Mnnilaring~f R I iz V 1~~ , J L~'1 V Qceup hn~~'ScdS©iA9n}q~~cse~~ p~ V!OpY C) l4fifi Ala . s OF.4PLIES WITk COP1^RIrHT ~~ /' ~ , \/ °\ N 1 v.~~ .w,vni .npJlnMl,<JJJt `\J I `' • \ . . ~~ .TT„E40W CYTOMETR OF ACRIDINE ORANGE STYGINED SPERM IS A RAPID `AND :PRACTICAL METHOD .FOR MONITORINO OCCUPATI!ONAL .EX~POSURE TO 6ENOTOXICA!NTS . Donald P . Evenson ~ Depau:keenb-ef ChemL%*+-;,`~So(u/th Dakota . State [Oniv)ir&!~t,rl, Brookings, SsuLlr9eka6e, 57007 Public awareness i!s :growing coneerning .thereproduct.ive consequences of thenumerous .environmental and occupational chemibals .Ezposure of germ cells within the seminiferous tubulesof the mammalian testisto .chemical toxins often causes severe perturbation of cell .growth, division and differentiation . Quantitative and .qual'itverdcon .of sperm may have : adverse consequences for fertility, and :normaiity of fetus . . To detect toxic effectsof'chemicai exposure on germ cells, it iss of prime importancee todevelop : more sensitive and practicai .methodsl by which putative . Previously selected :alterionsgmc1Iaybeinvstgd (Overstreet, . 198A)cri!teria for male reproductive risk assessment required the tests be .aJ ob,jective,b), technica3lysound, c) biologically stable, . d) sensitive, and e) feasible . Flow cytometric measurement of acridine orange stained sperm . meets all of these criteria for monitoring occupational exposure to genotoxicants . This conclusion is based on two independent studiesof sperm obtained from : 1) toxin exposed mice, and 2) patients .attending .iafertility and cancer treatment clinics . FLOW CYTOM.ETRY 0F SPERM!OBTAINEDFROM TOXIN'EXPOSED .PIICE Previous work (Wyrobek :andlBruce, 1975 ;see Wyrobek :et al, 1983 and .Topham, 1983forreviews .) has shown that mouse sperm head morphology is sensitive to : toxic chemicall exposure . These .observations led to developmentt of the .sperm http://legacy.library.ucsf.edu/tid/xae80c00/pdf 122 lEncnson head morphology assay which has proven useful for reproductive toxicology because 100X .of'germ cell mutagens tested have demonstrated .a .positiveresponse (Hyrotiek et al, 1'983). . . This assay is currently performed by light microscope measurements of stained .epididymal sperm isolated from toxinexposed mice . . Consequently, theas .say suffers from some~e of the typi!cal limitations .of light microscopy ;e .g ., sSownessof measurement, Auman eye judgments, and often small numbers of observations which .limit statisticalsigpificance . Recently we .deveioped (Evenson et al, 1980 ;'. 1985a)) thee flow cytometric sperm~chromatin structure assay (SCSA) thatappears to be : as ;sensitive as the sperm head morphoiogyy as-say for detecting toxin,induced abnormali!ties in mouse sperm nuclei . The SCSA measures the susceptibility to acid- or thermal-induced denaturation of sperm nuclear DNA in situ . Our experimentall flow cytometric approach for the SCSA ' is il!lustrated by recent work.using ethylnitrosourea (ENID),, a powerful alkylating agent . : Groups .of F1 male.mice (C57s1/6J x C3H/'HeJ) were exposedi .p,d to phosphate-buffered saline (PBS ; :control) or dosagess of ENUU ranging,from 5 to . 75' mg/kg body weight in,PBSi, daily x .5!days .Twenty-eight days followingthe .last chemical exposure the .mice were killed: . The caudal epididymides were excised and placed into TNE : (0 .01IM TRIS., 0 .15 M!NaCl!, 1 mM!EDTA, pH .7 .4)', minced with scissors :and the sperm filtered through153 .um nylon mesh . Saerm Head Morphology Assay An aliquot of each epididymal sperm suspension wass stained with Eosin Y, smeared onto glass slides and mounted . Aminimum of 1000 sperm heads were scored!by theimorphoSogy criteria of Wyrobek and Bruce (1975) . Fig . 1 shows thatt sperm isolated from ENU exposed mice have a dose-related increase of headmorphol~ogy abnormalities . http://legacy.library.ucsf.edu/tid/xae80c00/pdf Flow C7tumctric Spxm Chrnmatin Stimclurc :#suy . ! 123 aP o 100 t0UOoo~ mg/kENl9Fiuret . . Effects of ENU oncaudal .epididymal sperm head morphology . Each point represents the meanfrom .threem4ce ; vertical lines showthe .standard deviations . . Arepeatexperiment .demonstrated the same .response ; :however, the standardldeviation at the highest dose was muchsmai!ler (from Evensan et al, 1985a) . Soerm Chromatim Structure A'ssavl'Acid (SCwSA/aeid)) of Fresh Eoididvmal Soerm, Epididymal .sperm were stained by the TSAO (two .orange) :technique .(IDarzynkiewiez ett al, . 1976 .stepoaridn ; Evenson et al, 1I9E5a) and measured by flow cytometry . This .procedure includes a 30sec treatment of the samplewithTriton .X-100, which,permealizes the cell membranes for dye penetrationy, and also 0 .061N HC1I which may cause partial denaturation of sperm nuclear DNA in situ . The extent of IDNA'denaturation was measured by the metachromatic shift .in acridine .orange .(.AO)staineitsperm from green fLuorescence . (AO intercalated into double-stranded DNA) to red fluorescence (AO associated with single .stranded DNA) . Fig .. 2 illustrates the effect .of ENU on total fluorescence vs1 t(redPred + green fluorescence) of AO stained epididymal sperm . The term at provides .a numerical index for thee extent of DNA denaturatiom im situ (Aarzynkiewicz et al ., 1975) . The total fluorescence .(green +red„ corresponding to dlxuble- and singRe-strandednucleic acids, respectively)) did not increasein response to ENU exposuree indicating ENU did not cause an abnormal increase or retention of DNA,orRNA. Furthermore, RNAse treatment http://legacy.library.ucsf.edu/tid/xae80c00/pdf 124 /E"nsan 70 0 mgPkd W 0 s 7 a m 25 mg/k 1,25'~mg/kd W U Z. Wl U y W O 0 LL TO J 0 mg/k g 250 F O F 0 0 0.6 - - -1 .0 0. 0.5 10 RED/RED+GREEN FLUORESCENCE ( .-a) Figure .2 .Relationship between exposure level to ENO and red and green fluorescence emission of epididymal .sperm analyzed by the SCSA/acid (from Evensos.et al, 19B5a), did not alter this staining pattern . Note .tke : shift towards higher a t values'indtt'cating an ENU dose-related alteration ofchromati~n structure asdefined by an increased .sensitivity toDNOI . denaturati~on . Soerm .Chromatin Structure kssay/Thermal (SCSA/thermaiiY of Fixed Eoididvmal Sverm .Nuclei Epididymal .sperm nuclei .were isolated by sonication http://legacy.library.ucsf.edu/tid/xae80c00/pdf Flbwq1omelri< Spcrun Chrnmxtin StrucDure Aissay 1' ] 25 of sperm samples represented in Fig . 2 . After fixation (ethanol :aeetone)) and rehydration,, thee nuclei were heated at 100°C for 5min andlthen stailned withAO (Evenson et a1, 1980, 1985a) . . Fi~g . 3shows ENIDD also caused an increased susceptibility to thermal denaturation of DNA .in situ . w< .rzn .< . . .o . , cr'---~w~ . . L_:-''' J 0 { 4 , . i r,s~o ',eao Figure 3 . Relationship between exposure .level to ENA and red andlgreea fluorescence emission of epididymal spermnuc'Lei analyzed by the .SCSA/therraL. Dwo independent sets of measurements on heated and non-heatedAmlstained'.nuclei'were made ; note the highlleveiof reproducibilitybetweenithe . .twoyse(frmEvnoetali,1985) http://legacy.library.ucsf.edu/tid/xae80c00/pdf 1?6 :1 Elrnson Fig . . 4 shows computer-plotted a t tY'equencyhis2ograms of'the .5'samplesf represented in Fig . 3, left-hand 'heated" columnw Note .the .increased shoulder seen even at the loWestt dosage . (25mglkg). . . 12 so Figure 4 . Effect of ENUlon mouse .sperm a t distributions : (fromEvenson et al, 1985a) . It is obviousfrom the data in Fig . . 4 that the coefficient of variation .(C . .V .)of a tincreases with dosage of ENU exposure ; this relationship is plotted in Fig . 5 . Interestingly ; the curve: inF'Sg .5isvrtualydeni-ctohC .V .otfwhle'sprmvautdbyhe9CSAYacid(r=0 .9, . data not shown .) . Apparently both .the .acid and heat treatment prior to AOistaining resolve the same toxin-induced :.abnormaLity of chromatin structure . . N; Compari son o f data i n Fi g . 11 w i th that in Fi g . 5 shows ~ that the flowcytiometric-based SCSA is as sensitive as the ~ . ..n 6neA .. .u n ..,. .. . _ ..__ . . .nr.nnAnln _ . . . ..__,.w . _ . . ....Y . nalc innlM1,dineg C. .. . .nnU n n4An .. nl.e.e< ._ . . .wh6n .. . . . . . . .. . . . .. .. . ., .. . . . .. . . .. . ..._-,!~ lhinhnnn_ --- . e ____r ._, ______ ._, ______ . , -,~ - hydroxyurea, methylmethanesulfonate, ethylmethane GIl- http://legacy.library.ucsf.edu/tid/xae80c00/pdf ~ ~' Fluw'Cytomclrfc .Sprrm C6rol Structure axay 1127 t`-T---t-T 0 100 200 300 mg/kg ENU Figure 5 .Relationship.tietween exposure level to ENU and coefficient of variation .of ;,t of epididymal sperm nuclei evaluated by the SCSA/thermal (from .Evenson et al, 19B5a) . sulfonate and others (E4~enson etal!, 19850 . oonfirmsthis point . Regres ion an lyse showl one minorexcepr tion, the .slopese of the :curves of sperm morphology abnormalities and standard deviation of s t vs chemical dosage were not different fromleack other .Thus we conclude that. ourSCSA is measuring a .factor of chromatin structure thatis highly related to sperm head .morphology abnormalities . FLM . tMEASUREMENTS :OF Ai0 STAINED HUMAN SEMEN SAMPLES Toxin exposuree can cause immature germ cells to be .released into semen . Fig . 6 shows FCM derived .data on fresh TSAOstai'ned semen aliquots obtained from a normal volunteer and twoo cancer patients exposed (ornot)r to chemotherapeutic drugs . The TSAD,procedure apparently does not denature .histone-associated!DNAin situ . FDowcytometer photomultiplier gains,weresete toinclude .cell types ranging in DNA stainability from sperm to di.pl'oiid cells . This.difference is 10-fold since: the peak ofthenormal .sperm population is at channel 8!and the diploid cell!s peakat channel 80 (patient .B) . . Thecontrol samplehasa homogeneous distribution withninimal .green and red fluorescence . Patient A .hadl http://legacy.library.ucsf.edu/tid/xae80c00/pdf q'© stained semen ce11B __ no+nexr B' Figure 6 . Computer drawn two-parameter (F'530,, green fluorescence vs F)600, red fluorescence) histogramss representing distribution of TSAO stained human semencells A . Normal voSunteer„ B . Cancer patient exposed to ehemotherapyy, C .Testicular cancer patient treated'by unilateral orchiectomy ( :fromEvenson and Melamed, 1983) . been exposed to chemotherapy drugs 3 months prior to semen sample collection . The sperm~count was 55 x 11OU/m1 . Note . that thesee sperm had & hi~gherlevel ofDNA .stainbly(IDe-twnchals10 .- 30) indicating a .Lackof normal chromatin condensation . . Theima3pri'ty of ceilspresents in the semen were : round spermatids verified by their identical staining pattern to the round .spermatids .in minced testiculiar biopsy samples . By light .microscopy,it .is difficult .which could be .todisnguhr pematidsfrolukcye present from infections . FCHlmeasurementseasily and positively distinguish between these tvocell types . . Patient B with testicular cancer had unilateral .orchiectomy 11 months prior to collection .of a semen sample thus showing that disease alone apparently caused~a deteri~orationn of spermatogenesis . ln this sample, ce11sranging in maturiity from diploid cellsto nearly mature sperm (but with abnormal morphology) were present . . The majority of patients at infertility ciinicsdo not show such, gross semen abnormalities as represented in Fig, 6 . To detect .lessor levels of chromatin,abnormalities, sperm .nuclei have been, isolated, heated, stained with AOO and http://legacy.library.ucsf.edu/tid/xae80c00/pdf Flnx• C>lomctric . SMTm Chromatin Structurc AssaF /1?9 measured .by FCH!according to the SCSA/thermal dbscribed above for the mouse .studies . Fig . 7 shows data~ on semen from .& fertile donor and 5'pati~ents attending an infertility cLinffc .Note the dramatic' .heterogeneity betwe n samples oEsusceptibility to DNA denaturation . ul..r.rto. rrc ..en v.e•+.> i i . I ,. /~~ .i I I I I I't 1 U I~I ifJ'1 AETT] Ll Figure .7 . Computer drawn scattergramsof the distribution of sperm nuciei .from one control (fertile donor) and 5 pa-tients attending an infertilitycli'nic, according to their green (F530)' and red (P)600) fluorescence intensities after heating (or notheating) and staining with AO (SCSA/ thermal) . Patients'were diagnosed as followsc. (1) . Morphologypoor to borderl'ine~ ; Infection (S-) . ; (2)' Prostatitis/Nonrspeci :.ic urethritis (NSU), morphology http://legacy.library.ucsf.edu/tid/xae80c00/pdf 130 / Ewnsun minimal„ Ir ; . (3) . ProstatitisLNSU4I good specimen, I* ;, (4) Morphology and motility gpod, .2 miscarriages, I- ;, (5) Excellent .specimen in all respects .(from Evenson et aID, U9U5b) . . Perhaps themost .interestinge samples are those that are scored!as 'excellent' by .theclasirtofspemcunt ., morphology„ and motility but have a significant abnormality of chromatin structure (e .g, patient 5,Fig .. 7)1. Thus ;, our technique wi11 nott onlyrapidly screen ..for toxininduced orotherabnormalitiesr present in semen but wi1L detectiabnormalities not found by other techniques .SimlarFCMesunt .havebeen made on semem samples obtainedfrom,testicular carcinoma patients (Evenson et al, 1984a) and leukemiapati .ents successfully treated by .chemotkerapy ('.Evenson .et al, 1984bY. Ftowcytometric measurement of AO stained semen eells ; or epididg,mal sperm providesanew, sensitive, objective, biologicaylystable, technically sound and feasible method to : monitor for genotoxicants ; thatt may bee found in occupar tionaL environments . Since semen samples may bee collected, frozen and sent toan FCM laboratory for measurements, thiss method is .indeed practical for screening programs . I ACKNONLEDGEMENTS Thiswork was supported .by National .Institute of Health Grant No . R01ES03035 and US EnvironmentaSProtection. Agency Grant. No . H810986' . Although the research described in this article has been funded in part by the .HealthEffectsReseareh Laboratory, US EhvironmentalProtection .Agency through Grant No . CR870991 toSouth Dakota .State University it has .not .been subjected to the Agency peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred . . Publication No . 2077 from the SouthhDakota State UhiversityExperiment Station . I .thank Rebecca Baer for help Ln preparation of manuscript . http://legacy.library.ucsf.edu/tid/xae80c00/pdf N ~ N W ~ C17 ~ O C W Lluw Ctgomcti~ic $twrm Chromatin Structurc~ .]sczg.l~ 131 REFERENCES Darzynkiewicz Z,, ThaganosF, Sharpless .T, . Melamed :.MR (1975) . Thermal denaturation of'DNA,in situ as studied by acridtine orange staining .and automated cytofluorometry .ExpCelRs9D :4q 1 .Darzynkiewi~ez Z, Traganos. F, SharplessT,, Melamed MR (1976) . Lymphocyte stimulatiionc: a rapid multiparameter analysis :2881-2884' .ProcNatlA~dSiU73 .EvensoDP'(1985t) . Male germ cell analysis by flow cytometry :Effects of cancer, chemotherapy and other factors on testicul'ar function .and sperm chromatin structure . In Andreef MA ('.ed)'. : Proceedingsof the Conference on Clinical Cytometry . New YorkAcad of'Sai .(i~n press) . Evenson DP, Arlin Z, We1t .S,, C1apsML, Melamed MR'(19g4a), . Male .reproductive capacity may recover following drug treatment,w2tfi the L-10 protocol for acute lymphocytic leukemia(ALL) . Cancer 53 :30-36 . Evenson DP, Darzynkiewicz Z, Melamed MR ('19 .80) .Relation of mammalian sperm .cfiromatin heterogeneity to fertility . Science 210 :1131'~1133 . Evenson DP„ Higgins PH, GruenebergD, Ballachey B( :1985a) . . 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Principles and methods for their detection, Vol 8, pp. 2o1-23U . http://legacy.library.ucsf.edu/tid/xae80c00/pdf Wyrobek .AJ, Bruce WR(1!975).« Chemical .inductofspermabnlits .inmice . Proc : Natl Acad Sei 72 :4425-44'29 . . Wyrobek AJ, Gordon,LA, Burkhart .FG, Francis MW, Kapp RW, Letz G, Malling HN, Topham JC, . Whorton D(1983) . . An . evaluation of human sperm .asm indicators of chemically induced alterationsof spermatogenic funet'ion : A report of the B :S . Environmental .Protection Agency Gene-Tox Program . Mutat Res .115 :73-1u8 . http://legacy.library.ucsf.edu/tid/xae80c00/pdf