A comparative study of chemically induced DNA damage in isolated human and rat testicular cells

Reproductive Toxicology, Vol. 10, No. 6, pp. 509-519, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238196 $15.00 + .OO ELSEVIER

PI1 SOS90-6238(96)00138-4

A COMPARATIVE STUDY OF CHEMICALLY INDUCED DNA DAMAGE IN ISOLATED HUMAN AND RAT TESTICULAR CELLS CHRISTINE BJ@RGE,* GUNNAR BRUNBORG,* RICHARD WIGER,* JBRN A. HOLME,* TIM SCHOLZ,~ ERIK DYBING,* and ERIK J. SPIDERLUND* *Depan:ment of Environmental Medicine, National Institute of Public Health, Oslo, Norway SInstitute for Surgical Research, The National Hospital, Oslo, Norway

Abstract - Testicu.lar cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxlcants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxlcants (i.e., reacting with DNA either spontaneously or vla metabolic activation). In addition, a few indirect genotoxlc and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 pM, human and rat), benomyl(10 to 100 pM, human and rat), thiotepa (10 to 1000 pM, human and rat), cisplatln (30 to 1000 pM, human; 100 to 1000 @I, rat), Cd’+ (30 to 1000 pM, human; 100 to 1000 pM, rat), and acrylonitrile (30 to 1000 pM, human; 30 to 300 pM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (2 100 pM, rat and human), 1,2-dibromoethane (EDB) (2 100 @Z, rat; 1000 pM human), thlram (2 30 pM, rat; 2 100 pM, human), and chlordecone (300 pM, rat; 2 300 pM, human). Finally, flve chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dlbromo-3-chloropropane (DBCP) (2 10 pM), 1,3tiitrobenzene (1,3-DNB) (2 300 @i), Cr6+ (1000 pM), and aflatoxln B, (2 100 PM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 CM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity. 0 1996 Elsevier Science Inc. Key Words: DNA damage; testicular cells; humans and rats; reproductive toxicants.

INTRODUCTION

including exposure to environmental pollutants have been suggested. So far nearly 100 different xenobiotic exposures have been evaluated for their effects on sperm production in the human male. About 50 of such exposures have been suggested to be detrimental to sperm production, including ionizing radiation, different pesticides, heavy metals, and life style factors (6-8). Recently, a hypothesis linking the decrease in sperm quality and other disorders of the male reproductive tract to an increased embryonic exposure to endocrinedisrupting environmental contaminants, has received considerable attention (9,lO). However, other possible mechanistic links exist. The germinal epithelium of the testis is one of the most proliferative active tissues in the body and is, therefore, potentially susceptible to the DNA damaging effects of environmental toxicants, drugs, or radiation (11). It is well documented from studies on patients treated with cytostatic agents that genotoxic exposure can cause reduced sperm count and infertility (12,13). The nematocide 1,2-dibromo-3-

Compared to most animal species, the human male is of low fertility. It is estimated that one in five couples experience fertility problems, and among this group the male has been shown to be the infertile partner in 30% of the cases (1). When fertilization does occur, at least onethird of the early embryos die (2), and 20 to 30% of all developmental defects appear to have genetic origin (3). During the past 50 years there has been an increased incidence of testicular anomalies including testicular cancer, and, furthermore, human semen quality has apparantly declined (4,5). A number of possible causes

Address correspondence to Christine Bjorge, Department of Environmental Medicine, National Institute of Public Health, P.O. Box 4404 Torshov, N-0403 Oslo, Norway. Received 25 March 19!)6; Revision received 3 June 1996; Accepted 3 June 1996. 509

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chloropropane (DBCP) is one of the few occupational/ environmental chemicals that has been shown to cause reduced fertility in men. Based on studies with different species (14), and a number of halogenated (15), and deuterated and methylated analogs of DBCP (16), we have previously suggested that DBCP-induced ssDNA breaks are important for its testicular toxicity in experimental animals (17). This finding indicates that the reduced sperm count observed after occupational exposure to DBCP may have been caused by its DNA damaging properties (14-20). DNA damage in testicular cells may also result in cancer and heritable defects. Evaluation of the level of DNA damage is, therefore, of prime significance in understanding the response of male germ cells to toxic agents. The evaluation of human reproductive risk from exposures to drugs and chemicals is to a large extent based on extrapolation from animal data. To provide a better basis for risk extrapolation from animal studies, we have studied suspensions of human and rat testicular cells and compared them with regard to their sensitivity to DNA damage after treatment with different known reproductive toxicants. Most of the chemicals are known to react with DNA, whereas others cause DNA damage by indirect mechanisms or they are nongenotoxic. Crude single cell suspensions of human or rat testicular cells were treated in vitro with pesticides: benomyl, chlordecone, DBCP, 1,2_dibromoethane (EDB), methoxychlor, and thiram; industrial chemicals: acrylamide, acrylonitrile, cadmium dichloride (Cd2’), 1,3_dinitrobenzene (1,3DNB), sodium dichromate (Cr6’), and styrene oxide (metabolite of styrene); cytostatic agents: cisplatin and thiotepa; and mycotoxins: aflatoxin B,. Single-strand breaks and alkali-labile sites (ssDNA breaks) were assessed using the alkaline filter elution technique.

Volume 10, Number 6, 1996

27-l), cytosine- 1-B-o-arabinofuranoside (Ara-C), 1,3-dinitrobenzene (1,3-DNB) (CAS No. 99-65-O), 1,Zdibromoethane (EDB) (CAS No. 106-93-4), hydroxyurea (HU), methoxychlor (CAS No. 72-43-5), and trypsin (III-S) from Sigma Chemical Co., St. Louis, MO; acrylamide (CAS No. 79-06-l) and styrene oxide (CAS No. 96-09-3) from Koch-Light, Coinbrook Bucks, UK; acrylonitrile (CAS No. 107-13-l) and thiram (CAS No. 137-26-8) from Aldrich, Steinheim, Germany; chlordecone (CAS No. 143-50-o) and benomyl (CAS No. 17804-35-2) from Chem Service, West Chester, UK; and thiotepa (CAS No. 52-24-4) from Lederle, Puerto Rico. Other chemicals were analytical grade from commercial suppliers. Animals Sexually mature male Wistar rats (MOL: WIST, 200-300 g) were obtained from Mollegaard, Ejby, Denmark. The animals were housed in plastic cages on hardwood bedding and were given RMI(E) standard pelleted feed (Special Diet Services, UK) and water ad libitum.

MATERIALS AND METHODS

Human testes Human testes were obtained immediately after death of the organ transplant donors, supplied by the Norwegian National Hospital. After decapsulation, the tissue of one testis was minced and kept in cold RPM1 1640 medium at 4°C until cell preparation (8 to 12 h later). DNA damage was measured by alkaline filter elution of preparations from seven donors after in vitro treatment with different chemicals. These donors were: donor-l (29 years), donor-2 (31 years), donor-3 (41 years), donor-4 (51 years), donor-5 (68 years), donor-6 (70 years), and donor-7 (71 years). Due to limited capacity of the alkaline filter elution equipment it was not possible to test all chemicals at all concentrations using cells from the same donors.

Chemicals DBCP (CAS No. 96-12-8) (> 99% pure by GC analysis), prepared by bromination of ally1 chloride (21), was kindly supplied by Dr. Sidney D. Nelson, University of Washington, Seattle, WA; collagenase (CLS II, 352 U/mg) from Worthington Biochemical Corp., Freehold, NJ; dimethyl sulfoxide (DMSO) from Rathburn, Walkerbum, Scotland, UK; fetal calf serum (FCS) and RPM1 1640 cell culture medium from Gibco, Grand Island, NY; Hoechst 33258 from Calbiochem-Boehringer, La Jolla, CA; cadmium dichloride (CdCI,) (CAS No. 10108-64-2), sodium dichromate (Na,CrG,) (CAS No. 10588-01-9) and proteinase K from Merck, Darrnstadt, Germany; aflatoxin B, (CAS No. 1162-65-Q, bovine serum albumin (V) (BSA), cisplatin (CAS No. 15663-

Preparation and characterization of testicular cells Rat testicular germ cells were prepared as described by Bradley and Dysart (22) with some modifications (16). In short, testes were decapsulated and incubated at 32°C in Hank’s HEPES buffer with collagenase (100 to 150 U/mL) for 20 min. Trypsin (0.25 mg/rnL) was then added, and the tubular suspension was further incubated for 12 to 15 min. Trypsination was stopped by adding fetal calf serum (FCS) (l%), the resulting cell suspension was filtered, centrifuged three times (270 x g for 5 min), resuspended in Hank’s HEPES buffer with 1% bovine serum albumin (BSA), and then filtered through a nylon mesh (0.25 mm). The total yield per gram wet testis tissue was about 70*106 cells with cell viability greater than 95%, as measured by trypan blue exclusion.

DNA damage in human testicular

Human Testicular Germ Cells. Crude cell suspensions were prepared as dlescribed by Bradley and Dysart (22), and Soderlund and coworkers (16) with some modifications (20), including an increased collagenase concentration (200 U/~-IL) and an extended incubation time (30 min). Furthermore, all centrifugations were at 740 x g for 5 min. The total yield per gram wet testis tissue was about 15*106 cells with a viability greater than 95%, as measured by trypan blue exclusion. Minced human testis tissue was kept at 4°C for 8 to 12 h in RPM1 1640 medium prior to the preparation of single cell suspensions. Characterization o~‘Germ Cells by Flow Cytometry.

DNA was stained by adding 1.2 pg Hoechst 33258 and 0.004% Triton X-100 in 1 mL aliquots of cells (5*105), suspended in Hank’s Balanced Salt Solution (HBSS) supplemented with 2% BSA. The samples were incubated in the dark at room temperature, whereafter they were placed on ice. Blm fluorescence was measured after 15 min using an Argus 100 Flow cytometer (Skatron, Lier, Norway). The percentages of haploid, diploid, and tetraploid cells were estimated from DNA histograms using the Multicycle Program (Phoenix Flow Systems, San Diego, CA). It was possible to distinguish different cell types on the basis of their DNA content (Hoechst fluorescence) and cell size (forward light scatter). Microscopic

Characterization

of Germ

Cells.

Smears containing testicular cells in FCS were air dried quickly, fixed in methanol, and stained with 2% Giemsa in neutral distilled water. Cell morphology was evaluated using a Nikon Optiphot microscope (1000x). Smears of human and rat testicular cells were also stained with Hoechst 33258 for fluorescence microscopic analysis. These techniques were used to confirm the determinations of the various populations as analyzed with flow cytometry. DNA damage Alkaline Filter El&ion. ssDNA breaks in human and rat testicular cells exposed to different testicular toxicants were measured by an automated alkaline filter elution system (19), based on the method of Kohn and co-workers (23). Crude single-cell suspensions of human or rat testicular cells (4.010~cells in 2 mL) were treated with the following agents-pesticides: benomyl (10 to 100 pM, human and rat), chlordecone (10 to 1000 pM, human; 10 to 300 p,M, rat), DBCP (10 to 300 yM, human; 10 to 100 p,M, rat), EDB (30 to 1000 ~.LM,human and rat), methoxychlor (10 to 100 pM, human and rat), and thiram (10 to 100 ~_LM, human; 10 to 300 FM, rat); industrial chemicals: acrylamide (30 to 1000 FM, human; 100 to 1000 I.LM,rat), acrylonitrile (30 to 1000 pM,

cells 0 C. BJP~RGEET AL.

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human; 30 to 300 p,M, rat), Cd*+ (30 to 1000 PM, human; 100 to 1000 p,M, rat), 1,3-DNB (10 to 300 p,M, human; 30 to 300 ~_LM, rat), Cr6’ (30 to 1000 pM, human and rat), and styrene oxide (10 to 300 pM, human and rat); cytostatic agents: cisplatin (30 to 1000 PM, human; 100 to 1000 pM, rat), and thiotepa (10 to 1000 PM, human and rat), and the mycotoxin aflatoxin B, (10 to 300 PM, human; 30 to 300 yM, rat) for 30 min at 32°C in HEPES buffer with 1% BSA. The concentration ranges were choosen to allow the detection of both potent and less potent DNA damaging agents. In some cases the highest nominal concentration could not be achieved due to limited solubility (i.e., precipitation in the culture medium) of the test compound (benomyl, chlordecone, and methoxychlor). The test chemicals were dissolved in DMSO (final concentration 0.25%), except aflatoxin B, and cisplatin, which were dissolved in methanol (final concentration 0.25%) or cell culture medium, respectively. Control cultures contained the same concentrations of solvents. Cells were centrifugated and resuspended in 2 mL Hank’s HEPES buffer with 1% BSA, their viability was estimated with trypan blue after 30 min incubation with the different test chemicals, and samples (4010~ cells in 2 mL) were loaded onto 25 mm polycarbonate filters (2 pm, Nucleopore, Cambridge, UK). After lysing and deproteinization, DNA was eluted (0.03 mL/min) with 20 mM Na,EDTA at pH 12.50 + 0.04. Two-hour fractions were collected and DNA determined fluorimetrically with the Hoechst 33258 dye, using an automated setup (19,24). The damage levels were calculated from elution profiles and are expressed as “Normalized Area Above Curve” (NAAC) (25). Statistics

Statistical comparisons using the mean NAAC value were performed using the Wilcoxon two-sample distribution test. A P-value of less than 0.05 was considered significant. RESULTS Composition of human and rat testicular germ cells The interindividual variation in the composition of

testicular cells from seven humans was considerable as estimated by flow cytometry and microscopy: haploid cells (round/elongated/elongating spermatids) 56 f 15%, diploid cells 28 + 14%, and tetraploid cells (mostly primary spermatocytes) 14 + 9%, whereas comparable numbers from 18 rats were 71 f 4%, 20 -c 3%, and 9 f 3%, respectively. The compositions of the human testicular cell preparations used in this study, before chemical treatment (representing seven donors) are shown in Figure 1.

diploid

51 years

Donor 4

tetraploid

tetraploid

haploid

.

haploid

diploid

DNA

dipbid

tetrsploid

Donor 6 70 years

DNA

diploid

A

DNA

diploid

71 years

Donor 7

tetraploid

tetrapbid

41 years

Donor 3

Fig. 1. Variations in the composition of testicular cells isolated from human organ transplant donors and analyzed by flow cytometry. Data are shown for six human organ transplant donors [donor 5 (not shown) was very similar to donor 71. The variability in cell composition among seven rats were: haploid cells, 71.7 + 4.2%; diploid cells, 19.7 k 3.2% and tetraploid cells, 8.7 rf: 2.5%.

DNA

diploid

saploid

plaid

29 years

Donor 1

Donor 2 31 years

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DNA damage in human testicular cells 0 C. BJP~RGE ET AL.

menced. To investigate whether delayed preparation would influence chemically induced DNA damage, rat testicular cells prepared with or without a delay were exposed to DBCP. The results showed that a delay of 8 to 24 h did not lead to any substantial changes in the level of DBCP-induced ssDN.4 breaks as measured in the rat cells (data not shown). DNA single-strand breaks were analyzed by alkaline filter elution of crude suspensions of human and rat testicular cells after in vitro exposure to 15 known reproductive toxicants. Six. of the chemicals did not induce significant levels of ssDNA breaks in either human or rat testicular cells: metboxychlor (10 to 100 PM, human and rat), benomyl (10 to 100 yM, human and rat), thiotepa (10 to 1000 p,M, human and rat), cisplatin (30 to 1000 p,M, human; 100 to lOtDOp,M, rat), Cd2+ (30 to 1000 p,M, human; 100 to 1003 FM, rat), and acrylonitrile (30 to 1000 PM, human; 30 to 300 PM, rat) (Figure 2). Four of the chemicals induced significant levels of ssDNA breaks in both species (Figure 3). Styrene oxide was clearly positive at concentrations 2 100 PM in both human and rat testicular cells. EDB was clearly positive at concentrations 2 100 PM in the rat; however, in human testicular cells, only the highest concentration tested (1000 FM) produced a positive (P c 0.05) but moderate response. Thiram was clearly positive at concentrations 2 30 ~,LMin the rat ~~~11s; however, only the highest concentration (100 yM) caused a significant increase in ssDNA breaks in human cells. The response at 100 PM was similar in the two species. Chlordecone showed a marked positive response at 300 p,M in both humans and rats, but no effect at lower concentrations. Five chemicals induced statistical significant levels of ssDNA breaks in one species bat not in the other (Figure 4). For only one chemical (DBCP) this species difference was clearly evident. This co:mpound induced a clear concentration-dependent increase in ssDNA breaks in rat testicular cells (10 to 100 PIM), whereas in human testicular cells no response was observed even at the highest concentration tested (300 ELM). Acrylamide induced a low but significant level of ssDNA breaks in human testicular cells at the highest concentration tested (1000 p,M), whereas no response was observed in rat testicular cells. 1,3-DNB, Cr6* and aflatoxin B, induced very low levels of ssDNA breaks in rat testicular cells at 300 p,M, 1000 p,M, and 1 100 p,M, respectively, whereas no significant increase was observed in human testicular cells. In an attempt to increase the sensitivity of the assay, the accumulation of ssDNA breaks in human and rat testicular cells were assayed for strand breaks after ex-

Human

Rat

DNA damage For practical reasons, the decapsulated and minced human testis tissue was kept at 4°C for 8 to 24 h in RPM1 1640 medium until cell preparation could be com-

140 Thiotepa 100

- Cisplatin 100. 60. 20.

nnn

140 c& 100

I

140 Acrylonitrile 100 60.

Concentration

(cIM)

Fig. 2. Chemically induced ssDNA breaks in isolated human and rat testicular cells as measured by alkaline filter elution. Cells isolated from the testes of either human organ transplant donors or rats (4010~ in 2 mL) were exposed to methoxychlor (10 to 100 FM, human and rat), benomyl (10 to 100 p,M, human and rat), thiotepa (10 to 1000 FM, human and rat), cisplatin (30 to 1000 FM, human; 100 to 1000 PM, rat), Cd2+ (30 to 1000 FM, human; 100 to 1000 PM, rat), or acrylonitrile (30 to 1000 PM, human; 30 to 300 (*M, rat) for 30 min at 32°C. Values are from three organ transplant donors or three rats.

posure to the different reproductive toxicants in the presence of the repair inhibitors cytosine- 1-P-D-arabinofuranoside (Ara-C) and hydroxyurea (HU). However, this did not lead to any significantly increased levels of ssDNA breaks compared to those measured in the absence of Ara-C and HU (data not shown). The viability of both rat and human testicular cells were similar to control cells (95%) after 30 min incubation with acrylamide, acrylonitrile, aflatoxin B,, beno-

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Human

Rat

Volume 10, Number 6, 1996 Rat

t 140.

100.

Human

t DECP

t l

60.

140

- Acrylamide 100.

EDB -I A= ‘0

CO20. 140.

2

a

-

100,

1,3-ONE

140.

Chlordecone

l

TOOBO-

rfiflfl

20. 0

10

30

100

300

,

1000

:, ,:” 0

10

30

100

300

1000

140

Atlatoxln8, 100

Concentration

(FM)

Fig. 3. Chemically induced ssDNA breaks in isolated human and rat testicular cells as measured by alkaline filter elution. Cells isolated from the testes of either human organ transplant donors or rats (4010~ in 2 mL) were exposed to styrene oxide ( 10 to 300 FM, human and rat), EDB (30 to 1000 PM, human and rat), thiram (10 to 100 p,M, human; 10 to 300 p,M, rat) or chlordecone (10 to 1000 PM, human; 10 to 300 PM, rat) for 30 min at 32°C. Values are from three organ transplant donors or three rats. *Significantly different from the control. P < 0.05.

myl, cisplatin, DBCP, 1,3-DNB, EDB, ide, and thiotepa. On the other hand, Cd’+, chlordecone, methoxyclor, and decrease in the viability from control about 70% at the highest concentrations.

Cr6’, styrene oxincubation with thiram caused a values (95%) to

DISCUSSION Compared to rat testicular cells, the testicular cells obtained from organ donors varied considerable in their composition (Figure 1) (20). Interindividual physiologic differences such as age, previous medical treatment, drug abuse, exposure to environmental chemicals, or severe trauma are likely to influence the composition of cells within the testis (26-28). It is not known whether the variation in the present material reflect temporary or more permanent differences among the donors. The compositions of the human samples shown in Figure 1 are well within the range of our total human material. No

Concentration

(jdvll

Fig. 4. Chemically induced ssDNA breaks in isolated human and rat testicular cells as measured by alkaline filter elution. Cells isolated from the testes of either human organ transplant donors or rats (4010~ in 2 mL) were exposed to DBCP (10 to 300 PM, human; 10 to 100 PM, rat), acrylamide (30 to 1000 PM, human; 100 to 1000 PM, rat), 1,3-DNB (10 to 300 PM, human; 30 to 300 PM, rat), Cr6+ (30 to 1000 p,M, human and rat) or aflatoxin B, (10 to 300 PM, human; 30 to 300 p,M, rat) for 30 min at 32°C. Values are from three organ transplant donors or three rats. *Significantly different from the control. P

< 0.05.

obvious correlation between the composition of cells and age of the donor was observed. Despite the observed relative large compositional differences among the donors, only minor differences in induced ssDNA breaks in crude human testicular cell preparations were observed after exposure to the different test chemicals. In the present investigation 15 chemicals with known testicular toxicity were selected from different categories of use. Genotoxic as well as nongenotoxic compounds were chosen. Most of these compounds have been shown to interact with DNA either spontaneously or after metabolic activation (acrylamide, acrylonitrile, aflatoxin B i, Cd2+, Cr6+, cisplatin, DBCP, 1,3-DNB,

DNA damage in human testicular cells 0 C. BIBRGE ET AL.

EDB, styrene oxide, Warn, and thiotepa), whereas one chemical has been reported to cause DNA damage by indirect mechanisms (be:nomyl). Two nongenotoxic testicular toxicants postulated to act via a hormonal mechanism were included (chlordecone, methoxychlor). Based on the results from testing these chemicals with human and rat testicular cells, they may be grouped into three categories (I, II, and II& depending on the response in one or both species. Category I (Figure 2) represents the testicular toxicants that showed no induction of ssDNA breaks in human or rat testicular cells. It appears that the toxicity of these compounds to testicular cells is not mediated via direct interaction with DNA. Some of the effects on the reproductive system observed after in vivo exposure to chemicals may, in part, be due to hormonal effects, as seems to be the case w:lth the chlorinated hydrocarbon insecticide methoxychlor. This compound has been reported to be metabolized to a monophenol metabolite with estrogenic properti.es (29,30). Indirect interaction with DNA may be relevant for benomyl. Recent reports indicate that this fungicide causes genotoxicity (31-33) by disturbing the intracellular precursor pool of deoxyribonucleotides (34). However, some of the other testicular toxicants that were negative in our assay have been reported to interact with DNA. The cytostatic agent thiotepa kills and/or severely disrupts proliferation and differentiation of mouse testicular germ cells, and an increase in chromatin structure alteration after exposure has been detected (35). Thiotepa has been reported to induce dominant lethal mutations (36) and chromosomal aberrations in spermatogonia (37), and spermatocytes (38). Thiotepa has also been shown to induce DNA interstrand crosslinks (39,400).Most probably, the presence of DNA crosslinks will reduce or mask any transient increase in the level of ssDNA breaks formed during DNA-repair (40), thus explaining the lack of increase in ssDNA breaks after exposing human and rat testicular cells to thiotepa. In most studies no ssDNA breaks are found in mammalian cells after exposure to the cytostatic and direct acting mutagen cisplatin (41). However, increased levels of ssDNA breaks may be detected using a long exposure time followed by further incubation with inhibitors of DNA polymerization (42). The failure of cisplatin to induce an:y significant levels of ssDNA breaks in human and rat testicular cells after a short exposure time may, as with thiotepa, be explained by its ability to induce intra- and interstrand DNA crosslinks (43). It is, however, interesting to note that cisplatin has marked effects on P450-dependent drug metabolism and steroid hydroxylation activity (44), and thus, a possible general feminization of steroidogenesis could contribute to cisplatin’s testicular toxicity. Cd’+ is only a weak DNA damaging agent (45). Increased levels of ssDNA breaks

515

after exposure to Cd*+ in vitro have been observed (46); however, negative findings have also been reported (47,48). The apparent lack of genotoxicity observed after exposure of rat and human germ cells to Cd*+ in the present study is in agreement with the latter findings. Cd*’ treatment has been found to increase the permeability of the testicular endothelium, with subsequent hemorrhage into the interstitial area of the testis (49,50); this may constitute a mechanism for Cd*+-induced toxicity in the testis. The last testicular toxicant that showed no increase in ssDNA breaks, acrylonitrile, has been reported to induce ssDNA breaks in mammalian cells in vitro (51), and to be genotoxic after exposure in vivo (52-54). Acrylonitrile is toxic to the testis (55), and has been reported to bind covalently to testicular cell DNA and to inhibit DNA synthesis in testicular cells after in vivo exposure (53). On the other hand, acrylonitrile was not found to induce unscheduled DNA synthesis (UDS) in rat spermatocytes (56,57), and is also negative with respect to the induction of dominant lethal mutations (58,59) and reciprocal translocations (60) in male germ cells. In the present study we did not detect any increase in ssDNA breaks in human and rat testicular cells after exposure to acrylonitrile in vitro. Among the compounds that were shown to induce ssDNA breaks in both human and rat testicular cells (category II), several are known genotoxicants (Figure 3). DNA damage may, hence, constitute a possible mechanism for the toxic effects of these compounds in the testis. Styrene oxide induces ssDNA breaks, point mutations, chromosome aberrations, micronuclei, sister chromatid exchanges (SCE), and UDS in cultured mammalian cells (61-63). Furthermore, styrene oxide has been shown to bind covalently to DNA (64-66) and to induce ssDNA breaks in a number of organs in mice including the testis (67). EDB appears to be activated by glutathione conjugation to a reactive mutagenic intermediate (68,69), and ssDNA breaks in rat testicular germ cells are induced after both in vitro (Figure 3) (22) and in vivo exposure (22). Thiram induces micronuclei and polyploidy in mouse spermatocytes in vivo (70), morphologically abnormal sperm in mice (71,72), and direct DNA damage judged from increased levels of SCE and UDS in human lymphocytes in vitro (73), and ssDNA breaks in testicular germ cells (Figure 3). The remaining testicular toxicant that was found to induce DNA damage in both species was chlordecone. This compound is generally considered to be a nongenotoxic carcinogen (74); however, DNA strand breaks in rat hepatocytes have been reported (75). In the present study chlordecone was found to increase the level of ssDNA breaks at relatively high concentrations (300 to 1000 FM), an exposure that was associated with some cytotoxicity in both human and rat testicular cells. To study if heavily damaged cells

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could contribute to DNA damage measured by alkaline filter elution, we used the plasma membrane damaging agent Triton X-100 to cause cell death. This treatment, however, caused only minor increases in ssDNA breaks (data not shown). This indicates that the DNA damage observed after exposure to chlordecone does not reflect DNA damage formed during the process of cell death but seems to be due to effects on the DNA not secondary to cell death. However, because chlordecone may cause cell death via different mechanisms than Triton X- 100, which may affect DNA differently to a greater/lesser extent, a definite conclusion cannot be drawn. It is also interesting to note that chlordecone has been reported to have a weak estrogenic effect (76) and, consequently, its testicular toxicity could have been mediated via hormonal mechanisms. The third category of known testicular toxicants comprises chemicals that induced ssDNA breaks in either human or rat testicular cell preparations (Figure 4). DBCP induced significant levels of ssDNA breaks in rat testicular cells, which is in accordance with our earlier studies (16-18,25,77). Conjugation of DBCP with glutathione and the subsequent formation of a reactive episulfonium ion is suggested to be a major activation pathway in these cells (16,21,22,78-80). DNA has been suggested as a target for DBCP-induced toxicity (16,8183). However, no ssDNA breaks were induced by DBCP in isolated human testicular cells, even though these cells were able to metabolically activate DBCP to species binding irreversibly to macromolecules (20). DBCP and EDB have been reported to induce ssDNA breaks in rat testicular cells via a similar mechanism (16,22,69,79,80), whereas in the present investigation DBCP and EDB varied with respect to their genotoxic potential in isolated human testicular cells (Figure 4). The apparent lack of induction of ssDNA breaks in human testicular cells could indicate that different reactive DBCP metabolites are involved in binding to cellular macromolecules (e.g., proteins) compared to DNA damage, or that DBCPinduced DNA damage may be repaired at different rates in the two species (20). Some of the men who became sterile after occupational exposure to DBCP revealed seminiferous tubules that contained Sertoli cells only (84-86). Thus, spermatogonia may be an important target for DBCP, and inherently, crude cell suspensions contain few spermatogonia. Further experiments are needed, however, to clarify this apparant discrepancy. Acrylamide induces dominant lethal mutations in male rats (59) and mice (87), heritable translocations in mice (88), and UDS in rat spermatocytes (56,57), probably through interaction of its electrophilic vinyl group with DNA. However, the yield of the alkylated DNA in testicular cells was found to be low (89). Accordingly, we observed a low, but statistically significant level of

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ssDNA breaks induced in human cells, while in the rat no significant increase was seen (Figure 4). 1,3-DNBinduced testicular toxicity is rodent species specific, with mouse and hamster showing less damage than the rat (90,9 1). Application of BrdU immunohistochemistry indicates that 1,3-DNB exerts no effects on DNA synthesis in rat spermatogonia and preleptotene spermatocytes (92). On the other hand, an abnormal chromatin structure of mouse sperm following exposure to 1,3-DNB has been reported (90). It has been proposed that differences in pharmacokinetics could offer at least a partial explanation of the rodent species differences of this compound (91), and similar arguments apply to the present observations with rat vs. human cells. In rodent testicular cells reductive metabolism, which is the primary route of biotransformation of 1,3-DNB (93), results in numerous potentially toxic metabolites (94). Cr6+ is reported to induce a spectrum of DNA damage in mammalian cells such as ssDNA breaks, DNA-protein crosslinks, and chromiumDNA adducts (95-99). In the present study we observed a low level of Cr6’-induced ssDNA breaks in rat testicular cells, and an insignificant increase of ssDNA breaks in human testicular cells (Figure 4). This may indicate that there are differences in the cellular redfox levels in the two species, which could be important for the resulting DNA damage. Another possibility could be differences in the repair of Cr6+-induced DNA damage, as previously reported for CHO cells (98,99). The last chemical in this group, aflatoxin B i, induced statistically significant levels of ssDNA breaks in rat testicular cells only. Aflatoxin B 1 does not induce UDS in rat spermatocytes (56), dominant lethal mutations (58), or reciprocal translocation (60) in male germ cells. Metabolic activation by P450 is required to form the DNA reactive epoxide (100,101). Taking into consideration that the compound is negative in several germ cell test systems for genotoxicity and the fact that the P450 enzyme activities of germ cells are low (102) compared to the liver, the positive response observed in rat testicular cells after exposure to aflatoxin B, (Figure 4) was somewhat unexpected. However, it is important to remember that testicular cells other than the germ cells, such as Leydig or Sertoli cells, may be important target cells for the chemicals tested. These and previous results obtained with alkaline filter elution of germ cells from rats are in good agreement with results obtained with other genotoxicity assays. Being aware that the positive response in rat testicular cells obtained with Cr6’ and aflatoxin B, are only marginal, there seems to be a close correlation between responses obtained in rat and human testicular cells in vitro. The only clear species difference was found with DBCP, and as discussed above, this may be due to species differences in target cells. The studies illustrate that

DNA damage in human testicular

isolated human and rat testicular cells, combined with alkaline filter elution, constitute a useful experimental approach for studying chemically-induced testicular DNA damage and toxiciQ. Data obtained in this way are valuable in human risk assessment. Acknowledgments - The study was supported by the Research Council of Norway and NIH Grant ESO2728. We thank Bente Trygg and Kirsti Haug for their skilled technical assistance.

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