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Induction of Transitional Cell Hyperplasia in the Urinary Bladder and Aberrant Crypt Foci in the Colon of
Rats Treated with Individual and a Mixture of Drinking Water Disinfection By-Products
Kevin S. Mcdorman, Sundeep Chandra, Michelle J. Hooth, Susan D. Hester, Robert Schoonhoven and Douglas C. Wolf
Toxicol Pathol 2003 31: 235
DOI: 10.1080/01926230390183733
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TOXICOLOGIC PATHOLOGY, vol 31, no 2, pp 235–242, 2003
C 2003 by the Society of Toxicologic Pathology
Copyright °
DOI: 10.1080/01926230390183733
Induction of Transitional Cell Hyperplasia in the Urinary Bladder
and Aberrant Crypt Foci in the Colon of Rats Treated with Individual
and a Mixture of Drinking Water Disinfection By-Products
KEVIN S. MCDORMAN,1 SUNDEEP CHANDRA,2 MICHELLE J. HOOTH,3 SUSAN D. HESTER,3
ROBERT SCHOONHOVEN,4 AND DOUGLAS C. WOLF1,3
1
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709
3
Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory (NHEERL),
U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, and
4
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599
2
ABSTRACT
Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these
cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to
determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of
DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate,
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H )-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months.
The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using
immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were
identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined
using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based
on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in
the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of
them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.
Keywords. Rat; urinary bladder; colon; aberrant crypt foci; water; disinfection by-products; cell proliferation.
INTRODUCTION
Although chlorination of drinking water as a means of disinfection has often been considered one of the most effective
public health measures instituted, it produces hundreds of
disinfection by-products (DBPs), many of which are toxic
and carcinogenic in experimental animals (5). Chronic exposure to disinfected surface waters is considered one of the
contributing factors to the development of urinary bladder
and colon cancer (7, 8, 54). Consistent evidence from epidemiological studies has suggested an increase in the risk of
urinary bladder, colon, and rectal cancer among people consuming disinfected drinking water that contains high levels
of trihalomethanes (54).
Mutagenic activity of chlorinated water is due to the presence of chemicals produced from reactions of chlorine with
natural humic substances released by the breakdown of vegetation in the source water (5). The chlorinated hydroxyfuranone (eg, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone, commonly referred to as MX) have been shown
to be responsible for a majority of this mutagenic activity
(5, 30). MX is reported to occur at much lower concentra-
tions in drinking water than other DBPs, yet may pose a
significant public health concern because it is the most potent mutagen currently identified in drinking water (30). Two
years of treatment with MX in drinking water resulted in a
multiorgan carcinogenic response in both male and female
Wistar rats (29). MX did not cause neoplasms of the colon
or urinary bladder with our chronic exposure, but did cause
individual transitional epithelial cell hypertrophy with karyomegaly in the urinary bladder in both sexes (29).
Cancer of the urinary bladder and colon are significant
human health problems. Cancer of the urinary bladder was
the first cancer identified as being associated with industrialization (26). Subsequently, specific chemicals (eg, aromatic
amines), chemical mixtures (eg, cigarette smoke), and environmental agents have been identified as causes of urinary
bladder malignancies in humans (11, 26). The majority of urinary bladder neoplasms in humans (approximately 80%) are
superficial and papillary at first presentation, and 10–15% of
these will progress to a muscle-invasive carcinoma (3). Men
are affected approximately 3 times more often than women
(3, 11).
Colon cancer is the second most common cancer in men
and women from developed countries (21, 23). Human populations exposed to concentrations of trihalomethanes of
50 µg/L or more for at least 35 years were 1.5 times more
likely to develop colon cancer (28). Exposure to chlorinated
Address correspondence to: Dr Douglas C. Wolf, U.S. EPA MD-68,
86 TW Alexander Drive, Research Triangle Park, North Carolina 27711;
e-mail: [email protected]
235
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MCDORMAN ET AL
surface water has also been associated with a statistically significant increased relative risk of developing rectal
cancer (24). While not all the epidemiologic studies have
shown this correlation, the majority do suggest a positive
association (35). There is even a concentration-dependent
increase in risk of developing colon and rectal cancer when
chloroform in used as an indicator of the concentration of
total trihalomethanes (15). These epidemiologic studies are
supported by animal studies where specific disinfection byproducts, when given to rats for up to 2 years, resulted in
development of colon adenomas and adenocarcinomas (16).
It has been proposed that the majority of colon carcinomas
develop via an adenoma-carcinoma sequence with sequential
genetic alterations accompanying the morphologic progression. The earliest precursor lesions identified in experimental
colon cancer are aberrant crypt foci (ACF). ACF have also
been identified in patients with familial adenomatous polyposis and sporadic colon cancer as well as in patients with
benign colonic diseases (2, 18).
ACF are thought to be markers of carcinoma development
(2, 18, 40, 45). The number of lesions is reported to increase
with age in humans and after treatment with carcinogens in
rodents. Chemically induced carcinogenesis in the rat colon
may follow distinct pathways depending on the part of the
colon from which they arise. In human patients, ACF are
thought to be an intermediate stage in colon cancer development only in differentiated tumors of the distal portion of the
colon whereas tumors of the proximal colon may arise without developing this intermediate stage. ACF also appear to
predict cancer development in a subset of rat colon neoplasia.
The frequency of ACF can be increased in rodents exposed
to carcinogens and have been decreased with dietary manipulation (1, 18, 22, 39–43).
Few laboratory studies have examined the potential health
risks associated with prolonged exposure to DBP mixtures.
Current default risk assessments for chemical mixtures assume additivity of carcinogenic effects, but there is little
evidence that this method accurately reflects biological response. A study was designed to examine the potential carcinogenicity of a mixture of DBPs that act on similar target
organs and to characterize the nature of the interaction of
these compounds. Male and female Long-Evans rats heterogeneous for a germline insertion mutation in the Tsc2
tumor suppressor gene (Eker rats) were treated with low
or high doses of potassium bromate (KBrO3 ), 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H )-furanone (MX), chloroform, and bromodichloromethane (BDCM) alone or in a
mixture. Previously, we reported that treatment with this mixture produced on average no more renal, splenic or uterine
tumors than the individual compound with the greatest effect, suggesting that the default assumption of additivity may
overestimate the carcinogenic effect of chemical mixtures in
drinking water (25). The present study was designed to determine if preneoplastic or neoplastic lesions of the colon
and urinary bladder would develop in rats treated with drinking water DBPs administered alone or as a mixture. Results
of this study suggest that there is evidence that DBPs can
influence the development of preneoplastic lesions in the
colon (ACF incidence) and enhance transitional epithelial cell
proliferation in the urinary bladder of rats.
TOXICOLOGIC PATHOLOGY
MATERIALS AND METHODS
Animal Dosing
Complete animal study details, including in-life and chemical consumption data, are described elsewhere (25). Male
and female Long-Evans rats from a mutant Tsc2 carrier
colony (Eker rats, University of Texas, MD Anderson Cancer
Center, Smithville, TX) were exposed via drinking water to
individual or a mixture of disinfection by-products (DBPs).
KBrO3 (CAS 7758-01-2; Aldrich Chemical Co., Milwaukee,
WI), MX (CAS 77439-76-0; Radian International, Austin,
TX), chloroform (CAS 67-66-3; Sigma, St. Louis, MO), and
BDCM (CAS 75-27-4; Aldrich Chemical Co, Milwaukee,
WI) were administered at low concentrations of 0.02, 0.005,
0.4, and 0.07 g/L, respectively and high concentrations of 0.4,
0.07, 1.8 and 0.7 g/L, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either
low concentrations or high concentrations, respectively. The
low doses were chosen as the concentration of chemical that
produced no observable effect for cancer in the liver or kidney after chronic exposure in drinking water to normal rats in
previously published reports (25). The high doses were chosen as the concentration of chemical that produced the lowest
observable effect level for cancer in the liver or kidney after
chronic exposure in drinking water to normal rats in the same
series of studies (25).
Animals were euthanized by CO2 asphyxiation after 10
months of treatment. There were 10 control animals, 8 in
each individually treated group, and 14 in each mixture
group. 5-Bromo-20 -deoxyuridine (BrdU; Sigma, St. Louis,
MO, CAS#59-14-3) was administered to 6 animals in each
group, via drinking water solutions at a final concentration of
20 mg/100 ml, for 3 days before necropsy in order to assess
alterations in cell proliferation. All aspects of these studies
were conducted in facilities certified by the Association for
Assessment and Accreditation of Laboratory Animal Care
(AALAC-International) in compliance with the guidelines
of that organization and those of the NHEERL Animal Care
and Use Committee.
Urinary Bladder Histology and Cell Proliferation
At necropsy, the urinary bladders were removed, tied off
below the trigone, inflated with approximately 0.25 ml of
10% formalin, then immersed in 10% formalin for fixation.
Midsagittal sections of fixed bladder were taken through the
apex and trigone, placed in tissue cassettes, and processed
to block using routine methods. A 5-µm thick, hematoxylin
and eosin (H & E) stained slide was prepared for each animal
and examined using a Nikon Eclipse E400 light microscope
(Southern Micro Instruments, Atlanta, GA).
Urinary bladders from control rats and rats treated with
low and high doses of MX and the mixture were analyzed
using BrdU incorporation as a marker for cell proliferation. Additional 5 µm thick, unstained sections were prepared and treated with antibodies directed against BrdU (BD
Biosciences, San Jose, CA). Immunostaining was performed
based on previously described methods (17, 26). Bladder sections were examined for the number of transitional epithelial
cells positive for BrdU immunoreactivity. Cell proliferation
counts were performed using Bioquant version BQTCW98
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Vol. 31, No. 2, 2003
RAT BLADDER AND COLON LESIONS FROM DBPS
(R&M Biometrics, Knoxville, TN). Cell counting was limited to intact epithelium with a minimum of 1,000 total cells
counted. The labeling index (LI) was calculated based upon
the number of BrdU positive cells [LI = (BrdU positive
cells/total number of cells counted) × 100]. Data were analyzed with a 1-way analysis of variance (ANOVA) followed
by the Tukey-Kramer multiple comparisons test using Instat
(GraphPad, San Diego, CA).
Identification of Aberrant Crypt Foci (ACF)
Formalin fixed segments of proximal, middle, and distal
colon were stained with 0.2% methylene blue for approximately 2 minutes. Whole mounts of the stained colon segments were prepared and then placed flat on glass slides.
ACF were visualized by examination of the mucosal surface using a compound microscope (Figure 1). The numbers
of ACF were counted at 40X magnification. When an ACF
was identified, the number of individual crypts in each focus was counted at 100X magnification. A grid square was
counted as occupied if a methylene blue positive crypt or
crypts occupied at least 50% of a grid square. The eyepiece grid was calibrated using a stage micrometer. Each
grid square measured 170 µm2 at 100X magnification. After
ACF were counted, representative foci (2 per section of colon)
and adjacent uninvolved tissue were placed in cassettes, fixed
in neutral buffered 10% formalin, and processed by routine
methods to a 5-µm H & E stained section and examined
microscopically.
Determination of Colon Crypt Cell Proliferation
Colons from control rats (n = 10) and all male rats treated
with high-dose KBrO3 (n = 8), MX (n = 8), CHCl3 (n = 8),
BDCM (n = 8), and the high-dose mixtures (n = 12) were
analyzed using proliferating cell nuclear antigen (PCNA)
as a marker for proliferation. The 2 transverse sections described above from the proximal third, middle third, and
distal third of the large intestine were stained for PCNA
immunoreactivity, totaling 6 sections per rat. Immunohistochemical staining for PCNA was performed using a commercially available antibody kit (PCNA kit; Zymed Labs,
South San Francisco, CA). Slides were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity with 3% H2 O2 in methanol. After blocking, the slides
were incubated with biotinylated mouse anti-PCNA antibody for 60 minutes at room temperature, rinsed with PBS,
then incubated with streptavidin-peroxidase for 10 minutes
at room temperature, and rinsed again with PBS. Slides
were stained with diaminobenzidene (DAB) for 5 minutes, counterstained with Mayer’s hematoxylin for 1 minute,
FIGURE 1.—Microscopic view of ACF in the rat colon stained with 0.2%
methylene blue (×40 original magnification). 2.—An example of a complete
colonic crypt selected for measuring cell proliferation in a PCNA stained Eker
rat colon (×400 original magnification). 3.—Urinary bladder from an untreated
12-month-old female Eker rat. The transitional epithelium is 2–3 cell layers
thick. H&E (×400 original magnification). 4.—Urinary bladder from a 12month-old female Eker rat treated with 0.07 g/L MX. The transitional epithelium is hyperplastic, consisting of 4–5 layers of cells. H&E (×400 original
magnification).
Figures 1–4
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237
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MCDORMAN ET AL
rinsed with tap water, dehydrated and coverslipped. After
staining, 20 crypts were counted from each segment of the
colon. Only complete crypts were selected for counting,
characterized by an open and complete crypt lumen from
surface to base and also with the base of the crypt extending to the muscularis mucosa (Figure 2). All stained nuclei and mitotic figures were counted within the crypt and
indexes were calculated based on mean labeling per crypt
and, separately, percent mitoses among the labeled cells.
The labeling index (LI) was calculated based upon the number of PCNA positive cells [LI = (PCNA positive cells/20
crypts) × 100]. Data were analyzed using JMP (SAS, Cary,
NC) with Dunnett’s multiple comparison to control, TukeyKramer test for multiple comparisons and Student’s t-test for
individual comparisons.
RESULTS
The rat urinary bladder is lined by transitional epithelium that normally is no more than 2 to 3 cell layers thick
(Figure 3). Chronic administration of MX at the high dose
caused a moderate, locally extensive to multifocal hyperplasia of the transitional epithelium (Figure 4). This histologic
change was observed in the urinary bladders of both male and
female rats, and was often accompanied by marked hypertrophy of individual transitional cells (Table 1). These cells
contained very large nuclei with otherwise normal morphology. One-half of the male rats treated with high dose MX had
hyperplastic lesions accompanied by individual cell hypertrophy. Lesions were more pronounced in females where the
incidence was 100%. The lesions were not present in control
animals and only rarely observed in animals exposed to the
high-dose DBP mixture. The incidence of both lesions was
much lower in both male and female rats treated with the
high-dose DBP mixture, even though this mixture contained
the same concentration of MX.
Cell proliferation data were collected from urinary bladders of control rats and rats treated with low and high doses
of MX and the DBP mixture (Table 2). Treatment with MX at
the high dose caused roughly a 2-fold increase of the labeling
index in both male and female urinary bladders compared to
control rats. This increase was more pronounced in the female rats. All other treatment groups, including the high dose
mixture, showed no significant change compared to control.
TABLE 1.—Lesion incidence in urinary bladders of 12-month-old Eker rats
treated with DBPs.
Treatment (g/L)
Incidence of
epithelial
hyperplasia
in males
Incidence of
individual cell
hypertrophy
in males
Incidence of
epithelial
hyperplasia in
females
Incidence of
individual cell
hypertrophy
in females
Control
0.02 KBrO3
0.4 KBrO3
0.4 CHCl3
1.8 CHCl3
0.07 BDCM
0.7 BDCM
0.005 MX
0.07 MX
Low-dose mix
High-dose mix
0/10
0/8
0/8
0/7
0/8
0/8
0/8
0/8
4/8
0/14
1/12
0/10
0/8
0/8
0/7
0/8
0/8
0/8
0/8
5/8
0/14
1/12
0/10
0/8
0/8
0/0
0/8
0/8
0/8
0/8
7/7
0/14
3/14
0/10
0/8
0/8
0/0
0/8
0/8
0/8
0/8
7/7
0/14
1/14
TOXICOLOGIC PATHOLOGY
TABLE 2.—Cell proliferation in urinary bladders of 12-month-old Eker rats
treated with DBPs.
Treatment (g/L)
Labeling index (LI)∗
in males
Labeling index (LI)∗
in females
Control
0.005 MX
0.07 MX
Low-dose mixture
High-dose mixture
2.47 ± 1.07
1.31 ± 0.45
4.59 ± 2.38a
1.75 ± 1.09
2.82 ± 2.44
1.61 ± 0.83
2.00 ± 0.75
4.93 ± 2.17b
2.11 ± 0.84
1.57 ± 0.48
∗
(BrdU positive cells/total number of cells counted) × 100; numbers are group mean
LI ± SD.
a
Significantly different compared to low-dose MX ( p < 0.05).
b
Significantly different compared to control, low dose MX, and high-dose mixture
( p < 0.05).
A number of male rats from all treatment groups developed
aberrant crypt foci (ACF), whereas none of the control male
rats had ACF (Table 3). The ACF were somewhat uniformly
distributed throughout the large intestine. Rats treated with
the high dose of MX had the highest total number of ACF as
well as the highest total number of crypts contained within
ACF. A dose-dependent increase in total numbers of ACF,
crypts within ACF, and mean size of ACF was present in
rats treated with KBrO3 . A dose-dependent increase was also
observed in total numbers of ACF and crypts within ACF in
MX treated rats, and total numbers of ACF in chloroformtreated rats.
When examined by Dunnett’s multiple comparison test
to control values, high-dose chloroform and high- and lowdose MX had significantly increased mean size of ACF, and
high-dose MX and low-dose chloroform had significantly
increased numbers of total crypts in ACF (Table 3). When
examined by the Tukey-Kramer multiple comparison test,
rats treated with the high dose MX had greater numbers of
ACF than controls and rats treated with high-dose KBrO3 ,
BDCM, and the high-dose mixture. High-dose chloroform
treated rats also had greater numbers of ACF than control
or high-dose mixture-treated rats. As before, rats treated
with the high-dose of MX also had greater total numbers
of crypts in ACF than control rats or rats treated with highdose chloroform, KBrO3 , BDCM or the high-dose mixture
(Table 3). Out of the animal groups that actually developed ACF, only the low-dose mixture group had increased
mean ACF size compared to low-dose BDCM and high-dose
mixture. No other parameters were statistically significantly
different.
Cell proliferation data were generated from the high dose
male rats (Table 4). A labeling index and a mitotic index were
analyzed, both for the entire colon and for each defined segment of the colon. The labeling index in the proximal colon
from control animals was significantly lower than the labeling index in the middle and distal colon. This pattern was also
present in all treatment groups. Compared to control animals,
the labeling index was significantly increased only in rats
treated with the high-dose mixture. This increase was apparent in each individual segment and the entire colon overall.
The percent of cells in mitosis was increased in the middle and distal segments and entire colon in bromate treated
rats, while mitoses were decreased in the middle segment of
MX treated rats and the entire colon of rats that received the
mixture (Table 4).
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Vol. 31, No. 2, 2003
RAT BLADDER AND COLON LESIONS FROM DBPS
239
TABLE 3.—Aberrant crypt foci in 12-month-old male Eker rats treated with DBPs.
Treatment (g/L) (n)
Incidence
Total ACF
Mean ACF/colon
Total crypts
in ACF
Mean crypts/ACF
Mean size
ACF mm2
% ACF
in proximal colon
% ACF
in middle colon
% ACF
in distal colon
Control (10)
0.02 KBrO3 (8)
0.4 KBrO3 (8)
0.4 CHCl3 (7)
1.8 CHCl3 (8)
0.07 BDCM (8)
0.7 BDCM (8)
0.005 MX (8)
0.07 MX (8)
Low-dose mix (14)
High-dose mix (12)
0/10
3/8
6/8
7/7
6/8
7/8
6/8
7/8
8/8
3/14
7/12
0
6
11
11
25b
9
10
15
34b
11
8
0
0.75
1.38
1.57
3.13
1.13
1.25
1.88
4.25
0.79
0.67
0
17
60
95a
69
29
27
60
166a,b
72
46
0
2.83
5.45
8.64
2.76
3.22
2.7
4.0
4.88
6.55
5.75
0
0.31
0.63
1.02
0.36a
0.36
0.26
0.43a
0.45a
1.14
0.49
0
33.33
18.18
18.18
40
66.66
30
13.33
38.23
73
25
0
33.33
45.45
45.45
28
0
40
53.33
32.35
0
50
0
33.33
36.36
36.36
32
33.33
30
33.33
29.41
27
25
a
b
Significantly different from control by Dunnett’s test at p < 0.05.
Significantly different from control by Tukey-Kramer test at p < 0.05.
DISCUSSION
The present study reports that disinfection by-products
(DBPs) can initiate cell proliferation in the urinary bladder
and the formation of aberrant crypt foci (ACF) in the large
intestine of rats. The halogenated furanone MX appeared to
be the predominant chemical that induced the development of
urinary bladder epithelial hyperplasia and cell proliferation,
and had the greatest effect on ACF incidence and number.
MX has potent bacterial mutagenicity (14, 33, 34), and is a
direct acting mutagen in vivo (46) as well as in mammalian
cells in vitro (4, 31). It has been shown to cause DNA adduct
formation when reacted with isolated bases in vitro (19, 36),
although these adducts have not been found in tissue samples. MX has also been shown to be a multisite carcinogen
in the rat following 2 years of administration of doses as low
as 0.4 mg/kg (29). In addition, MX can function as a tumor
promoter when administered to rats during the postinitiation
phase in a 2-stage model of glandular stomach carcinogenesis (38). However, exposure to MX had not previously been
shown to cause neoplastic lesions in the urinary bladder or
intestinal tract of rodents (29, 48).
In a population-based case-control study of bladder cancer in Iowa, associations were found for duration of chlorinated surface water consumption and total and average
lifetime DBP intake, as represented by estimated total trihalomethanes (8). Positive findings were restricted to men
and to ever-smokers. In the men, smoking and exposure
to chlorinated surface water mutually enhanced the risk of
bladder cancer (8). Most urinary bladder carcinogens exert
their action by direct contact with bladder epithelium (37).
However, increased cell proliferation can occur either as a
result of a direct mutagenic effect or by cytotoxicity with
ensuing cell regeneration (6). Activation of cell proliferation and changes at the DNA level in a target organ are considered essential for initiation and cancer development (6).
Sequential administration of single urinary bladder carcinogens, followed by other complete or incomplete carcinogens,
has been shown to result in an increase in urinary bladder tumor incidence compared to carcinogens administered alone
(10, 51).
The presence of urothelial hyperplasia and neoplasms of
the rat urinary bladder is unusual in control animals (10).
Extensive experimental efforts have successfully identified
several bladder specific carcinogens in rodent species (9, 10,
50, 53). A continuum of stages in urinary bladder carcinogenesis has been established. Neoplastic processes typically
begin with simple hyperplasia, progress to papillary or nodular (PN) hyperplasia, and sometimes papilloma and invasive
carcinomas. The model of initiation-promotion is also relevant to urinary bladder carcinogenesis (10). Observations of
experimental urinary bladder carcinogenesis have suggested
that 2 types of simple hyperplasia may exist, one reversible
and the other nonreversible (20, 50). However, it is difficult to distinguish benign reversible hyperplastic changes
from preneoplastic and neoplastic bladder epithelial proliferations (50). A study with the rat urinary bladder carcinogen N -butyl-N -(4-hydroxy-butyl)nitrosamine (BBN)
for 12 weeks showed increases in S-phase labeling from 1.2 ±
0.3 in control animals to 38.2 ± 6.1 in bladders with simple hyperplasia (50). The increase in cell proliferation after
BBN treatment was substantially greater than the increase in
urothelial proliferation present after exposure to MX. This
may contribute to the difference in the urinary bladder tumor
response between BBN and MX.
TABLE 4.—Proliferative features of male Eker rat colons after 10 months of exposure to DBPs in the drinking water.
Treatment (g/L) (n)
LI proximal
% Mitoses proximal
LI middle
% Mitoses middle
LI distal
% Mitoses distal
LI colon
% Mitoses colon
Control (10)
0.4 KBrO3 (8)
1.8 CHCl3 (8)
0.7 BDCM (8)
0.07 MX (8)
High-dose mix (12)
7.8 ± 4.6c
8.2 ± 4.5
8.7 ± 3.5
10.8 ± 3.4
9.2 ± 2.3
11.7 ± 4.6a
2.3 ± 1.2
4.1 ± 4.0
3.7 ± 2.2
4.1 ± 3.5
3.3 ± 2.4
2.3 ± 1.2
15.0 ± 4.1
12.7 ± 3.0
16.2 ± 5.5
16.0 ± 4.6
15.3 ± 6.3
19.6 ± 4.4a
2.1 ± 1.0
4.1 ± 2.0a,b
2.2 ± 2.5
2.4 ± 1.6
0.6 ± 0.5a
0.9 ± 0.8
15.5 ± 2.6
13.1 ± 4.8
15.3 ± 2.7
19.0 ± 5.6
16.2 ± 4.0
20.0 ± 6.4a
2.2 ± 1.4
3.8 ± 1.8a,b
2.0 ± 1.2
1.5 ± 0.8
1.3 ± 0.8
1.2 ± 1.0
12.8 ± 2.0
11.3 ± 3.1
13.4 ± 3.4
15.2 ± 3.3
13.6 ± 4.2
16.9 ± 4.6a,b
2.5 ± 1.1
3.8 ± 1.1a
2.5 ± 1.8
2.4 ± 1.4
1.5 ± 0.6
1.3 ± 0.6a
Significantly different from control by Student’s t-test at p < 0.05.
Significantly different from control by Dunnett’s test at p < 0.05.
c
Significantly lower ( p < 0.05) than LI in middle or distal colon in control animals by Tukey-Kramer multiple comparison test, middle and distal not different from each other in control
animals.
a
b
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MCDORMAN ET AL
A less than additive lesion response was observed in the
urinary bladders of rats exposed to the high-dose mixture.
Chemical analysis of the DBP mixture demonstrated that the
relative concentrations of each chemical in the individual
chemical solutions and the solutions composed of the mixtures of DBPs were similar (25). This indicates that limited
interaction of the chemicals occurred in the actual drinking
water solutions that were administered. However, interactions
that are uncontrollable and difficult to predict based on available literature may have occurred after the mixture was consumed. The data presented in the present study suggests that
a significant chemical interaction may have occurred following the administration of the mixture of DBPs, but only at the
high doses. It is possible that smaller amounts of MX and/or
metabolic derivatives of MX reached the urinary bladder in
rats treated with the high-dose mixture of DBPs. However,
the exact interaction and cause of the less than additive lesion
response are largely unknown.
Numerous chemicals have been identified as actual or
probable DBPs in drinking water. The trihalomethanes,
haloacetic acids, and haloacetonitriles are the most prevalent classes of contaminants in finished drinking water (35).
Within these classes of halogenated compounds, BDCM and
bromoform have been shown to produce colon tumors in rats
after 2 years of treatment (16), and dibromoacetic acid also
induced ACF in rats (35). This is the first report of DBPs
other than trihalomethanes or haloacetic acids causing ACF
(ie, MX), and the first report of a DBP from a process other
than chlorination causing ACF (ie, KBrO3 ). In addition to
individual DBPs, the mixture of DBPs enhanced ACF development as well as increased proliferation of colon crypt
epithelium. Our results are consistent with previous reports
for BDCM, however others have not found ACF in chloroform treated F344 rats (13). This difference may be due to the
genetic predisposition of tumorigenesis in the Eker rats used
for our studies, although a higher incidence of colon tumors
has not been associated with this rodent model.
The significance of ACF is based on the supposition that
preneoplastic changes occurring in single crypts will lead
to altered crypt morphology and ultimately tumors. ACF are
considered preneoplastic lesions that are present in the colons
of carcinogen treated rodents and in humans with a high risk
for developing colon cancer (2, 40, 49, 52). Colon carcinogenesis is believed to begin with genetic and morphological
alterations in individual ACF (40).
Normal colonic crypt structure includes stem cells that lie
within the basal layers and function to repopulate the entire
crypt. As cells within the crypt divide, new cells are pushed
towards the lumen replacing dead or sloughed cells. Each
stem cell repopulates a defined portion of the tissue, the socalled turnover unit (23). The turnover unit is used in colon
cancer modeling as the susceptible target population for initiation and subsequent genetic changes in the pathogenesis to
cancer (23). However, it is practically impossible to morphologically identify a turnover unit on a standard microscopic
section. The smallest functional unit that is easily identifiable is the entire crypt. In the present study the entire colonic
crypt was used as the basic functional unit for quantitation of
proliferation.
In the present study, only rats that received the mixture
of DBPs had enhanced cell proliferation within the colon
TOXICOLOGIC PATHOLOGY
crypts. This may suggest that although any of the DBPs can
initiate the development of ACF, a mixture of DBPs could
promote the growth of these lesions making it more likely
for them to develop into tumors. In addition, KBrO3 treated
rats had increased mitoses in the middle and distal colon and
MX treated rats had decreased mitoses in the middle colon.
These differences in mitotic rates suggest an alteration in cell
cycle times (47). Enhanced mitotic rates, as seen following
treatment with KBrO3 , would suggest shortened cell cycling
because more mitotic figures are present at any 1 point in
time. The decreased mitotic rates after MX treatment may
suggest a cell cycle arrest, perhaps to allow for DNA repair
since MX is a direct acting mutagen (4, 30).
ACF and colon tumors arise more commonly in the distal
than the proximal portion of the large intestine in humans and
rodents (2, 12, 18, 32, 44). In human patients 38% of colon
cancers arise in the cecum and ascending colon, 18% in the
transverse colon, and 43% in the descending and sigmoid portions of the large intestine (12). In the rat and mouse colon,
tumors are more frequently diagnosed in the distal third to
half of the large intestine than the proximal third (32, 44).
However, in some cases they are reported as being scattered
throughout (16). Growth kinetics of ACF have been shown to
be different in the different regions of the colon (2). In one report, ACF and tumors arose earliest and in greater numbers in
the distal colon but with repeated treatment eventually greater
numbers of ACF and tumors developed in the proximal large
intestine (2). In the present study the proximal portion of the
colon consistently had a lower proliferation rate than either
of the other segments of the colon, but there appeared to be
no difference in overall distribution of ACF throughout the
colon. This study did not result in tumor development, so it
was not determined whether any 1 segment was more likely
to develop adenomas or carcinomas.
In summary, the experimental evidence is not inconsistent
with human epidemiological studies where disinfected drinking water enhances the development of lesions in the urinary
bladder and colon that are associated with cancer development. MX was the chemical that caused epithelial hyperplasia and increases in cell proliferation in the urinary bladder,
whereas all of the DBPs, as well as a mixture of them, caused
the development of the putative preneoplastic lesion of colon
cancer.
ACKNOWLEDGMENTS
The authors wish to acknowledge Rebecca Debolt for assistance in establishing cell proliferation counts of PCNA, as
well as technical assistance from EPL (EPA contract), and
animal care from staff at EPA/NHEERL. K.S. McDorman
was supported by EPA training agreement CT827206 with
the UNC curriculum in toxicology, and by NIH toxicology training grant ES07126. The authors also wish to thank
Dr. James Swenberg and Dr. Anthony DeAngelo for scientific
review of the manuscript. This manuscript has been reviewed
internally by the EPA, but does not necessarily reflect EPA
policy nor does mention of trade names reflect endorsement.
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