* Ottawa Health Research Institute, Hormones, Growth, Development, and Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9; The Ottawa Hospital, and Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 1J8;
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6; and
Environmental Health Research Division, Health Canada, Tunney's Pasture, Ottawa, Canada K1A 0L2
Received May 21, 2004; accepted August 26, 2004
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ABSTRACT |
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Key Words: trichloroethylene; chloral hydrate; fertilization; spermegg binding; trichloroethanol.
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INTRODUCTION |
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Data from previous studies indicated that TCE exposure is associated with impairment of male fertility. In a study that evaluated 55 occupational categories for occupation-related infertility, men employed as mechanics and involved in degreasing of engine parts appeared to have the highest risk of idiopathic infertility (Rachootin and Olsen, 1983). In other studies, TCE exposure was associated with reduced sperm density in a cohort of men exposed occupationally to TCE (Chia et al., 1996
). Multigenerational studies in mice and rats exposed orally to TCE identified reduced fertility and increased incidence of abnormal sperm in some but not all generations (NTP, 1985
, 1986
). Inhalation exposure to TCE has also been shown to cause male infertility, reduced testis size, reduced epididymal sperm number and motility, reduced serum testosterone levels (Kumar et al., 2000
, 2001
), and a significant increase in the proportion of misshapen sperm nuclei (Land et al., 1981
). More recent studies reported a decrease in the percentage of zona-free eggs fertilized by sperm from rats exposed to TCE in drinking water (DuTeaux et al., 2004
).
The metabolism of TCE is mediated through two major pathways: conjugation with glutathione and oxidation via cytochrome P450 (Fig. 1). Conjugation with glutathione produces S-(1, 2-dichlorovinyl) glutathione and subsequently S-(1, 2-dichlorovinyl)-L-cysteine (Bruckner et al., 1989; Lash et al., 2000a
). Oxidative metabolism of TCE occurs in the liver and produces the primary transient metabolite TCE oxide in one pathway, which is then converted to dichloroacetyl chloride. Chloral is the major metabolite in another pathway where it is rapidly converted to its hydrate (chloral hydrate, CH), which then undergoes reduction and oxidation to form trichloroethanol (TCOH) and trichloroacetic acid (TCA), respectively (Kimmerle and Eben, 1973
). TCOH and TCA are major circulating and urinary TCE metabolites (Lash et al., 2000a
). Dichloroacetyl chloride metabolism generates dichloroacetic acid, which can also be formed via dechlorination of TCA. The toxicity of TCE is ascribed to its bioactivation, although the specific metabolites implicated in particular outcomes have not been fully resolved (Bull, 2000
; Lash et al., 2000a
; Odum et al., 1992
). However, it has been proposed that CH, dichloroacetic acid, and TCA are TCE metabolites associated with liver and lung toxicity, whereas S-(1, 2-dichlorovinyl)-L-cysteine is implicated in kidney toxicity (Lash et al., 2000b
).
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In this study, we have investigated the potential effects of TCE exposure on sperm function. We have examined the impact of TCE inhalation on the capacity of mouse spermatozoa to bind to mature zona-intact eggs in vitro and to fertilize eggs in vivo. In addition, we have determined the toxic effects of TCE and its metabolites on spermegg binding. Our results showed that spermegg binding and fertilization are decreased in male mice exposed to TCE, and this decrease is likely due to the direct effects of CH and TCOH on sperm.
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MATERIALS AND METHODS |
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Animals. Male CD-1 mice (8090 days of age at the start of exposure) were purchased from Charles River Canada (St. Constant, Quebec), and were individually housed in polycarbonate cages with free access to Purina rodent chow 5010 (Ralston-Purina, St. Louis, MO) and water under constant photoperiod (12:12 h light/dark). Sexually mature female CF-1 mice were purchased from Charles River Canada and were used for mating studies and for egg retrieval in the spermegg binding studies. All procedures relating to animal treatment adhered to Canadian Council on Animal Care guidelines and were approved by Animal Care Ethics Committees at Health Canada and the Ottawa Health Research Institute.
Inhalation exposure. After acclimation to the facility for 1 week, mice were transferred to 2.5 m3 inhalation chambers and housed individually in suspended stainless-steel wire-mesh cages with an integral food dish and a water spigot. The chambers were operated at an air flow of 500 liters/min of HEPA and activated charcoal filtered air (ca. 10 chamber volumes/h), with an internal chamber temperature of 19°24°C, and relative humidity of 3050%. Mice were housed in these cages until the completion of all exposures.
Animals were exposed by inhalation to atmospheres containing a TCE concentration of 1000 ppm (5.37 mg/l) for 1 to 6 weeks (6 h/day, 5 days/week). The atmospheres were generated by evaporating TCE through a glass evaporative system, with the resulting vapor being carried by an air stream into the chamber inlet and mixed with the incoming air. Concentrations of TCE in air from both chambers (i.e., TCE and control) and from the surrounding room were monitored every 6 min throughout the exposure period by gas chromatography (X-Tra Process gas chromatography, Amscor, U.S.) connected to the chambers through a multi-valve system (Douglas et al., 1999). At the end of the exposure period, the flow of TCE was terminated without changing the flow rate of the incoming air stream. Control animals were used for each experiment and were treated identically in an adjacent chamber, except that the TCE evaporating system was not connected to the air intake. Food containers were removed from both TCE and control chambers for the duration of the exposure to prevent oral exposure to TCE by consumption of TCE-laden food.
Assessment of body weight, testis and epididymis weight, and sperm number and motility. After each TCE exposure regimen, body weights of both control and TCE-exposed mice were recorded. Mice were sacrificed by cervical dislocation, and right testis and epididymis were dissected, freed of fat pads, blotted on tissue paper, and weighed. The left cauda epididymides were longitudinally scored once with a surgical blade and the sperm-containing epididymal fluid was squeezed out from this incision into HEPES-buffered Krebs Ringers bicarbonate (KRB-HEPES) medium supplemented with 0.3% bovine serum albumin (BSA) (KRB-HEPES: 119.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 21 mM HEPES, 5 mM NaHCO3, 25 mM sodium lactate, 1 mM sodium pyruvate, 5.6 mM glucose, 2.8 µM phenol red, pH 7.4). Sperm were also squeezed from the vas deferens and combined with the caudal epididymal sperm suspension. An appropriate dilution of the sperm suspension was placed into a hemocytometer, and the number of total sperm and motile sperm were immediately counted under a Nikon inverted microscope (DIAPHOT-TMD, Nikon Canada, Mississauga, ON) at 200x magnification.
Assessment of sperm spontaneous acrosome reaction. Cauda epididymal and vas deferens sperm, prepared as described above, were washed once with KRB supplemented with 0.3% BSA (KRB-BSA): (KRB: same ingredients as KRB-HEPES except that 21 mM HEPES + 5 mM NaHCO3 buffering system was substituted with 25 mM NaHCO3) by centrifugation (350 g, 10 min, 27°C) resuspended in KRB-BSA at 10 million/mL and incubated for 30 min at 37°C under 5% CO2. These conditions have previously been shown to allow sperm capacitation (Tanphaichitr et al., 1993). Spontaneous acrosome reaction was determined by evaluating the sperm acrosomal status following a previously described method (Bleil and Wassarman, 1990
). Briefly, an aliquot of the capacitated sperm suspension (
1 million sperm) from each sample was fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Sperm were washed twice with 100 mM ammonium acetate (pH 9.0) and resuspended in the same buffer, after which an aliquot of the sperm suspension was placed onto a microscope glass slide coated with gelatin and air dried. Sperm on the slides were incubated in freshly made Coomassie blue dye (0.22% Coomassie blue G-250 (Bio-Rad, Hercules, CA), 50% methanol, 10% glacial acetic acid, 40% water) for 2 min. Slides were washed thoroughly with distilled water to remove unbound dye. Slides were air-dried, topped with Permount mounting medium (Fisher Scientific, Nepean, ON), and covered with coverslips. Stained sperm were examined under a brightfield microscope (Zeiss Canada, Toronto, ON) at 400x or 630x. Specifically, aldehyde-fixed acrosome-intact sperm are stained with Coomassie blue at their head convex ridge (the site of the acrosome), whereas acrosome-reacted sperm show negative staining.
In vitro spermegg binding. Spermegg binding was determined using procedures described previously (Tanphaichitr et al., 1993). Cumulus masses containing mature eggs were collected from superovulated female mice into KRB-HEPES-BSA using established procedures (Hogan et al., 1994
). Zona pellucida (ZP)intact eggs were freed from cumulus cells by 0.1% hyaluronidase digestion and washed in KRB-BSA, pre-warmed at 37°C under 5% CO2. Capacitated sperm from mice exposed to air (controls) and TCE were prepared as described above. Eggs (n = 2030) were then incubated with 60,000 motile capacitated sperm from control or TCE-treated mice, in a 60 µl droplet of KRB-BSA for 30 min at 37°C under 5% CO2. Subsequently, spermegg complexes were gently washed 4 times in KRB-BSA through a drawn Pasteur pipette (inner diameter 200 µm) to remove loosely attached sperm, placed into a well of a sera culture slide, and overlaid with mineral oil. The number of sperm bound to the ZP per egg was counted under an inverted microscope. Because numerous sperm bound to the ZP, only those in the focal plane of the zona diameter were counted. The nonspecific binding level of sperm to the egg ZP was determined by incubating sperm with fertilized eggs (Hogan et al., 1994
). This nonspecific binding level was 510% of the positive control value (i.e., when mature eggs were incubated with capacitated sperm) (Tanphaichitr et al., 1993
). The average number of sperm bound per egg from each control or TCE-treated mouse was then calculated, and the data from all control or TCE-treated mice were expressed as mean ± S.D., except for the data from the two control mice at week 4 and week 6 time points, which were simply expressed as an average of the two animals.
In alternate experiments where the direct effects of TCE and its metabolites were investigated, capacitated sperm were first incubated with various concentrations of TCE, CH, or TCOH for 30 min. Control sperm were those incubated in KRB-BSA, containing vehicle at the same concentration as that used for TCE solubilization (0.5% ethanol in the case of TCE and TCOH), or those incubated simply in KRB-BSA (in the case of CH). In each experiment, sperm from the same animal were used for control and treatments with TCE, CH, and TCOH. Both control and pretreated sperm were washed by centrifugation with KRB-BSA prior to incubation with mature eggs and the numbers of sperm bound per egg were determined as described above. Data of the number of sperm bound per egg were expressed as mean ± S.D. of means obtained from the three replicate experiments performed on different days.
In Vivo fertilization. Immediately after the TCE exposure, control and TCE-exposed mice were individually caged with a single female CF-1 mouse that had been superovulated by sequential i.p. injections of PMSG (5 IU) and hCG (10 IU), with a 48 h interval (Hogan et al., 1994). Caging of each female with a male was done immediately after the hCG injection. The females were examined for the presence of copulation plugs 14 h after the initiation of cohabitation. Females showing the copulation plugs were sacrificed, and cumulus masses containing mature eggs were collected from the oviducts into KRB-HEPES-BSA. Eggs were freed from cumulus cells by hyaluronidase digestion (Hogan et al., 1994
), washed in KRB-HEPES containing 0.1% polyvinylpyrrolidone, and fixed in 4% paraformaldehyde in PBS. Fixed eggs were stained with 5 µg/mL Hoechst 33258 and examined for fertilization, based on the presence of 2 pronuclei, under a Zeiss IM35 epifluorescence microscope. The number of eggs retrieved from each mated female that had two pronuclei was recorded, and the percentage of eggs fertilized was calculated setting total eggs retrieved as 100%. Data were then expressed as mean ± S.D. of percent eggs fertilized from all females mated one-to-one with control or TCE-treated males.
Statistical analysis. Data of percent eggs fertilized per male from fertilization studies were analyzed by 2-way factorial analysis of variance (ANOVA) using treatment (control vs. TCE) and weeks of exposure as tested factors. Significant differences indicated by ANOVA were further evaluated by Dunnett's method. Similar analyses were performed for mean spermegg binding data per male for sperm collected from animals exposed for 1 or 2 weeks, where the number of control and TCE-treated mice was 3 or more. However, spermegg binding data were available for only 2 control males each for the 4- and 6-week exposures, precluding ANOVA for these time points. Therefore, spermegg binding data for these time points were analyzed separately using Student's t-test. Data from the studies on the effects of sperm pretreatment with TCE or its metabolites (TCOH, CH) on in vitro spermegg binding were also subjected to 2-way ANOVA with pretreatment dose and experimental repeat number being the two tested factors. Significant effects were evaluated using Dunnett's method. The analyses were performed using SigmaStat (SPSS, Chicago, IL).
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RESULTS |
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DISCUSSION |
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It is well established that bioactivation of TCE is a prerequisite for evoking toxicity (Lash et al., 2000a). Although several P450 enzymes have been implicated in TCE metabolism, CYP2E1 appears to be the major P450 enzyme catalyzing the oxidation of TCE with the highest efficiency (Lash et al., 2000a
; Lipscomb et al., 1998
). In the male reproductive system of mice, CYP2E1 is localized to the epididymal epithelium and testicular Leydig cells (Forkert et al., 2002
), and to the efferent ductules (DuTeaux et al., 2003
). CYP2E1-dependent catalytic activity, as assessed by p-nitrophenol hydroxylation, was twofold higher in the epididymis than in the testis. CYP2E1 has also been identified as a P450 enzyme involved in TCE metabolism in the male reproductive tract (Forkert et al., 2002
). Incubations of microsomes from the epididymis and testis produced concentration-dependent formation of chloral that was considerably higher in the microsomes than in the epididymis and testis. These findings indicated that CYP2E1 is responsible, in part, for TCE bioactivation in the male reproductive tract. Hence, it was of interest to investigate the direct effects of the TCE metabolites CH and TCOH on the sperm ability to bind to the zona-intact eggs. Although previous results revealed a 40% decrease in in vitro fertilization in mouse gametes incubated in medium containing TCOH at a concentration 75-fold higher than that used in the current study, it was not clear whether the sperm, the eggs, or both were affected (Cosby and Dukelow, 1992
). Our results showed that both of these TCE metabolites caused significant decreases in the ability of washed epididymal sperm to bind to the egg ZP in vitro, with CH exerting a far more potent effect than TCOH (Fig. 4). It should be noted that the concentrations of TCE, TCOH, and CH used in our in vitro studies are comparable to their described levels in the epididymis homogenate of mice exposed to TCE by inhalation for 2 weeks or more (Forkert et al., 2003
). Our observed reactivity of CH on mouse sperm corroborates previous findings describing abnormalities of spermatogenic cells in mice treated with CH (Allen et al., 1994
; Nutley et al., 1996
; Russo et al., 1984
). In addition, treatment of rats with dichloroacetic acid produced abnormal sperm morphology and decreased motility (Linder et al., 1997
). These findings, in addition to the lack of an effect by the parental compound, TCE (Fig. 4), support the concept that bioactivation of TCE to its metabolites is responsible for the adverse effects of the compound on the sperm maturation process in the epididymis. This may be one of the causes of the observed decreases in spermegg binding and fertilization observed in TCE-inhaling mice. The mechanisms of how CH and TCOH affect the sperm plasma membrane and metabolism are under investigation in our laboratory. We are also examining whether the defensive mechanism, exerted by the animals at a certain time period after TCE exposure, is via secretion of inhibitors of the TCE metabolites' action into the epididymal fluid.
Substantial data have accrued revealing the adverse effects of occupational exposures to chemicals including TCE on male reproductive health. In a study of occupational exposure, sperm density was decreased and abnormal sperm increased in subjects exposed to TCE (Chia et al., 1996). A more recent study reported that exposure to chlorinated solvents, including TCE, decreased semen concentration and sperm motility and increased the number of abnormal sperm (Tielemans et al., 1999
). These findings indicated that TCE exposure has adverse effects on sperm production and quality. Other studies have revealed that paternal exposure to solvents increases the time for achieving conception (Sallmén et al., 1998
), decreases the rates of successful implantation after in vitro fertilization (Tielemans et al., 2000
), and increases the relative risk and incidence of spontaneous abortions (Lindbohm et al., 1991
; Taskinen et al., 1989
). Furthermore, a case control study examining the link between occupation and infertility revealed that men employed as auto mechanics were about 9 times more likely to have idiopathic infertility (Rachootin and Olsen, 1983
). Our current results, as well as our previous findings describing the presence of TCE metabolites (chloral, TCOH, TCA, and DCA) in seminal plasma of mechanics exposed to TCE in the workplace, as well as in the epididymis of mice exposed to TCE (Forkert et al., 2003
), strongly suggest that the observed adverse effects of TCE inhalation on male reproductive health are caused by the reactivity of TCE metabolites.
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ACKNOWLEDGMENTS |
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NOTES |
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3 Present address: Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
1 To whom correspondence should be addressed at PL 0803D, Tunney's Pasture, Ottawa, K1A 0L2, Canada. Fax: 613-957-8800. E-mail: Mike_Wade{at}hc-sc.gc.ca.
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