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An unidentified peak in a drug substance or drug product chromatogram raises many questions. What is the impurity? What is the source of the impurity? How much impurity is there? Can the impurity level be reduced or eliminated? Is the impurity toxic? What do we do now? The answers to these questions are typically provided by scientists in chemistry, manufacturing and controls (CMC) and nonclinical toxicology with the single objective of assuring that unavoidable drug impurities induce no risk or an acceptable level of risk for the intended indication and the stage of development. To help address these issues, the International Council for Harmonisation (ICH) guidelines for impurities in drug substance (Q3A) and drug product (Q3B), and for genotoxic impurities (M7) have been adopted and implemented in the United States, Europe, and many other countries around the world. The guidelines address how impurities in drug substances and drug products should be reported, identified, and/or qualified. This Mini Review offers practical insights regarding drug substance and drug product impurities and next steps for the toxicologist when impurity levels exceed the ICH reporting, identification, and/or qualification thresholds. The information presented is largely derived from the following ICH Harmonised Tripartite Guidelines:Q3A(R2) Impurities in New Drug Substances (October 2006), Q3B(R2) Impurities in New Drug Products (June 2006), and M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (June 2015) and/or the referenced literature. Insights regarding acceptible amounts of residual solvents and the calculation of permitted dialy exposures will be the subject of another review.

The Food and Drug Administration’s (FDAs) Medical Countermeasures Initiative was launched in 2010. The initiative is intended to coordinate health care responses to potential chemical, biological, radiological, nuclear, and emerging infectious disease threats by ensuring that the medical countermeasures (eg, drugs, vaccines, and diagnostic tests) that are developed are safe, effective, and secure. The incentives for developing these medical counter-measures include the potential to secure a Priority Review Voucher under the 21st Century Cures Act and the potential for an extended 5 years of exclusivity for the chemical entity. These incentives have increased interest in medical countermeasures and in the use of the Animal Rule to demonstrate substantial evidence of efficacy. This Mini Review offers practical insights into the purpose, challenges, and benefits of using of the Animal Rule as the regulatory route for new drug approval. The information presented is derived from the FDAs Guidance document [1].

Wildlife populations from contaminated ecosystems display a variety of reproductive alterations, including cryptorchidism in the Florida panther, small baculum in young male otters, small penises in alligators, sex reversal in fish, and altered social behavior in birds. The formation of biologically plausible hypotheses regarding disruption of reproduction in wildlife can be facilitated by mechanistic studies on laboratory animals. To this end, we are investigating the in vivo and in vitro effects of endocrine-disrupting toxicants in rodents. In vitro studies have used receptor binding and transfected cell assays to confirm the suspected mechanism of action, whereas in vivo rodent studies examine altered sexual differentiation. Antiandrogenic pesticides compete with the natural ligands for both rat and human androgen receptors, block androgen-induced gene expression in vitro and in vivo, delay puberty, reduce sex accessory gland size, and alter male rat sex differentiation. In contrast, xenoestrogens affect female central nervous system sex differentiation and fecundity without producing malformations or infertility in male offspring. Prenatal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or the TCDD-like polychlorinated biphenyls produce yet another profile of effects in the offspring, reducing numbers of ejaculated sperm in male progeny and inducing urogenital malformations in females. Although phthalates are reported to be estrogenic in vitro, in vivo exposure causes developmental alterations that more closely resemble antiandrogenic activity. The mammalian data indicate that exposure to endocrine-disrupting chemicals produces effects that are pathognomonic for mechanisms by which they act. Mechanistic information derived from mammalian studies can enhance our ability to predict toxicant effects on reproduction in fish and wildlife.

Male rats exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) display reduced fertility as a consequence of the direct action of TCDD on the epididymides, as well as delayed puberty and altered reproductive organ weights. The current study provides dose-response data for the reproductive effects of TCDD, administered during pregnancy, with an emphasis on the effects of TCDD on testicular, epididymal, and ejaculated sperm numbers. Long Evans Hooded rats were dosed by gavage with 0, 0.05, 0.20, or 0.80 microg TCDD/kg on Day 15 of gestation. After birth, growth, viability, and developmental landmarks were monitored in both male and female offspring. Shortly after puberty (49 and 63 days of age) and at 15 months of age, male offspring were necropsied. Growth and viability of the pups were reduced only at 0.80 microg TCDD/kg, eye opening was accelerated (all dosage groups), and puberty was delayed (at 0.20 and 0.80 microg TCDD/kg). Treated progeny displayed transient reductions in ventral prostate and seminal vesicle weights, while epididymal sperm reserves and glans penis size were permanently reduced. Ejaculated sperm numbers were reduced (45% in the 0.8 and by 25% in the 0.05 and 0.2 microg TCDD/kg dosage groups) to a greater degree than were cauda or caput/corpus epididymal or testicular (unaffected) sperm numbers. In conclusion, administration of TCDD on Day 15 of pregnancy at 0.05 microg/kg altered eye opening and reduced ejaculated sperm counts, while higher dosage levels also delayed puberty and permanently reduced cauda epididymal sperm reserves.

Vinclozolin and p,p'-DDE induce antiandrogenic developmental effects in vivo and are potent inhibitors of androgen receptor (AR) binding and AR-dependent gene expression in vitro. To determine whether this molecular mechanism is operative in vivo, the effects of these compounds on two androgen-regulated prostatic mRNAs were studied. Rats were sham operated or castrated and immediately implanted with one or two empty 2.5-cm silastic capsules or with one (1x) or two (2x) 2.5-cm capsules containing testosterone (T). T-implanted rats were treated by gavage for 4 days with vehicle (corn oil), vinclozolin (200 mg/kg/day), p,p'-DDE (200 mg/kg/day), or the antiandrogen flutamide (100 mg/kg/day) as a positive control. Vinclozolin, p,p'-DDE, and flutamide all induced a reciprocal decline in seminal vesicle (p < 0.01) and prostate (p < 0.01) weight as well as a reduction in immunohistochemical staining of AR in epididymal nuclei compared to vehicle-treated T-implanted controls. Specific AR antagonism was assessed by determining the ability of these chemicals to induce a testosterone-repressed prostatic message (i.e., TRPM-2) and/or repress a testosterone-induced prostatic message (i.e., prostatein subunit C3). Densitometry scans of Northern blots indicated that vinclozolin, p,p'-DDE, and flutamide each induced TRPM-2 mRNA and repressed C3 mRNA compared to vehicle-treated T-implanted controls. These antiandrogenic effects were competitively reduced in castrate rats implanted with two 2.5-cm T capsules (2x), where serum T levels were elevated more than twofold above physiological levels. Taken together, these data indicate that vinclozolin and p,p'-DDE act as antiandrogens in vivo by altering the expression of androgen-dependent genes.

Vinclozolin, a dicarboximide fungicide, alters morphological sex differentiation in male rats following perinatal exposure. The occurrence of these abnormalities correlates with the in vivo formation of two antiandrogenic metabolites of vinclozolin, (i.e. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (Mt) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2)), which are potent inhibitors of rat androgen receptor binding. As steroid hormone receptors exhibit promiscuity in their ability to bind different ligands, the present study evaluated the ability of these vinclozolin metabolites to bind to the estrogen (ER) and progesterone (PR) receptors in vitro, and to alter ER and PR function following in vivo exposure. To this end, in vitro ligand binding assays demonstrated that both M1 and M2 can compete with endogenous ligand for binding to the PR (Ki = 400 and 60 microM, respectively). In contrast, neither metabolite exhibited the ability to bind ER. Subsequent in vivo studies to evaluate the potential of vinclozolin to alter ER or PR function demonstrate that, (1) the estrogen-dependent increases in uterine weight and PR induction were not altered by vinclozolin; (2) the distribution of nuclear and cytosolic PR was not altered following short-term vinclozolin exposure; and (3) vinclozolin did not disrupt ovulation in cycling female rats. These studies indicate that although vinclozolin metabolites can compete for binding to the PR in vitro, concentrations of these metabolites do not reach sufficient levels to disrupt female reproductive function following short-term in vivo exposure to vinclozolin. In addition, these studies demonstrate the importance of correlating in vitro receptor binding data with in vivo studies in order to understand the physiological consequences of exposure to environmental toxicants.

The increase in the number of reports of abnormalities in male sex development in wildlife and humans coincided with the introduction of 'oestrogenic' chemicals such as DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) into the environment. Although these phenotypic alterations are thought to be mediated by the oestrogen receptor, they are also consistent with inhibition of androgen receptor-mediated events. Here we report that the major and persistent DDT metabolite, p,p'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), has little ability to bind the oestrogen receptor, but inhibits androgen binding to the androgen receptor, androgen-induced transcriptional activity, and androgen action in developing, pubertal and adult male rats. The results suggest that abnormalities in male sex development induced by p,p'-DDE and related environmental chemicals may be mediated at the level of the androgen receptor.

The ability of foreign compounds to affect the functioning of various endocrine systems is currently thought responsible for a wide variety of effects. The presentations in this Symposium reviewed the evidence for and against the involvement of endocrine systems in several different aspects of reproduction. The mechanism behind the ability of a triazine herbicide to cause enhanced appearance of mammary tumors in one strain of female rats is reviewed by Stevens. The data suggest that enhanced aging, not direct mammary modulation, is responsible. Dietary phytoestrogens, the mediators of their actions, their effects in various biological systems, and the relationships between phytoestrogen producers and consumers are all provocatively and succinctly reviewed by Hughes. Kelce presents the strategy used to dissect the mode and mechanisms of action of a fungicide that opened a new awareness in reproductive toxicology: the possibility of xenobiotics being antiandrogens. Finally, to heighten our understanding of the interplay among hormonal systems in vivo, Hess reviews the data that show that androgens are not the only hormones important in the development of the male reproductive system: the pituitary is shown to play a critical role at specific stages of development. The breadth of these presentations, and the implications of their findings, should make us pause and realize how much there is still to discover about the interaction between the reproductive system and anthropogenic compounds.

Decades ago it was reported that when male rats were exposed to chloroethylmethanesulfonate (CEMS) for 5 days prior to weekly matings with untreated females, the second mating resulted in reduced litter size. Since fertility was not assessed at earlier time points, it was not possible to determine whether CEMS exerted any effects on sperm in the epididymis. In this study, we used a 4-day exposure and assessed multiple reproductive endpoints on Day 5 to characterize effects of CEMS exposure (6.25-25 mg/kg) on Leydig cells and the epididymis. Exposure to CEMS caused a dose-related decline in serum testosterone (T) levels. This occurred at a dose lower than that required to decrease T production in vitro by testicular parenchyma. The in vitro decline was not attributed to a decrease in maximal hCG-stimulated T production, but to a decrease in unstimulated T production. CEMS was 5-fold less sensitive than ethane dimethanesulfonate (EDS) in reducing maximal hCG-stimulated T production. To control for alterations in the epididymis resulting from decreased serum T alone, T was implanted in CEMS-treated animals to maintain serum T at a concentration similar to that found in normal rats. This exogenous T failed to prevent the CEMS-induced decrease in the weight of the caput/corpus epididymidis but did prevent the CEMS-induced decrease in seminal vesicle weight. Implantation of T failed to prevent the CEMS-induced reduction in sperm reserves in the cauda epididymidis, and it failed to prevent the CEMS-induced alterations in the histology of both the corpus and proximal cauda epididymidis. The height of the epithelium in both of these regions was increased, and clear cells disappeared from the proximal cauda epididymidis. These results demonstrate that CEMS might alter the ability of the Leydig cell to respond to LH stimulation in vivo, and that alterations in the structure and function of the epididymis occur even when the serum concentration of T is maintained.

Recent studies with vinclozolin, a dicarboximide fungicide, demonstrate that perinatal exposure to 100 mg vinclozolin/kg/day from Gestational Day 14 through Postnatal Day 3 inhibits morphological sex differentiation. At 1 year, treated male rats exhibited hypospadias, cleft phallus, suprainguinal ectopic testes, a vaginal pouch, epididymal and testicular granulomas, and atrophic seminal vesicles and ventral prostate glands. This pattern of malformations suggests that this fungicide possesses antiandrogenic activity. To test this hypothesis, we examined the ability of vinclozolin to inhibit the conversion of testosterone to the more potent androgen 5 alpha-dihydrotestosterone via 5 alpha-reductase (EC 1.3.1.22) and to compete with androgen for binding to the androgen receptor. The results indicate that neither vinclozolin nor its degradation products, 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), inhibit 5 alpha-reductase activity. Although the ability of vinclozolin to compete with androgen for binding to the androgen receptor was weak (Ki > 700 microM), the two vinclozolin metabolites, M1 and M2, were effective antagonists of androgen receptor binding (Ki = 92 and 9.7 microM, respectively). As the concentrations of M1 in the serum of pregnant rats and their pups on Postnatal Day 3 meet or exceed the in vitro Ki for androgen receptor inhibition, we suggest that the demasculinizing effects of vinclozolin exposure in vivo also may be mediated via the antiandrogenic metabolites M1 and/or M2.

Ethane-dimethanesulfonate (EDS) has been shown to selectively kill Leydig cells and depress testosterone production in adult rats. A recent study has shown that immature rat Leydig cells are less sensitive to EDS exposure. There is evidence that the rabbit metabolizes EDS to methane sulfonic acid more rapidly than does the rat, reducing exposure to the parent compound. In the study reported here, we examined the effects of EDS on the Leydig cells in both adult and immature rabbits and compared the effects found with those previously reported in the rat. In vivo, EDS exposure demonstrated that Leydig cells from adult rabbits were affected, with both serum and interstitial testosterone production depressed. EDS effects in adult rabbits and rats were compared by exposing explants of testicular parenchyma to EDS in vitro and evaluating testosterone production. With this procedure, the rabbit testis was less sensitive to EDS treatment than the rat, with a 50% reduction rate (EC5 0 ) achieved with 2026 ItM EDS for the rabbit and with 336 tIM EDS for the rat. Perfusion of adult and immature rabbit testis with 430 M EDS demonstrated the insensitivity of the immature testis to EDS exposure: adult testosterone production was reduced 50% in 3.5 h, whereas no diminution was found in the immature rabbit. EDS exposure of interstitial cell preparations further demonstrated the insensitivity of immature rabbit Leydig cells, with an EC50 of 4397 FLM compared to an EC5, of 1137 ptM EDS in adult preparations. These and previously reported data in the rat provide evidence for the following order in Leydig cell sensitivity to EDS exposure: adult rat > adult rabbit > immature rat >> immature rabbit.

Yang et al. (2011) used the in vitro E-SCREEN assay to infer that health risks from “estrogenic” plastics can be eliminated by using their proprietary materials, processes, and products to manufacture plastics. An in vitro cell proliferation assay such as the E-SCREEN is a sensitive indicator of in vitro estrogen agonist activity and potential estrogenic activity in vivo (e.g., in the rat uterotrophic assay). However, in vitro properties may not manifest in in vivo activity, and neither demonstrates a health risk. Without definitive evidence that in vivo activity leads to adverse health effects, the results of Yang et al. are unconvincing and fail to support changing current manufacturing processes for plastics. The value of in vitro and in vivo estrogenic assays for predicting adverse health effects is largely untested but would need to account for actual exposure levels, metabolism, distribution, excretion, and the affinity of parent compounds and metabolites for estrogen receptor binding and transcriptional activation relative to and in competition with physiological levels of potent endogenous hormones. The combined effects of these exposures would also need to be assessed in the context of dietary (e.g., milk, cheeses, vegetables, meats, and other foodstuffs) and environmental estrogens. An excellent in vitro/in vivo study of combined effects (Charles et al. 2007) showed that while relatively high levels of a putative synthetic estrogen mixture increased the estrogenic action of common dietary phytoestrogens, low levels were without effect. Thus, sensitive in vitro detection may not portend estrogenic effects amid the endogenous and dietary hormonal milieu. Yang et al. (2011) made inferences about the safety of plastic food packages, but it is unfortunate that they did not use an extraction method that was approved by the U.S. Food and Drug Administration (FDA 2007). This would have improved the reliability and applicability of their results. Although food typically contacts only the inside surface of containers, Yang et al. extracted materials from 4-mm squares of cut plastic, exposing the inside, outside, and cut surfaces to the extraction medium. Substances may leach into food from the exposed surface of a plastic container but do not typically migrate through the plastic layer (Franz and Welle 2009); thus Yang et al.’s extraction method differs from FDA-approved methods and the way foods normally contact containers. Experimental error was not reported, making comparison of these results with standard methods impossible. In the study by Yang et al. (2011), irradiation methods for simulating “stress” were not well characterized, but they appear to have involved all surfaces of the plastic squares. However, even clear plastics can filter ultraviolet (UV) rays, reducing the potential irradiation of inside container surfaces. Similarly, colorants were added to the extraction mixture; however, during the production of plastics, colorants are embedded and tightly linked. The extent to which these procedures may have confounded the data cannot be known, but the resulting tested extracts may be substantially different from residues that could enter food from plastic containers. Yang et al. (2011) indicated that without increasing production costs, they can identify and/or have developed monomers, additives, and processing agents that lack estrogenic activity. This conclusion appears to derive from data for resins P1, P2, P3, P4, P19, and maybe P18 in their Table 3. In the text the authors noted six MCF-7 assays, but it is unclear whether a single assay was conducted for each of the six stressor and extraction combinations (microwave, UV, autoclave, saline, and ethanol) or whether the whole series was completed six times. Regardless, the authors provided no estimate of assay variance, making it difficult to differentiate real differences from experimental error. In addition, the relative safety of these new agents, particularly antiandrogenic potential, has yet to be resolved. In conclusion, Yang et al. (2011) provided interesting observations but failed to acknowledge the significant limitations of their observations to human health risk assessment. They relied on a very limited in vitro screen to model a very complex system, and those reviewing the study should be aware of the limitations of the approach and the interpretation of such data.

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

Steroid hormone receptors regulate embryonic development and sex differentiation by acting as ligand inducible transcription factors. Disrupting these processes can result in transient yet irreversible developmental defects. Several environmental chemicals have recently been identified with antiandrogen activity that have the potential to disrupt normal male sex development in utero. Detection of chemicals with the potential to disrupt normal androgen action is critical to protect human and ecological health. The molecular mechanisms by which several of these environmental chemicals act have been characterized and yielded insight into the development of in vitro and in vivo screening assays to detect chemicals with antiandrogen activity. These mechanisms and efforts to develop screening assays are discussed in the context of current endocrine disruptor screening and testing strategies.

Androgens secreted by the testes bind the androgen receptor in developing target tissues to induce the expression of genes required for male sexual differentiation and development. Androgen concentration and androgen receptor levels vary in male reproductive target tissues during development. Exposure to environmental androgen antagonists during critical windows of fetal and postnatal development can inhibit male sexual development by blocking transcription of androgen-dependent genes. As the sensitivity to androgen antagonists under conditions of varying androgen concentrations and varying androgen receptor levels is unknown, we used a luciferase reporter assay to investigate the transcriptional effects of a known androgen antagonist (the vinclozolin metabolite M2) at different androgen concentrations and different androgen receptor levels. The ability of M2 to inhibit transcription was greater at lower concentrations of androgen (5alpha-dihydrotestosterone) and androgen receptor. The data were modeled to determine the dose-response surface of M2 and androgen receptor concentrations at different 5alpha-dihydrotestosterone levels and the relationship of the 3 components to the response. The model and hypothesis testing results suggest that, at 0.01 and 0.1 nM 5alpha-dihydrotestosterone concentrations within the expected in vivo range of free androgen levels during development, the response-surface shapes were similar and the interaction of the androgen receptor and M2 concentrations to the response were similarly antagonistic. Thus, two components of the developmental stage, androgen and androgen receptor concentrations, are critical for sensitivity to the inhibitory effects of an androgen antagonist.

An enriched fraction of mesenchymal-like cells was isolated from the testes of 21 day old rats. Testosterone production (ng/10(6) cells.24 hours) by these cells when cultured in vitro was measured by radioimmunoassay of HPLC-purified extracts of culture medium. In the presence of LH + DHT there was a significant increase in testosterone secretion from 22 +/- 10 ng after day 1 of culture to 284 +/- 75 ng on day 3 (P less than 0.01). By contrast, LH or DHT alone were without significant effect. We conclude that LH alone is insufficient but that androgen and LH induce mesenchymal-like Leydig cell precursors from 21 day old rats to produce testosterone.

Industrial chemicals and environmental pollutants can disrupt reproductive development in wildlife and humans by altering the synthesis, transport, action, or elimination of gonadal steroid hormones. Steroid hormones control fundamental events in embryonic development and sex differentiation by binding to their cognate nuclear receptors, which act as steroid-inducible transcription factors to activate or repress transcription of target genes. The consequences of disrupting these events can be especially profound during embryonic development because the role of steroid hormones is crucial in controlling transient and irreversible developmental processes. Recent studies suggest that certain industrial pollutants and environmental pesticides have the potential to alter male sex development and reproductive processes in wildlife and human populations by acting as environmental antiandrogens (1–3). In some cases, laboratory studies have confirmed abnormalities of reproductive development observed in the field and have provided mechanisms to explain the disruptive effects of these environmental chemicals.

The effects of ‘endocrine disrupters’ on the health of human and wildlife populations are currently receiving a great deal of interest from the popular press and the scientific community. At the present, scientists are grappling with research needs in this new area and the need for new test protocols to screen for endocrine effects. The impetus for this new approach began at a Work Session in July of 1991 on ‘Chemically Induced Alterations in Sexual Development: the Wildlife/Human Connection’ attended by a multidisciplinary group of experts (Colborn and Clement, 1992). A consensus statement from this Work Session (Colborn and Clement, 1992: 1–6) concluded that ‘Many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals, including fish, wildlife, and humans. The consequences of such disruption can be profound because of the crucial role hormones play in controlling development.’ In addition, one portion of the consensus statement indicated that many wildlife populations were already affected by such compounds, with effects (relevant to this chapter) including demasculinization and feminization of male fish, birds and mammals and defeminization and masculinzation of female fish and birds.

Publisher Summary This chapter provides an overview of a study that determines the biochemical mechanism responsible for the antiandrogenic effects of vinclozolin exposure. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) are degradation products, detectable both within the soil and leaves of treated plants and in the serum of treated rats. The study suggests that some of the detrimental effects of vinclozolin exposure are likely to be mediated via the formation of the antiandrogenic vinclozolin metabolites M1 and M2. It is demonstrated in this chapter that vinclozolin, M1, or M2 was unable to alter the ability of epididymal 5α-reductase to convert testosterone to 5α-dihydrotestosterone and/or 5α-androstane-3a, 17β-diol (IC50 > 1000μM). The primary vinclozolin degradation products, M1 and M2, act as pure competitive antagonists of androgen receptor binding with Ki values of 92 and 9.7 μM, respectively. Vinclozolin, the parent compound, does not appear to be capable of androgen receptor binding at concentrations that are likely to exist in vivo.

Distribution of [14C]Ethane Dimethanesulfonate in Immature and Adult Male Rats Following an Acute Exposure. Laskey, J. W., Kelce, W. R., Klinefelter, G. R., Gray, L. E., Jr., and Ewing, L. L. (1994). Fundam. Appl. Toxicol. 22, 319-327. In the adult rat, ethane dimethanesulfonate (EDS) reduces testosterone (T) production by killing Leydig cells. Studies have also shown that acute EDS administration produces transient infertility and epididymal effects. Although these later effects were believed to be indirect results of the reduced Leydig cell T production, it was recently found that the epididymal effects were partially a direct result of in vivo EDS treatment. In contrast to the Leydig cells of the adult rat, immature Leydig cells are affected by EDS only at doses four- to sixfold higher than those that affect mature Leydig cells. In fact, the Leydig cells of the adult rat seem to be uniquely susceptible to the cytotoxic effects of EDS. Steroidogenesis in other organs, like the adrenal and ovary, are unaffected in vivo at doses that eliminate T production in males. In addition, studies have shown that doses of EDS that kill Leydig cells in vitro , isolated from the testes of adult rats, have no effect on similarly exposed hepatocytes. Hence, it was the objective of this study to describe the distribution and temporal fate of EDS in target (testes and epididymides) and nontarget tissues in immature and adult male rats and to determine if this information would explain either the age- or tissue-related susceptibility to EDS. We have concluded from this study that tissue distribution, integrated in vivo EDS dose, and differences in EDS metabolism are not the only factors contributing to the difference in sensitivity. The information collected in this study will enable us to use in vitro EDS concentrations for examination of the mechanism of action at doses relevant to those produced in vivo.

In this study the gonadotoxic effects of 2-hexanone administered as a pretreatment and/or 1,2-dibromo-3-chloropropane administered as a challenge on the activity of several key steroidogenic enzymes in the rat testis were examined. Despite the absence of an effect when either treatment was administered individually, the pretreatment/challenge combination inhibited the steroidogenic capacity of the rat testis. The specific activity of testicular 17 alpha-hydroxylase was significantly reduced (P less than 0.05), with respect to the control, following the pretreatment/challenge combination. There were no significant differences between the control and the individual treatments. The inhibition of 17 alpha-hydroxylase activity occurred in the absence of significant differences in testis weight and testicular protein content. This inhibition of testicular steroidogenic enzyme activity was specific as the activity of another testicular enzyme, namely C17,20-lyase, was not affected by any treatment. The decline in rat testicular 17 alpha-hydroxylase activity 18 h after the 1,2-dibromo-3-chloropropane challenge precedes the reported alterations in the seminiferous epithelium following this same treatment. The results of the present study indicate that the steroidogenic cell of the testis, i.e. the Leydig cell, is a potential site for the primary toxic effects of these agents in the rat testis.

A reliable isocratic high performance liquid chromatography (HPLC) procedure has been developed for the separation and subsequent radiometric quantitation of delta 4-3-ketosteroid-5 alpha-oxidoreductase enzyme activity. The specificity of this HPLC procedure has been confirmed through the use of authentic androgen radioisotopes, linearity of detector response, and mass spectral analysis. In conjunction with this we have also developed a simple Sep-Pak sample preparation procedure which allows the uniform complete isolation of these androgens from epididymal tissue homogenates. Utilizing these procedures we have determined the regional distribution of 5 alpha - reductase in the epididymis of the CD-1 mouse. Regional differences in enzyme activity were found between the caput-corpus and cauda regions. Enzyme activity (specific activity) was higher in the caput-corpus region (28.98 pmol 5 alpha - reduced androgens formed/h/mg protein) than the cauda region (6.20 pmol 5 alpha - reduced androgens formed/h/mg protein).