0.276 93.01 0.988 94.54 0.766 91.30 1.163 No. of pins 29.05 0.992 29.76 0.838 30.58 0.832 1.73 0.245 2.92 0.318 93.47 1.005 89.67 1.212* error 5.19 0.161 5.89 0.143 5.58 0.121 41.61 1.122 41.41 0.917 Error % accuracy Card Sorting Face (Time in sec.) Mental Control (45”) 116.51 0.409 118.14 1.351 115.96 0.306 1.24 0.183 1.41 0.252* 99.38 0.157 98.79 0.216 98.93 0.157 39.29 1.129 40.08 0.769 8.95 0.576 9.35 0.398 9.08 0.286 Error 0.10 0.061 0.38 0.102 0.27 0.08 % accuracy 98.88 0.546 96.23 0.825* 97.32 0.643 Time 16.23 0.998 20.43 1.547 18.73 1.040 Error 2.45 0.533 3.00 0.523 3.30 0.465 90.36 2.003 88.82 1.919 87.84 1.669 Forward 6.08 0.176 4.96 0.227*** Backward 4.73 0.164 3.47 0.216*** 4.37 0.128 66.15 1.816 58.0 1.731*** 65.24 1.238 Total Digit Symbol Test 0.62 0.123 98.42 0.392 39.65 1.179 % accuracy Memory 0.51 0.124 98.95 0.244 Time Hand Dynamometer Mental Control (30”) 0.49 0.168 98.93 0.351 0.72 0.183 Error % accuracy 2.61 0.244 90.99 0.871 41.31 1.347 Hand Steadiness Card Sorting Design (Time in sec.) Exposed (n=71) 39.58 1.086 Maintenance (n=49) 41.71 0.988 % accuracy Error % accuracy also been evaluated (Table 3). A positive correlation was observed between plasma ChE and RBCs ChE in maintenance group (r = 0.459; p < 0.001) and in exposed group (r = 0.402; p < 0.001). However, the activities of RBCs cholinesterase were significantly inhibited (p < 0.05) in exposed group subjects, indicating appreciable exposure to the pesticides from their working environment (Figure 1). The depression in ChE activity is an effect biomarker of pesticide load and in accordance to our previous published studies amongst pesticide formulators and with other studies conducted on sprayers of 6,7 pesticides in field conditions . The exposure of OP compounds manifests as a cholinergic crisis and diagnosis is based on the clinical signs and symptoms as well as the measurement of inhibition of erythrocyte (RBC) and/or plasma cholinesterase (ChE) activities. The correlation between plasma ChE and RBCs ChE in three groups have Maintenance (n=50) No. of pins Plasma ChE Results and Discussion Control (n=40) Tweezer Dexterity Table 3: Correlation between RBCs and Plasma ChE in study population relationships among continuous variables. Two tailed tests were used for all comparisons with level of significances as 5% (p < 0.05). Control (n=40) 42.13 1.238 Neurobahavioural Test Battery >5 Maintenance Indices Annual Report 2007-08 0.90 0.352 1.82 0.375 2.13 0.398 98.75 0.505 96.79 0.661 96.66 0.671* Error % accuracy 5.79 0.104 Data represented as mean ± SE; *p < 0.05; **p < 0.01; ***p < 0.001 Table 5: Levels of immunological profile in study population Table 6: Levels of thyroid hormones in study population Tests Tests IgA (mg/dL) IgG (mg/dL) IgM (mg/dL) Control (n=12) Maintenance (n=28) Control (n=12) Maintenance (n=28) Exposed (n=11) 272.42 ± 15.52 T3 (nmol/L) 2.36 ± 0.14 2.41 ± 0.17 2.42 ± 0.10 1473.86 ± 56.55 1374.10 ± 30.68 T4 (nmol/L) 138.58 ± 7.29 131.46 ± 10.92 131.11 ± 5.61 TSH (mIU/L) 3.14 ± 0.55 4.08 ± 1.36 3.06 ± 0.41 281.88 ± 19.27 291.39 ± 11.69 1455 ± 55.32 Exposed (n=11) 160.13 ± 10.13 191.19 ± 16.56 192.08 ± 9.18* Data presented as mean ± SE Data presented as mean ± SE *p < 0.05 51 Annual Report 2007-08 2. 3. 4. Figure 1. Level of RBCs cholinesterase in study population 5. In a subset of samples, the serum level of IgM reflected significant increase, indicating impairment of immune system of subjects at the level of exposure studied by us (Table 5). The levels of thyroid functional tests (T3, T4 and TSH) were within normal range (Table 6). The findings on significantly low RBCs ChE activity in exposed group in our study may indicate that the operational activities pertaining to production and / or formulation of these chemicals in industrial settings should be done with utmost care e.g. minimizing the risk by using protective devices to avoid absorption of these chemicals into the body and by instituting educational preventative modules on hygienic measures at periodic interval in order to ensure the safety to the workers. The analysis of data pertaining to clinical examination along with ECG of each subject of study population is in progress. 6. 7. 8. 9. Conclusion 10. The findings indicated that the workers involved in production of technical grade material and their formulation had comparatively significantly low level of RBCs cholinesterase activity. Serum level of IgM reflected significant increase, indicating impairment of immune system of subjects at the level of exposure studied by us. The levels of thyroid functional tests (T3, T4 and TSH) were within normal range. The neurobehavioral test battery indicated that maintenance and exposed group workers showed significant elevation in finger dexterity error test. Further, a significant decrease in neurobehavioral tests like forward and back memory, total digit symbol test, and increase in hand steadiness test was observed in maintenance group workers. 11. 12. 13. Acknowledgment: We are thankful to MR Variya, Yogesh Shah, SN Yadav, KA Patel, Raksha Agarwal, Divija Patel, Safia M Arab, Mukesh Vakharia, SV Upadhyay, Atul Shah, NJ Vaghela, Yamini Panchal for the technical assiatance during the study. 14. References 15. 1. 16. Bergmeyer H.U., Bowes G.N. Jr., Horder M. Jr., Moss D.W. Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 2. 52 IFCC method for aspartate aminotransferase. Clin. Chem. Acta. 1976; 70: F19F42. Empfehlungen der Deutschen Gesellschaft fur Klinische Chemie. Z Klin Chem u Klin Biochem 10 Jg 1972, 182-192. Mathieu MM, Artur Y ,Albury C et al. les members de la commission Enzymologie de la Societe Francaise de Biologie Clinique (1982): Recommandations pour la mesure de la concentration catalytique de la lactate deshydrogenase dans le serum humain d 30C. Ann.Biol.Clin.,40,123-125. Ellman GL, Courtney KD, Andres V Jr, Featherstone RH. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88-95 Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta 1999; 288:73-90 Srivastava AK, Gupta BN, Mathur AK, Mathur N, Mahendra PN, Bharti RS. The clinical and biochemical study of pesticide sprayers. Hum Exp Toxicol 1991; 10: 279-283. Bhatnagar VK, Karnik AB, Suthar AM, Zaidi SSA, Kashyap R, Shah MP, KulkarniPK, Saiyed HN. Biological indices in formulators exposed to a combination of pesticides. Bull Environ Contam Toxicol 2002; 68:22-28. Ames R, Steenland K, Jenkins B, Chrislip D, Russo J. 1995. Chronic neurologic sequelae to cholinesterase inhibition among agricultural pesticide applicators. Arch Environ Health 50:440-444. Davignon LF, St Pierre J, Charest G, Tourangeau FJ. 1965. A study of the chronic effects of insecticides in man. Can Med Assoc J 92:597-602. London L, Myers JE. 1998. Use of a crop and job specific exposure matrix for retrospective assessment of long-term exposure in studies of chronic neurotoxic effects of agrichemicals. Occup Environ Med 55:194-201. Steenland K, Dick RB, Howell RJ, Chrislip DW, Hines CJ, Reid TM, et al. 2000. Neurologic function among termiticide applicators exposed to chlorpyrifos. Environ Health Perspect 108:293-300. Anger W, Moody L, Burg J, Brightwell WS, Taylor BJ, Russo JM, et al. 1986. Neurobehavioral evaluation of soil and structural fumigators using methyl bromide and sulfuryl fluoride. Neurotoxicology 7:137-156. Van Wendel de Joode B, Wesseling C, Kromhout H, Monge P, Garcia M, Mergler D. 2001. Chronic nervous-system effects of long-term occupational exposure to DDT. Lancet 357:1014-1016. Kamel F, Rowland A, Park L, Anger W, Baird D, Gladen B, et al. 2003. Neurobehavioral performance and work experience in Florida farmworkers. Environ Health Perspect 111:1765-1772. Korsak RJ, Sato MM. 1977. Effects of chronic organophosphate pesticide exposure on the central nervous system. Clin Tox 11:83-95. Cole DC, Carpio F, Julian J, Leon N, Carbotte R, De Almeida H. 1997. Neurobehavioral outcomes among farm and nonfarm rural Ecuadorians. Neurotoxicol Teratol 19:277-286. Annual Report 2007-08 Determination of Urinary t,t-MA and S-Phenyl-Mercapturic Acid by Liquid Chromatography Tandem Mass Spectrometer in Footwear and Diamond Industry Workers VN Gokani, PB Doctor, HS Trivedi, RR Teli and PR Mansuri Abstract The urinary t,t-MA and S-PMA are the specific and sensitive biomarkers for the determination of low levels of benzene exposure. A sensitive LC-MS/MS method was developed for the simultaneous determination of t,tMA and S-PMA from human urine samples in a single run using dual column of two different ID to reduce the matrix effect. The recovery of spiked standard of t,t-MA (40ng/ml) in urine was 95.4±12.3% ranging from 79.4-114% and for SPMA (20ng/ml) was 69.7 ± 9.5% ranging from 60.1 - 89%. The limit of quantitation (LOQ) for t,t-MA was 5 ng/ml and that of SPMA was 0.03 ng/ml at the S/N ratio of 10. This method was applied on spot urine samples of 26 exposed subjects of footwear industry and 52 subjects of diamond industry, and 12 control subjects. The observed values of t,t MA and SPMA in these subjects are below the values of biological exposure indices (BEI) described by ACGIH of benzene exposure . However, both t,t- MA and SPMA values in one exposed subject, and SPMA in two subjects of the foot wear industry were found higher than BEI. Keywords: Trans, trans muconic acid, S- phenylmercapturic acid, benzene, LC MS/MS Introduction many as 25000 children between the age of 10 and 15 yrs are mainly employed in shoe making both for the domestic and international markets. Children work on soling (fixing upper portion of shoe to leather or rubber soles) with glue. The children working are exposed to benzene that is used as a solvent in glues. Most children suffer from respiratory problems, lung diseases and skin infections through constant exposure to glue and fumes. There is strong evidence that benzene is hematotoxic and causes various types of leukemia. It has been classified as a group - I 1 carcinogen by IARC . Two minor urinary metabolites, trans, trans muconic acid (t,t-MA) and s-phenylmercapturic acid (SPMA) are specific biomarkers of benzene exposure and can be used to monitor the benzene exposure in working environment2-7. Materials and Methods SPMA is considered to be a more specific biomarker than t,t MA for low level exposures to benzene, because excretion of t,t MA might be influenced by the uptake of sorbic acid (a widely used food preservative). While SPMA is formed after conjugation of the highly reactive benzene oxide with glutathione. Dietary or no other sources for excretion of SPMA than exposure to benzene have been reported so far. Chemicals: t,t-MA and SPMA standards were procured from SigmaAldrich, Methanol (MeOH), Acetonitrile (ACN) and HPLC water of LC grade were obtained from JT Baker ltd, USA, Iso propanol and disodium hydrogen phosphate (Merck (I) Ltd), Ethyl acetate, Creatinine, Formic acid from Fluka, Acetic acid and Hydrochloric acid were obtained from Across Organic, USA. Durapore Membrane filter from Milipore were used for the fitering of mobile phase. QSAX, 100 mg/3cc cartridge for sample clean up and extraction of analytes was used from Analchem Ltd, India. In this study, we have used a liquid chromatography / electrospray tandem mass spectrometry method for the determination of t,t-MA and SPMA in the same analytical run from the urine samples. This method was applied to monitor benzene exposure among child laborers of footwear industry of Agra and diamond industry workers of Surat. Thefootwear industry is a significant segment of the leather industry in India and ranks second among footwear producing countries next to china. According to estimate, as Sample collection: Spot urine samples from randomly selected 30 exposed child labourers working in footwear manufacturing at Agra, 15 matched control groups from school children studying in government school located in the neighboring area, 60 workers 53 Annual Report 2007-08 (CXP) were optimized by continuous infusion mode and the source dependent parameters like Nebulising gas (NEB), Auxilliary gas (heater gas), Curtain gas (CUR), Ion spray voltage, and source temperature were optimized by flow injection analysis (FIA) mode. engaged in the different processes (20-Planners, 20-Grinders, 10Markers, 10-Laser operators) of Diamond Manufacturing industries at Surat were collected in polyethylene container in cool condition. Frozen samples were transported in dry ice and preserved at -20 oC until analysis. Calibration curve: Calibration curves were prepared by processing spiked urine sample of healthy, non-smoker individual. Calibration standards were prepared by spiking urine to final concentrations of 5.0, 10.0, 20.0, 25.0, 40.0, 50.0 and 100.0 ng/ml of t,t-MA and SPMA mix prepared from reference stock standards. Calibration curves were prepared by plotting peak areas versus concentrations of standards. The calibration curves were generated using linear regression analysis. Sample clean up and extraction of analytes from urine samples: One ml urine sample of each subject was passed through a QSAX cartridge (100 mg/3cc) that was preconditioned with 1ml MeOH and 1 ml 2mM phosphate buffer (pH 6.8). Urine sample was applied at slow flow rate without drying the cartridge. Cartridge was rinsed with 1ml HPLC water followed by 1ml 2mM phosphate buffer (pH 6.8) and 1ml 0.1% aqueous acetic acid under full vacuum for 1 min. The analytes were eluted with 2x0.5 ml of 10% aqueous acetic acid. Final eluate was transferred to 10 ml centrifuge tube. One ml ethyl acetate was added to the eluate and vortex for 2-3 minutes. Upper organic layer (0.5 ml) was separated into a vacelute tube and evaporated it to dryness under nitrogen stream at room temperature. Dry residues were reconstituted in 0.5 ml of mobile phase and 5.0 µl was injected for LC-MS/MS analysis. Limit of detection (LOD) and limit of quantitation (LOQ) : The LOD and the lower LOQ were define as three and ten times respectively the standard deviation of the LC-MS/MS peak areas detected at the retention times of the analytes of interest in blank urine samples. Recovery: The recovery of t,t-MA and SPMA was calculated by comparing the area responses of extracted and non extracted standard solutions containing t,t-MA at 40 ng/ml and SPMA at 20 ng/ml, each analyzed for six times. Liquid chromatography: Perkin Elmer series 200 system equipped with quarternary pump, autosampler and peltier column oven was used for the analytes separation. HPLC separation was performed with dual column in series to minimize the matrix suppression on ionization. Altima C18- 50 x 2.1 mm id, 3µm particle size (Altech) and C18- 50 x 4.6 mm id, particle size 5 µm (Analchem ltd.) were used at the flow rate of 400 µl/min. A phenomenex universal guard column 4.0 mm Lx 3.0 mm id was used to protect the columns. Precision and accuracy: The inter-day and intra-day accuracy and precision were determined from the analysis on three independent QC samples tested on the same day and the five days of the study. Precision is expressed as the relative standard deviation (RSD) of the values found over the mean for each concentration (% CV). FDA acceptance criteria for precision are RSD < 20%. Gradient profile of mobile phase, started with 40% aqueous formic acid (0.1%) and 60% methanol with 0.1% formic acid was held for 3 mins. It was then increased from 40% to 80% in 4 min. and held for further 2 min. It was then reduced from 80% to 40% in 2 mins, where it was held for 3 min. to wash the column. The total run time was 14 min. Matrix effect on ionization: Pot column infusion experiments were performed to assess ion suppression. By using an infusion pump, a continuous posts column infusion of standard solution was introduced into the analytical LC system through a T-connector, during injection of an extract of urine. Ion suppression of analyte's signal by the urinary matrix was examined as negative chromatographic peak from the elevated baseline. Mass spectrometry: An API 3000 triple quadrupole mass spectrometer from Applied Biosystem was used for MS/MS analysis. The mass spectrometer was equipped with a turbospray (ESI) interface. Mass and product ion spectra were acquired in negative ionization mode.MRM transitions were monitored for quantitative analysis of t,t-MA and SPMA. Mass to charged ion ratio (m/z) of 141 and 238 were selected as precursor ions (Q1) for t,t-MA and SPMA respectively, while m/z 97 and 109 were selected as product ions (Q3). Creatinine measurement: 24 hours creatinine clearance is a measure of kidney function. Creatinine of spot or random urine sample is a measure of concentration or dilution of the urine. Urinary creatinine provides a good adjustment for the variations in concentration of spot urine samples due to differences in fluid intake among workers. Therefore creatinine of all the spot urine samples was measured by the alkaline picrate method8. All the results were expressed as t,t-MA and SPMA µg/g creatinine. For each analyte the compound dependent parameters like Declustering potential (DP), Focusing potential (FP), Enterance potential (EP), Collision energy (CE), and Collision exited potential 55 Annual Report 2007-08 Annual Report 2007-08 Table 1 Urinary concentrations of t,t-MA and SPMA (µg/g creatinine) in footwear industry workers Metabolite Exposed N t,t -MA Controls Mean± SEM Range 61.16±11.86 5.61 -259.84 N 9.41 ± 0.31 0.07 -91.7 26 SPMA Mean ± SEM Range 40.2 ± 8.05 13.91 -118.41 0.51 ± 0.15 0.04 -1.83 12 N= number of samples 141.9 96.9 Table 2 Urinary concentrations of t,t-MA and SPMA in diamond industry workers Type of Operations N Concentration of t,t MA and SPMA (µg/g creatinine) t,t MA 238.3 Planning 18 Grinding 14 Marking 10 Laser operation 10 101.1 Figure 1: Extracted Ion chromatogram of t,t-MA and SPMA 24.62 ± 2.57 (3.22 – 36.44 ± (7.71 – 13.15 ± (ND – 17.03 ± (5.61 – Values are Mean ± SEM; Figures in parentheses indicate the range; N= number of samples concentrations (Mean ± SEM) for t,t MA and SPMA were found 61.16 ± 11.86, ranging from 5.61 - 259. 84 µg/g creatinine and 9.41 ± 0.31, ranging from 0.07 ± 91.7 µg/g creatinine, respectively. In control workers, out of 15 urine samples the creatinine values of 3 samples were found below the normal range. In 12 samples, the concentrations (Mean ± SEM) for t,t- MA and SPMA were found 40.2 ± 8.05 µg/g creatinine, ranging from 13.91 to 118.41 and 0.51 ± 0.15 µg/g creatinine, ranging from 0.04 to 1.83 µg/g creatinine, respectively. of SPMA was 0.03 ng/ml, while Limit of Quantitation for tt-MA was 5 ng/ml and for SPMA was 0.1 ng/ml at the S/N ratio of 3 and 10, respectively. Results selected as the quantitative transitions for t,t-MA and SPMA. The extracted ion chromatogram of t,t-MA and SPMA is shown in Figure 1. Total run time of analysis was 14 minutes. t,t-MA was eluted at 3.1 min. and SPMA was eluted at 5.3 min. The product ions mass spectrum for t,t-MA and SPMA were recorded in negative ionization mode. The most intensive fragment (product ion) for t,t-MA was detected at m/z 97, while for SPMA it was detected at m/z 109. These transitions were The linearity of the calibration curve was determined over the ranges 10-100 ng/ml and 5-50 ng/ml for t,t-MA and SPMA 2 respectively. For each analyte, the coefficient determination (r ) was greater than 0.99. The detection limit for t,t-MA was 1 ng/ml and that 56 70.69) 6.56 101.97) 2.92 18.89) 4.38 64.84) SPMA 0.21 ± 0.04 (0.02 – 0.81) 0.08 ± 0.01 (0.01 – 0.27) 0.07 ± 0.02 (ND – 0.17) 0.16 ± 0.04 (0.04 – 0.47) The recovery (Mean ± SD) of spiked standard of t,t-MA (40ng/ml) in urine was 95.4 ± 12.3% (n=6), ranging from 79.4-114% and for SPMA (20ng/ml) it was 69.7 ± 9.5% (n=6) ranging from 60.1-89%. The intraday and interday coefficient variation (% CV) were below 20 % for both t,t-MA and SPMA, which met the FDA acceptance criteria. During the experiment of ion suppression effect of urine, the negative peaks revealed that the matrix effect was not the same for all retention times. Therefore, dual column of different diameter were used to set the retention time of analytes and reduced the matrix effect on elution. Quantitation of urinary t,t-MA and SPMA in diamond Industry: Out of total 60 samples, two urine samples of planner and 6 samples of grinder did not process because of the lower values of creatinine than the normal range. Urinary concentrations of t,t-MA and SPMA of workers are given in Table 2. The concentration of SPMA was found higher in planner and laser operators compared to grinders and markers. According to survey carried out by NIOH, all other groups except grinders and laser operators use solvents. Usually markers and planners sit in the same room. Quantitation of Urinary t,t-MA and SPMA in Footwear Industry: Urinary concentration of t,t-MA and SPMA of exposed and control workers are given in Table 1. Out of total 30 urine samples of exposed workers, the creatinine values of 4 urine samples were found below the normal range. In 26 samples of exposed subjects the 57 Annual Report 2007-08 Discussion 3 A sensitive and specific LC-MS/MS method has been standardized for the simultaneous measurements of two urinary metabolites, t,t-MA and SPMA, which are known to be good biomarkers of low levels of benzene exposure in humans. Previous studies have indicated that the exposure level could influence the ratio of benzene metabolites. At low levels of exposure to benzene, excretion of t,t-MA can be relatively high even when SPMA excretion is very low9,10. Urinary SPMA in workers exposed to 0.5-ppm benzene has been reported in the range from 7.2to 25 µg/g creatinine. Similarly, the predicted excretion of t,t-MA ranged from 0.39 to 1.1 mg/g creatinine for 0.5 ppm benzene exposure11. As t,t-MA is a metabolite of sorbic acid12 the values of t,t-MA in urine are strongly influenced by the consumption of food or drinks containing sorbic acid which is used as a preservative, while SPMA is a specific biomarker of benzene exposure at low level. It is observed by Wiesel et al13 that at relatively low benzene exposure levels, the complexity of the urine matrix suppressed the ionization. The suppression of monitored analytes by co-eluting matrix compound was also observed in our method. Therefore, in HPLC separation dual columns of different dimensions were used to reduce the matrix suppression on analytes at their elution times. In a study, among Chinese footwear workers, it was reported that these workers were exposed to high levels of benzene, toluene and other toxic solvents contained in the adhesive used in the shoe making process and many workers have been affected with aplastic anemia, leukemia and other health problems14. The observed values of t,t MA and SPMA in our study are below the values of Biological Exposure Indices (BEI) i.e. 500 µg/g creatinine for t,t-MA and 25 µg/g creatinine for SPMA, described by ACGIH15 of exposure to benzene. Except in one exposed subject in the footwear industry the values of t,t-MA and SPMA, and in two subjects the values of SPMA were found higher than BEI described by ACGIH. These results also support that the diamond manufacturers have stopped the use of benzene in diamond processing, as exposure is very low. Observed values might be due to exposure through other sources of environmental pollution. 4 5 6 7 8 9 10 11 12 13 References 1 2 14 IARC Monograph on the evaluation of carcinogenic risk to humans: Occupational exposure in petroleum refining, crude oil and major petroleum fuels, IARC; Lyons, France 1989:45. Inoue O, Seiji K , Nakatsuka H, et al. Urinary trans, trans muconic acid as an indicator of exposure to benzene. Br. J. Ind. Med. 1989; 46:122-127. 58 15 Ghittori S, Maestri L, Rolandi, L. Lodola L, Fiorentino ML, Imbriani M. The determination of t,t-MA in urine as an indicator of exposure to benzene. Appl. Occ. Env. Hyg., 1996; 11:187-191 Ruppert T, Scherer G, Tricker AR, Adlkofer T. trans, transmuconic acid as a biomarker of non-occupational environmental exposure to benzene. Int. Arch. Occup. Environ Health, 1997; 69:247-251. Van Sittert NJ, Boogard PJ, Beulink GD. Application of the urinary S- phenylmercapturic acid test as a biomarker of low level of exposure to benzene in industry. Br. J. Ind. Med. 1993; 50:460-469. Ghittori S, Imbriani, M Mestri L, Capodaglio E, cavallen A. Determination of s-Phenaymercapturic acid in urine as an indicator of exposure to benzene. Tox. Lett. 1999; 108: 329-34. Melikian AA, Qu Q, Shore R. et al. Personal Exposure to different levels of benzene and its relationships to the urinary metabolites s-Phenylmercapturic acid and trans, trans- muconic acid. J chromatogr. B Analyst Technol. Biomed. Life Sci. 2002; 778: 211-221. Harold Varley Determination of creatinine in urine. Practical Clinical Biochemistry, Vol 1, Allan H Gowenlock and Maurice Bill th (Eds.), 5 Edition, 1980; 484-485. Subourin PJ, Bechtold WE, Griffit WC Bimbaum LS, Lucier and Henderson RF. Effect of exposure concemntration, exposure rate and route of administration on metabolism of benzene by F344 rats and B6C3F1 mice. Tox. Appl. Pharm.1989; 99: 421. Henderson RF, Subourin PJ, Bechtold WE, Griffith WC, Medinsky MA, Bimbaum LS and Lucier GW. The effect of dose, dose rate, route of administration and species on tissue and blood levels of benzene metabolites. Environ. Health Perspect. 1989; 82:9. Melikian A, O'Concor, R, Prahalad AK , Hu P, LI H, K agan M Thompson S. Determination of the urinary benzene metabolites S-phenylmercapturic acid and trans,trans-muconic acid by liquid chromatography/electrospray tandem mass spectrometry. Carcinogenesis, 1999; 20 (4): 719-726. Barbieri A, Sabatini L, Accorsi A, Roda A & saverio Violante F. Simultaneous determination of t,t-muconic,Sphenylmercapturic and S-benzylmercapturic acids in urine by a rapid and sensitive liquid chromatography /electrospray tandem mass spectrometry method. Rapid Communication in Mass spectrometry. 2004; 18: 1983-1988. Weisel c, Y u R, Roy A, Georgopoulos P. Env. Hlth Persp. 1996; 104:1141. Chen MS, Chan A China's “ Market economics in Command” Footwear workers Health in Jeopardy. Int. J. Health Serv., 1999; 29 (4) : 793-811. American conference of Governmental Industrial Hygienists. Threshold Limit Values and Biological Exposure Indices for 2001, American Conference of Govt Industrial Hygienists, Cincinnati, OH, 2001. Annual Report 2007-08 Occupational Work Stress Among Cycle Rickshaw Pullers CK Pradhan, B Dalal, K Nayak and S Thakur Abstract The study was undertaken to evaluate the effect of rickshaw pulling on physiological and biochemical parameters of cycle rickshaw pullers. Fifteen cycle rickshaw pullers and eight control subjects, in the age group of 20-39 years were selected randomly from K olkata. Each subject participated for two consecutive days for determination of maximal oxygen uptake (VO2max) and energy expenditure study for doing work in three phases. Pre and post exercise blood samples were collected and analysed for biochemical parameters. VO2max of the rickshaw pullers was significantly higher than those of the control subjects. Average working heart rate, peak heart rate and energy expenditure of the cycle rickshaw pullers and the control subjects showed that the workloads of both the groups are heavy to very heavy. The energy requirement was same for both the groups while doing heavy work. Significant difference in glucose level was observed between rickshaw pullers and control subjects at pre-exercise condition. The Lactate Dehydrogenase (LDH) in preexercise condition of cycle rickshaw pullers was higher than normal range. Increasing trend of Creatine K inase (CK ) was noted after exercise in pullers. Cycle rickshaw pulling is categorised as heavy to very heavy job. Keywords: Rickshaw pullers, maximal heart rate, energy expenditure, creatine kinase Introduction Approval of the ethical committee of the institution was obtained. Written consent was obtained from all subjects prior to study. A large population is engaged in pulling cycle rickshaw as occupation in India and other South Asian countries. Strenuous physical activities influence their physiological and metabolic responses. The workload 1 of the cycle rickshaw pullers has been categorized as heavy . It is reported that significant fall in blood glucose takes place after heavy 2 exercise . Earlier study indicated that serum creatine kinase (CK ) increased during first hours after exercise3. Heavy exercise increases lactate production. Lactate dehydrogenase (LDH) activities enhances after heavy physical exercise or submaximal exercise for a long duration4,5. However, data on difference in physiological responses during various work phases and biochemical characteristics induced by cycle rickshaw pulling were not available. The objective of the study is to evaluate the effect of rickshaw pulling on physiological and biochemical parameters of cycle rickshaw pullers and compare the data with that of control subjects. Experimental Design: Each rickshaw puller participated for two consecutive days. On the first day, body height, weight and resting heart rate were recorded. Body surface area (BSA)6 and body mass index (BMI) were calculated from the height and weight of the subjects. Subsequently, the subjects were asked to pull rickshaw with two passengers for a period of 15 minutes according to their own habit of pedalling frequency on the same route in order to follow a standard road condition. After completion of this work, they are allowed to take rest for 15 minutes. This phase of 30 minutes (work plus rest) was considered as Phase I. Again the cycling was continued and designated as Phase II and Phase III. During pulling cycle rickshaw, heart rates were monitored every minute by heart rate monitored (Polar, Finland). The average working heart rate (AWHR) was calculated from the fourth minute of work7. The peak working heart rate of the subjects was recorded. Recovery heart rates were also recorded every minute and sum of recovery heart rate was calculated. The energy expenditure for cycle rickshaw pulling was calculated from the peak working heart rate (PWHR) using the equation of Datta and Ramanathan8. On the second day, the subject was allowed to do graded exercise in a bicycle ergometer (Biodex, USA) in the laboratory in order to determine maximal oxygen uptake (VO2max) and maximal heart rate (HRmax). Materials and Methods Subjects: Fifteen cycle rickshaw pullers in the age group of 20-39 years, were selected randomly. Eight control subjects (non rickshaw pullers) of same age group were selected from the adjoining area of K olkata. They were engaged in jobs like gardening and farming. 59 Annual Report 2007-08 were 134 ± 12.4, 133 ± 14.9 and 134 ± 16.7 bpm respectively suggesting that pulling cycle rickshaw is a job of heavy category17. Control subject also participated in this study for two consecutive days. On the first day, physical characteristics and resting heart were noted. HRmax and VO2max were determined in the laboratory. Next day, the subject was asked to pedal a bicycle ergometer (similar to cycle rickshaw pullers) in three phases of 15 min of work and 15 min of rest, each. The ergometer load was adjusted to produce heart rates in the range of 80% to 90% of their maximal heart rate. PWHR was noted and AWHR and recovery heart rates were also recorded every minute. During experiments the dry bulb (DB) temperature was 30.6 ± 3.09 ° C and wet bulb (WB) temperature was 27.2 ± 1.46 ° C. The VO2max study was conducted in the laboratory in comfortable environment at DB 25.7 ± 1.54 ° C; WB 18.7 ± 2.16 ° C. The mean PWHR of rickshaw pullers were found to be 145 ± 18.2 to 146 ± 14.9 bpm. This observation also substantiated that the job 18,19 was very heavy . As the sum of recovery heart rate (beats) did not vary (Phase I-1250 ± 110.8, Phase II 1254 ± 117.4 and Phase III 1258 ± 130.8), it could be interpreted that the work stress imposed on the rickshaw pullers were same in three phases. Repetition of work has no effect on physiological functions. In case of the control subjects, the values in three phases were 1339 ± 267.10, 1331 ± 248.50 and 1277 ± 251.98 beats respectively, which were not significantly different from each other as also with those of rickshaw pullers. Biochemical analysis: In case of rickshaw pullers, on the first day, 2 ml of blood sample was collected from antecubital vein one hour after completion of three phases of exercise. The state was designated as post-exercise condition. Next day, blood was also collected when the subject reported to the laboratory and was at rest for one hour. This state was described as pre-exercise condition.The control subjects, on the first day, were allowed to take rest for one hour after reporting at the laboratory followed by collection of blood samples (2 ml) from antecubital vein. This was pre-exercise condition. Next day, it was collected after work (pedalling a bicycle ergometer). This stage was defined as post exercise condition. The energy expenditure (EE) of rickshaw pullers in three phases -1 were 21.7 ± 2.27.and 21.7 ± 2.82 and 21.3 ± 3.43 kJ.min respectively and therefore the job of cycle rickshaw pulling can be 17,19 categorized as heavy . The results of the control subjects were almost same with the rickshaw pullers. Relative costs of work of the cycle rickshaw pullers and control subjects as percentage of maximal energy expenditure have been presented in Table 1. Result showed that there is an decreasing trend of the values in rickshaw pullers compared to control subjects in all the three phases of work. Significantly less value (p<0.05) was observed in rickshaw pullers in phase I of work. Each sample of blood was separated in two tubes: one containing fluoride (1 ml) for glucose; and the other (1 ml) was allowed to clot for serum separation. The following parameters were assayed by Chemwell analyser (USA) using standard commercial kit following methods of (i) Trinder9 for plasma glucose; (ii) Bakkar10 for Serum LDH; (iii) Ellis11 for Serum CK ; (iv) Bowers12 for Alkaline Phosphatase and (v) Bergmeyer13 for SGOT (vi) Fossati et al14 for Serum uric acid. Table 1. Results The mean age and BMI of rickshaw pullers was 27.8 ± 5.95 years and 21.76 ± 3.12 kg m-2 and that of control subjects were 25.6 ± 5.53 years and 22.20 ± 3.56 kg m-2 respectively. There was no significant difference in age as well as BMI. BMI values indicated that both the groups belonged to normal category15,16. Relative cost of work as percentage of maximal energy expenditure Work Phase Control Subjects (n = 8) Rickshaw puller (n = 15) Phase I 81.44 ± 9.58 74.18 ± 7.72* Phase II 13.20 83.25 ± 74.26 ± 10.44 Phase III 79.88 ± 6.90 73.15 ± 12.35 The biochemical variables in pre-exercise condition were found to be out of standard reference range, as manifested by elevated level of LDH (53% cases in rickshaw pullers, 38% cases in control subjects), plasma glucose (13% cases in rickshaw pullers, no cases in control subjects), CK (33% cases in rickshaw pullers, 25% cases in control subjects). Elevation of LDH was observed in 80% cases and that of CK was noted in 60% cases as an effect of exercise in tested population of rickshaw pullers. Among the control subjects, rise in LDH was noted in 50% cases as an effect of work and elevated CK was observed in 25% cases like pre-exercise condition. The average HRmax of the rickshaw pullers was 187 ± 6.2 bpm and it was significantly higher (p <0.05) than the value of control subjects (179 ± 10.9). The VO2max of the rickshaw pullers (36.6 ± -1 -1 4.32 ml.kg .min ) were significantly higher (p<0.001) compared to control subjects (30.1 ± 3.14 ml.kg-1.min-1). The heart rates during work varied from subject to subject in different phases. The AWHR of rickshaw pullers in phase I, II and III 61 Annual Report 2007-08 Table 2 shows that effect of work on energy utilization among the cycle rickshaw pullers and control subjects. The plasma glucose level was found to be within normal range at both pre and post exercise session. The level of serum LDH increased at post-exercise session in comparison to that of pre-exercise condition among rickshaw pullers. Similar trend was also noted in control subjects. Similarly, the level of serum CK was also elevated after rickshaw pulling in comparison to that at resting condition. change in the mean values of different parameters in different phases. Energy requirement was same for both the groups while doing heavy work. Relative cost of work of the control subjects were significantly higher than those of the rickshaw pullers during work in phase I, suggesting the metabolic changes took place during course of work. In both rickshaw pullers and control subjects, the glucose level showed lower trend after post exercise session compared to that of pre-exercise condition. The interesting observation noted was significantly higher value (p<0.001) in post-exercise condition of cycle rickshaw pullers compared to control subjects. Exercise induced rise in LDH was noted both in rickshaw pullers and control subjects. Similarly the level of Serum CK was elevated after rickshaw pulling in comparison to that of pre-exercise condition. Decreasing trend of CK at post exercise session was noted in control subjects. The findings reveal that creatine phosphate, as coin of energy, during exercise was recovered by CK in rickshaw pullers only. The effect of work on serum alkaline phosphatase (ALK P) and serum aspartate transaminase (AST) is also given in Table 2. Pre and post exercise values of ALK P were within normal range both in case of rickshaw pullers and of control subjects. In case of AST, a significant (p<0.001) difference was observed between control and rickshaw pullers at both post and pre-exercise condition. In post exercise condition, uric acid was noted above normal range in case of control subject. On the other hand, uric acid was exhibited within the normal range among rickshaw pullers at both pre- and post- exercise session. Liver function was assayed by estimating the ALK P and AST. In case of control subjects, AST and ALP level in pre and post exercise session maintain normal range. Higher than normal value of AST was observed among rickshaw pullers both at pre and post-exercise condition. As the rickshaw pullers perform heavy work, the AST level, in pre and post-exercise session, might be maintained at normal level. Analysis of serum uric acid revealed that heavy work induces oxidative stress in control subjects as manifested by increase in uric acid in post exercise session compared to pre exercise session whereas the rickshaw pullers habituated with this profession may not face such stress. Discussion The study indicated that the present rickshaw pullers had similar work capacity to that of those engaged in other occupations like steel mill workers20, Indian soldiers21 and agricultural workers22. VO2max of the rickshaw pullers were significantly higher than those of the control subjects. The job of cycle rickshaw pulling is considered as heavy to very heavy based on peak working heart rate and energy expenditure17,19. Data of different phases of work showed that there was no significant Plasma Glucose (mg/dL) Pre exercise Post exercise Control (n=8) Cycle Rickshaw Pullers (n=15) Control (n=8) Cycle Rickshaw Pullers (n=15) 69.3±12.16 81.8* ±15.13* 61.1±10.32 75.4 ± 7.43* * * ** ** Serum LDH (U/L) 373.1±76.02 469.1 ±61.66 411.1±113.93 477.0 ± 58.20 Serum CK (U/L) 151.5±90.74 182.9±69.96 133.9±69.57 202.9 ± 42.96 Serum ALK P (U/L) 243.63±68.17 227.00±150.58 236.25±82.93 222.37 ± 92.77 Serum AST (U/L) 29.00±8.68 44.50±6.28* * 29.88±14.28 44.10 ± 8.35 Serum uric acid (mg/dL) 5.69±1.78 6.30±1.81 5.79±1.35 5.30 ± 1.23 Values are Mean ±SD for each group. Values in parenthesis indicate number of subjects * = p<0.05, * * = p<0.01, * * * = p<0.001 (compared between control and rickshaw pullers 62 References 14. Fosatti P , Precipe L, Berti G Use of 3,5-dichloro 2 hydroxy benzene sulpfonic acid/ aminophenazone choromogenic system in direct enzymatic assay of uric acid in serum and urine. Clin. Chem 1980; 26:227-231 1. Pradhan CK , Thakur S, Mukherjee AK and Roy Chowdhury A: Physiological assessment of cycle rickshaw pullers. Ind J Physiol & Allied Sci.2004; 58 :113-118. 15. World Health Organisation (WHO) Physical status: the use and interpretation of anthropometry. Technical Report Series No. 854. Geneva : WHO 1995. 2. Mamus RT, Santos MGD, Campbell B and K reider R Biochemical effects of carbohydrate supplementation in a simulated competition of short terrestrial duathlon. J Int Soc Sports Nutrition 2006; 3: 6-11. 16. Weisell RC. 2002, Body mass index as an indicator of obesity. Asia Pacific J Clin Nutr 2002; 11(Suppl) : S681 - S684. 17. Astrand PO and Rodahl K . Textbook of Work Physiology, 3rd edn. New Y ork : Mc-Graw Hill Book Company; 1986. 3. Havas E, K omulainen J and Viko V Exercise induced increase in serum creatine kinase is modified by subsequent bed rest. Int J Sports Medicine 1997; 18: 578-582. 18. Brouha L: Physiology in Industry. New Y ork : Pergamon Press. 1960. 4. Costill DL, Fink WJ, Pollack ML Muscle fiber composition and enzyme activities in elite distance runners. Med. Sci. Sport 1976; 8 : 96 100. 19. Sen RN and Nag PK Work organization of heavy load handling in India. J Human Ergology 1975; 4, 103-113. 20. Saha PN. Aerobic capacity of steel workers in India. Ergonomics 1978; 21:1021-1025. 5. Wu HJ, Chen K T, Shee BW, Chang HC, Y i-Jen Huang Y J, Rong-Sen Y ang RS Effects of 24 h ultra-marathon on biochemical and haematological parameters. World J Gastroenterol 2004; 10 : 2711-2714. 21. Sengupta J, Joseph NT and Srinivasulu N Ergonomics studies of a digging tool. Ind J Physiol & Allied Sci. 1974; 28 : 22-28. 6. Banerjee S and Sen RN. Determination of the surface area of the body of Indians. Journal of Applied Physiology 1955; 7: 585 - 588. 22. Nag PK and Pradhan CK : Ergonomics in the hoeing operation. Int J Industrial Ergonomics 1992; 10: 341-350. 7. Nag PK , Sen RN and Ray US. Cardiorespiratory performance of porters carrying loads on a treadmill. Ergonomics 1979; 22 : 897907. 8. Datta SR and Ramanathan NL. Energy expenditure in work predicted from heart rate and pulmonary ventilation. J Appl Physiol 1969; 26 : 297 - 302. 9. Trinder P Determination of Glucose in blood using glucose oxidase with an alternative oxygen acceptor. Anal Clin. Biochem 1969; 6:24 Table 2. Effect of exercise on biochemical changes among control and cycle rickshaw pullers Variable (Unit) Annual Report 2007-08 ** * 10. Bakker AJ, Bakker A, Bierma-Ram A, Dijkstra JT, Renting-Wiering H, Syperda H, Z ijlstra A. Improved reliability of measurement of lactate dehydrogenase by IFCC method in heparin plasma. Clin Chem 2005; 51: 215-217. 11. Ellis G, Gold berg DM Automation of a kinetic spectrophotometric assay for ATP: creatine phosphotransferase activity suitable for human serum and muscle extracts. Enzymologia 1972; 42:407422. 12. Bowers GN Jr., McComb RB. A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin Chem 1966; 12:70-89. 13. Bergmeyer HU, Bowers GN, Horder M, Moss DW IFCC method for aspartate aminotransferase. Clin Chim Acta 1976; 70:F31-40. 63 Annual Report 2007-08 Blood Methemoglobin Levels in the Dye-Stuff Workers AB Patel, R Beniwal, BC Lakkad and VK Shivgotra Abstract A number of reports from the developed countries indicate that drugs, cosmetics or substances of abuse are the commonest causes of acquired methemoglobinemia. There are very few reports of occupational methemoglobinemia from these countries that may be due to better regulation of manufacturing processes. In a developing country like India, the chemical industry is growing rapidly and is highly concentrated in some parts of the country including Gujarat. The present study was done on 865 workers of various departments of a chemical manufacturing unit from Gujarat, which is engaged mainly in the production of dye or dye intermediates. A complete history was recorded with respect to duration and nature of occupation, demographic data, clinical symptoms etc on predesigned proforma. Samples were collected for routine analysis of urine and blood methemoglobin levels. The blood methemoglobin (MeHb) levels were not found to change significantly with the increase in the duration of employment. Keywords: Dye stuff-workers, dye and dye intermediates, blood methemoglobin Introduction Materials and Methods There are larger numbers of dye and dye intermediate units in India and Gujarat accounts for about three fourths of them. These units are engaged in the production or utilization of various chemicals including aromatic amines like p-aminophenol, p-phenylenediamine, chlorine derivatives of o- and p-toluidine, toluenediamine, diaminodiphenylmethane and p-nitroaniline. Various pathological effects by these chemicals have been documented most frequent being methemoglobinemia. Subjects: A total of 865 subjects enrolled for the study from an industry in Gujarat manufacturing and exporting over 500 chemical products including dyes and dye intermediates bulk drugs and intermediates, cresol and cresol derivatives, epoxy resins and epoxy hardeners, formaldehyde etc. were included in the study. These workers were employed in different departments and worked in three shifts during a day. A written consent was obtained from each subject. A complete history was recorded with respect to duration and nature of occupation, demographic data, etc. on a predesigned proforma. Hemoglobin can accept and transport O2 only when the iron atom is in its ferrous form. When hemoglobin becomes oxidized, 3+ it is converted to the ferric state (Fe ) or methemoglobin (MeHb) resulting in to a condition called methemoglobinemia in which there is inhibition of binding and delivery of oxygen by a red blood cell1. MeHb lacks the electron that is needed to form a bond with oxygen and thus is incapable of O2 transport. Exposure to chemicals such as certain dyes and dyestuff intermediates with nitrate and nitrite groups may cause rise in MeHb levels. This condition is mainly caused by the intentional or non-intentional exposure of oxidizing agents such as nitrates and nitrites 2 including occupational exposures . This study was conducted to assess the blood MeHb levels in the dyestuff workers. The levels were correlated with the duration of employment. Analysis of methemoglobin (MeHb) from blood: The blood samples of the workers were collected from antecubital vein in the vacutainer tubes containing EDTA. The MeHb levels from the blood samples were analyzed immediately by the method of Evelyn and Mallay3 as modified by Henry4. Hemoglobin estimation was carried out 5 by using the Drabkins method and the levels of MeHb were expressed as % of hemoglobin. 54/837 subjects with non-detectable levels of blood MeHb were excluded from statistical analysis. Routine analysis of urine: Analysis of urine samples for routine parameters including bilirubin, urobilinogen, glucose, ketone, urinary blood and proteins were carried out by using the reagent strips (Multistix SG, Bayer Ltd). 65 Annual Report 2007-08 In the present study, certain cases showed microscopic hematuria but it did not show any significant difference with respect to the duration of employment. When blood in urine is not visible by a naked eye but visible only under the microscope is called microscopic hematuria. The potential causes of this condition includes urinary tract infection, bladder or kidney stone, enlarged prostrate, glomerulo-nephritis, cancer of kidney, bladder or prostrate and strenuous exercise. Exposure to exogenous oxidizing drugs (benzocaine, trimethoprim, sulphonamides etc) and chemicals such as certain dyes and dyestuff intermediates with nitrate and nitrite groups (like aniline dyes, nitrobenzene, nitroaniline etc) may accelerate the rate of formation of methemoglobin up to one-thousandfold, overwhelming the protective enzyme systems and acutely increasing methemoglobin levels. Though blood MeHb is one of the good biomarkers to assess the exposures of the workers of the dyestuff industry. The study did not show any significant change in the blood MeHb levels of the workers indicating that the workers are not significantly exposed to methemoglobinemia inducing compounds. Table 1: Blood methemoglobin levels of the workers in relation to their total period of employment Duration of Range Blood MeHb No. of employment (%) of Hb (% of Hb) * workers (yrs) # 0.09 -2.78 0.83 0.041 # 0.02 -3.15 0.79 0.051 # 0.04 -3.44 0.80 0.045 # 0.02 -3.20 72 0.88 0.082 10 to <20 281 20 to <30 199 > 30 225 Total 777 0.82 0.025 Environmental and Biological Monitoring of Chromium Exposure Near Dumping Sites of Non-Recyclable Waste by Chromium Based Industry Discussion Necessary ethical clearance was obtained from the Institutional Ethical Committee for the study. Statistical Significance for difference among the groups was analyzed by using one-way ANOVA. <10 Annual Report 2007-08 Acknowledgment: Ms SK Patel, Mr BS Solanki and Ms H Ahir are acknowledged for their assistance in the work. References 1. Wright RO, Lewander WJ, Woolf AD: Methemoglobinemia: etiology, pharmacology and clinical management Ann. Emerg. Med 1999; 34: 646-56. 2. Dewan A, Patel AB and Saiyed HN: Acute methemoglobinemia- A common occupational hazard in an industrial city in western India. J. Occup. Hlth 2001; 43(3): 168-171. 0.02 -3.44 * The values are Mean SEMs; 3. Evelyn K A and Malloy HT: Micro-determination of oxyhemoglobin, methemoglobin and sulfhemoglobin in a single sample of blood. J. Biol. Chem 1938; 126: 655-662. # F (3, 776)=0.38, p> 0.05 Results 4. Henry R, Cannon DC, Winkleman JW: Clinical Chemistry, Principles and Practice. New Y ork, Harper and Row, 1974; pp1149. The occupational groups with limited numbers and those with higher mean age are excluded from further analysis. Urine examination showed microscopic hematuria among 4.17 % (35/837) of the workers with no significant difference among different departments of the industry. 5. Drabkin DL, Austin JH: Spectrophotometric studies: Preparation from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 1935; 112: 51-65 Blood MeHb levels did not change significantly (F=0.38, p > 0.05) with the increase in the duration of employment (Table 1). No significant change was found in blood MeHb of workers working in different departments. LJ Bhagia, NG Sathawara, RA Rathod, JB Vyas, MI Shaikh, P Sekhar Abstract The aim of this study was to evaluate total chromium content in biological (blood, urine and hair) and environmental (soil, air and water) samples collected from dumping and control sites using Flame Atomic Absorption spectroscopy (AAS) technique. The chromium levels in air, water and hair samples of both control and exposed sites/subjects were within normal limits. Chromium levels in soil samples were found to be much higher in exposed (dumping) sites as compared to control sites but lower than the normal values reported for residential localities. In exposed subjects the concentrations were higher (statistically not significant) than those in control subjects. Nevertheless, higher values observed in blood and urine samples might be due to combined exposure to chromium from soil and food. Keywords: Chromium exposure, Biological and Environmental Samples, AAS Introduction Collection of biological and environmental samples: About four ml venous blood samples of 634 subjects (373 exposed and 261 control), 349 hair samples (269 exposed and 80 control) from nape of the neck region and 707 urine samples (412 exposed and 295 control) 11were collected and analyzed for chromium using standard methods 13 . Similarly, 35 drinking water samples (8 control and 27 exposed) and 34 soil samples (11 control and 23 exposed) were also collected using standard methods. A total of 122 air samples were collected using both high volume sampler (outdoor) and personal sampler (indoor) from various locations. 25 air samples (8 control and 17 exposed) were collected on glass fiber filter paper EPM 2000 (24 cm X 20 cm) 3 using PM10 high volume sampler at the rate of 1.1 m /min for 8 hrs. A total of 97 samples (29 control and 68 exposed) were collected on membrane filter (37mm, 0.8mm pore size) and in 1M HNO3 (analytical grade) using personal sampler (SK C) at flow rate of 2 LPM for 8hr/day. Chromium is a naturally occurring element found in rocks, animals, plants, soil, and in volcanic dust and gases1. Chromium exists in the environment primarily in two valence states 1) trivalent (chromium 3+ ), which is biologically active, occur naturally in environment and in food and 2) hexavalent (chromium 6+ ), is a toxic2 are generally produced from industrial pollution and anthropogenic activities3,4. Chromium (III) in trace amount is an essential nutrient. In the chemical industry, chromium is used primarily in pigments, metal finishing, leather tanning and wood preservatives. Chromium pigments are used in paints, inks and plastic coloring. Workers employed in such industries may have a higher chance of exposure to chromium than the general population. Breathing high levels of chromium (VI) can cause irritation to the nose, such as runny nose, nosebleeds, and ulcers and holes in the nasal septum. Ingesting large amounts of chromium (VI) can cause stomach upsets and ulcers, convulsions, kidney and liver damage, 1 and even death . Epidemiological studies have shown that workers occupationally exposed to chromium (VI) are considered to be at risk for developing lung cancer5-10. Pre-treatment / Digestion of samples: Biological samples: 2ml of whole blood was digested in wet digestion system (Ethios 1600, advanced Microwave lab station, Italy) using a mixture of 2ml of conc. HNO3 (ultra pure) and 0.2ml H2O2. The digested sample was diluted and make up the volume for 4ml with triple distilled water and centrifuged14. Materials and Methods Selection of sites: The exposure sites were selected where dumping of non-recyclable waste was done. Control sites were selected 5 kilometers away from the exposure sites. 66 Hair samples were washed twice with acetone and triple distilled water and dried in dust free oven at 50 º C. Approx 0.2 g hair sample 67 Annual Report 2007-08 was digested with 2ml conc. HNO3 (ultra pure), 1 ml triple distilled water and 0.2ml of H2O2 by wet digestion (Ethios 1600, advanced Microwave lab station, Italy) and make up the volume for 4ml with triple distilled water11,15,16. Table 1: Mean chromium level of environmental samples Environmental Samples: Approx. 2g sieved and dried soil samples were digested using 4ml of conc. HNO3 (ultra pure) and 5ml of distilled water on a hot plate at about 60-70 º C. The samples were evaporated to dryness and the final volume was made up to 10ml with 1M HNO3 solution (ultra pure). Air (mg/m3) The collected air samples were wet digested using 4ml of conc. HNO3 (ultra pure) and 5 ml of distilled water on a hot plate at about 60-70 º C and the filtrate was made to 4ml volume using 1M HNO3 (ultra pure). Calibration/analysis: All the digested samples were analyzed by flame Atomic Absorption Spectrophotometer (Perkin Elmer, USA, double beam with background corrector, model 3100) at the wavelength 283.3 nm. The method was standardized by preparing solutions of different standard chromium concentrations like 0.5,1 and 4 ppm. Samples Water ( mg/L) Exposed Indoor (Filter Paper media) Indoor (HNO3 in bubbler) Outdoor (High volume sampler) Tube well Municipality Soil ( mg/g) Control 0.26 0.03 (34) 0.2 0.05 (8) 0.63 0.06 (34) 0.09 0.5 (21) 0.07 0.01 (17) 0.018 0.002 (8) 30.53 4.98 4.36 0.53 (8) (10) 6.43 0.55 (17) 234.77 54.22 33.37 3.68 (11) (23) Values expressed as Mean SE, Values in parenthesis indicate number of samples Annual Report 2007-08 Biological Samples: The mean blood Cr level of the exposed subjects (4.34 ± 0.21 mg/dL) was higher as compared to control subjects (3.71 ± 0.18 mg/dL) (Table 2) and values reported by ATSDR1 (i.e. 2.0 to 3.0 mg/100ml). The mean urine Cr concentration of exposed subjects (27.93 ± 0.82 mg/L) was slightly higher than that of control subjects i.e. 21.12±0.51 mg/L (Table2). Assuming that urine excreted per day to be one liter, the values for both control and exposed subjects are higher than the value recommended by ATSDR (10mg/day)1. Cr from soil in exposed sites may be responsible for high concentrations of Cr in blood and urine of exposed subjects. But high Cr concentration in urine and blood samples of subjects from control sites cannot be explained from lower values of Cr observed in soil samples from control sites. It seems that Cr from food also might contribute to Cr concentration in blood and urine samples. Samples Blood (g/g) Urine The mean hair chromium level of exposed subjects (0.68 ± 0.06 mg/g) was slightly higher as compared to control subjects (0.53 ± 0.08 mg/g). The populations with no known exposure to chromium reportedly have hair levels ranging from 50-100 ppm1. (g/L) Exposed 4.34 0.21 (373) 0.68 0.06 (269) Control 3.71 0.18 (261) 0.53 0.08 (80) 13. Jenkins, DW. Toxic trace metals in mammalian hair and nails. 1979; EPA-600/4-79-049 14. Sathawara NG, Patel K G, Vyas JB,et al. Chromium exposure study in chemical based industry. J. Environ. Biol. 2007; 28(2): 405408 27.93 0.82 (412) 21.12 0.51 (295) References 1. 2. Values expressed as Mean SE, Values in parenthesis indicate number of sample The mean soil Cr levels in the exposed samples 234.77±54.22 mg/g were much higher than control samples 33.37±3.68 mg/g. Canadian Soil Quality Guidelines for the Protection of Environmental and Human Health17, reported that normal soil samples have Cr concentration of 64 mg/g. Samples collected from control sites have concentrations lower than 64 mg/g whereas exposed sites had much higher concentration. High levels of Cr in soil of exposed sites were expected because of dumping of non-recyclable waste of the industry. Results and Discussion Table 1 depicts the mean chromium levels of indoor air samples taken by personal samplers in the exposed and control sites. The Cr concentrations in indoor air samples are much lower than TLV18 of 0.5 mg/m3. The mean chromium levels of outdoor air 3 samples in the control site was 0.018±0.002 mg/m while that at exposed site was 0.07±0.01 mg/m3. There is no specific standard 1 for Cr in community environment . 68 Mancuso TF and Hueper WC. Occupational cancer and other health hazards in a chromate plant: A medical appraisal. I. Lung cancer in chromate workers. Ind. Med. Surg. 1951; 20: 358-363. 9. Mancuso TF. International Conference on Heavy Metals in the Environment. Toronto, Ontario, Canada. 1975; (Cited in U.S. EPA, 1991; and in U.S. EPA, 1984a) 10. Sano T. and Mitohara I. Occupational cancer among chromium workers. Jap. J. Chest Disorders 1978;37: 90-101 (Cited in U.S. EPA, 1984a) 11. Harrison, WW, Y urachek JP and Benson CA: The determination of trace elements in human hair by atomic absorption spectroscopy. Clin. Chem. Acta, 1969; 23: 83-91 12. Petering HG, Y eager DW and Wither SO: UP Trace metal content of hair\ -II Cadmium and lead of human hair in relation to age and sex. Arch. Environ Hlth., 1993; 27: 327- 330 Table:2: Mean chromium level of biological samples (g/dL) Hair Environmental Samples: The mean chromium level was found to be higher in water from exposed sites in both tube well water (30.53±4.98mg/L) and municipality water (6.43±0.55mg/L) as compared to control samples (4.36 ±0.53mg/L) (Table 1). However these values were lower than the prescribed standards i.e. 100 mg/L in exposed as well as control samples1. 8. 3. 4. 5. 6. 7. 15. Buckey, RA, Chem. HCA and Dreosti, IE. Radio-isotopic studies concerning the efficacy of standard washing producers for the cleansing of hair before zinc analysis. Amer. J.Clin. Nutr., 1984; 40:840-846. Agency for Toxic Substances and Disease Registry (ATSDR). Chromium Toxicity. U.S. Department of Health and Human Services, Atlanta, G.A. 2000 US EPA (US Environmental Protection Agency). Health Assessment Document for Chromium. Environmental Criteria and Assessment Office, US Environmental Protection Agency, Research Triangle Park, NC. EPA 600/8-83-014F. NTIS PB 85115905; 1984b. World Health Organization. Chromium Environmental health Criteria 61. Geneva, Switzerland. 1988 Hertel RF. Sources of exposure and biological effects of chromium. In: Environmental Carcinogens Selected Methods of Analysis, International Agency for Research on Cancer, Lyon, 1986; 8:79-92. IARC Scientific Publication No. 71. Leonard A. and Lauwerys RR. Carcinogenicity and mutagenicity of chromium. Mutat. Res. 1980; 76: 227-239 Langard S. The carcinogenicity of chromium compounds in man and animals. In: Burrows, C., Ed. Chromium: Metabolism and Toxicity. CRC Press, Inc., Boca Raton, FL, 1983; 13-30. Mackison FW, Stricoff RS and Partridge LJ. Occupational Health Guidelines for Chemical Hazards. National Institute for Occupational Safety and Health, DHHS (NIOSH) Washington, DC. 1981; Pub. # 81-123. 16. Y ung-Y ung T, Chin- Thin W, Wei-Tun C, Chin-Wang H. Determination of Trace Elements in Hair Specimens of Workers in Paint Factory. Analytical Science 1997; Vol. 13 Supplement 17. Canadian Soil Quality Guidelines for the Protection of Environmental and Human Health, 1997; Cepgrcqe.ccmc.ca/download/en/262 18. ACGIH: Threshold Limit Values for chemical substances and physical agents and biological exposure indices, Cincinnati, 2008. 19. Mertz, W. Chromium-Occurrence and function in biological systems. Phys. 1969; Rev 49,163-239. 69 Annual Report 2007-08 On-line Simultaneous Detection of Tri- and Hexa-valent Chromium in Urine by Ion Chromatography SSA Zaidi, SJ Gandhi and KA Patel Abstract Online separation, post-column derivatization and spectrophotometric determination of chromium species (chromium III and chromium VI) in human urine were carried out by ion chromatography. Study groups included healthy volunteers control, and occupationally exposed workers (welders, spray painters and coach manufacturers). Spot urine samples during the work-shift were collected and analysed within 24 hrs. Prior to analysis pH of all the urine samples was adjusted to 6.7 to 6.8. Calibration graph for chromium III and chromium VI revealed coefficient determination 0.998 and 0.994 with RSD 3.979 and 9.213%, respectively. The average level of Cr III (mean + S.D.) in control group (N=20) was found to be 37.54 + 21.63 µg/l or ppb. Chromium VI was also detected in 6 of the 20 control subjects and the values were 0.414 + 0.194 ppb. Chromium III content in welders (N=17), spray painters (N=13) and coach manufacturers (N=11) were registered to be 56.83 + 37.32, 34.06 + 19.55, and 92. 35 + 53.47 ppb, respectively. Chromium VI content in welders (N=15) and coach manufacturers (N=8) was measured to be 0.58 ± 0.39, and 0.35 ± 0.22 ppb respectively. Only 2 of the 13 spray painters exhibited chromium VI levels in their urine, and the values were 0.19 and 2.18 ppb. The level of Cr III was significantly higher (p<0.01) in coach manufacturers and welders (p<0.05) as compared to control. Keywords: Trivalent and hexavalent chromium, ion chromatography, welders, spray painters Introduction was reported, however for unexposed population it ranges > 1-2 µg/L or ppb. NIOSH considers all chromium VI compounds (including chromic acid) to be potential occupational carcinogens and recommends an exposure limit of 1 µg chromium (VI)/m3 for a 10-hour workday, 40-hour workweek. Worldwide reports are very scanty on the excretion of chromium in unexposed population and occupationally exposed workers. Even recent reports of ATSDR 2008 do not provide much information on the excretion of chromium species in urine of unexposed and chromium workers. In one of the recent studies1 conducted in 2004 by “ Eco Friends” in a village of Noraiakheda, K anpur, North India poisoning of ground water was reported 100 to 1000 times higher than the desirable limits for drinking water. The concentration of hexavalent chromium has been reported up to 16.3 mg/l that is 1630 times more than the WHO standards2 (0.01 mg/l) for drinking water at a depth of 140 ft in this village. With the aims of chromium toxicity background and health effects and the scarcity of the data in context of present Indian scenario it is urgently needed to measure chromium levels in our Indian environment. Total chromium has been reported by few investigators, however, chromium species i.e. Cr III and Cr VI level have hardly been attempted by researchers earlier. The present study describes the levels of dissolved chromium compounds (Cr III and Cr VI) in urine using ionchromatography in healthy control and occupationally exposed workers. India is one of the third largest producers of chromium compounds and contributes about 10% of the total world's production. People may also be exposed to higher levels of chromium if living near to the landfill sites with chromium-containing wastes, industrial facilities that manufacture or use chromium and chromium-containing compounds, cement-producing plants, industrial cooling towers that previously used chromium as a rust inhibitor, waterways that receive industrial discharges from electroplating, leather tanning and textile industries and busy roadways. Breathing in higher levels of chromium VI (greater than 2 µg/m3) can cause irritation to the nose, such as runny nose, sneezing, itching, nosebleeds, ulcers, and holes in the nasal septum. Long-term exposure to chromium has been associated with lung cancer. Breathing in chromium III does not cause irritation to the nose or mouth in most people. In the same way, small amounts of chromium VI that swallowed will not hurt, however, accidental or intentional swallowing of larger amounts has caused stomach upsets and ulcers, convulsions, kidney and liver damage, and even death. The concentration of total chromium in air (both Cr III and Cr VI) generally ranges between 0.01 and 0.03 µg/m3. Chromium concentrations in drinking water (mostly as Cr III) are generally very low, less than 2 ppb. A wide variation in the urinary level of chromium 71 Annual Report 2007-08 Statistical analysis: Instrument IC 2500 is equipped with Chromeleon software (6.70 version programme) for automated data processing and quantifying the analytes. This facility was used during this study. Materials and Methods Sample preparation: Spot urine samples about 25 to 50 ml were collected in fresh 60 ml vial without using any preservatives. Fresh vials were washed with 0.1 M HNO3 followed by RO water and then finally with ultra pure water (Millipore). All urine samples were passed through 0.45 µm syringe filter (ACRODISC® ). After adjusting pH of the samples to 6.7-6.8, 1 ml of the each filtered urine sample was applied on Dionex ion-chromatograph. Due to the relatively low levels of biological matrix in urine compared to blood, urine samples were directly applied. + 3 1 - Cr - 3.517 US EPA method 218.6 as modified by Dionex in its Technical Note No. 24 was employed to determine chromium III and chromium VI in urine samples. We, very first time employed this method for the simultaneous detection of trivalent chromium and hexavalent chromium in urine. It provides a beautiful mean of separating triand hexavalent-chromium from the other species and from the other biological constituents present in the sample. This method allows sufficient sensitivity to determine chromium (Cr III) as chromic ion (Cr3+ ) and Cr VI as chromate (CrO42-) ion. After on-line simultaneous separation of Cr III and Cr VI, these chromium species are allowed to react with post column reagent, diphenylcarbazide (DPC) to form derivatives, which are read spectrophotometrically at 520 nm employing PDA-100 detector. Assay Conditions CG5A Guard column 2 mM PDCA 2 mM Na 2HPO4 10 mM NaI 50 mM CH 3COONH4 2.8 mM LiOH Flow rate 1.0 ml/min Post - column reagent 2 mM DPC, 10% CH 3 OH 0.9 N H 2 SO4 Reagent flow rate Mixing device Detector wavelength Sample loop volume 0.5 ml/min Reaction Coil 520 nm 500 μL + 6 - 5.592 3+ 6+ Figure 1: Chromatogram of standards (trivalent Cr ; 2 ppm) and hexavalent (Cr ; 10 ppb) On-line simultaneous separation and detection of Cr III and Cr VI is shown in Figure 1. Figure 2 shows absorption spectra. Figure 3 indicates five-point calibration graph. Coefficient determination for Cr III and Cr VI were 0.998 and 0.994 with RSD 3.979 and 9.213 %, respectively. Good reproducibility of the results with consistent retention time was obtained (Figure 4). The above calibration of chromium III and chromium VI was used for the quantification of chromium species. CS5A Analytical column Eluent 2 - Cr Results Chemicals: All the chemicals and solvents used in this study were of high purity grade. Milli Q water of 18 mega-ohms was used throughout the study. Reference standards of chromium III and chromium VI, were purchased from Fluka (Germany). Other chemicals used in this study were of highly purified grade (more than 99.5 % pure). Dionex Ion-chromatograph IC 2500 was used for the chromatographic studies. Columns Annual Report 2007-08 Urinary Levels of Chromium III and Chromium VI in study population: The levels of chromium III and chromium VI are summarized in Table 1. The average level of Cr III (Mean + S.D.) in control group (N=20) was found to be 37.54 + 21.63 µg/l or ppb. Chromium VI was also detected in 6 of the 20 control subjects and the values were 0.414 + 0.194 ppb. Chromium III content in welders (N=17), spray painters (N=13) and coach manufacturers (N= 11) were registered to be 56.83 + 37.32, 34.06 + 19.55 and 92. 35 + 53.47 ppb respectively. Chromium VI content in welders (N=15) and coach manufacturers (N=8) was measured to be 0.58 ± 0.39, and 0.35 ± 0.22 ppb respectively. Only 2 of the 13 spray painters exhibited chromium VI levels in their urine, and the values were 0.19 and 2.18 ppb. Some of the occupational workers in each group did not show either Cr III or Cr VI content in their urine. The level of Cr III was significantly higher (p<0.01) in coach manufacturers and welders (p<0.05) as compared to control. Calibration curve: A mixture of standards (Cr III and Cr VI) at varying concentrations as mentioned below was prepared to obtain a five points calibration curve. Solutions were prepared fresh each time of calibration. Concentrations were: A) 100 ppb Cr+ 3 + 0.5 ppb Cr+ 6, B) 200 ppb Cr+ 3 + + 6 + 3 + 6 + 3 1.0 ppb Cr , C) 400 ppb Cr + 2.0 ppb Cr , D) 1000 ppb Cr + 5.0 ppb + 6 + 3 + 6 + 3 + 6 Cr , E) 2000 ppb Cr + 10 ppb Cr , F) 5000 ppb Cr + 25 ppb Cr . One ml of the above standard mixture of solution was injected and the instrument received 500 mL as injecting volume of sample/ standard. 72 Figure 2: Spectral representation of standards: Chromium III and Chromium VI 73 Annual Report 2007-08 Annual Report 2007-08 Table 1. Determination of Cr III and Cr* VI in urine by ion chromatography 4.00 Cr+3 Area [mAU*min] External UV_VIS_1 4.00 Cr+6 Area [mAU*min] External UV_VIS_1 Control Parameter Subject studied 3.00 3.00 2.00 2.00 20 Cr 3+ (ppb) 17 (N=17) (N=13) (N=11) (10.18 -77.75) (31.9 -225.8) Cr 6+ (ppb) 0.414 + 0.196 0.58 + 0.397 0.194 & 2.17 0.35 + 0.22 Detected (N=15) (N=15) (N=2) (N=8) (0.008 -1.43) -- (0.099-0.675) 0 1,000 2,000 3,000 4,000 5,000 6,000 * pH of all urine samples was adjusted to 6.7-6.8 ; Value represents Mean + SD; Figure in parenthesis indicate range # p<0.05 when compared with control group; @ p<0.01 when compared with control group ppb 0.00 0.0 5.0 10.0 15.0 20.0 25.0 30.0 2 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #9 1 - Cr+3 [B] Chromium VI 0.57 Figure 3: Calibration curve of standard chromium. [ A] Chromium III; [ B] Chromium VI mAU 3.17 CALIBRATION OF CR+3 AND CR+6 mAU AN 7 AUGUST 2008 SCALE #36 Urine sample44 dil 1:1 UV_VIS_1 WVL:520 nm -0.13 2.50 1- WVL:520 nm 33 2.3 -0.25 -0.38 2.00 -0.49 -0.8 1.50 10.0 1 r+3 -C 92 3.4 2 r+6 -C -0.00 2-3.033 UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1 . , std Cr+3 100 ppb+Cr+6 0.5 ppb std Cr+3 200 pp+Cr+6 1 ppb std Cr+3 400 ppb+ Cr+6 2 ppb std Cr+3 1ppm + Cr+6 5 ppb std Cr+3 2ppm+Cr+6 10 ppb std Cr+3 5ppm+Cr+6 25 ppb 4-Cr+6-5.692 23.5 SCALE #3 SCALE #4 SCALE #8 SCALE #9 SCALE #5 SCALE #6 chromium III and chromium VI were found quite high as compared to other studies reported from abroad. However, our results correlate with the previous results reported earlier from this Institute from the same place of this study. Higher levels may reflect indiscriminate disposal of chromium waste and poor hygiene system and occupational condition besides many other factors that may affect exposure and measurement. Several methods, as summarised by ATSDR3 are available for the analysis of chromium in different biological media and many of them are approved by federal agencies and organisation such as EPA and NIOSH. Earlier methods described total concentration of chromium in environmental and biological samples, which seems not to be appropriate from the toxicological implementation point of view, as Cr III is nutritionally required by the body while Cr VI is highly toxic and carcinogenic. Therefore it is of particular interest to explore toxicity associated with Cr VI. Earlier it was difficult to distinguish Cr III and Cr VI but now methods are available to quantify both forms of chromium species simultaneously. Therefore, estimation of Cr III and Cr VI is now gaining popularity to assess chromium toxicity and health effects associated with particular forms of chromium. EXT541NM 0.38 0.12 AN 7 AUGUST 2008 AN 7 AUGUST 2008 AN 7 AUGUST 2008 AN 7 AUGUST 2008 AN 7 AUGUST 2008 AN 7 AUGUST 2008 5 ppb WVL:520 nm 0.25 1 - CALIBRATION OF CR+3 AND CR+6 2 - CALIBRATION OF CR+3 AND CR+6 3 - CALIBRATION OF CR+3 AND CR+6 4 - CALIBRATION OF CR+3 AND CR+6 5 - CALIBRATION OF CR+3 AND CR+6 6 - CALIBRATION OF CR+3 AND CR+6 std Cr+3 1ppm + Cr+6 2 - Cr+6 mAU 1-2.417 [A] Chromium III 67 5.5 1.00 2 r+3 -C 00 - 3.7 3- 6 Cr+ min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.3 Figure 6: Overlay of urine sample with standard trivalent chromium (1 ppm) and hexavalent chromium (5 ppb) 50 - 5.4 0.50 0.0 -0.00 -13.1 -0.4 min 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 -0.82 -0.4 10.2 min 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.2 Figure 5: Chromatogram of urine sample of study subject indicating presence of trivalent and hexavalent chromium Figure 4: Standard chromatogram of Cr III and Cr VI showing reproducibility of retention time in overlay of standards Discussion level of analytes. Analytical column CS5A has unique ability to separate anionic and cationic species in single run of column and both species were found well separated by the standard procedure used in this study. Though chromium species are highly pH dependent and are inter-convertible; at acidic pH, chromium III dominates while alkaline pH favours chromium VI form. However at pH 6.8 both forms of chromium could be eluted with high degree of Automated on-line separation, derivatization and detection of chromium species (chromium III and chromium VI) by Dionex ionchromatograph IC 2500 provide excellent opportunity to separate chromium III and chromium VI species which is not possible in many of the sophisticated instruments used to quantify chromium at ultra low 74 13 92.35 + 53.47 @ (21.90 -152.24) 3-Cr+3-3.842 ppb 21 34.06 + 19.55 (N=20) Range 0.00 Coach manufacturers (8.5 - 46.70) Range 1.00 Spray painters 56.83 + 37.3 # 37.54 + 21.63 Detected 1.00 Welders accuracy. Decline or rise in pH affects the estimation. Therefore chromium in this study was estimated at pH 6.8. All urine samples were adjusted to p H 6.7 to 6.8 prior to the analysis and no sample treatment was given. Some urine samples were diluted wherever required. Results of this study indicated that chromium III was detected in all control and exposed subjects. Concomitant level of hexavalent chromium in control group was detected to excrete chromium VI in urine, however it presumed that chromium when entered the body is detoxified to chromium III that is excreted from the body. But we provide evidence that concomitant excretion of hexavalent chromium does also appear in the urine (Figures 5 & 6), which has not been reported earlier. Available reports indicate that excretion of hexavalent chromium in urine is rare and has almost not been described earlier. This needs further investigations. Levels of References 75 1. Eco Friends; Environmental Education Protection and Security: http:/www.ecofriends.org/reports /043Groundwater.htm 2. WHO, 1988 World Health Organisation. Environmental Health Criteria 61: Chromium. Geneva: WHO 197. 3. ATSDR 2002. ATSDR Tox Profiles: U.S. Department of Health & Human Services, Agency for Toxic Substances and Disease Registry, Division of Toxicology, U.S.A. Annual Report 2007-08 Chemically Induced Methemoglobinemia from Acute Nitrobenzene Poisoning AB Patel, A Dewan, KJ Upadhyay,* SA Patel * and JK Patel* Abstract Methemoglobinemia is an unusual and potentially fatal condition in which hemoglobin is oxidized to methemoglobin lowering its ability to bind and transport oxygen. The most common cause of methemoglobinemia is the ingestion or inhalation of oxidizing agents such as nitrates and nitrites. A rare case of nitrobenzene poisoning after oral ingestion which presented with altered sensorium and cyanosis is reported. Timely identification of the compound and the estimation methemoglobin (MeHb) were helpful in saving the patient. The importance of laboratory support of a poison information center in evading death due to unknown poisoning has been highlighted. Keywords: Nitrobenzene poisoning, methemoglobinemia, poison information centre Materials and Methods A worker (20 yrs) associated with screen printing work, ingested an unknown liquid used for screen printing in an intentional poisoning at around 6:00 hours in the morning. He was admitted to the Civil Hospital, Ahmedabad at 12:00 hours. Physical examination; routine examination; routine hematological and urine examination, blood gas analysis,ECG X-ray chest were performed on admission. The blood samples were analyzed for routine biochemical investigations which included blood Hb, SGPT, RBS, blood urea, serum-billirubin,-sodium and-potassium and urine for drug screening. Unknown pale liquid used for ingestion by the patient was scanned through UV-Visible wavelengths on a spectrophotometer (Cary-100, Varian). Serum and * B J Medical College, Civil Hospital. Ahmedabad 76 Arterial blood gas analysis showed pH 7.34, PaO2 70.6 mm of mercury, PaCO2 31.8 mm of mercury, HCO3 16.4 meq/l. The patient was given oxygen with venti-mask at the rate of 6-8 liters/min. The unknown pale yellow liquid consumed for suicide was sent to the Acknowledgment: We are thankful to K S Shah, GB Jingar, AA Pandya, SK Patel and BS Solanki for the technical assistance and help during the study. Hemoglobin can accept and transport O2 only when the iron atom is in its ferrous form. When hemoglobin becomes oxidized, it is converted to the ferric state (Fe3+ ) or methemoglobin resulting in to a condition called methemoglobinemia in which there is inhibition of 5 binding and delivery of oxygen by a red blood cell . MeHb lacks the References 1. Gunnell D, Eddleston M, Phillips MR, K onradsen F. The global distribution of fatal pesticide self-poisoning: Systematic review. BMC Public Health. 2007;7: 357. 2. ATSDR (Agency for Toxic Substances and Disease Registry). Toxicological profile for nitrobenzene. Prepared by Life System, Inc. under subcontract to Clement Associates, Inc., for ATSDR, US Public Health Service under contract 205-88-0608. ATSDR/TP90-19,1990. 3. Ellman GL, Courtney K D, Andres V Jr, Featherstone RH. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88-95. 4. Henry R, Cannon DC, Winkleman JW. Clinical Chemistry, Principles and Practice. New Y ork, Harper and Row, 1974; pp1149. 5. Wright RO, Lewander WJ, Woolf AD. Methemoglobinemia: etiology, pharmacology and clinical management. Ann. Emergency Med. 1999; 34: 646-56. 6. Dewan A, Patel AB, Saiyed HN. Acute methemoglobinemia- A common occupational hazard in an industrial city in western India. J Occ Hlth. 2001;43,168-71 7. Goldstein RS, Chism JP, Sherill JM, Hamm Jr. TE. Influence of dietary pectin on intestinal microfloral metabolism and toxicity to nitrobenzene. Toxicol Appl Pharmacol. 1984;75: 547-553. 8. Gupta G, Poddar B, Salaria M, Parmar V. Indian Pediatrics. 2000; 37: 1147-1148. 4 Results and Discussion A worker (20 yrs) associated with screen printing work, ingested an unknown liquid used for screen printing in an intentional poisonin, had vomiting and epigastric burning sensation after about 40 minutes of ingestion was admitted to the Civil Hospital, Ahmedabad for treatment. Physical examination of fingers and tongue revealed a cyanosis with poor respiratory effort; pulse rate 120/min; blood pressure 100/70 mm of mercury and pupils bilaterally dilated. On admission, the hemoglobin level was 13.7 g/dl (normal: 14-18 g/dl) and total leucocyte count (20,000/cu mm) showe polymorphonuclear leucocytosis. There was no jaundice and toxicity profile for cannabis, morphine, cocaine, amphatamine, benzodiazepine and phenobarbitone gave negative results. Routine urine examination, ECG, X-ray chest and biochemical profile which included SGPT, RBS, blood urea, serum billirubin, sodium and potassium were within normal limits. Although not completely understood, reduced nitrobenzene metabolites are believed to be responsible for nitrobenzene-induced methemoglobinemia. Studies with laboratory animals demonstrated that orally administered nitrobenzene is reduced in the intestine and that intestinal microfloral metabolism is essential for the production of methemoglobin7. We have observed extremely high levels of methemoglobin (66.7%) and these levels reduced near to normal after a dose of methylene blue (100 mg, i.v.). The levels again rose after 18 hrs of antidote administration. Delayed release of nitrobenzene from stores in the adipose tissue and gastrointestinal tract is commonly seen after severe poisoning8. This delayed rise in the MeHb levels may be attributed to the release of nitrobenzene. Serum and RBC cholinesterase levels were within normal limits excluding the involvement of organophosphate pesticide. The MeHb levels from the blood samples were analyzed on the same day and the levels were found to be 66.7 % of Hb (normal: 0-3 % of Hb). On confirmation of chemically induced methemoglobinemia, he was administered 100 mg methylene blue i.v. over 10 minutes at 16:10 hrs. His MeHb level reduced to 5.4 % after 1 hour of methylene blue administration and gained consciousness at 22:00 hrs. Serial MeHb levels at different time intervals are shown in Figure 2. He was absolutely asymptomatic at the time of discharge. 3 258nm 2 Nitobenzene,pure Abs Suicide is a major cause of premature mortality globally and 113914 suicides are recorded annually from India1 for which variety of chemicals are used. The treatment of poisoning caused by an unknown compound is a challenge to a treating physician and the situation becomes graver when the laboratory support for certain specific poison is unavailable. Nitrobenzene, an aromatic nitro-compound, occurs as a pale yellow moderately water-soluble oily liquid with an odor resembling that of bitter almonds. It is used in the manufacture of aniline, benzidine, quinoline, azobenzene, rubber chemicals, pharmaceuticals and dyes2. It is also used as a solvent in shoe and metal polishes and in screen-printing. Its toxic effects are due to its ability to induce methemoglobinemia. A case study of acute nitrobenzene poisoning in a suicidal attempt is discussed. RBC cholinesterase levels determined by spectrophotometry on a RA50 chemistry analyzer by modified Ellman's were method using acetyl thiocholine as substrate3. The MeHb levels from the blood samples were analyze by the method of Evelyn and Mallay as modified by Henry4. Analysis of the unknown liquid, cholinesterase and serial estimations of blood MeHb were performed at poison Information center of the institute. electron that is needed to form a bond with oxygen and thus is incapable of O2 transport. This condition is mainly caused by the intentional or non-intentional exposure of oxidizing agents such as nitrates and nitrites including occupational exposures6. Poison Information Centreof the Institute for identification and the blood samples for MeHb analysis. From the absorption spectra (Unknown liquid and Nitrobenzene, pure were diluted 1:14,000 and 1: 30,000 respectively with ethanol 99.9% for scanning) which showed wavelength peak of 258 nm matching with the pure nitrobenzene (Figure 1) and from its typical bitter-almond smell; it was identified as nitrobenzene. 1 Unknown Liquid (Poison) 0 Ethanol,pure(base Line) 200 250 300 350 350 400 Wavelength(nm) Fig.1: Absorption spectra of the unknown liquid used by the patient for ingestion MeHb levels (% of total Hb) Introduction Annual Report 2007-08 80 70 60 50 40 30 20 10 0 66.7 15.8 5.4 6 1.63 6h50m 11h10m 28h 33h30m 53h30m Duration after ingestion (h:m) Fig.2: Methemoglobin levels during first 55 hours after nitrobenzene ingestion 77 Annual Report 2007-08 Annual Report 2007-08 Assessment of Human Exposure to PCBs Through Biological Monitoring VK Bhatnagar, N Shukla, VK Shivgotra, SSA Zaidi, R Kashyap, S Kumar Abstract The blood samples from the general population of Ahmedabad (urban, n=70; and rural, n=80) and fatty food samples (fish, n=50 and chicken, n=50) were analysed for the congeners of polychlorinated biphenyls. Sum of CB congeners (∑CB) in Ahmedabad (urban) and Ahmedabad (rural) had a mean of 5.78 ng/mL and 2.69 ng/mL respectively. There was significant positive correlation between age and CB 118 (r=0.46; p<0.05) and age and ∑CB (r=0.28; p <0.05). Average mean value of ∑CB in fish and chicken samples was 15.58 and 15.47 ng/gm-wet tissue respectively. Key words: PCB, congeners, human blood, biological monitoring Introduction Polychlorinated phenyls (PCBs) are the complex mixtures of chlorinated biphenyls that vary in degree of chlorination. These are used as coolants and lubricants in electrical equipment such as capacitors and transformers due to their general inertness and heat stability. These are also used in rubber, synthetic resin, ink and paint industries. However, upon searching the existing database on the residue level of these contaminants in our country, we find paucity of information. Therefore, the present study has been designed with the following objectives: Evaluate the levels of PCBs congeners in blood samples of general population of Ahmedabad (urban and rural settings); and Determine the levels of PCBs congeners in fatty food samples (chicken and fish) collected from the local markets of Ahmedabad. Materials and Methods The representative human blood samples from the general population Ahmedabad (urban area; n=70 and rural area; n=80) have been collected. It was ensured that the subjects have no occupational exposure to pesticides and other persistent pollutants. The objective of the study explained and informed consent received. Subjects were requested to provide information on their demographics, dietary habits and smoking status. These samples were extracted and clean up for the analysis of PCBs congeners. The representative fish samples (n=50), and chicken samples (n=50) collected from the various markets of Ahmedabad. Serum samples were extracted following the procedure as mentioned by Burse et al1. Serum sample denatured with methanol and extracted with nhexane-diethyl ether (1:1). 78 Extracts are eluted with hexane through deactivated silica gel in adsorption chromatographic colum. Fish and chicken samples were extracted following conventional procedures. Analyte was dissolved in appropriate volume of n-hexane and suitable volume injected into GC Agilent 6890N equipped with micro-ECD. GC conditions were: Capillary Column (J& W Scientific; 122-1262; DBXLB, 60 m X 0.25 mm X 0.25 m nominal). Oven temp programming was set to 150 0C for 1 min, then with 2 0C/min to 250 0C, kept for 20 min. N2 was used as carrier gas (1.1 ml/min). The injector (mode: 0 0 split-less) and detector temp kept at 270 C and 300 C respectively. Standard chromatogram of various CB congeners has been displayed in Figure 1. Correlation coefficient of various CB congeners at level (0.5, 1, 5, 10, 25, 50, 100 pg/mL) was observed 0.99. Recovery experiments carried out by spiking serum sample with mixture of various congeners (# 16) at 0.1 ppb level and % recovery ranged from 54% to 163%. Statistical calculation was performed using SPSS 16.0 for window. Figure 1: Standard Chromatogram of PCB's congeners on GC-ECD Figure 2: Lavels of PCB congeners in human blood of Ahmedabad (Urban) 79 Figure 3: Lavels of PCB congeners in human blood of Ahmedabad (Rural) Annual Report 2007-08 Annual Report 2007-08 Cancer in North East India - Understanding the Role of Pesticides VK Bhatnagar, U Mishra, MR Variya, VK Shivgotra, P Sivaperumal and R Kashyap Abstract The serum samples of breast cancer and NHL subjects and control subjects have been received from various cancer registries of NE region. The blood serum samples have been analyzed for the residues of DDT, HCH and Endosulfan and metabolites. These serum samples (n=201) have reflected the presence of pp'-DDE. Analysis of serum samples of control subjects is in progress. Keywords: Organochlorine pesticides, serum, brest and NHL Cancer Introduction Figure 4: Levels of PCB congeners in fish samples Figure 5: Levels of PCB congeners in chicken samples Results and Discussion CB 138, CB 180 and CB 170 were present in all fish samples (100%), whereas CB 28 and CB 74 in 49 fish samples (98%) and CB 153 in 48 fish samples (96%). Sum of CB congeners (∑CB) in Ahmedabad (urban) and Ahmedabad (rural) had a mean of 5.78 ng/mL and 2.69 ng/mL respectively. There was significant positive correlation between age and CB 118 (r=0.46; p<0.05) and age and ∑CB (r=0.28; p <0.05). Average mean value of ∑ CB in fish and chicken samples was 15.58 and 15.47 ng/gm-wet tissue respectively. Data in Figure 2 reflect the presence of various congeners in blood samples (n=70) collected from Ahmedabad (urban). ΣCB (sum of all congeners) ranged from 0.10 to 38.24 ng/mL with a mean of 5.78 ng/mL in urban samples. CB 199 was not detected in these serum samples. Serum Level of CB congeners in samples (n=80) collected from Ahmedabad (rural) has been depicted in Figure 3. ΣCB in rural serum samples was 2.69 ng/mL, which ranged from 0.15 to 13.3 ng/mL. However, the congeners e.g. CB 46, CB 105, CB 167 and CB 199 were not detected in rural samples. Acknowledgment: We are thankful to MR Variya, Y ogesh Shah, SN Y adav, Ashok Parmar, NA Suthar and CM Makwana for their technical assistance. Fifty samples each of fish and chicken were analyzed for CB congeners and levels have been depicted in Figure 3 and 4 respectively. Overall mean of Σ CB in fish and chicken was 15.58 ng/gm (Range: 6.42 - 31.57 ng/gm) and 15.47 ng/gm (Range: 2.51 30.15 ng/gm). CB 138, CB 180 and CB 170 were present in all fish samples (100%), whereas CB 28 and CB 74 in 49 fish samples (98%) and CB 153 in 48 fish samples (96%). CB 167 and CB 199 were not present in fish samples. Similarly, the congeners e.g. CB 153 were present in 49 chicken samples (98%) followed by CB 28, CB 138 and CB 170 in 48 chicken samples (96%). Three congeners e.g. CB 46, CB 99 and CB 199 were not detected in chicken samples. Conclusion Overall mean of Σ CB in fish and chicken was 15.58 ng/gm (Range: 6.42 - 31.57 ng/gm) and 15.47 ng/gm (Range: 2.51 30.15 ng/gm). 80 Reference 1. Burse VW, K orver MP, Needham LL, et al. (1989) Gas chromatographic determination of polychlorinated biphenyls (as Aroclor 1254) in serum. Collaborative study. J Assoc Anal Chem 72, 649-659. North Eastern (NE) states of the country have reported high incidence of cancer of all anatomical associated with use of tobacco and some sites, which have been related to pesticide exposures. NE region has diverse ethic groups with different customs, food habits and different life style. Moreover, there is extensive use of pesticides in tea gardens in NE region that can lead to widespread occupational and environmental exposures. A multi-institutional project involving RMRC - Dibrugarh; NIOH - Ahmedabad; IOP - New Delhi; ICPO - Noida; NCRPBangalore; and six population based cancer registries of NE region located at Aizal, Guwahati, Gangtok, Imphal, Dibrugarh and Silchar have been attempted to envisage the link between the magnitude of exposure, causative factor and genetic variants involved. Results and Discussion Materials and Methods The concentration of different organochlorine pesticide residues detected in the breast cancer and NHL subjects has been given in Tables 2 & 3 respectively. Preliminary analysis of serum samples of breast cancer and NHL subjects have reflected the presence of ppDDE, pp-DDT and -HCH in 96%, 78% and 29% of the samples respectively. The analysis of control subjects (n=125) and remaining serum samples is in progress. A total of blood serum samples (n=201) of breast cancer and NonHodgkin's lymphoma subjects from various cancer registries of NE region have been received. Serum samples have been processed for 1 extraction and clean up following the procedure of Dale et al for the analysis of persistent pesticide residues (-HCH, -HCH, -HCH, HCH, op'-DDE, pp'-DDE, op'-DDD, pp'-DDD, op'-DDT, pp'-DDT, Endosulfan, -Endosulfan, Endosulfan Sulfate and Dicofol). Final analysis was done on Shimadzu GC- 2010 equipped with ECD. GC conditions were: Capillary column 60 m x 0.2 m x 0.2 mm; Oven 0 0 0 0 0 100 C, 20 C/min 200 C hold 25 min; 1 C/min to 240 C; Injector Temp. 270 0C (spilt mode); Detector Temp-300 0C; Carrier gas (N2) Flow rate = 1.5 ml/min. For the purpose of validation of method for analysis of pesticide residues, the data on linearity, line of regression, correlation coefficient, and coefficient of determination at a concentration (Range: 1-100 pg) is given in Table 1. Acknowledgment: We are thankful to Y ogesh Shah, SN Y adav, Tejal Manvar, CM Makwana and Chander Parmar for the technical assistance. Reference 1. Dale WE, Curley A, Cueto C Jr. 1966. Hexane extractable chlorinated pesticides in human blood. Life Sciences 5: 47-54. 81 Annual Report 2007-08 Table 3: Levels of organochlorine pesticides in NHL cancer subjects from NE Region (ng/mL) Table 1: Regression equation, correlation coefficient and determination of coefficient of various analytes -1169.44+ 1119.85* x Correlation Coefficient (r) 0.99933 Coefficient of determination (r 2 ) 0.99865 1220.50+ 1316.03* x 0.99986 0.99973 Compounds Concentration (pg) Regression Equation -HCH 1, 5, 10, 25, 50, 100 -HCH 1, 5, 10, 25, 50, 100 Annual Report 2007-08 -HCH 1, 5, 10, 25, 50, 100 2625.07+ 1218.55* x 0.99723 0.99447 -HCH 1, 5, 10, 25, 50, 100 -16 08.18+ 1143.82* x 0.99972 0.99945 - Endosulfan 1, 5, 10, 25, 50, 100 796.13+ 1609.84* x 0.99988 0.99977 - Endosulfan 1, 5, 10, 25, 50, 100 381.51+ 1616.74* x 0.99989 0.99979 Endosulfan Sulphate 1, 5, 10, 25, 50, 100 - 5230.98+ 936.72* x 0.99857 0.99714 op' -DDE 1, 5, 10, 25, 50, 100 -704.79+ 1834.58* x 0.99988 0.99975 pp' -DDE 1, 5, 10, 25, 50, 100 -1254.13+ 2408.38* x 0.99957 0.99914 op' -DDD 1, 5, 10, 25, 50, 100 953.76+ 1438.72* x 0.99977 0.99953 pp' -DDD 1, 5, 10, 25, 50, 100 516.88+ 1520.97* x 0.99986 0.99972 op' -D DT 1, 5, 10, 25, 50, 100 1074.49+ 1692.76* x 0.99988 0.99976 pp' -DDT 1, 5, 10, 25, 50, 100 -826.15+ 1335.59* x 0.99995 0.9999 Dicofol 1, 5, 10, 25, 50, 100 -420.28+ 769.00* x 0.99996 0.99991 Centre n -HCH -HCH -HCH Aizwal 01 7.97 (1)* ND ND -HCH pp'-DDE pp'-DDT -DDT -Endo -Endo 7.97 (1)* 1.82 (1)* 114.68 (1)* 54.13 (1)* 168.81 (1)* -HCH -HCH -HCH pp'-DDE pp'-DDT - DDT 16 2.83 ± 0.85 (5)* 1.32 (1)* 1.12 (1)* 3.32 ± 0.90 (5)* 15.57 ± 3.68 (15)* 11.23 ± 2.60 (14)* 26.05 ± 6.12 (15)* 15 2.69 ± 0.51 (8)* ND ND 2.69 ± 0.51 (8)* 8 ± 3.17 (13)* 4.99 ± 2.373 (5)* 9.92 ± 4.21 (13)* 65 1.64 ± 0.12 (12)* 1.25 (1)* ND 1.61 ± 0.11 (13)* 15.88 ± 1.91 (64)* 11.06 ± 2.05 (52)* 24.87 ± 3.44 (64)* Imphal 09 1.59 ± 0.19 (3)* ND ND 1.59 ± 0.19 (3)* 26.49 ± 7.31 (8)* 11.66 ± 4.83 (7)* 32.62 ± 10.66 (9)* Silchar 15 2.05 ± 0.44 (2)* ND ND 2.05 ± 0.44 (2)* 18.01± 5.66 (12)* 8.97 ± 1.94 (12)* 26.98 ± 7.41 (12)* Dibrugarh Gangtok Guwahati n -HCH 24.94 8.54 (4)* ND ND 04 1.82 (1)* ND ND Guwahati 57 2.81 0.52 (23)* 3.65 (1)* ND 2.85 0.50 (24)* 19.81 3.05 (56)* 7.92 1.39 (48)* 26.59 4.22 (56)* 1.1 (1)* 1.1 (1)* Imphal 15 1.19 (1)* ND ND 1.19 (1)* 29.24 5.90 (15)* 12.71 2.00 (9)* 36.87 7.57 (15)* ND ND Silchar 04 ND ND ND ND 21.95 10.84 (4)* 3.48 0.79 (4)* 25.43 11.41 (4)* ND ND ND=Not detected; * Number of positive samples ND=not detected; * Number of positive samples 82 ND 8 4.11 (3)* Dibrugarh Table 2. Levels of organochlorine pesticides in breast cancer subjects from NE Region (ng/mL) Centre ND 18.94 5.89 (4)* 83 Annual Report 2007-08 Cancer in North East India: Understanding the Role of Tobacco PB Doctor, S Sedha, HS Trivedi, RR Teli and PR Mansuri Abstract Determination of cotinine provides a useful tool for estimating uptake of nicotine and tobacco related toxicants. We measured the level of cotinine in urine samples of tobacco related cancer cases as well as controls received from the six cancer registries of North East region of India. The urinary cotinine levels were measured by liquid chromatography coupled with triple quadrupole tandem mass spectrometer (LC/MS/MSAPI 3000). Urine samples to which deuterium labeled internal standard had been added were extracted with solid phase extraction (SPE) procedure using Drug test 1 SPE cartridge. Instrumental linearity/calibration curves were prepared by processing various concentrations i.e. 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0, 150.0 and 200.0 ng/ml (ppb) of cotinine, each containing 25ng/ml of deuterated internal standard (Cotinine d3). The linear correlation coefficient (r2) was 0.9998 for cotinine. The recovery percentage (Mean±SD) of cotinine was obtained in the range of 88.42 % ± 2.9 % to 91.36 % ± 1.33 % with less than 4% RSD. Cotinine was measured in 406 urine samples (295 male and 111 female subjects) of cancer cases and 185 samples (87 male and 98 female subjects) of controls. Key words: LC-MS/MS, Tobacco exposure, cotinine, cancer cases Introduction Materials and Methods The consumption of tobacco products is responsible for a significant portion of human cancer at various sites, in particular lungs, oral cavity, larynx, oesophagus, stomach etc.1 In India about 60 to 70% of cancers in males and 30 to 40% cancers in females have been associated to tobacco consumption. Tobacco products contain a diverse array of chemical carcinogens which are responsible for these cancers. More than 60 known carcinogens have been identified among constituents of tobacco smoke. These include polynuclear aromatic hydrocarbons (PAHs), aromatic amines, aldehydes, alkenes, metals and N-nitroso compounds. The most prevalent N-nitroso compounds in tobacco smoke and unburned tobacco products are the so called “ tobacco specific nitrosamines” (TSNAs)2-4. They are formed from nicotine and the other tobacco alkaloides during fermentation of tobacco. Two of the TSNA, 4-(Methylnitrosamine)-1-(3-pyridyl) 1-butanone (NNK ) and 4(Methylnitrosamine)-1-(3-pyridyl) 1-butanol (NNN) are present relatively in high concentrations and are the strongest carcinogens 5. This study is a case control epidemiological design at selected six cancer registries of the North East Region of India. All incidence cases of tobacco related cancers of the esophagus, lung, stomach and oral cavity and pesticide related Breast and Non-Hodgkin's Lymphoma (NHL) cancers, diagnosed and confirmed by microscopy between 1st February, 2006 to 19th June, 2008 from cancer registries located at Civil Hospital Aizwal, Regional Institute of Medical Sciences, Imphal, BB Baruah Cancer Hospital, Guwahati, Silchar Medical College and Hospital, Silchar, Sir TNM Hospital, Gangtok and Assam Medical College, Dibrugarh have been included. A specially designed questionnaire has been used to record tobacco habits and occupational history. Urinary cotinine level is the most widely used and reliable biochemical marker to quantify exposure to tobacco products6. Carcinogen biomarkers are important in establishing carcinogen dose in people who are exposed to tobacco products and in understanding mechanism of carcinogenesis and might ultimately be useful in predicting cancer risk. 84 Sample Collection: Collection of the biological samples is hospital based and samples of controls matched for age, sex and place of residence were also collected from 5 centers. The spot urine samples around 50-60 ml. were collected in polyethylene cryo containers from selected patients and controls and preserved in 0 (-20 C) deep-freezer at selected hospitals and were transported in frozen condition to IOP, New Delhi from Gangtok (Sikkim), Guwahati (Assam) and Aizwal (Mizoram) and to ICPO, Noida from Silchar (Assam) and Imphal (Manipur) and then were air-shipped under dry Annual Report 2007-08 ice to the NIOH for the estimation of cotinine - metabolite of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its Glucoronides (NNAL-Gluc) - metabolites of NNK , 1-hydroxypyrene metabolite of pyrene, exposure marker of PAHs, t,t-MA - metabolite of benzene and 8-OHdG as a DNA damage marker by HPLC and LCMS/MS techniques. Chromatographic Conditions: Aliquots of urine samples were prepared in sterile centrifuge tubes for each analytes and sediments of each sample were sent to Institute of Cytology and Preventive Oncology (ICPO) for further investigation. A total of 953 urine samples of cancer cases and 432 urine samples of controls were received. Out of 953 samples of cancer cases, 695 urine samples of tobacco related and 258 samples of pesticide related cancer cases were received. Measurement of Creatinine: Creatinine of spot or random urine sample is a measure of concentration or dilution of the urine. Urinary creatinine provides a good adjustment for the variations in concentration of spot urine samples due to differences in fluid intake. Therefore, creatinine of all the spot urine samples was measured by the Alkaline Picrate (Jaffe's) Method7. HPLC Perkin Elmer Series 200 with Quaternary pump, Auto sampler and Peltier Column Oven Column X Terra MS C 18, Length 100mm, ID 2.1, 5 µm Particle size Guard Column X Terra 20 x 2.1 mm Column Oven 40 ° C Flow Rate 300 µl Injection Volume 10µl Mobile Phase (Gradient) A: 5mM Ammonium Formate B: Acetonitrile C: HPLC water Instrumental linearity/calibration curves: Instrumental linearity/calibration curves were prepared by processing various concentrations i.e. 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0, 150.0 and 200.0 ng/ml (ppb) of cotinine, each containing 25 ng/ml of deuterated internal standard (Cotinine d3) prepared from reference stock standards in mobile phase. Calibration standards were prepared by spiking 2ml of blank urine with suitable amounts of different concentrations. All the samples were cleaned up and extracted by SPE. The peak areas of analyte were plotted versus the original concentrations and evaluated by linear least square regression analysis using Analyst, Version 1.4.1. The acceptable value of the correlation coefficient (r2) should be equal to or more than 0.99. Estimation of Cotinine from Urine Samples by LC-MS/MS: Measurement of human urinary cotinine level is a practical approach for obtaining important information about tobacco exposure. Cotinine is the major metabolite of nicotine and most widely used and reliable biomarker of tobacco exposure. Extraction of Urine Samples: Drug test 1 mixed mode solid phase extractions columns from Anal Chem (I) Ltd, Allahabad were used for the clean up and extraction of analyte from urine samples. Instrumental limit of detection (LOD) and limit of quantification (LOQ): LOD & LOQ were defined as three and ten times the standard deviation of the LC-MS/MS peak areas detected at the retention times of the analyte of interest in blank urine sample (S/N ratio ³3 and ³10 respectively). Recovery was evaluated by adding five different concentrations- 10, 50, 100, 200, 500 ng/ml of cotinine in the urine of non-tobacco user. Five replicates of spiked urine samples were extracted by SPE and same number of concentrations of cotinine prepared in mobile phase were analysed on LC-MS/MS. Ratio of the results of unextracted and extracted samples were used to estimate recovery percentages. Mass Spectrometry: Optimization of compound and source dependent parameters: For analysis of urinary cotinine the MS analysis was performed with API 3000 triple Quadrupole Mass spectrometer (Applied Biosystems/ MDS Sciex) equipped with electrospray ionization interface. The electronization interface was operated in positive ionization mode with an ion spray voltage of 5000 K V. Full scan mass spectra were recorded in order to select the most abundant mass to charge ratio (m/z). Full scan product mass spectra was obtained with continuous infusion of cotinine standard (Sigma, USA) (500 ng/ml) in product ion scan mode keeping Q1 constant on the m/z value. The most abundant product ion for cotinine was chosen for LC-MS/MS analysis in the multiple reaction-monitoring (MRM) mode. Optimization of compound and source dependent parameters have been carried out by continuous infusion mode and flow injection analysis (FIA). Results and Discussion The extracted ion chromatogram (XIC) of cotinine is shown in Figure-1. Under the conditions of LC-MS/MS selected, the linear range of 85 Annual Report 2007-08 Annual Report 2007-08 Table 1 Percentage wise distribution of cancer cases and controls according to urinary cotinine level Percentages of cotinine levels (ng/ml) Types of cases <5 5-19.9 20 - 49.9 50 - 999 1000-8000 > 8000 Lung (78) 26.92 (21) 24.36 (19) 3.85 (3) 42.31 (33) 1.28 (1) 1.28 (1) Stomach (75) 24.00 (18) 20.00 (15) - 41.33 (31) 14.67 (11) - Oesophagus (84) 15.48 (13) 29.76 (25) 3.57 (3) 40.48 (34) 10.71 (9) - Oral (98) 33.67 (33) 17.35 (17) 7.14 (7) 30.61 (30) 11.22 (11) - Others (71) 26.76 (19) 21.13 (15) 2.82 (2) 36.62 (26) 12.68 (9) - 18.38 (34) 4.86 (9) 24.32 (45) 34.05 (63) - Control (185) 18.38 (34) 8,9 Number in parentheses indicate the number of cases, <5.0 ng/ml – Non Tobacco user (Non Smoker) 5.0 -19.9 ng/ml – Passive exposure, 20.0 -49.9 ng/ml – Abstinent user for greater than 2 weeks 50.0 - 999 n g/ml – Light to Moderate Tobacco users (smoker), 1000 -8000 ng/ml – Active tobacco user or heavy smoker Figure 1. Extractedion chromatograph of cotinine cotinine was determined. As shown in linear correlation coefficient (r2) was 0.9998 for cotinine. Instrumental Limit of Detection (LOD) and Limit of Quantification (LOQ) for cotinine were 0.2 ppb & 0.5 ppb respectively. The recovery percentage (Mean±SD) of cotinine was obtained in the range of 88.42 % ± 2.9 % to 91.36 % ± 1.33 % with less than 4% RSD. 3. 4. 5. Quantitation of Urinary Cotinine: Out of 1127 urine samples of cancer cases and controls, 1040 samples were processed for the measurement of creatinine. The creatinine of 807 urine samples (488 Cancer cases and 319 Controls) was found in the normal range. Out of 488 samples of cancer cases (365 male and 123 female) and out of 319 samples of controls (139 male and 180 female), cotinine was measured in 406 urine samples (295 male and 111 female subjects) of cancer cases and 185 samples (87 male and 98 female) of controls (Table 1). 6. 7. Acknowledgment: The investigators are grateful to PM K handvi and V Patani for the help rendered during the study. 8. References: 1. 2. 86 IARC: Tobacco smoking, In IARC Monographs on the evaluation of carcinogenic risk of chemicals to humans. Lyon, International Agency for Research on cancer, 1986; pp. 199-298. Hoffman, D, Adams, JD, Lisk D, Fisenne, Brunnermann and K D, I. Toxic and carcinogenic agents in dry and moist snuff. J. Nat Cancer Inst. 1987; 79: 1281-1286. 9. 87 Hoffman D, Hecht, SS. Advances in tobacco carcinogenesis, In Cooper CS, Grover PL (eds): Handbook of Experimental Pharmacology, Heidelberg, Springer Verlag, 1990; pp. 63-102. Hecht, SS. Tobacco carcinogens, their biomarkers and tobacco induced cancer. Nature Rev. Cancer, 2003; 3: 733-744. Hecht, SS. Human urinary carcinogen metabolites: Biomarkers for investigating tobacco and cancer. Carcinogenesis, 2002; 23: 907922. Lee PN. Uses and abuses of cotinine as a marker of tobacco smoke exposure. In: Gorrod JW, Jacob P III (eds). Analytical determination of nicotine and related compounds and their metabolites. Amsterdam: Elsevier Science, 1999, pp. 669 719. Harold Varley Determination of creatinine in urine. Practical Clinical Biochemistry, Vol 1, Allan H Gowenlock and Maurice Bill (Eds.), 5th Edition, 1980; 484-85. Moyer TP, Charlson, JR, Enger, RJ, Dale, LC, Ebbert, JO, Schroeder, DR and Hurt, RD. Simultaneous analysis of nicotine, nicotine metabolites and tobacco alkaloids in serum or urine by Tandem Mass spectrometry with clinically relevant metabolic profiles. Clin. Chem. 2002, 48: 1460 - 1471. Nicotine and Metabolites, Urine. “ Arup's Guide to Clinical Laboratory Testing.” 2006, 0092356, CPT codes 83887. Annual Report 2007-08 Experimental Studies on Geno- and Feto-Toxicity of Panmasala Sunil Kumar, Archana Kumari, BN Mojidra, AK Gautam, Y Verma and BC Lakkad Abstract The study aimed to assess the possible effects of panmasala in somatic as well as reproductive cells and its modification by natural compounds. For in vitro study lymphocyte culture was treated with 3 different concentrations of aqueous extract of panmasala to find out panmasala induced chromosomal aberrations and micronuclei induction (MN), if any. Adult Swiss albino male and female mice were exposed to three different doses of panmasala plain (PMP) and panmasala with tobacco (PMT) (0.5, 1.5 and 3%) through feed for a period of 3 and 6 months and mating was allowed in order to study reproductive performance. In another experiment, 3 and 6% of both PMP and PMT were given through feed from gestation day (GD) 0, 6 and 14 till weaning to assess the fetotoxic potential of panmasala. To assess the modifying role of curcumin against panmasala induced mutagenicity, 2.5 g curcumin /kg feed for 8 weeks was given after 12-week panmasala exposure. The preliminary data showed an increase in MN induction in binucleated cells of human lymphocytes while observations on CA is in progress. Higher dose of PMT (1.5 and 3.0%) for 3 and 6 months adversely affected the reproductive performance while 3% dose of both types of panmasala for 6 months also negatively influenced the pregnancy outcome. In utero exposure from GD 0 with both doses of PMT resulted in pre term birth. Lower birth weight was observed among the offspring of dams treated from GD 0, 6 and 14. Curcumin has showed its efficacy against panmasala induced clastogenecity and spermatotoxicity. Panmasala is having spermatotoxic and genotoxic potential and more damage is seen with gutkha as compared to panmasala plain. Keywords: Gutkha; panmasala; fetotoxicity, micronuclei; reproductive performance Introduction Previously, significant increase in sperm head shape abnormality was observed after short term (12 week) and long term (6 months) exposure of panmasala. Sperm and spermatid count as well as daily sperm production was found to be significantly low after 3% PMT treatment for 6 month2. In dominant lethal test, 4 months treatment with 3 and 6% of panmasala reduced the fertility potential of treated mice. Data summarized for one spermatogenic cycle revealed significant reduction in total and live implants / female in PMT treated mice. Preimplantation loss was increased among all treated groups while postimplantation loss was higher only in 6% dose level3. Panmasala plain (PMP) is a dry mixture of areca nut, catechu, lime, cardamom and unspecified flavouring agents. Gutkha (PMT) is another popular variant of panmasala containing tobacco. Most of the ingredient of panmasala are reported to possess cytotoxic, mutagenic and genotoxic properties1. Even though data are available on mutagenicity and carcinogenecity of its major ingredients i.e., areca nut and tobacco, there is need to study the geno- and fetotoxic potential of their complex mixture like panmasala and its modification by natural compounds. Therefore, study is in progress with the following main objectives: In vivo and in vitro evaluation of mutagenicity of panmasala. To study fetotoxic potential of panmasala. To study the role of -carotene & curcumin in panmasala induced Frequency of micronuclei (MN) induction in bone marrow cells of mice was significantly high at the dose of 3% PMP and PMT after 6 and 12 week exposure as compared to control. Further, chromosomal aberrations (CA) was significantly elevated after both types of panmasala treatment with higher doses (1.5 and 3%) given for 6 weeks and at all the three doses given for 12 weeks with respect to control. mutagenicity. 89 Annual Report 2007-08 -carotene has been shown to have protective role against panmasala induced toxicity as it lowered the sperm head shape abnormality caused by 3% of both types panmasala and also reduced significantly the abnormal morphology at 6% PMP as compared to panmasala exposed animals maintained on standard diet. The present study conducted with following objectives: In vitro genotoxic potential of aqueous extract of panmasala To note the effect of panmasala treatment on reproductive performance To evaluate fetotoxic potential of panmasala To assess the modifying property of curcumin against the mutagenic potential of panmasala Materials and Methods In vitro mutagenicity of panmasala: For in vitro study, aqueous extract of panmasala was prepared4. The final yield of extracted material was 2.66 gm and 2.89 gm from PMP and PMT respectively. Stock solution (1 mg/ml) was prepared in triple distilled water and added to standard blood lymphocyte culture at the concentration of 7.5, 15 and 30 µg/ml of PMP and PMT extract. After 72 hours, cells were harvested for CA and MN study. In vivo mutagenicity of panmasala: Swiss-albino mice were maintained in Institute's animal house under controlled conditions of photoperiod, temperature and humidity in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). Animals were provided with standard mice feed and water ad libitum. Ethical clearance for the study th was obtained from the Institute's animal ethical committee held on 4 March 2005. Reproductive performance /fertility study: In order to evaluate reproductive fertility potential, adult Swiss albino male and female mice were treated with 1.5 and 3% of both PMP and PMT through feed for 3 and 6 months. In addition, a separate group was treated with lower dose i.e., 0.5% of both types of panmasala for six months. Thereafter they were allowed to mate for 5 days. Mating was confirmed by the presence of vaginal plug and considered as day 0 of pregnancy. Pregnant dams were maintained on standard mice feed. All the relevant parameters such as mating, fertility and pregnancy indices, gestation length, litter size, sex ratio and weight, survival, mortality etc. of the offspring were recorded. Fetotoxicity: To note the fetotoxic potential of panmasala at different stages of gestation, pregnant mice were treated with two different doses of panmasala (3 and 6% PMP and PMT) through feed from GD 0, 6 and 14 of pregnancy till weaning. Body weight of dam was taken every 3rd day. Pregnancy outcome was documented. 90 Annual Report 2007-08 Modifying role of curcumin against panmasala induced mutagenicity: To study the modifying property of curcumin, it was given at the dose of 2.5 g/kg feed for 8 weeks post panmasala treatment with 3 & 6% PMP and PMT for 12 weeks. Another group was allowed to maintain on basal diet (standard mice feed) post panmasala exposure. Control group was also maintained for 20 weeks without any exposure. Body weight was monitored weekly. After the scheduled treatment, animals were sacrificed. Slides were prepared for MN and sperm morphology assay. Statistical analysis One-way ANOVA and Tukey's post-hoc test, student t- test and Fisher exact test were performed to compare differences between control and treated groups. Results Figure 1: Body weight gain (% SE) of panmasala treated male and female for 3 months In vitro mutagenicity of panmasala: Preliminary observation on in vitro study revealed an increase in MN induction in binucleated cells of human lymphocytes culture after panmasala treatment. Further analysis and observation on CA is in progress. Reproductive performance/ fertility study: Data collected on weight gain indicated that panmasala treatment for 3 and 6 months reduced the body weight gain non-significantly in both the sexes as compared to control (Figure 1; Table 1). Mating indices were considerably declined at both the doses of PMT (1.5 and 3%) while it was comparable at PMP treatment groups after 3 and 6 months exposure with respect to control (Figure 2a). Fertility and pregnancy indices were non-significantly reduced at all the doses of PMT at both the durations of panmasala exposure while pregnancy index was lowered only at 3% of PMP (Figure 2b) but all these changes were statistically non significant. However negative effects indicated the potential adverse effects of panmasala on reproductive performance. All the panmasala treated pregnant dams (resulting from the mating of panmasala exposed male and female) had almost similar body weight gain during pregnancy. The results on pregnancy outcome of 3 and 6 months panmasala treated group are presented in Table 2 and 3 respectively. Gestation length, litter size and live pups within a litter were more or less similar in all the panmasala treated group as well as non treated group. Birth weight of pups born to 3 and 6 months panmasala treated parents were decreased as compared to control and it was significantly lowered at 3% PMT treatment for 3 months with respect to control (Table 2). Live birth and viability index were more or less similar among 3 months panmasala treatment at both the dose level while it reduced after six months exposure at 1.5% PMT and 3% of both PMP and PMT treated group with respect to control (Table 2 & 3). Weanling index was also lowered at 3% dose of PMP and PMT in both 3 and 6 months exposure and also decreased at 1.5% of PMP and PMT after 6 months exposure group. However, all these changes were statistically non significant. Figure 2: Reproductive performance in panmasala treated male and female a) Mating index b) Fertility and Pregnancy index dams from gestation day 0, 6 and 14 as compared to control. Sex ratio declined marginally in all the panmasala treated dams from GD 0 with respect to control. However, exposure from other gestation days had more or less similar sex ratio in treated and non treated groups. Fetotoxicity: Results on fetotoxicity showed that body weight gain during pregnancy was more or less similar in all the treated group i.e., 3 and 6% of both PMP and PMT from GD 0 and 6 with respect to control (Fig. 3 a,b) except slightly lower weight in 6% PMT treated group on day 18 of pregnancy. Results of the pregnancy outcome in panmasala treated dams from GD 0, GD 6 and GD 14 upto weaning are presented in Table 4-6. The results indicated that PMT exposure at both the dose level i.e., 3 and 6% from GD 0 significantly decreased the gestational length with respect to control (Table 4). Weight of the offspring born from 3 and 6% PMT treated dam from GD 0 and GD 6 was significantly lower as compared to control. It was also decreased significantly at 3% PMT treated dam from GD 14. In comparison to control, live birth index was reduced in GD 0 and GD 14 groups treated with 6% of both types of panmasala i.e., PMP and PMT while viability index was decreased at both the dose of PMT i.e., 3 and 6% and high dose of PMP i.e., 6% among all the groups i.e., GD 0, 6 and 14. Weanling indices were considerable lowered at 6% of both types of panmasala treated Results on the modifying role of curcumin against panmasala showed that treatment with curcumin for 8 week was able to reduce the sperm head shape abnormality in all the panmasala treated groups with respect to group maintained on basal diet indicating the efficacy of curcumin towards panmasala induced spermatotoxicity (Table 7). Data on micronuclei induction among curcumin treated (for 8 weeks) after panmasala treatment for 12 weeks and control animals is shown in Table 8. Curcumin has reduced percentage of micronucleated polychromatic erythrocytes at lower dose (3%) of both types of panmasala treated groups as compared to group maintained on basal diet. However both the treatments viz. curcumin and basal diet showed more or less similar induction of micronuclei in PCE and NCE 91 Annual Report 2007-08 Annual Report 2007-08 Table 1: Body weight gain (% SE) of panmasala treated male and female for 6 months Male Female 90 Control 19.40 1.26 8.14 2.08 80 0.5% PMP 20.01 2.49 5.41 1.29 0.5% PMT 17.66 1.30 5.56 1.35 1.5% PMP 18.63 1.94 5.51 0.78 1.5% PMT 14.59 2.17 4.92 1.08 3% PMP 15.98 2.10 4.56 3.68 3% PMT 17.49 1.77 4.70 2.42 1.5% PMP 3% PMT 19.09 0.28 19.67 0.26 19.00 0.17 7.67 1.20 7.00 0.55 6.71 0.64 8.09 0.74 7.42 0.85 Live pups (n) 7.33 1.25 6.60 0.75 5.57 0.84 7.45 0.92 7.00 0.86 1.38 0.01* 19.71 0.29 1.44 0.02 1.47 0.02 1.47 0.02 1.46 0.01 Live birth index (%) 94.55 4.43 93.14 4.30 93.39 4.75 90.40 6.45 94.67 3.61 Viability index (%) 93.32 3.85 100.00 0.00 100.00 0.00 90.00 10.00 87.92 4.50 Weaning index (%) 93.32 3.85 97.14 2.86 96.88 3.13 88.18 9.87 82.41 8.61 * p<0.05; Data are expressed as mean SE Table 3: Pregnancy outcome of 6 months panmasala treated animals Control Gestation length (days) Litter Size (n) Live pups (n) Birth weight (g) Live Birth Index (%) Viability Index (%) Weaning Index (%) 19.40 0.24 6.20 0.49 5.80 0.66 1.51 0.02 90.00 10.00 100.00 0.00 100.00 0.00 0.5% PMP 19.40 0.24 6.00 0.71 5.00 1.10 1.61 0.03 90.18 6.08 95.83 4.17 95.83 4.17 0.5% PMT 20.40 0.75 5.60 1.08 4.40 1.33 1.40 0.02 88.19 6.84 100.00 0.00 100.00 0.00 80 3 PMT 70 % weight gain % weight gain 3% PMP Gestational length (days) Litter size (n) Birth weight (g) 6 PMT 60 50 40 30 1.5% PMP 1.5% PMT 3% PMP 3% PMT 19.22 0.36 6.44 0.94 6.11 1.06 1.44 0.02 91.01 7.38 94.87 2.48 91.05 4.79 19.20 0.25 6.60 0.82 5.40 1.02 1.47 0.04 82.65 3.00 80.83 12.37 76.67 13.69 19.89 0.20 7.33 0.58 6.33 0.87 1.46 0.02 83.64 7.47 84.13 10.48 80.00 14.58 19.27 0.14 6.22 0.70 4.89 0.84 1.47 0.03 82.29 5.55 78.13 7.54 77.08 12.07 3 PMT 60 50 40 30 0 0 3 6 9 12 15 18 (b) Gestation day (GD) 9 12 15 18 Gestation day (GD) Figure 3: Body weight gain during pregnancy in panmasala treated dams (a) GD 0 (b) GD 6. Table 4: Pregnancy outcome of dam treated with panmasala from GD 0 Control 3% PMP 3% PMT 6% PMP 6% PMT Gestational Period (days) 19.78 ± 0.28 19.00 ± 0.26 18.17 ±0.11* 18.83 ± 0.17 18.67 ±0.24* Litter Size (n) 9.89 ± 0.42 9.80 ± 0.70 9.67 ± 0.73 9.33 ± 1.05 9.11 ± 1.17 Live pups (n) 9.78 ± 0.43 9.60 ± 0.75 9.33 ± 0.75 9.17 ± 1.17 8.33 ± 1.08 Live birth index (%) 98.89 ± 1.11 97.42 ± 1.81 96.07 ± 2.25 97.22 ± 2.78 93.33 ± 4.57 Viability index (%) 97.84 ± 1.45 95.94 ± 2.38 93.88 ± 3.76 92.73 ± 3.4 95.48 ± 2.93 Weaning index (%) 93.58 ± 2.33 89.87 ± 4.39 89.34 ± 6.62 89.09 ± 4.45 77.62 ± 16.2 Sex ratio (M/F) 1.78 ± 0.43 1.32 ± 0.21 1.28 ± 0.15 1.24 ± 0.36 1.19 ± 0.16 Birth weight (g) 1.47 ± 0.01 1.43 ± 0.01 1.39 ± 0.01* 1.42 ± 0.02 1.39 ± 0.01* * p<0.05; Data are expressed as mean SE; Data are expressed as mean SE 92 3 PMP 6 PMT 10 10 (a) Control 6 PMP 20 20 1.5% PMT 19.20 0.20 3 PMP 6 PMP 70 Table 2: Pregnancy outcome of 3 months panmasala treated animals Control Control 93 Annual Report 2007-08 Table 7: Alterations in mice sperm morphology with curcumin (8 week) post 12 weeks panmasala treatment Table 5: Pregnancy outcome of dam treated with panmasala from GD 6 3% PMT 3% PMP Gestational Period (days) Litter Size (n) Live pups (n) 19.50 ± 0.5 18.71 ± 0.18 6% PMP 18.86 ± 0.26 6% PMT 8.14 ± 1.1 8.00 ± 0.82 9.14 ± 1.12 9.67 ± 0.56 9.14 ± 1.12 9.67 ± 0.56 Live Birth Index (%) 100.00 ± 0.0 98.41 ± 1.59 100.00 ± 0.00 100.00 ± 0.00 Viability Index (%) 96.06 ± 2.5 93.37 ± 4.61 90.28 ± 5.01 94.63 ± 3.75 93.98 ± 4.21 Sex ratio (male/female) 2.37 ± 1.20 1.45 ± 0.04 91.70 ± 4.42 1.68 ± 0.37 1.38 ± 0.07* 87.50 ± 4.69 Curcumin 0.17 0.23 4.35 0.22 6.04 0.83 3.81 0.39 3% PMP 3.95 6% PMP 4.9 3% PMT 6% PMT 3.83 0.49 4.88 0.68 4.06 0.32 1.96 ± 0.70 1.39 ± 0.06 2.38 ± 0.66 1.38 ± 0.06* Table 6: Pregnancy outcome of dam treated with panmasala from GD 14 3% PMP 3% PMT 6% PMP 6% PMT 19.00 ± 0.26 18.75 ± 0.25 18.67 ± 0.33 18.80 ± 0.49 8.67 ± 0.71 10.60 ± 0.51 9.33 ± 0.67 8.60 ± 0.51 8.67 ± 0.71 10.60 ± 0.51 8.67 ± 0.84 7.80 ± 0.86 Live Birth Index (%) 100.00 ± 0.00 100.00 ± 0.00 92.21 ± 5.11 91.11 ± 8.89 Viability Index (%) 97.92 ± 2.08 95.00 ± 5.00 88.48 ± 6.55 88.00 ± 12.00 97.92 ± 2.08 90.78 ± 4.60 83.05 ± 7.46 75.00 ± 25.00 1.59 ± 0.59 2.57 ± 1.37 1.60 ± 0.36 1.80 ± 1.10 1.46 ± 0.02 1.38 ± 0.01* 1.44 ± 0.01 1.45 ± 0.01 * p<0.05; Data are expressed as mean SE; 94 % MNPCE Basal Diet 91.57 ± 4.23 * p<0.05; Data are expressed as mean SE; Weaning Index (%) Sex ratio (male/female) Birth weight (g) Basal diet Table 8: Alteration in micronuclei in PCEs and NCEs after curcumin post treatment (8week) to panmasala (12week) Weaning Index (%) Litter Size (n) Live pups (n) Control 1.88 0.10 8.00 ± 1.09 Gestational Pd. (days) % Sperm head abnormality SE Pretreatment 18.67 ± 0.33 8.00 ± 0.82 Birth weight (g) Annual Report 2007-08 % MNNCE Curcumin Basal Diet Curcumin CONTROL 0.66 0.08 3% PMP 0.83 0.07 0.64 0.22 0.35 0.03 0.27 0.04 3% PMT 0.84 0.08 0.74 0.10 0.37 0.04 0.23 0.04 6% PMP 0.81 0.16 0.89 0.14 0.33 0.05 0.37 0.03 6% PMT 1.07 0.16 1.21 0.13 0.36 0.03 0.39 0.07 0.25 0.03 References after 6% of PMP and PMT exposure. All these change were nonsignificant. 1. Discussion The results obtained so far suggests that panmasala is having mutagenic and clastogenic potential as evident by the in vitro lymphocyte culture and in vivo increase in MN in swiss albino mice. Our study corroborates with earlier work done on in vitro CHO cell lines5 and among panmasala chewers6. In addition, exposure of panmasala mainly PMT is shown to have fetotoxic potential as the in utero PMT treated dams had small gestation period along with lower birth weight. It is well documented that smoking adversely affects the pregnancy outcome. Thus, tobacco consumption resulting in pre-term birth along with lower birth weight is a consistent finding irrespective of the mode of intake. Recently arecoline, major alkaloid of areca nut, have been associated with adverse pregnancy outcomes7. Further, both types of panmasala demonstrated reproductive toxic potential as revealed by the impairment in reproductive fertility performance and pregnancy outcome. Data so far obtained indicated the male and female reproductive toxic potential of panmasala in Swiss albino mice. The preliminary results on modifying role of curcumin suggest that the adverse spermatotoxic effect of panmasala can be reduced by curcumin treatment. 2. 3. 4. 5. 6. 95 K umar S (2008) Panmasala chewing induces deterioration in oral health and its implications in carcinogenesis. Toxicol Mech Methods 18: 665 - 677. K umar S, Archana K , Gautam AK , Mojidra BN, Verma Y , Lakkad BC. Reproductive toxicity of panmasala in male Swiss albino mice. In XXVII Annual STOX; 2007, Oct 6-7; Bangalore, p 54. Archana K , Gautam AK , Mojidra BN, K apurkar U, Joshi M, Lakkad BC, K umar S. Germ cell mutagenic potential of Panmasala in male Swiss albino mice. Natl Seminar on Teratology & Genetic Disorders; 2008 Feb 8-9, BHU, Varanasi; p 18. Jaju RJ, Patel RK , Bakshi SR, Trivedi AH, Dave SJ and Adhvaryu SG. Chromosome damaging effects of panmasala. Cancer Lett. 1992; 65, 221 -226. Patel RK , Jaju RJ, Bakshi SR, Trivedi AH, Dave BJ and Adhvaryu SG. (1994). Panmasala a genotoxic menace. Mutat. Res. 320, 245-249. Dave BJ, Trivedi AH and Adhvaryu SG (1991) Cytogenetic studies reveal increased genomic damage among 'panmasala' consumers. Mutagenesis 6, 159-163. Garcı a-Algar O, Vall O, Alameda F, Puig C, Pellegrini M, Pacifici R and Pichini S (2005) Prenatal exposure to arecoline (areca nut alkaloid) and birth outcomes. Arch. Dis. Child Fetal Neonatal Ed 90, F276F277. Annual Report 2007-08 Effect of Arsenic on Brain and Testicular Tissue: Histopathological and Biochemical Study A Roy Choudhury and Sanghamitra Sarkar Abstract Arsenic exposure to Swiss mice, in the present study, gradually decreased the testicular weight compared to control suggesting cellular regression of the testicular tissue. Testicular histology exhibited severe cellular damage in spermatogenic cell. Degeneration of interstitial (Leydig) cells was observed in the testis of arsenictreated mice. Moreover Leydig cell population significantly decreases in both the doses over a period of 60 days. The Leydig cell nuclear diameter increased significantly in both the doses in 30 days followed by gradual diminution of the Leydig cell diameter in 45 and 60 days. The exposure to experimental mice in two doses (30 & 40 mg/L) for 30, 45 and 60 days showed a significant alteration in the activity of many antioxidants. The GSH content of brain and testicular tissue in both arsenic-treated mice revealed significant alteration with the increase in duration. In both the dose group for 30 days, there was marked increase in GSH content that afterward decreased with the duration of 60 days. GSH is known to protect cell against oxidative stress and any alteration in GSH level (either increase or decrease) indicates a disturbed oxidative status. When cells are oxidatively challenged, GSH synthesis increases and this may a cause of increase in GSH content after initial treatment of arsenic in mice. As oxidative stress continues, GSH synthesis cannot efficiently supply the demand, therefore GSH depletion occur. This may be the possible cause of further diminution of GSH content in mice after arsenic exposure. GSH depletion can impair a cell's defense against the toxic actions of many compounds and may lead to cell injury and death. Arsenic exposure in both the dose for 60 days duration enhanced significantly the lipid peroxidation of the testis, suggesting the generation of reactive oxygen radicals and testicular oxidative stress. Keywords: Arsenic toxicity, mice, oxidative stress, Leydig cell, lipid peroxidation Introduction to drinking of arsenic contaminated water has been reported from many countries. Recently large population in West Bengal in India and Bangladesh has been reported to be affected with arsenic5,6. Arsenic (As) is a common environmental contaminant widely distributed around the world as a result of natural and man-made origin. Groundwater with elevated concentrations of Arsenic has been 1-3 recognized as a problem of global concern . Arsenic contamination of groundwater is one of the principal pathways of human exposure to inorganic Arsenic. Elevated concentrations of Arsenic have been reported from several regions of the world4 resulting primarily from natural sources, such as erosion and leaching from geological formations, although sometimes from anthropogenic sources, such as uses of Arsenic for industrial purposes, mining activities and metal processing, and application of pesticides and fertilizers containing As. The risk of Arsenic contamination is generally much higher in groundwater compared to surface water. Chronic arsenic toxicity due Arsenic causes acute and chronic toxicity in human. The neurological system is the major target for the toxic effect of arsenic. Various studies confirmed that arsenic could cross blood brain barrier and produces alteration in whole rat brain biogenic amines level in 7 animal chronically exposed to arsenic . Histopathological observation in rat brain after exposure to multiple gallium arsenides revealed mild 8 effect on cerebral cortex region . Male Sprague-Dawley rat exposed to arsenic showed decreased locomotor activity and behavioural disorders9. Studies on human fetal brain tissue indicated regression in morphometric development and disturbance in the glutathione cycle enzymes and neuronal development. Generation of reactive oxygen 96 Annual Report 2007-08 species (ROS), nitric oxide (NO) and apoptosis was also prominent in 10 fetal brain tissue . Arsenic could reduce neurological viability; nuclear fragmentation and condensation as well as DNA degradation in primary 11 culture of rat neuron . Significant changes in the steady state level of three major neurotransmitters, dopamine, norepinephrine and 5 hydroxytryptamine and monoamine-oxidase were observed following 12 chronic arsenic exposure (III) in male albino rats . Histological Study: The testes were fixed in Bouin's fixative, embedded in paraffin and 5m thick sections were stained with routine hematoxylin-eosin. Histopathological changes in the testes were examined under optical microscope. Morphometric Study: Quantitative analysis of spermatogenesis was carried out from 5 perfect transversely cut tubules at VII-stage of spermatogenesis from each testis of respective groups. The relative number of spermatogonia, resting spermatocyte, pachytene spermatocyte and step-7 spermatids per tubular cross section were quantified at 1000X magnification18. Correction for changes in nuclear diameter was made using Abercrombie formula19. Seminiferous tubular diameter was determined at 400X magnification by ocular micrometer. The morphometric measurement of Leydig cell nuclear diameter was done by using ocular micrometer at 600X magnification and Leydig cell population was analyzed per square cm area from sagital plane area of the section at same magnification20. Arsenic causes reproductive effect on human and animal species. Few studies have shown that arsenic exposure produces ovarian and uterine disorders13, testicular steroidogenic dysfunction14, and spermatogenic disorder in experimental animal15. The available data point to dose dependent effects on growth and viability of the conceptus and offspring but no effect on fertility16. In human study a correlation has been observed between arsenic exposure and incidence of abortion17. Although various tissue toxicity caused by arsenic has been intensively investigated, the arsenic induced testicular and brain tissue toxicity and generation of oxidative stress in testicular and brain tissue are still unclear. Therefore, the effect of arsenic (30 & 40 mg/L) in drinking water ad libitum in Swiss albino mice and the possible generation of oxidative stress and testicular and brain tissue histopathology were investigated. Lipid Peroxidation: Extent of lipid peroxidation was estimated by the method of Buege and Aust21. Brain and testes were homogenized in ice-cold 0.25 M phosphate buffer (pH 7.4). The homogenate and the 5000g supernatant were used. To this homogenate, TCA-TBA-HCl [Trichloroacetic acid (TCA) 15 % w/v, thiobarbituric acid (TBA) 0.375%, and hydrochloric acid (HCl) 0.25N] were added and mixed thoroughly. The solution was heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000g for 10 min. Levels of thiobarbituric acid reactive substances (TBARS) were measured spectrophotometrically at 532nm. Measurement of reduced glutathione: The reduced glutathione content of the tissue was measured by the method of Ball RC et al22. For calibration, a standard curve of reduced glutathione was prepared using varied concentrations of glutathione treated with DTNB. Tissue 23 protein was estimated by the method of Lowry et al . Materials and Methods Animals and Treatment: Adult Swiss male albino mice weighing approximately 25- 30 g were obtained from the animal facility of Central Research Institute (Ayurved), Ministry of Health and Family Welfare, Kolkata. They were housed in a room with ambient temperature of 0 24±1 C in 12 h light/darkness cycle. Mice were allowed standardized pellets supplied by NIN, Hyderabad and sterilised water ad libitum. Table 1: Effect of sodium arsenite on body weight (g) and organ weight (mg/g body wt) 30 days 60 days 45 days Body wt. 28.7 4.34 32.5 0.95 Control 35.5 5 Sodiummetaarsenite was dissolved in drinking water and was administered at 30mg/L and 40 mg/l (30ppm and 40ppm respectively) through drinking water ad libitum. Animals were mainly divided into major 3 groups: Group I: Control, drinking water ad libitum only, Group II: Sodiummeta-arsenite dissolved in drinking water at dose level of 30mg/L for 30, 45 and 60 days respectively. Group III: Sodium-meta-arsenite dissolved in drinking water at dose level of 40 mg/L for 30, 45 and 60 days respectively. After the experimental period, rats were weighed and sacrificed by cervical dislocation. Testes and brain were removed, cleaned of the adhering tissues and weighed. Tissues were immediately and subjected for biochemical and histopathologial study. 30 mg/l 35.1 10.6 38.5 2.3 36 4.6 40 mg/l 35.6 4.28 34.1 4.91 35 5.7 Results Body and Organ Weight: The body weight gain was not significantly changed after arsenic exposure in both the treated groups over a period of 60 days in comparison to the control (Group I) [Table 1]. Significant reduction in testicular weight in Group II (Arsenic with 30mg/L) was observed from 45 days onward. But more conspicuous 97 Annual Report 2007-08 Annual Report 2007-08 significant decrease in resting and pachytene spermatocyte was observed in 30, 45 and 60 days respectively. The population of step-7spermatid was conspicuously decreased from 30 day onward compared to the control in group II. Similarly in higher dose group (Group III), significant and gradual reduction in different stages of spermatogenesis i.e. resting and pachytene spermatocyte, 7spermatid was noted after 30, 45 & 60 days but spermatogonial cell count was unaltered (Table 3). decrease in testicular weight was noted in Group III (Arsenic with 40mg/L) after 30 days and significant diminution was found on day 60 (Table 2). In brain tissue, the weight was not significantly reduced after arsenic treatment with 30mg/l over a period of 60 days. Similarly insignificant change was noted in animals treated with 40mg/l arsenic for 30 and 45 days, but significant reduction was observed after 60 days (p<0.005) [Table 2]. Histological Finding: Control testis (Group I) showed normal histoarchitectural organization of germ cells and Leydig cell in seminiferous tubules on day 30 through 60 (Figure 1a). Lower dose group i.e. 30mg/L (Group II) for 30 days showed disintegration of peritubular membrane, with disorganization of germ cells. After day 45 of Group II, vacuolization of seminiferous epithelium, loss of germ cells population along with Leydig cell atrophy were noted (Figure 1b). In the same group on day 60, histopathological changes included degeneration and loss of germ cells, disarrangement of seminiferous epithelium and atrophy of seminiferous tubules. Leydig cell showed marked atrophy. Treatment with 40mg/L arsenic (Group III) produces similar effect on testicular tissue like Group II, but of higher intensity. The effect followed 30 day onward and on 45 (Figure 1c) and60 days. Arsenic exposure over a period of 60 days also exhibited appearance of giant cell in the seminiferous tubules (Figure 1d). In the cerebellar cortex, morphological and structural variation occurs after arsenic exposure. Histologically, the cerebellum of the control mice showed three layers: outer molecular layer, internal granular layer and between the two a single row of large flask- shaped cells, the cell of Purkinje (Figure 2a). The low dose of arsenic, 30 ppm has effect on the cerebellar region of brain. In group for 30 days, there was degeneration of the granular cell layer. The purkinje cell layer was intact with a small area of granule cell loss. In 45 days for the same group, moderate vacuolation in white matter occur and there is purkinje cell degeneration was observed along with granular cellular degeneration. In the group II for 60 days, numerous vacuolation occur and are very apparent in the white matter along with granular and purkinje cell loss (Figure 2b). Figure 1: a) Control testicular tissue in mice showing normal organization of germ cells and Leydig cell in seminiferous tubules. Õ (H& E, x 600). b) Treatment with 30mg/L arsenic on mice for 45 days showing disintegration (D) of seminiferous tubules, vacuolization of seminiferous epithelium (V ) & loss of germ cells population. (H & E, x 600). Seminiferous tubular diameter showed a dose and duration dependent reduction in both the treated groups over a period of 60 days (Table 3). Gradual significant reduction in Leydig cell population compared to that of control in both the treated groups (Group II & III) over a period of 60 days was observed (Table 3). The Leydig cell nuclear diameter showed a marked variation in the treated groups i.e. Group II & III. The nuclear diameter significantly increased in both the treated group initially in 30 days, but followed a slow and gradual decrease over 60 days. c) Treatment with 40 mg/L arsenic on mice for 45 days showing severe degenerative (SDG) changes in the testicular tissue where loss of germ cell is apparent in seminiferous tubule. (H & E, x 600). Treatment with 40mg/L for 60 days showing complete degeneration of spermatogenic (DGSP) cell with the appearance of giant cell in seminiferous tubules. (H & E, x 600). Testicular Tissue Biochemistry: testicular glutathione (GSH): GSH content varied markedly in both the arsenic treated groups with increase in duration. In both the groups, i.e. 30 and 40 mg/l arsenic, GSH content increase after 30 days of treatment (p<0.001) followed by gradual significant decrease in 45 and 60 days. A significant and conspicuous decrease in GSH content was noted in higher arsenic treated group i.e.40 mg/L (Figure 3). Testicular Total Thiol: Similar effect was noted in total thiol content of testicular tissue after arsenic exposure. The value varied markedly in both the arsenic treated groups i.e. 30 & 40 mg/l arsenic with increase in duration. In both the groups, total thiol content increase after 30 days of treatment but gradual significant decrease was observed in 45 & 60 days (Figure 4). Testicular Lipid Peroxidation: The result shown in fig. 5 indicate that the lipid peroxidation level increased significantly in testicular tissue after arsenic exposure in both the arsenic treated groups i.e. 30 & 40 mg/l arsenic than control with increase in duration. The increase was highly significant for 60 days (p<0.001) in both the arsenic treated groups. Similarly the higher doses, i.e. 40 ppm the effect are more severe than the lower dose. In 30 days for 30mg/L arsenic, the granular cell layer thickness decreases when compared with the control. There is granular cellular and purkinje cell degeneration and moderate but prominent vacuolation. In 45 and 60 days (Figure 2c), severe degeneration in white matter along with extensive granular cell and purkinje cell loss occur. Vacuolation in the molecular layer and cellular degeneration in the molecular layer was more prominent in 45 and 60 days of 40mg/L arsenic treatment. Brain Biochemistry; Figure 6 indicate that the GSH content in the brain after arsenic treatment revealed a significant increase in both the treated groups i.e. 30 & 40 mg/l arsenic after 30 and 45 days but a gradual decrease was noted with in duration of 60 days. Figure 2: a) The cerebellum showed normal structure with three layer- Molecular layer,Inner granular layer and in the middle of it the Purkinje cells. (H& E, x 10). b) The cerebellum of mice exposed to 30mg/L arsenic for 60 days showed numerous vacuolation occur and are very apparent in the white matter along with granular and purkinje cell loss. (H& E, x 10). Brain Lipid Peroxidation: The malondialdehyde content (a measurement of lipid peroxidation) in brain was significantly increased in both the arsenic treated groups i.e. 30 & 40 mg/l arsenic on 30 days, but significantly declined after 45 and 60 days of treatment in both the dose groups (Figure 7). Morphometric analysis: In Group II, spermatogonial count was not altered from that of control after 30 days through 60 days. But gradual 98 The cerebellum of mice exposed to 40mg/L arsenic for 60 days showed degeneration in white matter along with extensive granular cell and purkinje cell loss. (H& E, x 10). 99 Annual Report 2007-08 Annual Report 2007-08 Table 2: Effect of sodium arsenite on absolute organ weight (g) and relative weight (mg/g body weight) of brain and testicular tissue of experimental mice Exposure Testicular weight Brain weight Absolute weight Relative weight Absolute weight Relative weight Control 0.19 0.008 5.64 0.408 0.41 0.008 30mg/l 0.18 0.017 NA 4.91 0.226 0.39 0.036 40mg/l 0.16 0.023* 5.251.36 0.39 0.012 NA Control 30mg/l 0.22 0.008 5.3 0.66 0.42 0.007 10.95 1.23 0.18 0.017 NA 4.95 0.775 0.40 0.026 NA 10.08 1.36 40mg/l 0.17 0.022** 5.311.8 0.36 0.045 * 12.45 1.6 Control 0.22 0.007 5.9 1.02 0.46 0.008 11.8 1.57 30mg/l 0.19 0.034** 5.2 0.62 0.44 0.041 NA 9.67 2.316 40mg/l 0.16 0.019** 4.711.138 0.38 0.033 ** 12.12 1.65 30 days 10.57 0.18 NA 10.24 1.348 9.22 0.52 45 days Figure 6: Glutathione content in brain tissue after arsenic exposure Figure 3: Glutathione content in testicular tissue after arsenic exposure 60 days Table 3: Effect of arsenic at a dosage of 30 and 40mg/L arsenic for 30,45 & 60 days respectively on testicular tissue Seminiferous tubular diameter ( m) [n= 50] Spermatodynamic count per 6.4 sertoli cell in each case no. of observation was 10 at the 1000x magnification Spg Pachytene R-Spcyt 7- sptd Leydig cell nuclear diameter ( m) [n= 10] Leydig cell 2 count/cm (n= 10) Figure 7: Lipid peroxidation values in brain tissue after arsenic exposure Discussion 30 days Control 171.11 5.518 5.711.86 30mg/l 168.74 3.18* 5.95 1.76 NS 40mg/l 164.01 11.57 ** 5.76 1.09 NS 42.25 9.037 6.2 2.70 5.4 0.71 19.46 3.74 NA 20.37 2.57 NA 39.27 3.02 NA 8.73 2.84 ** 4.75 .0.45 ** 17.96 2.52 *** 24.15 5.76 *** 8.3 1.828 ** 4.48 0.48 * 23.06 1.86 21.06 2.53 14.26 3.53 *** Figure 4: Total thiol content in testicular tissue after arsenic exposure 45 days Control 182.6 3.75 5 1 1.86 30mg/l 161.87 3.20 *** 5.884 1.39 40mg/l 159.18 4.13*** 5.12 1.41 199.2 7.34 5.6 1.22 NS NS NS 22.34 1.56 22.4 0.95 45.6 7.45 6.5 2.43 5.3 0.52 11.51 2.96 ** 14.02 1.78 *** 19.15 8.08 *** 5.35 1.27 NA 4.43 0.57 *** 11.09 2.95 *** 13.82 2.05 *** 16.8 3.56 *** 7.7 1.946 NA 4.3 0.36 * 60 days 23.42 2.6 23.3 2.16 44.2 5.25 6.5 2.43 5.3 0.52 30mg/l 154.20 2.75 *** 5.82 1.23 NS 10.74 3.46 *** 10.5 3.22 *** 16.15 5.76 *** 6.0 1.61 NA 4.59 0.93 ** 40mg/l 157.85 4.96*** 5.03 1.30 NS 10.97 3.56 *** 9.79 3.89 *** 12.95 4.56 *** 5.57 1.27 NA 3.99 0.63 * Control Value are mean SEM, NS Non significant; * p<0.05; ** p<0.01; *** p<0.001;., Spg: Spermatogonia, R-Spcyt: Resting spematocytes, Pachyt: Pachytene Spermatocytes, 7-Sptd: Spermatids. Number of observation is in parenthesis. 100 Figure 5: Lipid peroxidation values in testicular tissue after arsenic exposure Arsenic is considered as a toxic metal, which reflects on human health. Various workers have observed systemic disorders 24-26, but male reproductive study in relation to arsenic toxicity is scanty. Earlier study indicated that heavy metals like lead, mercury and chromium 27 causes cytotoxic effect in the male reproductive function . Arsenic exposure to Swiss mice, in the present study, gradually decreased the testicular weight compared to control suggesting cellular regression of the testicular tissue. This observation is in corroboration with the earlier finding of Pant et al.28. Testicular histology in this study exhibited severe cellular damage in spermatogenic cell. Moreover, the appearance of eosinophilic multinucleated giant cell in the seminiferous tubule in higher treated group indicated cellular degeneration. A significant gradual dose dependent regression was observed in the number of resting spermatocyte, pachytene and round spermatid in 30 and 40 mg/L over a period of 60 days, whereas there was no significant decrease in the number of spermatogonia. These finding acts as an indicator that the maturation of spermatogonia through the process of meiosis has been severely distrupted following 101 Annual Report 2007-08 arsenic exposure. The above observation is in agreement with the recent finding of Omura et al.29. Degeneration of interstitial (Leydig) cells was observed in the testis of arsenic-treated mice. Moreover Leydig cell population significantly decreases in both the doses over a period of 60 days. The Leydig cell nuclear diameter increased significantly in both the doses in 30 days followed by gradual diminution of the Leydig cell diameter in 45 and 60 days. Inspite of a testosterone assay in this study, it may be suggested that the degeneration of Leydig cell with significant decrease in the Leydig cell population probably would have resulted in decreased synthesis of testosterone, which in turn disturb the process of spermatogenesis. It has already been established that Leydig cell plays an important role in the structural and functional integrity of seminiferous tubules and synthesis of testosterone, which is one of the main component of regulation the post meiotic stage of spermatogenesis 30-32. The exogenous arsenic exposure may cause a chemical stress on the cellular function. The initial increase in Leydig cell diameter may be a better indication to adopt the metal induced stress but due to continuous stress effect, cellular exhaust may be a result of Leydig cell atrophy. In the present investigation, arsenic exposure to experimental mice in two doses (30 & 40 mg/L) for 30, 45 and 60 days showed a significant alteration in the activity of many antioxidants. The GSH content of brain and testicular tissue in both arsenic-treated mice revealed significant alteration with the increase in duration. In both the dose group for 30 days, there was marked increase in GSH content that afterward decreased with the duration of 60 days. GSH is known to protect cell against oxidative stress and any alteration in GSH level (either increase or decrease) indicates a disturbed oxidative status. When cells are oxidatively challenged, GSH synthesis increases and this may a cause of increase in GSH content after initial treatment of arsenic in mice. As oxidative stress continues, GSH synthesis cannot efficiently supply the demand, therefore GSH depletion occur. This may be the possible cause of further diminution of GSH content in mice after arsenic exposure. GSH depletion can impair a cell's defense against the toxic actions of many compounds and may lead to cell injury and death33,34. Cellular toxicity of arsenic was found to be inversely related to intracellular GSH levels and thus may be enhanced by GSH depletion35. Acute administration of arsenic to rats produced a significant reduction in hepatic GSH36. Chronic exposure of rats or mice to arsenic via injections caused up to 35% depletion in hepatic GSH, 37,38 along with liver injury . Similar effect was observed in the total thiol concentration. Arsenic exposure in both the dose for 60 days duration enhanced significantly the lipid peroxidation of the testis, suggesting the generation of reactive oxygen radicals and testicular oxidative stress. Increased level of lipid peroxidation and consequent damage to the cell membrane due to arsenic and its compounds was reported by Annual Report 2007-08 39 a number of investigators . But in brain tissue, lipid peroxidation showed a variable response after arsenic exposure for 30, 45 and 60 days. The LPO content increased significantly in both the arsenictreated groups after 30 days but a significant decrease in LPO content in brain was found in arsenic treated mice after 45 and 60 days. Arsenic induced elevation of oxidative stress reported previously by 40,41 other workers but at this point of time, the possible reason of sudden decrease in LPO content of arsenic exposure is not understood. 11. 12. 13. References 1. Bhattacharya, P.; Chatterjee, D.; J acks, G.Occurrence of Ascontaminated groundwater in alluvial aquifers from the Delta Plains, eastern India: Option for safe drinking water supply. Int. J . Water Res. Dev. 1997, 13, 79-92. 2. Bhattacharya, P.; Frisbie, S.H.; Smith, E.; Naidu, R.; J acks, G.; Sarkar, B. Arsenic in the Environment: A Global Perspective. In Handbook of Heavy Metals in the Environment; Sarkar, B., Ed.; Marcel Dekker: New Y ork, 2002; 145-215. 3. Bhattacharya P.; J acks, G.M.; Khan, A.A. Arsenic in groundwater of the Bengal delta plain aquifers in Bangladesh. Bull. Environ. Cont. Toxicol. 2002, 69, 538-545. 4. Naidu, R.; Bhattacharya, P. Management and remediation of arsenic from contaminated water. In. Managing Arsenic in the Environment: From Soil to Human Health, Naidu, R.; Smith, E.; Owens, G.; Bhattacharya, P.; Nadebaum, P. Eds.; CSIRO Publishing Melbourne, Australia, 2006; 331-354. 5. Guha Majumdar DN, Das Gupta J , Santra A. Chronic arsenic toxicity in West Bengal- worst calamity in the world. J Ind .Med Assoc 1998; 96: 4-7. 6. Chowdhury U K, Biswas BK, Chowdhury RT, Samanta G, Mandal BK, Basu GK, Chandra GR, Lodh D, Saha KC, Mukherjee SC, Roy S, Kabir S, Quamruzzaman Q and Chakraborti D (2000): Groundwater arsenic contamination in Bangladesh and West Bengal, India. Environ Health Perspect, 108, 393-397. 7. Tripathi, N., kannan, G.M., Pant, B.P., J aiswal D.K., Malhotra, P.R., Flora, S.J .S., 1997. Arsenic induce changes in certain neurotransmitters level and their recoveries following chelation in rat whole brain. Toxicol. Lett. 92: 201-208. 8. Flora, S.J .S., Dube, S.N., Sachan, A.S., Pant, S.C., 1994. Effect of multiple gallium arsenide exposure on some biochemical indices in rat brain. Indust. Health. 32:247-252. 9. Rodriguez, V.M., Carrizales, L.J , J imenz-capdeville, M.E., Dufour, L., Giordano, M., 2001. Effect of sodium arsenite exposure on behavioural parameters in the rat. Brain. Res. Bull. 15: 301-308. 10. Chattoadhayay, S., Bhaumik, S., Nagchaudhuri, A., Das Gupta, S., 2002. Arsenic induced changes in growth development and 102 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. apoptosis in neonatal and adult brain cells in vivo and in tissue culture. Tox. Lett. (Special issue). 128. Namgung, U ., Xia, Z ., 2001. Primary cultures of rat cerebellar neurons were used to study mechanism of arsenic. Toxicol. Appl. Pharmacol. 174, 130-138. Kannan, G.M., Tripathi, N., Dube, S.N., Gupta M., Flora, S.J .S., 2001. Toxic effect of arsenic (III) on some hematopoietic and central nervous system variables in rats and guinea pigs. Clin. Toxicol. 39:675-682. Chattopadhyay S, Pal S, Ghosh D and Debnath J . Effect of dietary co-administration of sodium selenite on sodium arsenite induced ovarian and uterine disorders in mature albino rats. Toxicological Scie. 2003; 75: 412-422. Sarkar M, Chaudhuri G., Chattopadhayay A., Biswas NM. Effect of sodium arsenite on spermatogenesis, plasma gonadotrophins and testosterone in rats. Asian. J . Androl 2003; 5: 27-31. Shukla J P, Pandey K. Impaired spermatogenesis in arsenic treated freshwater fish, Colisa fasciatus (Bl. and Sch.).Toxicology Letters 1984; 21(2): 191-195. Schroeder, H.A and Mitchener, M., 1971. Toxic effects of trace elements on the reproduction of mice and rats. Arch. Env. Health. 23: 102-106. Tabacova, S., Baird, D.D., Balabaeva, I., Lolova, D., Petrove, I., 1994. Placental arsenic and cadmiun in relation to lipid peroxides ans glutathione levels in maternal-infant pairs from cooper smelter areas. 15: 873-881. Leblond CP and Clarmont Y . Differentiation of stages of the cycle of seminiferous epithelium. Ann.N.Y .Acad.Sci 1952; 55: 548-573. Ambercrombie M. Estimation of nuclear population from microtome section. Anat. Rec 1946; 94: 239-249. Chowdhury AR and Chatterjee AK. Effect of adrenalectomy on the testicular cell of immature rats. Ind J Expt Biol 1978; 16: 88. Buege J A, Aust SD (1978): Microsomal lipid peroxidation. Methods in Enzymology, 52: 302-310. Ball CR (1966): Estimation and identification of thiols in rat spleen after cysteine or glutathione treatment: Relevance to protection against nitrogen mustards. Biochem Pharmacol., 15(7): 809-816. Lowry OH, Rosebrough NJ , Farr AL, Randell RJ . Protein measurement from phenol reagent. J . Biol.Chem. 193: 265-275, 1951. Tchounwou, PB, Patlolla AK, Centeno J A. Carcinogenic and systemic health effects associated with arsenic exposure- A critical Review. Toxicol Pathol 2003; 31(6): 575-588. Guha Majumdar DN, Das Gupta J , Santra A. Chronic arsenic toxicity in West Bengal- worst calamity in the world. J Ind .Med Assoc 1998; 96: 4-7. Rossman TG. Arsenic. In: Environmental and occupational Medicine, Rom. W.N. Ed, Lippencott-Raven, Philadelphia. 1998; 1011-1019. 27. Roy Chowdhury A. Male Reproductive Toxicology New Perspective in Life Science. In: Life Science in Modern Perspective, U GC Academic Staff College, U niversity of Calcutta. 2004; 97-105. 28. Pant N, Murthy RC, Srivastava SP. Male reproductive toxicity of sodium arsenite in mice. Hum Exp Toxico 2004; 23 (8): 399-403. 29. Omura M, Y amazaki K, Tanaka A, Hirata M, Makita Y and Inoue N. Changes in the Testicular Damage Caused by Indium Arsenide and Indium Phosphide in Hamsters during Two Y ears after Intratracheal Instillations. J Occup Health 2000; 42: 196-204. 30. Bartke A. Pituitary-Testis relationship. Reprod. Biol 1991; 1: 136152. 31. Steinberger E and Duckett GE. Effect of estrogen and testosterone in the initiation and maintenance of spermatogenesis in the rats. Endocrinology 1965; 76: 1184-1189. 32. Sharpe RM, Maddocks S & Kerr J B. Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement. American J ournal of Anatomy 1990; 188: 3-20. 33. Deneke SM, Fanburg BL. Regulation of cellular glutathione. Am J Physiol 257(4 Pt 1):L163-173 (1989). 34. Anderson ME. Glutathione and glutathione delivery compounds. Adv Pharmacol 38:65-79 (1996). 35. Ochi T, Kaise T, Oya-Ohta Y . Gluthathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALC/c 3T3 cells. Experientia 50:115-120 (1994). 36. Maiti S, Chatterjee AK. Effects on levels of glutathione and some related enzymes in tissues after an acute arsenic exposure in rats and their relationship to dietary protein deficiency. Arch Toxicol 75:531-537 (2001) 37. Liu J , Liu Y , Goyer RA, Achanzar W, Waalkes MP. MetallothioneinI/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of chronic oral or injected inorganic arsenicals. Toxicol Sci 55:460467 (2000). 38. Flora SJ S. Arsenic-induced oxidative stress and its reversibility following combined administration of N-acetylcysteine and meso 2,3-dimercaptosuccinic acid in rats. Clin Exp Pharmacol Physiol 26:865-869 (1999). 39. Modi M, Flora SJ . Combined administration of iron and monoisoamyl-DMSA in the treatment of chronic arsenic intoxication in mice. Cell Biol Toxicol. 2007 40. Chattopadhayay, S., Bhaumik, S., Nagchaudhuri, A., Das Gupta, S., 2002. Arsenic induced changes in growth development and apoptosis in neonatal and adult brain cells in vivo and in tissue culture. Tox. Lett. (Special issue). 128. 41. Nag Chaudhuri,A., Basu, S., Chattopadhayay, S., Das Gupta, S., 1999. Effect of high arsenic content in drinking water on rat brain. Ind. J . biochem. Biophys. 36, 51-54. 103 Annual Report 2007-08 Load of Environmental Metals in Pregnant Women and Reproductive Outcome S Kumar, S Shaikh, V Mishra*, HU Doshi*, NG Sathwara, LJ Bhagia Abstract Developing fetus is vulnerable to the exposure to persistent chemicals, as developing organisms may not have adequate defense mechanism. Certain environmental chemicals including metals might influence the female reproductive/endocrine system. The study is in progress to find out the load of environmental metals in pregnant women and reproductive outcome. One hundred and sixty four pregnant women were enrolled from OPD of Obstetrics and Gynecology, of an hospital of Ahmedabad. Toxic heavy metals i.e. lead, cadmium and essential metals like zinc, iron and calcium were analyzed in the blood of pregnant women. They were followed in all trimesters and after delivery. Reproductive outcome was recorded in terms of neonatal anthropometric indices. The mean blood lead level in the sample analysed so far, 8.15 ± 0.63 mg/dL and the range was 1.21 to 28 mg/dL. The mean serum zinc, iron and ionic calcium level was 63.81 ± 2.53 mg/dL, 47.44 ± 2.72 mg/dL and 1.04 ± 0.01 mmol/L respectively. A slightly lower birth weight and birth length was observed in the offspring of subjects having higher lead levels i.e. > 10 mg/dL as compared to <10 mg/dL. There is considerable lead level in the women of this area and preliminary analysis revealed that there was slightly lower birth weight and body length among the offsprings of higher maternal blood lead levels. Key words: Pregnant women, metals, birth weight, body length, head circumference Introduction 50 45 40 35 30 44.26 Blood Pb < 10 40.75 33.3 33.5 Blood Pb > 10 25 20 15 10 5 2.67 2.34 0 Birth weight (kg) Body length (cm) Head circumference (cm) A number of occupations are being reported to be associated with 1 reproductive dysfunction in males as well as in females . Women are exposed to toxic environmental chemicals during their day-to-day activities often without their knowledge. General population or even pregnant women/fetuses or children are exposed to various toxic metals and their oxides in a number of ways. Metals are present in the environment because of their release from a wide variety of sources. Metals enter into the food chain often by way of bioconcentration, and some of them like mercury bio-accumulates in the tissues of aquatic organisms and enter into the food chain. Experimental studies indicated that both male and female reproductive system is vulnerable to the effects of some of the metals, which depend upon dose, duration, and timing of exposure2. Metals like lead, cadmium, and mercury have toxic effects on reproduction. However, data pertaining to reproductive outcome in human are less documented. Certain environmental chemicals including metals might influence the female reproductive/ endocrine system leading to increasing infertility as well as adverse reproductive outcome. The present study is in progress i) to find out the load of environmental toxic heavy metals in pregnant women and in cord blood samples of newborn and ii) the role of heavy metals if any in reproductive outcome. Dept. of Obstetrics and Gynecology, IKD*, Civil Hospital*, Ahmedabad Materials and Methods st One hundred and sixty-four pregnant women, in their 1 trimester of pregnancy have been enrolled randomly, from the OPD of Obstetric and Gynecology department, of a hospital at Ahmedabad. A written consent has been taken from each subject after explaining the objectives and possible benefit of the study to individual as well as to the society. Among enrolled subjects, follow-up study has been undertaken in 79 in 2nd, 60 in 3rd trimester of pregnancy and 45 women after delivery. The subjects were interviewed and information was recorded about personal characteristics such as name, age, income, education, habits such as tobacco chewing, smoking, drinking and occupational and environmental exposure especially, possible exposure to metals, pesticides and organic solvents, area of residence, medical and reproductive history of the woman and her partner on predesigned proforma. Blood samples were collected in each trimester of pregnancy and also after delivery. Routine hematological parameters like total erythrocyte, leucocyte and hemoglobin levels were estimated. Blood samples were digested with HNO3 and H2O2 for the estimation of metals like lead, cadmium and zinc using Atomic Absorption Spectrophotometer (AAS, Perkin Elmer Model No. 3100, U SA). The analysis of ionic cadmium was Carried out using electrolyte analyzer (AVL, 9181, Germany). Few cord blood samples were also collected for metal analysis. Information related to newborn i.e. birth 105 Annual Report 2007-08 Table 5: Pregnancy outcome of the study subjects according to area of residence Table 1: Subjects enrolled in first trimester and follow-up and obstetric history Age group (yrs) Annual Report 2007-08 Details of subjects Residential Area Birth weight (kg) Gravidity Table 6: Birth weight, body length and head circumference of newborn according to maternal blood lead levels Body length (cm) Head circumference (cm) 1st trimester 2nd trimester 3rd trimester After Delivery 1 2 3 4 5 Total Rural (5) 2.60 ± 0.16 43.2 ± 0.86 34.0 ± 0.54 Lead level (µ g/dL) Birth weight (kg) Body length (cm) Head circumference (cm) ≤ 25 108 49 35 25 63 29 15 1 - 108 U rban (20) 2.60 ± 0.07 44.23 ± 0.31 33.35 ± 0.17 10 2.67 ± 0.10 44.26 ± 0.43 33.30 ± 0.28 26 -30 44 23 18 16 15 14 11 3 1 44 Industrial (20) 2.59 ± 0.12 42.57 ± 0.6 33.26 ± 0.40 10 2.34 ± 0.54 40.75 ± 1.79 33.50 ± 1.25 ≥ 31 12 7 7 4 4 4 3 1 - 12 Total 164 79 60 45 82 47 29 5 1 164 Age group (yrs) n Spontaneous abortion MTP Total < 25 108 18 7 25 26 – 30 44 12 9 21 ³ 31 12 2 2 4 Total 164 32 18 50 MTP- Medical termination of pregnancy Table 3: Haematological parameters among study subjects Hb (g/dL) 6 3 RBC 10 /mm 3 WBC /mm Age group (yrs) Different trimester < 25 26 - 30 ? 31 I trimester II trimester III trimester 10.39± 0.10 10.43± 0.16 10.52± 0.27 10.41± 0.08 10.43±0.16 10.28± 0.20 3.93± 0.05 4.0± 0.08 4.14± 0.14 3.97± 0.04 4.11 ± 0.06 4.13 ± 0.07 7,350± 169 7,722± 296 7,283± 425 7,445±140 7,495 ± 173 7,283 ± 425 Values are expressed as mean ± SE Table 4: Metal level among study subjects Metals n Mean ± SE Range Lead (µ g/dL) 77 8.15 ± 0.63 1.21 - 28.0 Cadmium (µ g/dL) 7 0.94 ± 0.20 0.3 - 1.8 Z inc (µ g/dL) 42 63.81±2.53 37.8 - 108.07 Calcium (mmol/L) 164 1.04± 0.01 0.75 -1.19 Iron (µ g/dL) 62 47.44±2.72 18.2 -120.0 106 weight (soon after birth), and body length and head circumference were recorded within 24 hrs. after delivery. Results Table 2 : History of spontaneous and induced abortion of the study subjects Hematological parameters Values are expressed as mean ± SE Numbers in parenthesis indicate the number of subjects; Values are expressed as Mean ± SE ³10 µ g/dL and preliminary analysis revealed slightly lower birth weight and body length among the offspring of higher maternal blood lead groups (³10 µ g/dL) as compared to <10 µ g/dL (Table 6). Discussion The information about subjects enrolled, and their follow-up and obstetric information indicated that eighty-two women were in first pregnancy, rd forty-seven in second, twenty nine in 3 and five were in fourth pregnancy (Table 1). The mean age, weight and height of the study population was 24.8 ± 0.25 yrs, 51.16 ± 0.68 kg, and 153.6 ± 4.8 cm. respectively. Data pertaining to distribution of subjects according to residential area indicated that, 89 subjects were residing in urban area, 21 in rural area and 54 in industrial area. The socioeconomic status indicates that about 55% subjects belong to the lower economic group (Rs <3000/month). About half of the subjects were vegetarian. History of partner indicated that there were 7.3% smokers, 28.6% chewers and 5.5% occasional alcohol users. Most of the women were housewives (93.2%). Complication during previous/present pregnancy showed that about 21 (12.8%) of women, had complication in the present or previous pregnancy and 32 subjects having history of spontaneous abortion in the study group (Table 2). The haematological profile of the study population as per the age group and trimester is depicted in table 3. The mean haemoglobin level was more or less similar in all the groups. However slightly lower haemoglobin level was noted in the third trimester with respect to first and second trimesters. No significant changes were observed for RBC and WBC counts. The load of metals in the study subjects is shown in Table 4. The mean blood lead level in the sample analysed so far was 8.15 ± 0.63 mg/dL (range 1.21 to 28 mg/dL). The mean serum zinc and iron level in the study group was 63.81 ± 2.53 mg/dL (range 37.80 to 108.07 mg/dL) and 47.44 ± 2.72 mg/dL (range18.2 to 120 mg/dL) respectively. The mean serum ionic calcium level was 1.04 ± 0.01 mmol/L (range-0.75 to 1.19 mmol/L). Cadmiumcould be analysed only in few samples i.e. 7 subjects and the mean level was 0.94 ± 0.20 mg/dL. The preliminary data on birth weight, body length and head circumference of newborn according to area of residence indicated that no considerable difference in birth weight was found according to area of residence. However, body length of newborn was slightly lower in the industrial area as compared to rural and urban area (Table 5). The data further analysed as per the maternal blood lead level i.e. <10 µ g/dL and Exposure to lead for women of childbearing age can have adverse effect on their offspring as it is known that lead deposited in the bone can be mobilized during pregnancy and also transfer from mother to the fetus3. A considerable level of lead is observed in the study and preliminary analysis indicated slightly lower birth weight and body length of the newborn. There is a report, which indicated that prenatal lead exposure might reduce neonatal weight gain4. Earlier, Saxena et al., observed mean blood lead level of 22.53 among pregnant women with various adverse outcomes such as pre-term delivery, stillbirth and spontaneous abortion 5 as compared to 19.4 in normal delivery cases . In the present study mean blood lead level of 8.5 ± 0.63 µ g/dL (1.21±28.0 µ g/dL) was observed in the women of Ahmedabad, indicating that some of the pregnant women carried higher blood lead level and had the potential of adverse effects. The preliminary data indicated that birth weight and body length of the newborn were decreased slightly when maternal blood lead levels increases. Presently, we are following the enrolled subjects till delivery and also enrolling more women in first trimester of pregnancy. The relationship, if any, between metal level and reproductive outcome will be assessed in near future, after the analysis of metals in remaining and follow up subjects. References / 1. 2. 3. 4. 5. 107 Kumar S. Occupational exposure associated with reproductive dysfunction. J Occup Health 2004; 46: 1-19. Kumar S. Is environmental exposure associated with reproductive health impairments? J . Turkish-German Gynecol Assoc. 2008; 9(1): 60-69. Gulson BL, Mizon KJ , Korsch MJ , Palmer J M and Donnelly J B. Mobilization of lead from human bone tissue during pregnancy and lactation--a summary of long-term research. Sci Total Environ. 2003; 303: 79104. MDH (Minnesota Department of Health). Blood lead screening guidelines for pregnant women in Minnesota. www.health.state.mn.us/divs/eh/lead, U pdated 12/2007]. Saxena DK, Singh C, Murthy RC, Mathur N and Chandra SV. Blood and placental lead levels in an Indian city: a preliminary report. Arch Environ Health 1994; 49: 106-110.
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