- Email: [email protected]
DNA Strand breaks in testicular cells from humans and rats following in vitro exposure to 1,2-dibromo-3-chloropropane (DBCP)
Reproductive
Toxicology, Vol. IO, No. 1. pp. 51-59, 1996 Copyright 0 1996 Elsevier Science Inc.
Printed in the USA. All rights reserved 0890.6238196 $15.00 + .OO ELSEVIER
0890-6238(95)02018-7
DNA STRAND BREAKS IN TESTICULAR CELLS FROM HUMANS AND RATS FOLLOWING IN VITRO EXPOSURE TO 1,ZDIBROMO-3-CHLOROPROPANE (DBCP) CHRISTINEBJORGE,* RICHARDWIGER,* J@RNA. HOLME,* GUNNARBRUNBORG,* TIM SCHOLZ,~ERIK DYBING,* and ERIK J. SBDERLUND* *Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway tInstitute for Surgical Research, The National Hospital, Oslo, Norway
Abstract - Preparations of testicular cells from human organ transplant donors and from Wistar rats were compared with respect to their composition of the different testicular cell types, their ability to metabolize 1,2-dibromo-3-chloropropane (DBCP), and their relative sensitivity to induction of DNA single strand breaks and alkali labile sites (ssDNA breaks) after treatment with DBCP, 4-nitroquinoline N-oxide (4-NQO), and x rays. Flow cytometric and microscopic analysis demonstrated that the interindividual variation in the composition of testicular cell types was considerably greater in the human tissue than in that from rats. The in vitro metabolic activation of DBCP (50 to 250 pM), measured as radiolabel covalently bound to macromolecules, was three-fold faster in rat testicular cells compared to human testicular cells. X rays (1 to 10 Gy) and 4-NQO (0.5 to 2.5 pM) induced ssDNA breaks to a similar extent in both human and rat testicular cells as measured by single cell gel electrophoresis (SCGE) and alkaline filter elution. In contrast, 1,2-dibromo-3-chloropropane (DBCP) (3 to 300 pM) caused no significant DNA damage in human testicular cells, whereas in rats there was a clear concentration-dependent increase in ssDNA breaks. The data show that, compared to rats, testicular cells from humans are less efficient in activating DBCP to metabolites binding covalently to macromolecules. However, from the rate of covalent binding observed one would expect a significant degree of DBCP-induced SSDNA breaks in the human testicular cells. The low level of DBCP-induced ssDNA breaks in human testicular cells could indicate that different reactive DBCP metabolites are involved in binding to cellular macromolecules compared to DNA damage, or that different rates of DNA repair exist in human and rat testicular cells. Key Words: DNA damage;
human; rat; testicular
cells; DBCP, 4-NQO; x rays.
INTRODUCTION
(LH). In some cases the infertility appeared to be permanent (10-12). Apart from a female sex predominance found in children from fathers heavily exposed to DBCP, possibly due to a Y chromosome nondisjunction (13), no other toxic effects in the offspring have been reported (13-15). Indications of DBCP’s potential testicular toxicity was observed in rats in 1961 (16), and additional information concerning its testicular toxicity has been provided in a number of animal studies (17-20). Several testicular cell types have been suggested to be targets for DBCP-induced toxicity (17,21-25). Recently, attention has been given to DNA as a possible target for DBCP-induced organ toxicity (2631). Studies with cytostatic agents have indicated that DNA damage can result in death of spermatogonia leading to a reduction in the number of normal spermatozoa as well as to spermatozoa with genetic defects (32). Metabolism of DBCP appears to be a necessary step in the pathogenesis of its organ damage (33). DBCP may be activated to reactive metabolites by both P450 and glutathione (GSH) S-transferase-dependent pathways
During the past 40 to 50 years there have been increases in the incidences of testicular cancer, testicular anomalies, such as hypospadias and cryptorchidism, and there also appears to have been a decline in semen quality (1). A number of possible causes have been suggested, including exposures to ionizing radiation, various pesticides or heavy metals, life style factors, or increased embryonic exposure to endocrine-disrupting environmental contaminants (2-6). 1,2-Dibromo-3-chloropropane (DBCP) is one of the few chemicals that has definitely been shown to cause sterility or reduced fertility in humans (7-9). Laboratory findings among DBCP production workers included oligospermia, azoospermia, and elevated levels of follicle stimulating hormone (FSH) and luteinizing hormone Address correspondence to Christine Bj@rge, Department of Environmental Medicine, National Institute of Public Health, Geitmyrsvn. 75, N-0462 Oslo, Norway. Received 21 June 1995; Accepted 27 July 1995. 51
52
Reproductive
Toxicology
(24,34-36). In testicular cells, GSH-dependent metabolism seems to play an important role in causing DNA damage after in vitro exposure to DBCP (37,20). In order to further elucidate possible mechanisms involved in DBCP-induced testicular toxicity in humans, we have characterized and compared suspensions of human and rat testicular cells with regard to DBCP toxicity. This involved the study of in vitro metabolic activation of radidabelled DBCP to reactive metabolites covalently bound to cellular macromolecules and induction of DNA single strand breaks and alkali labile sites (ssDNA breaks) assessed by single cell gel electrophoresis (SCGE) (31,38,39) and alkaline filter elution (40,41). X rays and 4-nitroquinoline N-oxide (4-NQO), which cause DNA damage via different types of initial DNA lesions, were used to further characterize the testicular cells with respect to their response to DNA damaging agents. The present study indicates that comparative investigations using human and rat testicular cells could represent a valuable approach for risk assessment of germ cetI toxic&s. MATERIALS
AND METHODS
Chemicals DBCP (~99% pure by GC analysis), prepared by kx-omhation of ally1 chloride (42), was kindly supplied by Dr. Sidney D. Nelson, University of Washington, Seattle, USA. RadiolabelEed DBCP (3-14C-DBCP, 9.9 mCi/ mmol) was obtained by custom synthesis from Sigma Chemical Co. St. Louis, MO, USA. Other chemicals were from the following sources: collagenase (CLS II, 352 Ulmg) from Worthington Biochemical Corp., Freeb&l, w, USA; dimethyl sulfoxide (DMSO) from Rathburn, Walkerburn, Scotland, UK; RPM1 1640 cell culture medium and fetal calf serum (FCS) from Gibco, Grand Island, NY, USA; Hoechst 33258 from Calbiochem&&ringer, La Jolla, CA, USA; proteinase K from Merck, Darn&a&, Germany; bovine serum albumin (V) (BSA), ethidium bromide, N-lauryI-Na-sarcosinate, normal agarose (type I:low EEQ), 4-nitroquinoline N-oxide (4NQO}, triton X-100, and trypsin (III-S) from Sigma Chemical Co., St. Louis, MO, USA; and NuSieve GTG agarose from FMC Bioproducts, Rockland, ME, USA. Other chemicals were analytical grade from co,mmercial suppliers. Animals Sexually mature male W&tar rats (MOL: WIST, 200 to 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 (S.pecial Diet Services, Essex, UK) and water ad 1IbittIm.
Volume 10. Number
1, 1996
Organ donors Human testes were obtained from 18 organ donors, supplied by the Norwegian National Hospital. After decapsulation, the tissue 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 SCGE and alkaline filter elution in cells prepared from five of the donors after in vitro treatment with DBCP, 4-NQO, or x rays. These donors were: Donor 1 (25 years), donor 2 (70 years), donor 3 (47 years), donor 4 (48 years), and donor 5 (42 years). It was not possible to obtain detailed information on the health history of these donors. Preparation and characterization of testicular cells Rat testicular cells were prepared as described by Bradley and Dysart (37) with some modifications (20). 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/mL) was then added, and the tubular suspension was further incubated for 12 to 15 min. Trypsination was stopped by adding fetal calf serum (FCS) (1%). The resulting cell suspension was filtered, centrifuged three times (270 x g for 5 min) and resuspended in Hank’s HEPES buffer with 1% bovine serum albumin (BSA) and filtered through a nylon mesh (0.25 mm). The total yield per rat testis was about 2 . lo* cells with cell viability greater than 95%, as measured by trypan blue exclusion. Human testicular ceils. The preparation of a crude cell suspension was much the same as described for rats (37,20). Modifications include increased coilagenase concentration (200 U/r&) and an extended incubation time (30 min). Furthermore, all centrifugations were at 740 x g for 5 min. The total yield per gram wet human testis tissue was 10 to 15 . 106 cells with a viability greater than 95%, as measured by trypan blue exclusion. This yield is 3 to 4 fold lower than with rat testis. Characterization of germ cells by flow cytometry. DNA was stained by adding 1.2 Pg Hoechst 33258 and Triton X-100 (0.004% final) to one-ml aliquots of cells (5 . 105), suspended in Hank’s Balanced Salt Solution (HBSS) supplemented with 2% BSA. Blue 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, USA). It was possible to distinguish different cell types on the basis of their DNA contents (Hoechst fluorescence) and cell size (forward light scatter). Microscopic characterization of germ cells. Smears containing testicular cells in FCS were air dried quickly,
DNA damage
in human testicular
fixed in methanol, and stained with Giemsa in neutral distilled water. Cell morphology was evaluated using a Nikon Optiphot microscope (1000x). Smears of human and rat testicular cells were also made for fluorescence microscopic analysis after staining with Hoechst 33258. These techniques were used to confirm the determination of the various populations as analyzed with flow cytometry. Histologic examination. Testis samples were fixed in Bouin’s solution and paraffin embedded sections were stained with hematoxylin-eosin. Tissue sections were evaluated using a transmission microscope. Exposure to chemical mutagens Crude single cell suspensions of human and rat testicular cells (4 . 106/mL, 2 mL in scintillation vials) were incubated with 50 to 250 pM 14C-DBCP for 60 min at 32°C in Hank’s HEPES buffer with 1% BSA, for the measurement of radiolabel covalently bound to cellular macromolecules. For detection of ssDNA breaks by alkaline filter elution or SCGE, suspensions of human and rat testicular cells (2 . 106/mL, 2 mL in scintillation vials) were exposed to DBCP (3 to 300 pM), or 4-NQO (0.5 to 2.5 FM), for 30 min at 32°C in Hank’s I-IEPES buffer with 1% BSA. X rays Human and rat testicular cells (4 . lo6 in 4 mL Hank’s HEPES buffer with 1% BSA) were irradiated in 20 mL scintillation vials on ice, with x rays (1 to 10 Gy) delivered by a Phillips MG300 x-ray unit (Germany) operated at 10 rnA and 260 kV. Radiation was filtered with 0.5 mm Cu. The dose rate was 8.7 Gy/min as calibrated with iron sulphate dosimetry. Cells were kept on ice until analysis for ssDNA breaks with SCGE or alkaline filter elution. Metabolic activation The amount of radiolabel covalently bound to cellular macromolecules was determined by the method of Wallin and coworkers (43). After exposure to 14CDBCP, 50 PL of the testicular cell suspension was loaded onto glass microfiber filters followed by precipitation with ethanol and extensive washing with methanol, acetone, and n-hexane. The radioactivity bound to cellular macromolecules was determined by liquid scintillation counting, and protein was measured according to the method of Lowry and coworkers (44).
53
cells 0 C. BIBRGEET AL.
tion system (40), based on the method of Kohn and coworkers (41). Testicular cells exposed to DBCP or irradiated with x rays were centrifugated, resuspended in 2 mL Hank’s HEPES buffer with 1% BSA, and loaded onto 25 mm polycarbonate filters (2 pm, Nucleopore, Cambridge, UK). Cells were then lysed, deproteinized, and their 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 set-up (45,40). Single cell gel electrophoresis (SCGE). In testicular cells exposed to DBCP, 4-NQO, or x rays, ssDNA breaks were assayed using SCGE (38,39), as described by Bjorge and coworkers (31). In short, frosted microscope slides were prepared with a “sandwich” of normal melting agarose (0.5%), testicular cells (2.5 1 105) mixed 1:l (v/v) in 1.2% low melting agarose, and a final top layer of low melting agarose (0.6%). The slides were immersed in lysing solution at 4°C for at least 1 h, transferred to the electrophoresis chamber containing freshly made alkaline electrophoresis buffer, and left in this buffer (at 10°C) for 40 min to allow unwinding of DNA. Electrophoresis was carried out at 20 V (0.75 V/cm) and approximately 300 mA for 25 min at 10°C. The slides were removed, neutralized with 3 x 5 min washes with Tris (0.4 M pH 7.5), and stained with 75 pL ethidium bromide (20 p,g/mL). The entire procedure was carried out in dim light. Cellular DNA damage (comets) were visualized using a Leitz fluorescence microscope (Ortholux II) equipped with a solid state CCD video camera and analyzed using the Fenestra Comet image analysis system (Kinetic Imaging LTD, Liverpool, UK). Comets appearing in randomly selected fields were measured and only overlapping comets were omitted. The tail moment (i.e. the product of the tail length and the portion of the fluorescence in the tail in relative units) was chosen as the parameter for quantitating ssDNA breaks. Tail moments from 200 to 250 comets (i.e. from 4 to 5 slides) were analyzed for each concentration of DBCP, 4-NQO, and x rays. Statistics Statistical comparisons using the mean tail moment were performed using the Wilcoxon two-sample distriTable 1. Composition of testicular germ cell preparations from humans and rats Species
Haploid cells (So)
Diploid cells (%)
Tetraploid cells @o)
DNA damage
Humana Ratb
512 15 71+4
28* 14 2Ok3
15*9 923
Alkaline filter e&ion. ssDNA breaks in testicular cells were measured by an automated alkaline filter elu-
“Means + SD of 18 human testes. “Means f SD of testes from seven rats.
Reproductive
Toxicology
Volume 10, Number
I, 1996
w
Human tes?is 3
Human testis 1 25 years
I
Human testis 2
haphd
70 years
47 ysars
ONA
Human testis 5 42 years
I
Human testis 4 48 years
Rat testls
Fig. I. The composition of testicular germ cells isolated from human organ transplant donors or from rats as analyzed by flow cytometry. Data are shown for 5 human organ transplant donors and one typical rat. bution test. A P value of less than 0.05 was considered significant. RESULTS Composition of human and rut testicular cetis Testis tissues from 18 donors, ranging in age from 25 to 70 years or from rats were analyzed by flow cytometry and histology. The interindividual variation in the composition of testicular cells from humans was considerable as estimated by these techniques. The percentage distribution of the various cellular stages (means + S.D) in preparations from a total of 18 human organ donors were as follows: haploid cells (round/elongating/ elongated spermatids) 57 + 15%, diploid cells 28 + 14%, and tetraploid cells (mostly primary spermatocytes) 15 f 9%. Comparable numbers from 7 rats were 7 1 f 4%, 20 + 3%, and 9 f 3%, respectively (Table 1). The individual composition of the 5 testicular cell preparations (representing 5 donors) used to assess DNA damage is shown in Figure 1, together with the mean testicular cell composition from one typical rat. Histologic analyses. Donor 1 (25 years) and 5 (42 years) revealed normal spermatogenesis with elongated spermatids occurring in more than 90% and spermiation
in approximately 10% of the seminiferous tubules. Donor 2 (70 years) was characterized by seminiferous tubules with variable diameters, some filled with loose spermatogenic epithelium. Primary spermatocytes often revealed degenerative signs such as pycnotic nuclei. No spermiation was observed in this particular donor. The testis of donor 3 (47 years) was characterized by large variations in the diameter of the seminiferous tubules, a number of which revealed degenerative changes in the seminiferous epithelium. In approximately 10% of the tubules the epithelium was very low and contained mostly Sertoli cells with some spermatogonia. Several tubules were surrounded by a layer of collagen-rich connective tissue just outside the basal lamina. Donor 4 (48 years) was characterized by degenerative changes including intracellular vacuolization in approximately half of the seminiferous tubules. Very few tubules contained elongated spermatids. These five human organ donors were randomly chosen and were representative regarding the testicular cell composition within the group of 18 donors analyzed in Table I. Activation of DBCP to reactive metabolites Incubation of crude cell suspensions of rat testicular cells with 14C-DBCP showed that the covalent binding of radiolabel to macromolecules was linear up to 90 min
55
DNA damage in human testicular cells 0 C. BJCI)RGE ET AL.
(data not shown). The binding of i4C-DBCP (50 to 250 FM) to macromolecules of human and rat testicular cells was compared at 60 min incubation (Figure 2). The highest rate of covalently bound radiolabel per mg protein per 60 min was seen in rat testicular cells (1.33 nmol i4CDBCP) compared to human testicular cells (0.42 nmol 14C-DBCP).
X-rays 100.
Human
Rat
80.
0 GY
0 GY
60. 40. 20.
DNA damage
DNA single strand breaks were analyzed by SCGE in crude suspensions of human and rat testicular cells after treatment with x rays (1 to 10 Gy), 4-NQO (0.5 to 2.5 p.M), or DBCP (3 to 300 p.M). The two former agents readily induced ssDNA breaks in both human and rat testicular cells in a concentration-dependent manner and no apparent species differences were observed. Furthermore, the relative differences in the sensitivity between the various donors were minor (Figure 3 and 4). In contrast, concentrations of DBCP up to 300 pM caused no significant ssDNA breaks in human testicular cells from various donors (Figure 5), whereas a large increase in ssDNA breaks was observed in rat testicular cells after exposure to DBCP above 3 p,M (Figure 5). The alkaline filter elution technique was used in parallel with SCGE. The two methods appeared to be equally sensitive in detecting ssDNA breaks in testicular cells after treatment with DBCP (3 to 300 pM) or x rays (1 to 10 Gy) (Figure 6). For practical reasons, the decapsulated and minced human testis tissue was kept at 4°C for 8 to 12 h in RPM1 1640 medium until cell preparation could be commenced. We investigated whether a comparable delay in the preparation of rat testis would influence the metaCOVALENT
BINDING
Human
0
50
100
DBCP
250
(pM)
Fig. 2. In vitro covalent binding of ‘%-DBCP to macromolecules. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to 14C-DBCP (50 to 250 FM). Values are means + SD of three experiments.
80
5 Gy*
60
5 Gy*
40
I
20. 100-l
3
60. 60.
5
7
10
Gy*
5
7
9
tt
13
15
1
3
5
7
10
Gy*
5
7
9
tt
13
15
a
11
13
15
.
40.
t
3
9
11
13 15
1
3
Tail moment Fig. 3. X-ray induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors, or from rats, were exposed to x rays (1 to 10 Gy) on ice and analyzed by SCGE. The tail moment distributions shown are means +SD from experiments with 5 organ transplant donors (donor 1-5) or 4 rats. The abscissa denotes the following tail moments values:l: I-10; 2: 11-20; 3: 21-30; 4: 3140; 5: 41-50; 6: 51-60; 7: 61-70; 8: 71-80; 9: 81-90; 10: 91-100; 11: 101-110; 12: 111-120; 13: 121-130; 14: 131-140; 15: 141-150. *Significantly different from the control. P < 0.05.
bolic activation of DBCP to reactive metabolites or the formation of DBCP-induced ssDNA breaks. An 8 to 12 h delay did not lead to any substantial change in the level of covalently bound radiolabel or DBCP-induced ssDNA breaks (data not shown) compared to rat testicular cells prepared without such a delay. DISCUSSION Compared to rat testicular cells, the interindividual variation in the composition of testicular cells was greater in humans (Figure 1 and Table 1). Among the approximately 40 organ donor testes that have been examined so far, a relatively high interindividual variation
56
Reproductive
Toxicology
4-NQO
Volume 10, Number 1, 1996 Human
Rat
100 60 60 40 E
20
r
loo
0”
60 60 40 20
Tail
moment
Fig. 4. 4-Nitroquinoline N-oxide (4-NQO)-induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to 4-NQO (0.5 to 2.5 pM) and analyzed by SCGE. See also legend to Figure 3.
in the cellular composition appears to be a common feature as determined by both flow cytometric and histologic analyses. Flow cytometric analyses of isolated human testicular cells revealed that donors number 3 and 4 both had unusual compositions compared to the normal composition. This was confirmed by histologic examination. The variation in cell type composition was observed in all age classes of the donors. Further studies may reveal if there is a relationship between sperm quality and testicular cell composition. Despite the observed compositional differences among the donors, only minor differences in induced ssDNA breaks in crude human testicular cell preparations were found. Metabolic activation of DBCP to DNA-damaging intermediates appears to be a necessary step in the DBCP-induced testicular atrophy in rats (33). DBCP may be activated to reactive metabolites both by a P450 (24,34,35,46) and/or a GSH S-transferase(s) (GSTs)dependent pathway (35,36,47). Experiments with diethyl maleate indicate that a GST-dependent activation of DBCP dominates in testicular cells (3547). There are good correlations between DBCP-induced ssDNA breaks and testicular atrophy, both in various rodent spe-
Tail
moment
Fig. 5. DBCP-induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to DBCP (3 to 300 FM), and analyzed by SCGE. See also legend to Figure 3.
ties and in rats exposed to DBCP analogs with different toxicities (20,48). An analysis of the structure of DBCP GSH conjugates is consistent with their formation via episulfonium ion intermediates (35,49). Recent data indicate that the rat testicular tissue contains relatively high GST activities (50,5 l), whereas in human testis low levels of GSTs have been observed (51,52). In the present study, a high rate of covalent binding of 14C-DBCP was observed in rat testicular cells compared to human testicular cells, the latter being threefold lower in activity (Figure 2). These results are in accordance with the reported levels of GSTs measured in human and rat testicular germ cells (50-52). Little is known about the induction of DNA damage and repair in human testicular cells. X-ray- and 4-NQOinduced DNA damage, representing different types of DNA lesions each with its specific pattern of repair, was used to compare the response of human and rat testicular
DNA damage in human testicular cells
l
C.
h3RFE
ET AL.
Alkaline elutlon Rat
1
Humrn
Human 250
.
200
-80
150
.
60
loo
-
40
-
20
'IOU
7 50
0
3
10
DBCP
30100300
IpM)
0
1
3
X-rays
5
10
(Gy)
01
0310301003oa
DBCP
[#A)
3
X-rays
5
10
(GyI
Fig. 6. DBCP- and X-rays-induced DNA damage in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transpiant donors or from rats were exposed to DBCP (3 to 300 (LM), or irradiated with x rays (1 to 10 Gy) and analyzed for ssDNA breaks with alkaline filter elution (NAAC) or SCGE (tail moments). Values are from 2 (means) or 3 (means + SD) organ transplant donors or rats.
cells to genotoxic treatments. X rays and 4-NQO induced ssDNA breaks in a dose-dependent manner, measured by SCGE in the testicular cells of both species (Figures 3 and 4). Comparable levels of ssDNA breaks in both species were hence observed after exposure to x rays and 4-NQO. The latter compound produces several types of DNA adducts, some of which are repaired by nucieotide excision repair pathways, similar to UV-induced DNA damage (53,54). On the other hand, marked differences were observed between rat and human testicular cells with regard to DBCP-induced ssDNA breaks, measured by SCGE or alkaline filter elution (Figures 5 and 6). The low level of ssDNA breaks observed in human testicular cells was very unexpected since humans appear to be highly sensitive to DBCP-induced testicular toxicity (7,l l), an effect that is related to DNA damage in laboratory animals (26-31). From the level of covalent binding (Figure 2) one would expect a significant level of ssDNA breaks in the human testicular cells. One explanation for the lack
of ssDNA breaks observed in human testicular cells may be that the spectra of DBCP-induced DNA lesions differ in rat and human testicular cells, and that such lesions are detected with varying efficiencies by the methods used. On the other hand, in rat testis a significant amount of DBCP-induced DNA damage appears to be alkali-labile; thus, one would expect to find a significant level of ssDNA breaks with alkaline filter elution or SCGE in human testicular cells. Differences in the nature of DBCP adducts formed and/or their repair are other factors that may influence the extent of ssDNA breaks induced. Other explanations for the severe testicular toxicity observed among DBCP exposed workers may be related to the exposure situation (7,12,55,56). Repeated exposure to low doses of DBCP may affect the activity of DBCP activating enzymes or DNA repair enzymes. In some studies GSTs have been shown to be induced in different tissues by a variety of xenobiotics (57,58). Histologic examination of the testes from humans and ani-
58
Reproductive
Toxicology
mals exposed to DBCP for longer periods resulted in a highly increased proportion of Sertoli cells (I 2,17,59) indicating that rapidly proliferating spermatogonia represent the cell type most sensitive to DBCP. This is in accordance with the assumption that DNA is a critical target molecule also in humans. To further explore a possible role of DNA damage in DBCP-induced testicular atrophy, experiments are in progress in which the effects of DBCP on DNA replication-synthesis is studied in spermatogonial cell cultures, using seminiferous tubules isolated from humans and rats. In the present paper we have shown that there are distinct differences in the response of human and rat testicular cells to DBCP, whereas the response to x rays and 4-NQO is similar. More generally, the use of crude single cell suspensions of testicular germ cells isolated from human organ transplant donors and laboratory animals constitute a valuable tool to study testicular genotoxicity. Such comparative studies may be helpful when assessing the risk associated with human reproductive toxicants. study was supported by the Research Council of Norway and NIH Grant ES02728. We thank Bente Trygg and Kirati Haug for their skilled technical assistance.
Acknowledgments-The
REFERENCES 1. Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence of decreasing quality of semen during past SO years. Br Med J. 1992; 305609-13. 2. Wyrobek AJ, Gordon LA, Burkhart JG. An evaluation of human sperm as indicators of chemically induced alterations of spermatogenie function. A report for the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res. 1983;115:73-148. 3. Wyrobek AJ. Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. Reprod Toxicol. 1993;7:3-16. 4. Olshan AF, Faustman EM. Male-mediated Reprod Toxicol. 1993;7: 191-202.
developmental
toxicity.
alterations in sexual 5. Colborn T, Clement C. Chemically-induced and functional development: The wildlife/human connection. Princeton; Princeton Scientific Publishing. 1992. 6. Sharpe RM, Skakkebaek NE. Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet. 1993;341:1392-5. 7. Whorton MD, Krauss RM, Marshall S, Milby TH. lnfertility m male pesticide workers. Lancet. 1977;2: 1259-61. 8. Federal Register, Occupational propane. 1977;45:536-49.
exposure to 1.2.dibromo-3-chloro-
9. Sandlifer SH, Wilkins RT, Loadholt CB, Lane LG, Eldridge JC. Spermatogenesis in agricultural workers exposed to dibromochloropropane (DBCP). Bull Environ Contam Toxicol. 1979;23:70310. 10 Potashnik A. A four-year reassessment of workers with dibromochloropropane-induced testicular dysfunction. Andrologia. 1983: 15:1&l-70. 11. Eaton M, Schenker M, Whorton D, Samuels S, Perkins E, Overstreet J. Seven-year follow-up of workers exposed to 1.2-dibromo3-chloropropane. J Occup Med. 1986;28: I 145-50. (DBCP): an 12. Potashnik G, Yanai-Inbar I. Dibromochloropropane
Volume
10, Number
1, 1996
R-year reevaluation of testicular function and reproductive mance. Fertil Steril. 1987;47:317-23.
perfor-
13. Goldsmith JR, Potashnik G, Israeli R. Reproductive outcomes in families of DBCP-exposed men. Arch Environ Health. 1984;39: 85-9. 14. Potashnik
G, Phillip M. Lack of birth defects among offspring
conceived during or after paternal exposure to dibromochloropropane (DBCP). Andrologia. 1988;20:90-4. 1s. Whorton
MD, Wong 0. Morgan
RW, Gordon N. An epidemio-
logic investigation of birth outcomes in relation to dibromochloropropane contamination in drinking water in Fresno County, California. USA. Int Arch Occup Environ Health. 1989;61:403-7. 16. Torkelson TR, Sadek SE, Rowe VK, Kodama JK, Anderson HH, Loquvam GS, Hine CH. Toxicological investigations of 1,2-dibromo-3-chloropropane. Toxicol Appl Pharmacol. 1961;3:545-59. 17. Kluwe WM. Acute toxicity of I ,2-dibromo-3-chloropropane in F344 male rat. II Development and repair of the renal epididymal, testicular. and hepatic lesions. Toxic01 Appl Pharmacol. 1981;59: x4-95 18. Rao KS, Burek JD. Murray FJ. John JA, Schwetz BA, Beyer JE, Parker CM. Toxicologic and reproductive effects of inhaled 1,2dibromo-3-chloropropane in male rabbits. Fundam Appl Toxicol. 1982;2:241-51. and re19. Whorton MD, Foliart DE. Mutagenicity, carcinogenicity productive effects of dibromochloroptopane (DBCP). Mutat Res. 1983: 123: 13-20. 20. Soderlund EJ, Brunborg G, Omichinski JG, Holme JA, Dahl JE, Nelson SD. Dybing E. Testicular necrosis and DNA damage caused by deuterated and methylated analogs of 1.2-dibromo-3chloropropane in the rat. Toxicol Appl Pharmacol. 1988:94:43747. 21. Miller GE, Welsh MJ, Brabec MJ. Alteration of lactate metabolism in Sertoli cells and spermatocytes by I ,2-dibromo-3-chloropropane. Toxicologist. 1985;5:120. 22. Warren DW, Wisner JR, Ahmad N. Effects of 1,2-dibromo-3chloropropane on male reproductive function in the rat. Biol Reprod. 1984;31:45&63. 23. Tofilon PT. Clemet RP, Piper WN. Inhibition of the biosynthesis of rat testicular heme by 1.2-dibromo-3chloropropane. Biochem Pharmacol.
1980;29:2563-6.
24. Kluwe WM. Lamb JC IV, Greenwell A, Harrington FW. 1,2-Dibromo-3chloropropane (DBCP)-induced infertility in male rats mediated by a post-testicular effect. Toxic01 Appl Pharmacol. 1983~7 1:294-g. 25. Greenwell A, Tomaszewski KE. Melnick RL. A biochemical basis for 1&dibromo-3.chloropropane-induced male infertility: Inhibition of sperm mitochondrial electron transport activity. Toxic01 Appl Pharmacol.
1987:9 1:274-80.
26. Lee IP, Suzuki K. Induction of unscheduled DNA synthesis in mouse germ cells following 1.2-dibromo-3-chloropropane (DBCP) exposure. Mutat Res. 1979;68: 169-73. 27. Amann RP, Bemdtson WE. Assessment of procedures for screening agents for effects on male reproduction: Effects of dibromochloropropane (DBCP) on the rat. Fundam Appl Toxicol. 1986;7: 244-55. 28. Teramoto S, Saito S, Aoyama H, Shirasu Y. Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP). Mutat Res. 1980;77:71-8. 29. Saito-Suzuki R, Teramoto S, Shirasu Y. Dominant lethal studies with I .2-dibromo-3chloropropane and its structurally related compounds. Mutat Res. 1982;101:321-7
DNA damage in human testicular
cells 0 C. BJBRGE ET AL.
59
30. Rao KS, Burek JD, Murray FJ, John JA, Schwetz BA, Bell TJ, Potts WJ, Parker CM. Toxicologic and reproductive effects of inhaled 1,2-dibromo-3-chloropropane in rats. Fundam Appl Toxi-
44. Lowry 0, Rosebrough NJ, Farr AL, Randall RL. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:26575.
col. 1983;3:104-10. 31. Bjorge C, Wiger R, Holme JA, Brunborg G, Andersen R, Dybing E, Soderlund EJ. In vitro toxicity of 1,2-dibromo-3chloropropane (DBCP) in different testicular cell types from rats. Reprod Toxicol. 1995;9:4611173. 32. Grosovsky AJ, De Boer JG, De Jong PJ, Drobetsky EA, Glickman BW. Base substitution, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc Nat1 Acad Sci USA. 1988;85: 185-8. 33. Dybing E, Omichinski JG, Soderlund EJ, Brunborg G, Lag M, Holme JA, Nelson SD. Mutagenicity and organ damage of 1,2-dibromo-3-chloropropane (DBCP) and tris(2,3-dibromopropyl)phosphate (T&BP): role of metabolic activation. In: Hodgson E, Bend JR, Philpot RM, eds. Reviews in biochemical toxicology. New York: Elsevier Science Publishing Co; 1989;10: 139-87. 34. Gingell R, Beatty PW, Mitschke HR, Mueller RL, Sawin VL, Page AC. Evidence that epichlorohydrin is not a toxic metabolite of 1,2-dibromo-3-chloropropane. Xenobiotica. 1987;17:22940. 35. Omichinski JG, Brunborg G, Holme JA, Soderlund EJ, Nelson SD, Dybing E. The role of oxidative and conjugative pathways in the activation of 1,2-dibromo-3-chloropropane to DNA damaging products in testicular cells. Mol Pharmacol. 1988;34:74-9. 36. Holme JA, Soderlund EJ, Brunborg G, Omichinski JG, Bekkedal K, Trygg B, Nelson SD, Dybing E. Different mechanisms are involved in DNA damage, bacterial mutagenicity and cytotoxicity induced by 1,2-dibromo-3-chloropropane in suspensions of rat liver cells. Carcinogenesis. 1989;10:49-54. 37. Bradley MD, Dysart G. DNA single-strand breaks, double-strand breaks and crosslinks in rat testicular germ cells: measurement of their formation and repair by alkaline and neutral filter elution. Cell Biol Toxicol. 1985;1:181-5. 38. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:189-91. 39. Olive PL, Durand RE, Wlodek D, Banath JP. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res. 1992;198:2.59-67. 40. Brunborg G, Holme JA, Soderlund EJ, Omichinski JG, Dybing E. An automated alkaline elution system. DNA damage induced by 1,2-dibromo-3-chloropropane in viva and in vitro. Anal Biochem. 1988;174:522-36. 41. Kohn KW, Erikson LC, Ewig RAG, Zwelling A. Measurement of strand breaks and crosslinks by alkaline elution. In: Friedberg EC, Hanawalt PC, eds. DNA repair: A laboratory manual of recent procedures. New York Marcel Dekker; 1981:379401. 42. Omichinski JG, Soderlund EJ, Bausano JA, Dybing E, Nelson SD. Synthesis and mutagenicity of selectively methylated analogs of 1,2-dibromo-3chloropropane and tris(2,3-dibromopropyl)phosphate. Mutagenesis. 1987;2:287-92. 43. Wallin H, Schelin C, Tunek A, Jergil B. A rapid and sensitive method for determination of covalent binding of benzo(a)pyrene to proteins. Chem-Biol Interact. 1981;38:109-18.
45. Sterzel W, Bedford P, Eisenbrand G. Automated determination of DNA using the fluorochrome Hoechst 33258. Anal Biochem. 1985;147:462-7. 46. Omichinski JG, Soderlund EJ, Dybing E, Pearson PG, Nelson SD. Detection and mechanism of formation of the potent direct-acting mutagen 2-bromoacrolein from 1,2-dibromo-3chloropropane. Toxic01 Appl Pharmacol. 1988;92:286-94. 47. Soderlund EJ. Meyer DJ, Ketterer B, Nelson SD, Dybing E, Holme JA. Metabolism of 1,2-dibromo-3chloropropane by glutathione S-transferases. Chem-Biol Interact. 1995;97:257-272. 48. Lag M, Soderlund EJ, Brunborg G, Dahl JE, Holme JA, Omichinski JG, Nelson SD, Dybing E. Species differences in testicular necrosis and DNA damage, distribution and metabolism of I ,2-dibromo-3-chloropropane (DBCP). Toxicology. 1989;58: 1334. 49. Pearson PG, Soderlund EJ, Dybing E, Nelson SD. Metabolic activation of 1,2-dibromo-3-chloropropane: evidence for the formation of reactive episulfonium ion intermediates. Biochemistry. 1990;29:4971-81. 50. Den Boer PJ, Poot M, Verkerk A, Jansen R, Mackenback P, Grootegoed A. Glutathione-dependent defence mechanisms in isolated round spermatids from the rat. Int J Androl. 1990;13:2638. 51. Dibiasio KW, Silva MH, Shull LR, Overstreet JW, Hammock BD, Miller MG. Xenobiotic metabolizing enzyme activities in rat, mouse, monkey, and human testis. Drug Metab Dispos. 1991;19: 227-3 1. 52. Klys HS, Whillis D, Howard G, Harrison DJ. Glutathione Stransferase expression in the human testis and testicular germ cell neoplasia. Br J Cancer. 1992;66:589-93. 53. Waters R, Jones CJ, Martin EA. Yang AL, Jones NJ. The repair of large DNA adducts in mammalian cells. Mutat Res. 1992;273: 145-55. 54. Mirzayans R, Paterson MC, Waters R. Defective repair of class of 4NQO-induced alkali-labile DNA lesions in xeroderma pigmentosum complementation group A fibroblasts. Carcinogenesis. 1985; 6:555-9. 55. Whorton D, Milby TH, Krauss RM, Stubbs HA. Testicular function in DBCP exposed pesticide workers. J Occup Med. 1979;21: 161-6. 56. Olsen GW, Lanham JM, Bodner KM, Hyhon DB, Bond GG. Determinants of spermatogenesis recovery among workers exposed to 1,2-dibromo-3chloropropane. J Occup Med. 1990;32:979-84. 57. Vos RME, Van Blanderen PJ. Glutathione tion to their role in the biotransformation Biol Interact. 1990;75:241-65.
S-transferases in relaof xenobiotics. Chem
58. Meyer DJ, Harris JM, Gilmore KS, Coles B, Kensler TW, Ketterer B. Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole-3-thiones. Carcinogenesis. 1993;14:567-72. 59. Biava CG, Smuckler EA. Whorton D. The testicular morphology of individuals exposed to dibromochloropropane. Exp Mol Pathol. 1978;29:448-58.
Toxicology, Vol. IO, No. 1. pp. 51-59, 1996 Copyright 0 1996 Elsevier Science Inc.
Printed in the USA. All rights reserved 0890.6238196 $15.00 + .OO ELSEVIER
0890-6238(95)02018-7
DNA STRAND BREAKS IN TESTICULAR CELLS FROM HUMANS AND RATS FOLLOWING IN VITRO EXPOSURE TO 1,ZDIBROMO-3-CHLOROPROPANE (DBCP) CHRISTINEBJORGE,* RICHARDWIGER,* J@RNA. HOLME,* GUNNARBRUNBORG,* TIM SCHOLZ,~ERIK DYBING,* and ERIK J. SBDERLUND* *Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway tInstitute for Surgical Research, The National Hospital, Oslo, Norway
Abstract - Preparations of testicular cells from human organ transplant donors and from Wistar rats were compared with respect to their composition of the different testicular cell types, their ability to metabolize 1,2-dibromo-3-chloropropane (DBCP), and their relative sensitivity to induction of DNA single strand breaks and alkali labile sites (ssDNA breaks) after treatment with DBCP, 4-nitroquinoline N-oxide (4-NQO), and x rays. Flow cytometric and microscopic analysis demonstrated that the interindividual variation in the composition of testicular cell types was considerably greater in the human tissue than in that from rats. The in vitro metabolic activation of DBCP (50 to 250 pM), measured as radiolabel covalently bound to macromolecules, was three-fold faster in rat testicular cells compared to human testicular cells. X rays (1 to 10 Gy) and 4-NQO (0.5 to 2.5 pM) induced ssDNA breaks to a similar extent in both human and rat testicular cells as measured by single cell gel electrophoresis (SCGE) and alkaline filter elution. In contrast, 1,2-dibromo-3-chloropropane (DBCP) (3 to 300 pM) caused no significant DNA damage in human testicular cells, whereas in rats there was a clear concentration-dependent increase in ssDNA breaks. The data show that, compared to rats, testicular cells from humans are less efficient in activating DBCP to metabolites binding covalently to macromolecules. However, from the rate of covalent binding observed one would expect a significant degree of DBCP-induced SSDNA breaks in the human testicular cells. The low level of DBCP-induced ssDNA breaks in human testicular cells could indicate that different reactive DBCP metabolites are involved in binding to cellular macromolecules compared to DNA damage, or that different rates of DNA repair exist in human and rat testicular cells. Key Words: DNA damage;
human; rat; testicular
cells; DBCP, 4-NQO; x rays.
INTRODUCTION
(LH). In some cases the infertility appeared to be permanent (10-12). Apart from a female sex predominance found in children from fathers heavily exposed to DBCP, possibly due to a Y chromosome nondisjunction (13), no other toxic effects in the offspring have been reported (13-15). Indications of DBCP’s potential testicular toxicity was observed in rats in 1961 (16), and additional information concerning its testicular toxicity has been provided in a number of animal studies (17-20). Several testicular cell types have been suggested to be targets for DBCP-induced toxicity (17,21-25). Recently, attention has been given to DNA as a possible target for DBCP-induced organ toxicity (2631). Studies with cytostatic agents have indicated that DNA damage can result in death of spermatogonia leading to a reduction in the number of normal spermatozoa as well as to spermatozoa with genetic defects (32). Metabolism of DBCP appears to be a necessary step in the pathogenesis of its organ damage (33). DBCP may be activated to reactive metabolites by both P450 and glutathione (GSH) S-transferase-dependent pathways
During the past 40 to 50 years there have been increases in the incidences of testicular cancer, testicular anomalies, such as hypospadias and cryptorchidism, and there also appears to have been a decline in semen quality (1). A number of possible causes have been suggested, including exposures to ionizing radiation, various pesticides or heavy metals, life style factors, or increased embryonic exposure to endocrine-disrupting environmental contaminants (2-6). 1,2-Dibromo-3-chloropropane (DBCP) is one of the few chemicals that has definitely been shown to cause sterility or reduced fertility in humans (7-9). Laboratory findings among DBCP production workers included oligospermia, azoospermia, and elevated levels of follicle stimulating hormone (FSH) and luteinizing hormone Address correspondence to Christine Bj@rge, Department of Environmental Medicine, National Institute of Public Health, Geitmyrsvn. 75, N-0462 Oslo, Norway. Received 21 June 1995; Accepted 27 July 1995. 51
52
Reproductive
Toxicology
(24,34-36). In testicular cells, GSH-dependent metabolism seems to play an important role in causing DNA damage after in vitro exposure to DBCP (37,20). In order to further elucidate possible mechanisms involved in DBCP-induced testicular toxicity in humans, we have characterized and compared suspensions of human and rat testicular cells with regard to DBCP toxicity. This involved the study of in vitro metabolic activation of radidabelled DBCP to reactive metabolites covalently bound to cellular macromolecules and induction of DNA single strand breaks and alkali labile sites (ssDNA breaks) assessed by single cell gel electrophoresis (SCGE) (31,38,39) and alkaline filter elution (40,41). X rays and 4-nitroquinoline N-oxide (4-NQO), which cause DNA damage via different types of initial DNA lesions, were used to further characterize the testicular cells with respect to their response to DNA damaging agents. The present study indicates that comparative investigations using human and rat testicular cells could represent a valuable approach for risk assessment of germ cetI toxic&s. MATERIALS
AND METHODS
Chemicals DBCP (~99% pure by GC analysis), prepared by kx-omhation of ally1 chloride (42), was kindly supplied by Dr. Sidney D. Nelson, University of Washington, Seattle, USA. RadiolabelEed DBCP (3-14C-DBCP, 9.9 mCi/ mmol) was obtained by custom synthesis from Sigma Chemical Co. St. Louis, MO, USA. Other chemicals were from the following sources: collagenase (CLS II, 352 Ulmg) from Worthington Biochemical Corp., Freeb&l, w, USA; dimethyl sulfoxide (DMSO) from Rathburn, Walkerburn, Scotland, UK; RPM1 1640 cell culture medium and fetal calf serum (FCS) from Gibco, Grand Island, NY, USA; Hoechst 33258 from Calbiochem&&ringer, La Jolla, CA, USA; proteinase K from Merck, Darn&a&, Germany; bovine serum albumin (V) (BSA), ethidium bromide, N-lauryI-Na-sarcosinate, normal agarose (type I:low EEQ), 4-nitroquinoline N-oxide (4NQO}, triton X-100, and trypsin (III-S) from Sigma Chemical Co., St. Louis, MO, USA; and NuSieve GTG agarose from FMC Bioproducts, Rockland, ME, USA. Other chemicals were analytical grade from co,mmercial suppliers. Animals Sexually mature male W&tar rats (MOL: WIST, 200 to 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 (S.pecial Diet Services, Essex, UK) and water ad 1IbittIm.
Volume 10. Number
1, 1996
Organ donors Human testes were obtained from 18 organ donors, supplied by the Norwegian National Hospital. After decapsulation, the tissue 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 SCGE and alkaline filter elution in cells prepared from five of the donors after in vitro treatment with DBCP, 4-NQO, or x rays. These donors were: Donor 1 (25 years), donor 2 (70 years), donor 3 (47 years), donor 4 (48 years), and donor 5 (42 years). It was not possible to obtain detailed information on the health history of these donors. Preparation and characterization of testicular cells Rat testicular cells were prepared as described by Bradley and Dysart (37) with some modifications (20). 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/mL) was then added, and the tubular suspension was further incubated for 12 to 15 min. Trypsination was stopped by adding fetal calf serum (FCS) (1%). The resulting cell suspension was filtered, centrifuged three times (270 x g for 5 min) and resuspended in Hank’s HEPES buffer with 1% bovine serum albumin (BSA) and filtered through a nylon mesh (0.25 mm). The total yield per rat testis was about 2 . lo* cells with cell viability greater than 95%, as measured by trypan blue exclusion. Human testicular ceils. The preparation of a crude cell suspension was much the same as described for rats (37,20). Modifications include increased coilagenase concentration (200 U/r&) and an extended incubation time (30 min). Furthermore, all centrifugations were at 740 x g for 5 min. The total yield per gram wet human testis tissue was 10 to 15 . 106 cells with a viability greater than 95%, as measured by trypan blue exclusion. This yield is 3 to 4 fold lower than with rat testis. Characterization of germ cells by flow cytometry. DNA was stained by adding 1.2 Pg Hoechst 33258 and Triton X-100 (0.004% final) to one-ml aliquots of cells (5 . 105), suspended in Hank’s Balanced Salt Solution (HBSS) supplemented with 2% BSA. Blue 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, USA). It was possible to distinguish different cell types on the basis of their DNA contents (Hoechst fluorescence) and cell size (forward light scatter). Microscopic characterization of germ cells. Smears containing testicular cells in FCS were air dried quickly,
DNA damage
in human testicular
fixed in methanol, and stained with Giemsa in neutral distilled water. Cell morphology was evaluated using a Nikon Optiphot microscope (1000x). Smears of human and rat testicular cells were also made for fluorescence microscopic analysis after staining with Hoechst 33258. These techniques were used to confirm the determination of the various populations as analyzed with flow cytometry. Histologic examination. Testis samples were fixed in Bouin’s solution and paraffin embedded sections were stained with hematoxylin-eosin. Tissue sections were evaluated using a transmission microscope. Exposure to chemical mutagens Crude single cell suspensions of human and rat testicular cells (4 . 106/mL, 2 mL in scintillation vials) were incubated with 50 to 250 pM 14C-DBCP for 60 min at 32°C in Hank’s HEPES buffer with 1% BSA, for the measurement of radiolabel covalently bound to cellular macromolecules. For detection of ssDNA breaks by alkaline filter elution or SCGE, suspensions of human and rat testicular cells (2 . 106/mL, 2 mL in scintillation vials) were exposed to DBCP (3 to 300 pM), or 4-NQO (0.5 to 2.5 FM), for 30 min at 32°C in Hank’s I-IEPES buffer with 1% BSA. X rays Human and rat testicular cells (4 . lo6 in 4 mL Hank’s HEPES buffer with 1% BSA) were irradiated in 20 mL scintillation vials on ice, with x rays (1 to 10 Gy) delivered by a Phillips MG300 x-ray unit (Germany) operated at 10 rnA and 260 kV. Radiation was filtered with 0.5 mm Cu. The dose rate was 8.7 Gy/min as calibrated with iron sulphate dosimetry. Cells were kept on ice until analysis for ssDNA breaks with SCGE or alkaline filter elution. Metabolic activation The amount of radiolabel covalently bound to cellular macromolecules was determined by the method of Wallin and coworkers (43). After exposure to 14CDBCP, 50 PL of the testicular cell suspension was loaded onto glass microfiber filters followed by precipitation with ethanol and extensive washing with methanol, acetone, and n-hexane. The radioactivity bound to cellular macromolecules was determined by liquid scintillation counting, and protein was measured according to the method of Lowry and coworkers (44).
53
cells 0 C. BIBRGEET AL.
tion system (40), based on the method of Kohn and coworkers (41). Testicular cells exposed to DBCP or irradiated with x rays were centrifugated, resuspended in 2 mL Hank’s HEPES buffer with 1% BSA, and loaded onto 25 mm polycarbonate filters (2 pm, Nucleopore, Cambridge, UK). Cells were then lysed, deproteinized, and their 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 set-up (45,40). Single cell gel electrophoresis (SCGE). In testicular cells exposed to DBCP, 4-NQO, or x rays, ssDNA breaks were assayed using SCGE (38,39), as described by Bjorge and coworkers (31). In short, frosted microscope slides were prepared with a “sandwich” of normal melting agarose (0.5%), testicular cells (2.5 1 105) mixed 1:l (v/v) in 1.2% low melting agarose, and a final top layer of low melting agarose (0.6%). The slides were immersed in lysing solution at 4°C for at least 1 h, transferred to the electrophoresis chamber containing freshly made alkaline electrophoresis buffer, and left in this buffer (at 10°C) for 40 min to allow unwinding of DNA. Electrophoresis was carried out at 20 V (0.75 V/cm) and approximately 300 mA for 25 min at 10°C. The slides were removed, neutralized with 3 x 5 min washes with Tris (0.4 M pH 7.5), and stained with 75 pL ethidium bromide (20 p,g/mL). The entire procedure was carried out in dim light. Cellular DNA damage (comets) were visualized using a Leitz fluorescence microscope (Ortholux II) equipped with a solid state CCD video camera and analyzed using the Fenestra Comet image analysis system (Kinetic Imaging LTD, Liverpool, UK). Comets appearing in randomly selected fields were measured and only overlapping comets were omitted. The tail moment (i.e. the product of the tail length and the portion of the fluorescence in the tail in relative units) was chosen as the parameter for quantitating ssDNA breaks. Tail moments from 200 to 250 comets (i.e. from 4 to 5 slides) were analyzed for each concentration of DBCP, 4-NQO, and x rays. Statistics Statistical comparisons using the mean tail moment were performed using the Wilcoxon two-sample distriTable 1. Composition of testicular germ cell preparations from humans and rats Species
Haploid cells (So)
Diploid cells (%)
Tetraploid cells @o)
DNA damage
Humana Ratb
512 15 71+4
28* 14 2Ok3
15*9 923
Alkaline filter e&ion. ssDNA breaks in testicular cells were measured by an automated alkaline filter elu-
“Means + SD of 18 human testes. “Means f SD of testes from seven rats.
Reproductive
Toxicology
Volume 10, Number
I, 1996
w
Human tes?is 3
Human testis 1 25 years
I
Human testis 2
haphd
70 years
47 ysars
ONA
Human testis 5 42 years
I
Human testis 4 48 years
Rat testls
Fig. I. The composition of testicular germ cells isolated from human organ transplant donors or from rats as analyzed by flow cytometry. Data are shown for 5 human organ transplant donors and one typical rat. bution test. A P value of less than 0.05 was considered significant. RESULTS Composition of human and rut testicular cetis Testis tissues from 18 donors, ranging in age from 25 to 70 years or from rats were analyzed by flow cytometry and histology. The interindividual variation in the composition of testicular cells from humans was considerable as estimated by these techniques. The percentage distribution of the various cellular stages (means + S.D) in preparations from a total of 18 human organ donors were as follows: haploid cells (round/elongating/ elongated spermatids) 57 + 15%, diploid cells 28 + 14%, and tetraploid cells (mostly primary spermatocytes) 15 f 9%. Comparable numbers from 7 rats were 7 1 f 4%, 20 + 3%, and 9 f 3%, respectively (Table 1). The individual composition of the 5 testicular cell preparations (representing 5 donors) used to assess DNA damage is shown in Figure 1, together with the mean testicular cell composition from one typical rat. Histologic analyses. Donor 1 (25 years) and 5 (42 years) revealed normal spermatogenesis with elongated spermatids occurring in more than 90% and spermiation
in approximately 10% of the seminiferous tubules. Donor 2 (70 years) was characterized by seminiferous tubules with variable diameters, some filled with loose spermatogenic epithelium. Primary spermatocytes often revealed degenerative signs such as pycnotic nuclei. No spermiation was observed in this particular donor. The testis of donor 3 (47 years) was characterized by large variations in the diameter of the seminiferous tubules, a number of which revealed degenerative changes in the seminiferous epithelium. In approximately 10% of the tubules the epithelium was very low and contained mostly Sertoli cells with some spermatogonia. Several tubules were surrounded by a layer of collagen-rich connective tissue just outside the basal lamina. Donor 4 (48 years) was characterized by degenerative changes including intracellular vacuolization in approximately half of the seminiferous tubules. Very few tubules contained elongated spermatids. These five human organ donors were randomly chosen and were representative regarding the testicular cell composition within the group of 18 donors analyzed in Table I. Activation of DBCP to reactive metabolites Incubation of crude cell suspensions of rat testicular cells with 14C-DBCP showed that the covalent binding of radiolabel to macromolecules was linear up to 90 min
55
DNA damage in human testicular cells 0 C. BJCI)RGE ET AL.
(data not shown). The binding of i4C-DBCP (50 to 250 FM) to macromolecules of human and rat testicular cells was compared at 60 min incubation (Figure 2). The highest rate of covalently bound radiolabel per mg protein per 60 min was seen in rat testicular cells (1.33 nmol i4CDBCP) compared to human testicular cells (0.42 nmol 14C-DBCP).
X-rays 100.
Human
Rat
80.
0 GY
0 GY
60. 40. 20.
DNA damage
DNA single strand breaks were analyzed by SCGE in crude suspensions of human and rat testicular cells after treatment with x rays (1 to 10 Gy), 4-NQO (0.5 to 2.5 p.M), or DBCP (3 to 300 p.M). The two former agents readily induced ssDNA breaks in both human and rat testicular cells in a concentration-dependent manner and no apparent species differences were observed. Furthermore, the relative differences in the sensitivity between the various donors were minor (Figure 3 and 4). In contrast, concentrations of DBCP up to 300 pM caused no significant ssDNA breaks in human testicular cells from various donors (Figure 5), whereas a large increase in ssDNA breaks was observed in rat testicular cells after exposure to DBCP above 3 p,M (Figure 5). The alkaline filter elution technique was used in parallel with SCGE. The two methods appeared to be equally sensitive in detecting ssDNA breaks in testicular cells after treatment with DBCP (3 to 300 pM) or x rays (1 to 10 Gy) (Figure 6). For practical reasons, the decapsulated and minced human testis tissue was kept at 4°C for 8 to 12 h in RPM1 1640 medium until cell preparation could be commenced. We investigated whether a comparable delay in the preparation of rat testis would influence the metaCOVALENT
BINDING
Human
0
50
100
DBCP
250
(pM)
Fig. 2. In vitro covalent binding of ‘%-DBCP to macromolecules. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to 14C-DBCP (50 to 250 FM). Values are means + SD of three experiments.
80
5 Gy*
60
5 Gy*
40
I
20. 100-l
3
60. 60.
5
7
10
Gy*
5
7
9
tt
13
15
1
3
5
7
10
Gy*
5
7
9
tt
13
15
a
11
13
15
.
40.
t
3
9
11
13 15
1
3
Tail moment Fig. 3. X-ray induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors, or from rats, were exposed to x rays (1 to 10 Gy) on ice and analyzed by SCGE. The tail moment distributions shown are means +SD from experiments with 5 organ transplant donors (donor 1-5) or 4 rats. The abscissa denotes the following tail moments values:l: I-10; 2: 11-20; 3: 21-30; 4: 3140; 5: 41-50; 6: 51-60; 7: 61-70; 8: 71-80; 9: 81-90; 10: 91-100; 11: 101-110; 12: 111-120; 13: 121-130; 14: 131-140; 15: 141-150. *Significantly different from the control. P < 0.05.
bolic activation of DBCP to reactive metabolites or the formation of DBCP-induced ssDNA breaks. An 8 to 12 h delay did not lead to any substantial change in the level of covalently bound radiolabel or DBCP-induced ssDNA breaks (data not shown) compared to rat testicular cells prepared without such a delay. DISCUSSION Compared to rat testicular cells, the interindividual variation in the composition of testicular cells was greater in humans (Figure 1 and Table 1). Among the approximately 40 organ donor testes that have been examined so far, a relatively high interindividual variation
56
Reproductive
Toxicology
4-NQO
Volume 10, Number 1, 1996 Human
Rat
100 60 60 40 E
20
r
loo
0”
60 60 40 20
Tail
moment
Fig. 4. 4-Nitroquinoline N-oxide (4-NQO)-induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to 4-NQO (0.5 to 2.5 pM) and analyzed by SCGE. See also legend to Figure 3.
in the cellular composition appears to be a common feature as determined by both flow cytometric and histologic analyses. Flow cytometric analyses of isolated human testicular cells revealed that donors number 3 and 4 both had unusual compositions compared to the normal composition. This was confirmed by histologic examination. The variation in cell type composition was observed in all age classes of the donors. Further studies may reveal if there is a relationship between sperm quality and testicular cell composition. Despite the observed compositional differences among the donors, only minor differences in induced ssDNA breaks in crude human testicular cell preparations were found. Metabolic activation of DBCP to DNA-damaging intermediates appears to be a necessary step in the DBCP-induced testicular atrophy in rats (33). DBCP may be activated to reactive metabolites both by a P450 (24,34,35,46) and/or a GSH S-transferase(s) (GSTs)dependent pathway (35,36,47). Experiments with diethyl maleate indicate that a GST-dependent activation of DBCP dominates in testicular cells (3547). There are good correlations between DBCP-induced ssDNA breaks and testicular atrophy, both in various rodent spe-
Tail
moment
Fig. 5. DBCP-induced ssDNA breaks in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transplant donors or from rats were exposed to DBCP (3 to 300 FM), and analyzed by SCGE. See also legend to Figure 3.
ties and in rats exposed to DBCP analogs with different toxicities (20,48). An analysis of the structure of DBCP GSH conjugates is consistent with their formation via episulfonium ion intermediates (35,49). Recent data indicate that the rat testicular tissue contains relatively high GST activities (50,5 l), whereas in human testis low levels of GSTs have been observed (51,52). In the present study, a high rate of covalent binding of 14C-DBCP was observed in rat testicular cells compared to human testicular cells, the latter being threefold lower in activity (Figure 2). These results are in accordance with the reported levels of GSTs measured in human and rat testicular germ cells (50-52). Little is known about the induction of DNA damage and repair in human testicular cells. X-ray- and 4-NQOinduced DNA damage, representing different types of DNA lesions each with its specific pattern of repair, was used to compare the response of human and rat testicular
DNA damage in human testicular cells
l
C.
h3RFE
ET AL.
Alkaline elutlon Rat
1
Humrn
Human 250
.
200
-80
150
.
60
loo
-
40
-
20
'IOU
7 50
0
3
10
DBCP
30100300
IpM)
0
1
3
X-rays
5
10
(Gy)
01
0310301003oa
DBCP
[#A)
3
X-rays
5
10
(GyI
Fig. 6. DBCP- and X-rays-induced DNA damage in human and rat testicular germ cells. Crude single cell suspensions isolated from the testes of either human organ transpiant donors or from rats were exposed to DBCP (3 to 300 (LM), or irradiated with x rays (1 to 10 Gy) and analyzed for ssDNA breaks with alkaline filter elution (NAAC) or SCGE (tail moments). Values are from 2 (means) or 3 (means + SD) organ transplant donors or rats.
cells to genotoxic treatments. X rays and 4-NQO induced ssDNA breaks in a dose-dependent manner, measured by SCGE in the testicular cells of both species (Figures 3 and 4). Comparable levels of ssDNA breaks in both species were hence observed after exposure to x rays and 4-NQO. The latter compound produces several types of DNA adducts, some of which are repaired by nucieotide excision repair pathways, similar to UV-induced DNA damage (53,54). On the other hand, marked differences were observed between rat and human testicular cells with regard to DBCP-induced ssDNA breaks, measured by SCGE or alkaline filter elution (Figures 5 and 6). The low level of ssDNA breaks observed in human testicular cells was very unexpected since humans appear to be highly sensitive to DBCP-induced testicular toxicity (7,l l), an effect that is related to DNA damage in laboratory animals (26-31). From the level of covalent binding (Figure 2) one would expect a significant level of ssDNA breaks in the human testicular cells. One explanation for the lack
of ssDNA breaks observed in human testicular cells may be that the spectra of DBCP-induced DNA lesions differ in rat and human testicular cells, and that such lesions are detected with varying efficiencies by the methods used. On the other hand, in rat testis a significant amount of DBCP-induced DNA damage appears to be alkali-labile; thus, one would expect to find a significant level of ssDNA breaks with alkaline filter elution or SCGE in human testicular cells. Differences in the nature of DBCP adducts formed and/or their repair are other factors that may influence the extent of ssDNA breaks induced. Other explanations for the severe testicular toxicity observed among DBCP exposed workers may be related to the exposure situation (7,12,55,56). Repeated exposure to low doses of DBCP may affect the activity of DBCP activating enzymes or DNA repair enzymes. In some studies GSTs have been shown to be induced in different tissues by a variety of xenobiotics (57,58). Histologic examination of the testes from humans and ani-
58
Reproductive
Toxicology
mals exposed to DBCP for longer periods resulted in a highly increased proportion of Sertoli cells (I 2,17,59) indicating that rapidly proliferating spermatogonia represent the cell type most sensitive to DBCP. This is in accordance with the assumption that DNA is a critical target molecule also in humans. To further explore a possible role of DNA damage in DBCP-induced testicular atrophy, experiments are in progress in which the effects of DBCP on DNA replication-synthesis is studied in spermatogonial cell cultures, using seminiferous tubules isolated from humans and rats. In the present paper we have shown that there are distinct differences in the response of human and rat testicular cells to DBCP, whereas the response to x rays and 4-NQO is similar. More generally, the use of crude single cell suspensions of testicular germ cells isolated from human organ transplant donors and laboratory animals constitute a valuable tool to study testicular genotoxicity. Such comparative studies may be helpful when assessing the risk associated with human reproductive toxicants. study was supported by the Research Council of Norway and NIH Grant ES02728. We thank Bente Trygg and Kirati Haug for their skilled technical assistance.
Acknowledgments-The
REFERENCES 1. Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence of decreasing quality of semen during past SO years. Br Med J. 1992; 305609-13. 2. Wyrobek AJ, Gordon LA, Burkhart JG. An evaluation of human sperm as indicators of chemically induced alterations of spermatogenie function. A report for the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res. 1983;115:73-148. 3. Wyrobek AJ. Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. Reprod Toxicol. 1993;7:3-16. 4. Olshan AF, Faustman EM. Male-mediated Reprod Toxicol. 1993;7: 191-202.
developmental
toxicity.
alterations in sexual 5. Colborn T, Clement C. Chemically-induced and functional development: The wildlife/human connection. Princeton; Princeton Scientific Publishing. 1992. 6. Sharpe RM, Skakkebaek NE. Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet. 1993;341:1392-5. 7. Whorton MD, Krauss RM, Marshall S, Milby TH. lnfertility m male pesticide workers. Lancet. 1977;2: 1259-61. 8. Federal Register, Occupational propane. 1977;45:536-49.
exposure to 1.2.dibromo-3-chloro-
9. Sandlifer SH, Wilkins RT, Loadholt CB, Lane LG, Eldridge JC. Spermatogenesis in agricultural workers exposed to dibromochloropropane (DBCP). Bull Environ Contam Toxicol. 1979;23:70310. 10 Potashnik A. A four-year reassessment of workers with dibromochloropropane-induced testicular dysfunction. Andrologia. 1983: 15:1&l-70. 11. Eaton M, Schenker M, Whorton D, Samuels S, Perkins E, Overstreet J. Seven-year follow-up of workers exposed to 1.2-dibromo3-chloropropane. J Occup Med. 1986;28: I 145-50. (DBCP): an 12. Potashnik G, Yanai-Inbar I. Dibromochloropropane
Volume
10, Number
1, 1996
R-year reevaluation of testicular function and reproductive mance. Fertil Steril. 1987;47:317-23.
perfor-
13. Goldsmith JR, Potashnik G, Israeli R. Reproductive outcomes in families of DBCP-exposed men. Arch Environ Health. 1984;39: 85-9. 14. Potashnik
G, Phillip M. Lack of birth defects among offspring
conceived during or after paternal exposure to dibromochloropropane (DBCP). Andrologia. 1988;20:90-4. 1s. Whorton
MD, Wong 0. Morgan
RW, Gordon N. An epidemio-
logic investigation of birth outcomes in relation to dibromochloropropane contamination in drinking water in Fresno County, California. USA. Int Arch Occup Environ Health. 1989;61:403-7. 16. Torkelson TR, Sadek SE, Rowe VK, Kodama JK, Anderson HH, Loquvam GS, Hine CH. Toxicological investigations of 1,2-dibromo-3-chloropropane. Toxicol Appl Pharmacol. 1961;3:545-59. 17. Kluwe WM. Acute toxicity of I ,2-dibromo-3-chloropropane in F344 male rat. II Development and repair of the renal epididymal, testicular. and hepatic lesions. Toxic01 Appl Pharmacol. 1981;59: x4-95 18. Rao KS, Burek JD. Murray FJ. John JA, Schwetz BA, Beyer JE, Parker CM. Toxicologic and reproductive effects of inhaled 1,2dibromo-3-chloropropane in male rabbits. Fundam Appl Toxicol. 1982;2:241-51. and re19. Whorton MD, Foliart DE. Mutagenicity, carcinogenicity productive effects of dibromochloroptopane (DBCP). Mutat Res. 1983: 123: 13-20. 20. Soderlund EJ, Brunborg G, Omichinski JG, Holme JA, Dahl JE, Nelson SD. Dybing E. Testicular necrosis and DNA damage caused by deuterated and methylated analogs of 1.2-dibromo-3chloropropane in the rat. Toxicol Appl Pharmacol. 1988:94:43747. 21. Miller GE, Welsh MJ, Brabec MJ. Alteration of lactate metabolism in Sertoli cells and spermatocytes by I ,2-dibromo-3-chloropropane. Toxicologist. 1985;5:120. 22. Warren DW, Wisner JR, Ahmad N. Effects of 1,2-dibromo-3chloropropane on male reproductive function in the rat. Biol Reprod. 1984;31:45&63. 23. Tofilon PT. Clemet RP, Piper WN. Inhibition of the biosynthesis of rat testicular heme by 1.2-dibromo-3chloropropane. Biochem Pharmacol.
1980;29:2563-6.
24. Kluwe WM. Lamb JC IV, Greenwell A, Harrington FW. 1,2-Dibromo-3chloropropane (DBCP)-induced infertility in male rats mediated by a post-testicular effect. Toxic01 Appl Pharmacol. 1983~7 1:294-g. 25. Greenwell A, Tomaszewski KE. Melnick RL. A biochemical basis for 1&dibromo-3.chloropropane-induced male infertility: Inhibition of sperm mitochondrial electron transport activity. Toxic01 Appl Pharmacol.
1987:9 1:274-80.
26. Lee IP, Suzuki K. Induction of unscheduled DNA synthesis in mouse germ cells following 1.2-dibromo-3-chloropropane (DBCP) exposure. Mutat Res. 1979;68: 169-73. 27. Amann RP, Bemdtson WE. Assessment of procedures for screening agents for effects on male reproduction: Effects of dibromochloropropane (DBCP) on the rat. Fundam Appl Toxicol. 1986;7: 244-55. 28. Teramoto S, Saito S, Aoyama H, Shirasu Y. Dominant lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP). Mutat Res. 1980;77:71-8. 29. Saito-Suzuki R, Teramoto S, Shirasu Y. Dominant lethal studies with I .2-dibromo-3chloropropane and its structurally related compounds. Mutat Res. 1982;101:321-7
DNA damage in human testicular
cells 0 C. BJBRGE ET AL.
59
30. Rao KS, Burek JD, Murray FJ, John JA, Schwetz BA, Bell TJ, Potts WJ, Parker CM. Toxicologic and reproductive effects of inhaled 1,2-dibromo-3-chloropropane in rats. Fundam Appl Toxi-
44. Lowry 0, Rosebrough NJ, Farr AL, Randall RL. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:26575.
col. 1983;3:104-10. 31. Bjorge C, Wiger R, Holme JA, Brunborg G, Andersen R, Dybing E, Soderlund EJ. In vitro toxicity of 1,2-dibromo-3chloropropane (DBCP) in different testicular cell types from rats. Reprod Toxicol. 1995;9:4611173. 32. Grosovsky AJ, De Boer JG, De Jong PJ, Drobetsky EA, Glickman BW. Base substitution, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc Nat1 Acad Sci USA. 1988;85: 185-8. 33. Dybing E, Omichinski JG, Soderlund EJ, Brunborg G, Lag M, Holme JA, Nelson SD. Mutagenicity and organ damage of 1,2-dibromo-3-chloropropane (DBCP) and tris(2,3-dibromopropyl)phosphate (T&BP): role of metabolic activation. In: Hodgson E, Bend JR, Philpot RM, eds. Reviews in biochemical toxicology. New York: Elsevier Science Publishing Co; 1989;10: 139-87. 34. Gingell R, Beatty PW, Mitschke HR, Mueller RL, Sawin VL, Page AC. Evidence that epichlorohydrin is not a toxic metabolite of 1,2-dibromo-3-chloropropane. Xenobiotica. 1987;17:22940. 35. Omichinski JG, Brunborg G, Holme JA, Soderlund EJ, Nelson SD, Dybing E. The role of oxidative and conjugative pathways in the activation of 1,2-dibromo-3-chloropropane to DNA damaging products in testicular cells. Mol Pharmacol. 1988;34:74-9. 36. Holme JA, Soderlund EJ, Brunborg G, Omichinski JG, Bekkedal K, Trygg B, Nelson SD, Dybing E. Different mechanisms are involved in DNA damage, bacterial mutagenicity and cytotoxicity induced by 1,2-dibromo-3-chloropropane in suspensions of rat liver cells. Carcinogenesis. 1989;10:49-54. 37. Bradley MD, Dysart G. DNA single-strand breaks, double-strand breaks and crosslinks in rat testicular germ cells: measurement of their formation and repair by alkaline and neutral filter elution. Cell Biol Toxicol. 1985;1:181-5. 38. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:189-91. 39. Olive PL, Durand RE, Wlodek D, Banath JP. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res. 1992;198:2.59-67. 40. Brunborg G, Holme JA, Soderlund EJ, Omichinski JG, Dybing E. An automated alkaline elution system. DNA damage induced by 1,2-dibromo-3-chloropropane in viva and in vitro. Anal Biochem. 1988;174:522-36. 41. Kohn KW, Erikson LC, Ewig RAG, Zwelling A. Measurement of strand breaks and crosslinks by alkaline elution. In: Friedberg EC, Hanawalt PC, eds. DNA repair: A laboratory manual of recent procedures. New York Marcel Dekker; 1981:379401. 42. Omichinski JG, Soderlund EJ, Bausano JA, Dybing E, Nelson SD. Synthesis and mutagenicity of selectively methylated analogs of 1,2-dibromo-3chloropropane and tris(2,3-dibromopropyl)phosphate. Mutagenesis. 1987;2:287-92. 43. Wallin H, Schelin C, Tunek A, Jergil B. A rapid and sensitive method for determination of covalent binding of benzo(a)pyrene to proteins. Chem-Biol Interact. 1981;38:109-18.
45. Sterzel W, Bedford P, Eisenbrand G. Automated determination of DNA using the fluorochrome Hoechst 33258. Anal Biochem. 1985;147:462-7. 46. Omichinski JG, Soderlund EJ, Dybing E, Pearson PG, Nelson SD. Detection and mechanism of formation of the potent direct-acting mutagen 2-bromoacrolein from 1,2-dibromo-3chloropropane. Toxic01 Appl Pharmacol. 1988;92:286-94. 47. Soderlund EJ. Meyer DJ, Ketterer B, Nelson SD, Dybing E, Holme JA. Metabolism of 1,2-dibromo-3chloropropane by glutathione S-transferases. Chem-Biol Interact. 1995;97:257-272. 48. Lag M, Soderlund EJ, Brunborg G, Dahl JE, Holme JA, Omichinski JG, Nelson SD, Dybing E. Species differences in testicular necrosis and DNA damage, distribution and metabolism of I ,2-dibromo-3-chloropropane (DBCP). Toxicology. 1989;58: 1334. 49. Pearson PG, Soderlund EJ, Dybing E, Nelson SD. Metabolic activation of 1,2-dibromo-3-chloropropane: evidence for the formation of reactive episulfonium ion intermediates. Biochemistry. 1990;29:4971-81. 50. Den Boer PJ, Poot M, Verkerk A, Jansen R, Mackenback P, Grootegoed A. Glutathione-dependent defence mechanisms in isolated round spermatids from the rat. Int J Androl. 1990;13:2638. 51. Dibiasio KW, Silva MH, Shull LR, Overstreet JW, Hammock BD, Miller MG. Xenobiotic metabolizing enzyme activities in rat, mouse, monkey, and human testis. Drug Metab Dispos. 1991;19: 227-3 1. 52. Klys HS, Whillis D, Howard G, Harrison DJ. Glutathione Stransferase expression in the human testis and testicular germ cell neoplasia. Br J Cancer. 1992;66:589-93. 53. Waters R, Jones CJ, Martin EA. Yang AL, Jones NJ. The repair of large DNA adducts in mammalian cells. Mutat Res. 1992;273: 145-55. 54. Mirzayans R, Paterson MC, Waters R. Defective repair of class of 4NQO-induced alkali-labile DNA lesions in xeroderma pigmentosum complementation group A fibroblasts. Carcinogenesis. 1985; 6:555-9. 55. Whorton D, Milby TH, Krauss RM, Stubbs HA. Testicular function in DBCP exposed pesticide workers. J Occup Med. 1979;21: 161-6. 56. Olsen GW, Lanham JM, Bodner KM, Hyhon DB, Bond GG. Determinants of spermatogenesis recovery among workers exposed to 1,2-dibromo-3chloropropane. J Occup Med. 1990;32:979-84. 57. Vos RME, Van Blanderen PJ. Glutathione tion to their role in the biotransformation Biol Interact. 1990;75:241-65.
S-transferases in relaof xenobiotics. Chem
58. Meyer DJ, Harris JM, Gilmore KS, Coles B, Kensler TW, Ketterer B. Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole-3-thiones. Carcinogenesis. 1993;14:567-72. 59. Biava CG, Smuckler EA. Whorton D. The testicular morphology of individuals exposed to dibromochloropropane. Exp Mol Pathol. 1978;29:448-58.