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Toxicology |
Department of Environmental Toxicology, University of California, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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environment, epididymis, male reproductive tract, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The male reproductive toxicity of TCE has been investigated in several animal studies. Mice exposed to TCE by inhalation had significantly increased percentages of abnormal sperm at the highest exposure concentration (
150 ppm) [8]. Because mice were exposed for fewer days than are required for an entire cycle of spermatogenesis, the authors concluded that the spermatotoxicity occurred during the first or second meiosis or during sperm maturation [8]. Inhalation of TCE also resulted in significantly decreased epididymal sperm count and motility in rats [9]. Oral dosing with a mixture of groundwater contaminants including TCE resulted in no quantitative changes of spermatogenesis in male rabbits, although significant increases were found in the numbers of abnormal sperm [10]. A majority of the defects in ejaculated sperm were acrosomal-nuclear abnormalities [10].
Acute and chronic TCE exposure induces several adverse effects, including liver and kidney toxicity, in experimental animals (for review, see [1]). It is believed that TCE must be bioactivated to elicit its toxic effects [11], and it is metabolized by glutathione conjugation and cytochrome P450-dependent pathways, both of which form reactive metabolites that covalently bind cellular macromolecules (Fig. 1) [12]. Glutathione conjugation of TCE results in the formation of dichlorovinyl glutathione, which is processed by 
-glutamyltransferase and dipeptidases to dichlorovinyl cysteine (DCVC) [13]. The DCVC can be bioactivated by cysteine-conjugate ß-lyase (ß-lyase), resulting in the formation of a reactive thiol or chlorothioketene [14, 15]. The kidney is sensitive to the toxicity of dichlorovinyl glutathione and DCVC [16], whereas the liver appears to escape the toxic effects of these glutathione-derived metabolites [17]. The reactive metabolites formed by kidney ß-lyase can bind cellular proteins, including proteins in kidney mitochondria [14, 18–20]. This may be the initiating event in TCE-induced nephrotoxicity [14].
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Additionally, TCE is metabolized by cytochrome P450-dependent oxidation, primarily by the cytochrome P450 2E1 (CYP2E1) isoform [21]. The first step in TCE oxidation may involve the formation of an unstable epoxide [21] or the generation of a TCE-P450 intermediate [22]. The first metabolite characterized in this pathway is chloral, which exists in equilibrium with chloral hydrate [23]. Chloral and chloral hydrate are rapidly oxidized to trichloroethanol, trichloroacetic acid, and dichloroacetic acid, all of which are eliminated in the urine [23]. Reactive metabolites derived from cytochrome P450-dependent oxidation of TCE bind to hepatic proteins and enzymes [24–28] and have been directly associated with liver toxicity [12].
The formation of reactive metabolites that bind cellular macromolecules has been proposed as an important initiating event in target-organ toxicity [29]. Because reactive metabolites of TCE can covalently bind proteins, the objective of the present study was to determine if bioactivation and protein binding might be involved in the reproductive toxicity of TCE. The present study was conducted as a series of three experiments. First, animals were dosed in vivo with TCE, and then TCE-protein adducts in the epididymis and efferent ducts were evaluated immunochemically using the liver and kidney as positive controls. Second, to ascertain the role of the glutathione conjugation pathway and bioactivation of TCE cysteine conjugates in adduct formation, we examined the presence of the enzyme ß-lyase in the epididymis and efferent ducts and the formation of protein adducts in the epididymis and efferent ducts following in vivo administration of DCVC. Third, to ascertain the role of cytochrome P450-dependent metabolism in adduct formation, the epididymis and efferent ducts were tested for the presence and activity of CYP2E1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague Dawley rats (weight, 350–400 g) were purchased from Charles River Laboratories (Hollister, CA) and housed under a 12L:12D photoperiod in a temperature- (22±2°C) and humidity- (40–70%) controlled facility. Rats were maintained on rat chow (Formulab Purina 5008, St. Louis, MO) ad libitum and deionized water treated by reverse osmosis. The University of California, Davis, Animal Use and Care Administrative Advisory Committee approved all animal use.
Chemicals and Antibodies
Analytical-grade TCE, chloral hydrate, NADPH, and Tris base (purity, 98%–99.6%) were purchased from Sigma (St. Louis, MO). High-performance liquid chromatographic-grade acetonitrile and ethyl acetate were purchased from Fisher Scientific (Fair Lawn, NJ). Diazomethane was synthesized in-house according to the method of de Boer and Baurer [30]. The DCVC and goat anti-rat ß-lyase antibody were generous gifts of Dr. James L. Stevens (Eli Lilly). Dr. Neil R. Pumford (University of Arkansas) generously provided rabbit anti-dichloroacetyl antibody. Rabbit anti-rat CYP2E1 used for immunodetection was purchased from Amersham Pharmacia (Piscataway, NJ). A specifically designed goat anti-rat CYP2E1 was used for immunoinhibition (Daiichi Pure Chemicals, Tokyo, Japan).
Chemical Treatment of Animals
For TCE dosing, rats were exposed to either 0%, 0.2%, or 0.4% TCE (v/v) in a solution of 3% ethoxylated castor oil in drinking water for 14 days (n = 3 per group). Glass water bottles were filled every 24 h with fresh solutions to minimize headspace and TCE volatilization. For DCVC dosing, rats (n = 4) were injected i.p. with a single dose of 25 mg/kg of DCVC dissolved to a final concentration of 10 mg/ml in sterile saline. Control animals (n = 3) were injected i.p. with equivalent volumes of saline. All animals were killed by CO2 asphyxiation 24 h after final dosing.
Fluorescent Immunohistochemistry
Dichloroacetyl adduct localization Liver, kidneys, epididymides, and efferent ducts were dissected from TCE- and DCVC-treated animals, immersion-fixed in 1% paraformaldehyde for 30 min, and frozen-embedded in TissueTek OCT Compound (Sakura, Torrance, CA). Cryosections (thickness, 10 µm) were fixed in -20°C acetone for 10 min and air-dried. Sections were rehydrated in PBS, blocked with 5% normal goat serum in 1% BSA-PBS, and incubated with 1:50 (DCVC tissues) or 1:250 (TCE tissues) rabbit antidichloroacetyl. Sections were rinsed thoroughly in PBS and incubated with 1:500 AlexaFluor488 goat anti-rabbit immunoglobulin (Ig) G (Molecular Probes, Eugene, OR).
ß-Lyase and CYP2E1 localization Epididymides, efferent ducts, kidneys, and liver were dissected from untreated rats and then fixed, embedded, and sectioned as described above. For ß-lyase immunohistochemistry, cryosections were blocked with 5% normal donkey serum in 1% BSA-PBS, then incubated with 1:50 goat anti-rat ß-lyase and 1:1000 AlexaFluor488 donkey anti-goat IgG. For CYP2E1 immunohistochemistry, cryosections were blocked with 5% normal goat serum in 1% BSA-PBS, then incubated with 1:100 rabbit anti-rat CYP2E1 and 1:500 AlexaFluor488 goat anti-rabbit IgG. Background fluorescence was tested by incubating duplicate sections without primary antibody. Slides were visualized using an Olympus AX70 Provis Microscope (Melville, NY), and images were captured digitally by a Zeiss (Thornwood, NY) AxioCam CCD camera.
Subcellular Fractionation
Mitochondrial fractions were prepared as described by Cain and Skilleter [31]. Briefly, kidney, epididymides, and efferent ducts were minced in 20 ml of sucrose buffer (0.25 M sucrose, 5 mM Tris-HCl, and 1 mM EDTA; pH 7.4), homogenized, and then centrifuged at 460 x g for 10 min. The retained supernatant was centrifuged at 12 500 x g for 7 min. The mitochondrial layer was removed, resuspended, and repelleted at 12 500 x g. Microsomes were prepared according to the method of Lake [32]. Briefly, liver, kidney, efferent ducts, and epididymides were homogenized in a 1:4 ratio of 0.05 M Tris buffer (0.05 M Tris/HCl with 1.5% NaCl; pH 7.4) with 0.1 M phenylmethylsulfonyl fluoride and 1% (v/v) aprotonin. Kidneys and reproductive tissues were disrupted for 15 sec using a Brinkman Polytron (Westbury, NY). Homogenates were centrifuged at 9000 x g for 20 min at 4°C. The retained supernatant was centrifuged at 100 000 x g for 60 min at 4°C. The resulting pellet contained microsomes, and the resulting supernatant yielded cytosol. Total protein concentration was determined by the bicinchoninic acid method (Sigma) using a BSA (10 mg/ml) standard. Fractions were stored at -80°C until use.
Western Blot Analysis
Proteins were separated on 10% SDS-PAGE according to the method of Laemmli [33] and electroblotted to 0.45-µm nitrocellulose using a wet-transfer apparatus (Bio-Rad, Hercules, CA). All membranes were blocked with 5% milk in Tris-buffered saline (50 mM Tris-HCl and 150 mM NaCl; pH 7.5) with 0.05% Tween-20 for 60 min at 22°C. For detection of ß-lyase, mitochondria and cytosol prepared from kidney, epididymis, and efferent ducts were incubated with 1:250 goat anti-rat ß-lyase. For detection of CYP2E1, liver, kidney, epididymis, and efferent microsomes were incubated with 1:500 rabbit anti-rat CYP2E1. Proteins from DCVC- and TCE-treated animals were incubated with 1:200 or 1:500 rabbit anti-dichloroacetyl, respectively. All membranes were incubated with species-specific 1:10 000 alkaline phosphatase-conjugate IgGs, and immunoreactive bands were visualized using Western Blue alkaline phosphatase substrate (Promega, Madison, WI). Replicate blots incubated with normal serum in place of the primary antibody served as negative controls.
TCE Metabolism
A TCE metabolism assay was modified from a previously published method [34]. Briefly, liver, epididymal, and efferent duct microsomes were prepared as described earlier. Because of the scarcity of tissue, four to six rats were used to prepare a single pooled sample of efferent duct microsomes. Three separate pooled samples were used for each incubation (n = 3). For other tissue types, individual microsomal preparations were made from each of three animals (n = 3). Microsomes (total protein, 0.25–2.00 mg) were incubated in 0.05 M Tris buffer for 5 min at 37°C with 2–10 mM TCE (final concentration). Reactions were initiated by adding NADPH (final concentration, 1.5 mM), allowed to proceed for 15 min at 37°C, and then terminated by flash-freezing in a dry ice-acetone bath. Thawed samples were extracted twice with 1 ml of ethyl acetate and methylated with 1 ml of diazomethane (13 mg/ml in ethyl ether). Microsomes incubated without NADPH served as experimental controls. To determine method recovery, microsomes were incubated in triplicate without TCE, fortified with 1 µg/ml of chloral hydrate, extracted, and methylated as described above.
Gas Chromatography/Mass Spectrometry
Methylated extracts were analyzed with a Hewlett-Packard Model 6890 Series gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a 5972 mass selective detector and a DB-5ms 30-m x 0.25-mm (inner diameter), 0.25-µm film thickness column (J&W Scientific, Folsom, CA). Five-point standard curves were used at the beginning and end of each set of samples. The average peak area measured at each concentration was used to generate the standard curve. Assay limit of detection was 0.009 µg/ml. Retention time for methylated chloral equivalents was approximately 8.09 min. Quantitation was performed by manually drawing baselines for each peak of interest using Hewlett-Packard ChemStation software (ver. B.02.06). Methylated extracts from microsomal incubations were injected in duplicate. Resulting peak areas were averaged and converted to picomoles using linear regression from the standard curve.
Immunoinhibition of TCE Metabolism
Varying concentrations of goat anti-rat CYP2E1 antibody (0–145 µg/ml) were preincubated with 500 µg of efferent duct and liver microsomes for 30 min at room temperature. Then, TCE (final concentration, 5 mM) was added to the incubation mixture and prewarmed in a 37°C shaking water bath for 5 min. The reaction was initiated by adding NADPH (final concentration, 1.5 mM) and continued for 15 min at 37°C. Reactions were terminated by flash-freezing, and samples were extracted, methylated, and analyzed using gas chromatography/mass spectrometry (GC/MS) as described earlier. Residual activity (i.e., level of immunoinhibition) was calculated as a percentage of product formed in antibody-adsorbed incubations versus incubations containing equivalent amounts of normal goat serum in place of the anti-CYP2E1 antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals were exposed to drinking water with concentrations of 0%, 0.2% (2.73 mg/L), or 0.4% (5.46 mg/L) TCE emulsified in 3% ethoxylated castor oil as their only source of fluid for the duration of the experiment. These concentrations are within the range of measurements obtained in formerly contaminated drinking-water wells [1]. Based on animal weight and average daily consumption of 28 ml, the calculated doses ranged from 1.6–2.0 mg kg-1 day-1 for the low-dose animals and 3.4–3.7 mg kg-1 day-1 for the high-dose animals. Protein adducts were immunolocalized in tissues of animals dosed with TCE (Fig. 2) using an antibody previously shown to detect dichloroacetyl adducts in the liver of TCE-treated rats [28]. Protein adducts were localized in the centrilobular region of the liver (Fig. 2A) and the proximal convoluted tubules of the kidney (Fig. 2B). The corpus contained the highest level of staining of all regions of the epididymis (Fig. 2C). Protein adducts were also localized to the efferent ducts (Fig. 2D). No protein adducts were detected in the initial segment, the caput or cauda segments of the epididymis of treated animals, or in any tissues from control animals (data not shown). Western blot detection of protein adducts was not conducted because of the scarcity of efferent ducts and corpus epididymis from treated animals, although protein adducts were examined by Western blot analysis following in vitro TCE exposure later in the present study.
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Detection of Soluble But Not Mitochondrial Cysteine-Conjugate ß-Lyase in the Epididymis and Efferent Ducts
The presence of ß-lyase was tested in the epididymis and efferent ducts using the kidney as a positive control. In a previously reported
[35], we showed that ß-lyase was immunolocalized in the kidney proximal tubules of untreated rats, whereas the epididymis and efferent ducts exhibited only slight, diffuse immunoreactivity. The proximal tubule localization of ß-lyase was previously reported using the same antibody [19]. Repeated immunolocalizations of ß-lyase in the present study yielded similar results (data not shown). Western blot analysis of subcellular fractions was used to further evaluate the presence of ß-lyase in the epididymis and efferent ducts (Fig. 3). ß-Lyase was detected as a single dominant protein band in kidney cytosol (Fig. 3, lane 1) and mitochondria (Fig. 3, lane 2). This is similar to previous studies that have detected the highest level of ß-lyase in kidney mitochondria and cytosol and the lowest level in kidney microsomal and nuclear fractions [19]. A similar-sized band was detected in epididymal cytosol (Fig. 3, lane 3) and efferent duct cytosol (Fig. 3, lane 5). Epididymal mitochondria contained a weakly staining band at a lower molecular weight (Fig. 4, lane 4). No immunoreactive bands were detected in efferent duct mitochondria (Fig. 3, lane 6) or in replicate blots incubated without ß-lyase antibody (data not shown).
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No Protein Adducts Detected in the Epididymis and Efferent Ducts Following In Vivo DCVC Treatment
ß-Lyase activation of the TCE cysteine-conjugate DCVC results in the formation of dichloroacetylated protein adducts in kidney mitochondria and cytosol [36]. In the present study, animals were given a single i.p. injection of DCVC or saline. Immunochemical methods were used to detect dichloroacetylated proteins in the epididymis and efferent ducts (Fig. 4) using the same antidichloroacetyl antibody used for detection of adducts following TCE dosing. Protein adducts were localized to the proximal tubules in kidney cryosections from DCVC-treated animals (Fig. 4A). No protein adducts were detected in kidney cryosections from vehicle controls (Fig. 4B). No protein adducts were detected in efferent ducts or epididymides from DCVC-treated animals (data not shown). Western blot analysis was used to confirm these results in subcellular fractions prepared from treated (Fig. 4C) and control animals (Fig. 4D). A single protein adduct (43 kDa) was detected in kidney cytosol (lane 1, Fig. 4C) and mitochondria (lane 2, Fig. 4C) from DCVC-treated animals that corresponded in size to dichloroacetylated protein adducts detected in kidney cytosol and mitochondria following in vivo dosing of rats with perchloroethylene (tetrachloroethylene), a related chlorinated solvent [36]. A weakly staining band was detected in epididymal cytosol from DCVC-treated animals that corresponded in size to the immunoreactive kidney band (Fig. 4C, lane 3). No adducts were detected in epididymal mitochondria from treated animals (Fig. 4C, lane 4) or in either subcellular fraction from the efferent ducts of treated animals (data not shown). In vehicle controls, no comparably sized bands were detected in kidney or epididymal fractions (Fig. 4D, lanes 5–8) or in efferent duct fractions (data not shown).
CYP2E1 Detected in Efferent Ducts and Epididymis
The presence of CYP2E1 was investigated to determine the role of cytochrome P450-dependent metabolism in adduct formation in the epididymis and efferent ducts. The CYP2E1 was localized in cryosections from untreated animals (Fig. 5). The detection of CYP2E1 was qualitatively high in the efferent ducts (Fig. 5A) when compared to the initial segment (Fig. 5B). The corpus segment of the epididymis was slightly immunoreactive (Fig. 5C). The caput and cauda were only weakly immunoreactive (data not shown). Liver sections from the same animals showed a centrilobular localization of CYP2E1 (Fig. 5D), as previously reported [37]. Western blot analysis confirmed the presence of CYP2E1 (Fig. 6). A single band was detected in liver (Fig. 6, lane 1) and kidney microsomes (Fig. 6, lane 2) prepared from untreated rats. A comparably sized band was detected in efferent duct microsomes (Fig. 6, lane 4). No protein band was visible in microsomes prepared from the whole epididymis (Fig. 6, lane 3) or in replicate blots incubated with normal serum in place of the CYP2E1 antibody (data not shown).
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Efferent Ductule Metabolism of TCE Demonstrated In Vitro
An in vitro assay was used to determine if epididymal and efferent duct microsomes are capable of metabolizing TCE. Detection by GC/MS of methylated chloral equivalents was used as an indication for the oxidative metabolism of TCE. Standard curves of methylated chloral equivalents were linear to 0.01 µg/ml (R2 = 0.9958). The rate of formation of chloral equivalents (Vmax) in the liver microsomes was 2288 ± 476 pmol min-1 mg protein-1 and is similar to previously published rates [34]. The Vmax for efferent duct microsomes was 19 ± 2 pmol min-1 mg protein-1. No chloral formation was detected in microsomes prepared from the whole epididymis (total protein, 0.25–10.0 mg) when incubated with 2–10 mM TCE (detection sensitivity, 0.009 µg/ml). Enzyme activities were normalized to total protein concentration and incubation time. Chloral formation was linear with protein over the range of 0.25–1.00 mg total protein in efferent duct microsomes (Fig. 7A) and liver microsomes (Fig. 7B).
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Immunoinhibition of TCE Metabolism in the Efferent Ducts
Preadsorbing efferent duct microsomes with increasing concentrations of a polyclonal anti-CYP2E1 antibody before TCE incubation inhibited oxidative metabolism and chloral formation. The resulting level of product formation was measured by GC/MS as described earlier. Anti-CYP2E1 inhibited TCE metabolism in the efferent ducts in a concentration-dependent manner to 77% ± 9% (Fig. 8A). The rate of liver microsomal metabolism of TCE decreased 32% ± 13% at a ratio of 0.05 mg of anti-CYP2E1 per 1 mg of microsomal protein (Fig. 8B) but did not decrease with the addition of more antibody.
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Dichloroacetyl Adduct Detection
In vitro TCE exposure resulted in the formation of several protein adducts in reproductive tissues, as detected by Western blot analysis (Fig. 9). Liver microsomes incubated with 5 mM TCE showed an overall diffuse staining and several visible protein bands (Fig. 9A, lane 2), including bands of approximately 45–56 and 97 kDa that were not detected in liver microsomes incubated without TCE (Fig. 9A, lane 3). Efferent duct microsomes incubated with TCE also showed a distinct pattern of protein adduction (Fig. 9B, lane 4). The most visible protein adducts in exposed efferent duct microsomes were approximately 56 and approximately 50 kDa. These adducts were not present in efferent duct microsomes incubated without TCE (Fig. 9B, lane 5). One protein band (56 kDa) was detected in TCE-exposed epididymal microsomes (Fig. 9C, lane 6) and was distinct from bands detected in unexposed epididymal microsomes (Fig. 9C, lane 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to TCE is associated with a variety of adverse reproductive effects in animals and humans. Several studies have reported reductions in sperm quality (count, motility, and morphology) [2, 3] and decreased fertility [6, 7] in men exposed to TCE. Associations between TCE exposure and decreased sperm quality have also been reported in animals [8–10], although the results appear to be dependent on dose and route of exposure. Two studies have reported testis pathology following inhalation exposure [9, 41], but to our knowledge, no data indicate pathological changes in the epididymis following TCE exposure by any route of administration. Even without overt pathology, alterations in sperm count, motility, and fertilizing ability may indicate that the epididymis is a target of the toxic action of TCE. That is, TCE may act in subtle subcellular or biochemical ways to alter the integrity and function of the epididymis and associated sperm maturation processes.
One mechanism by which TCE can elicit damaging effects in the epididymis is by the formation of protein adducts. The covalent binding hypothesis suggests that covalent modification of critical cellular proteins may be an initiating event in target organ toxicities [36]. Previous studies have shown that TCE exposure can result in the binding of an activated form of TCE to cellular macromolecules [29, 49]. Dichloroacetyl chloride, an early metabolite of TCE, has been found to bind lysine residues with a high affinity [33]. In addition, TCE-protein adducts have been shown to alter cellular structure and function in the liver and kidney [12, 14, 24]. Results from the present study show, to our knowledge for the first time, that TCE can adduct proteins in the epididymis and efferent ducts of treated animals.
Both glutathione and cytochrome P450-mediated metabolism of TCE can result in the formation of protein adducts (Fig. 1) [11]. Adducts that arise in the kidney are generally associated with glutathione-mediated metabolism of TCE and formation of reactive intermediates by the enzyme ß-lyase [17, 18]. The liver has a high concentration of cytochrome P450s, including CYP2E1, the major isoform responsible for the oxidative metabolism of TCE [21]. Adducts detected in the liver are generally associated with CYP2E1 activity [27, 42]. Because of the potential involvement of both cytochrome P450- and glutathione-mediated processes, it was important to investigate the formation of adducts by either pathway within the epididymis and efferent ducts as a way to explain the male reproductive effects of TCE.
Glutathione and glutathione S-transferases have been localized in both rat and human epididymides and are thought to be involved in detoxification processes that protect sperm during epididymal transport [43, 44]. Because of the presence of these macromolecules, it may be possible that the epididymis can metabolize TCE by a glutathione-mediated pathway. If the enzyme ß-lyase is present and active in the epididymis, TCE metabolites (i.e., cysteine conjugates) could be bioactivated and adduct epididymal proteins in an analogous manner to the kidney [14]. However, results from the present study show only a slightly positive localization of ß-lyase in the efferent ducts and epididymis. Western blot analysis detected a soluble form of ß-lyase in the epididymis and efferent ducts, whereas both mitochondrial and soluble forms of ß-lyase were detected in the kidney. When comparing the results of immunohistochemistry and Western blot analysis, it appears that the diffuse staining of ß-lyase in epididymal and efferent duct tissue can be explained by the soluble nature of the immunoreactive protein. The more distinct staining in kidney sections may be caused by the presence of at least the two forms of ß-lyase and their more specific cellular localization.
The soluble form of kidney ß-lyase has been identified as glutamine transaminase K and is expressed in the rat cytosol and mitochondrial matrix [16]. It is possible that the soluble epididymal protein detected with anti-ß-lyase antibody is a related protein. However, it is the mitochondrial form, rather than the soluble form, of ß-lyase that has been implicated in the nephrotoxicity of both TCE and DCVC [16, 25]. The lack of a mitochondrial form of ß-lyase in the epididymis or efferent ducts would suggest that the epididymis and efferent ducts may not be able to bioactivate cysteine S-conjugates. To test this hypothesis, the presence of protein adducts was investigated in tissues collected from animals dosed with a single i.p. injection of the cysteine-conjugate DCVC. No protein adducts were detected in tissue sections from any region of the epididymis or efferent ducts of treated animals. This is in contrast to the formation of adducts in the corpus of the epididymis and the efferent ducts following in vivo TCE dosing.
There may be several explanations for the difference in adducts found following in vivo TCE and DCVC treatment. First, TCE and DCVC have very different physicochemical properties that affect their distribution and metabolism in the body. The TCE is lipid soluble and is readily sequestered into adipose tissue [12]. Differential sequestration of TCE in tissues with high fat content [12] might be particularly relevant to the epididymis and efferent ducts because of the fat pad surrounding these structures. Conversely, DCVC is a water-soluble compound that does not diffuse across cell membranes or readily enter fat stores [45]. Additionally, DCVC is distributed in the body via plasma [45]. The low level of blood perfusion to the epididymis [46] could limit the amount of DCVC reaching the epididymis and efferent ducts. It is possible that the lack of protein adducts detected in the epididymis and efferent ducts following dosing with DCVC may be caused by the low level of DCVC reaching the target tissue.
Recent data indicate that the kidney uptakes DCVC across the brush border by a Na+-dependent cotransport system [47]. The nonciliated cells within the efferent ducts contain a brush border and several ion transporters that are similar to those in the kidney [48]. Because of the similarities in solute transport, it may be possible for the epididymis and efferent ducts to sequester DCVC in a manner similar to that of the kidney, although to our knowledge, this has never been investigated. Even if DCVC could enter ductal epithelial cells, it is unlikely that protein adducts could form, because our data indicate the lack of a mitochondrial form of ß-lyase, the enzyme considered to be necessary for the bioactivation of DCVC to protein-binding reactive intermediates [15], in the epididymis and efferent ducts.
There appears to be little evidence for the presence and activity of ß-lyase in the epididymis and efferent ducts, but data from the present study indicate that these tissues may be capable of oxidative metabolism of TCE. The CYP2E1, the major isoform responsible for the oxidative metabolism of TCE [21], was detected in the efferent ducts and the corpus of the epididymis. Western blot analysis confirmed these results in the efferent duct microsomes, although no positive band was detected in microsomes prepared from the whole epididymis. This is not consistent with the immunohistochemical results in epididymal tissue sections. It is possible that the slightly positive cells in the corpus were diluted by a larger population of nonreactive cells in the remainder of the epididymis and therefore could not be detected by Western blot analysis.
Because oxidative metabolism is implicated in TCE liver toxicity and protein adduction [11], it was important to determine whether the epididymis and efferent ducts could metabolize TCE. Chloral hydrate formation is strongly correlated with CYP2E1 metabolism of TCE [34], and this was used as a proxy for TCE metabolism in an in vitro system. Analysis by GC/MS showed that TCE metabolism occurs in the efferent ducts but not in the epididymis. These results are consistent with those of the Western blot analysis and may indicate that the rat epididymis as a whole might not be capable of metabolizing TCE. The CYP2E1 has been detected in the mouse epididymis [41], although no details were given about specific localization within the epididymis. Metabolism of TCE was measured in microsomal fractions prepared from whole-mouse epididymides (i.e., three individual pooled samples prepared from the epididymides of 50 mice each) [41]. The specific activity of TCE metabolism was approximately 0.2 pmol chloral min-1 mg protein-1 [41]. The present study indicates a significantly higher level of enzyme activity (19 ± 2 pmol chloral min-1 mg protein-) in the rat efferent ducts.
Recent studies indicate that other isoforms of cytochrome P450, including CYP1A1, 2B1, and 2C11, may be capable of metabolizing TCE [49]. The observed 100-fold difference in Vmax values between liver and efferent duct metabolism of TCE in the present study may be explained by differences in tissue-specific expression of CYP2E1 or perhaps by the presence of multiple enzymes capable of metabolizing TCE. To test the contribution of CYP2E1 to TCE metabolism, we measured chloral formation after immunoinhibiting efferent duct and liver microsomes with increasing concentrations of an antibody to CYP2E1. The addition of anti-CYP2E1 antibody decreased efferent duct metabolism of TCE by 77%. This suggests that the efferent ducts metabolize TCE and that a majority of the metabolism results from CYP2E1 activity. In contrast, TCE metabolism by the liver was only inhibited 32%, suggesting that TCE metabolism is only partially dependent on CYP2E1 in the liver.
Because TCE is known to form adducts following P450-mediated oxidation, we tested for the presence of protein adducts following microsomal metabolism of TCE. Microsomes contain a variety of cytochrome P450s, including CYP2E1 [32], and little or no ß-lyase [19]. Therefore, any adducts present would most likely result from oxidative metabolism of TCE. In the present study, several dichloroacetylated protein adducts were detected following in vitro TCE exposure. One adduct detected in exposed efferent duct microsomes corresponded in molecular weight to CYP2E1. This suggests that CYP2E1 may be one protein adducted within the efferent ducts. A single protein adduct (56 kDa) was detected in TCE-exposed epididymal microsomes that was different from unexposed control microsomes. Because of the higher molecular weight, this protein may not be CYP2E1, but its presence suggests that the epididymis may still be a target of protein binding following TCE bioactivation. Alternatively, epididymal proteins may be adducted by TCE metabolites that are formed upstream in the efferent ducts, or even in the testes, which have been shown to possess metabolically active CYP2E1 [50]. Work by Cai and Guengerich [42] show that the dominant pathway for TCE adduct formation is through oxidative metabolism. Specifically, they postulate a mechanism by which reactive TCE intermediates rearrange to chloroacetyls, which react with lysine residues in proteins and produce stable protein adducts [42].
The results from the present study are, to our knowledge, the first to show the formation of protein adducts in reproductive tissues following TCE exposure. These data support the hypothesis that TCE acts through an in situ bioactivation mechanism in the efferent ducts and epididymis to cause target-tissue toxicity and adverse reproductive effects. In a soon-to-be-published study from this laboratory, in vitro fertilization was conducted with sperm from TCE-dosed male rats and zona-free eggs from untreated female rats. Our data indicate that males exposed to the highest concentration of TCE had markedly reduced fertilizing rates in comparison to control males in the absence of any difference in sperm count or motility (unpublished results). Further work is exploring the possibility that oxidative mechanisms may be involved in the reduced ability of sperm from TCE treated animals to fertilize oocytes.
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FOOTNOTES |
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2 Correspondence: Marion G. Miller, Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, California, 95616. FAX: 530 752 3394; gmiller{at}ucdavis.edu
Received: 19 December 2002.
First decision: 15 January 2003.
Accepted: 23 April 2003.
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