Inter-laboratory control data for reproductive endpoints required in the OPPTS 870.3800/OECD 416 reproduction and fertility test.

BACKGROUND: The U.S. EPA revised the Reproduction and Fertility Effects Test Guideline (OPPTS 870.3800/OECD 416) in 1998, adding numerous endpoints in an effort to incorporate new methodologies, improve the sensitivity for detecting reproductive toxicants, and more efficiently utilize study animals. Many of these new endpoints have not been used in regulatory reproductive toxicology studies prior to their inclusion in the test guidelines; thus, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI) initiated the Reproductive Endpoints Project to examine the utility of these new endpoints. METHODS: This report provides a retrospective analysis of 43 multi-generation studies (16 in Wistar rats, 27 in Sprague-Dawley rats) conducted according to the latest version of the test guidelines. It focuses on vehicle (negative) control values (means and ranges) for the various endpoints to examine inter-laboratory variability. RESULTS: Based on the compiled data, the most variable endpoints across laboratories and their associated coefficients of variation (CV) for each generation were: percent abnormal sperm (166-205%), testicular spermatid concentration (126-147%), postimplantation loss (97-104%), primordial follicle counts (69%, only measured in P2 females), and epididymal sperm concentration (52-57%). Absolute and relative prostate and thymus weights, weanling uterine weights, and anogenital distance had CVs of 25-50%. Sources of variability included procedural differences between laboratories, inherent biological variability, and/or small sample sizes for some endpoints. CONCLUSIONS: These inter-laboratory control data provide a means for laboratories to review their performance on reproductive toxicity measures, and provide perspective for interpreting their own control data and data from treated animals.

Use of the laboratory rat as a model in endocrine disruptor screening and testing.

The screening and testing program the US Environmental Protection Agency (EPA) is currently developing to detect endocrine-disrupting chemicals (EDCs) is described. EDCs have been shown to alter the following activities: hypothalamic-pituitary-gonadal (HPG) function; estrogen, androgen, and thyroid hormone synthesis; and androgen and estrogen receptor-mediated effects in mammals and other animals. The value and limitations of mammalian in vivo assays are described that involve the use of the laboratory rat, the EPA Endocrine Disruptor Screening and Testing Advisory Committee species of choice. The discussion includes the evaluation of high-priority chemicals positive in the Tier 1 Screening (T1S) battery, and of subsequent testing in the Tier 2 (T2) battery, with additional short-term screening assays proposed for use in T1.5 to eliminate any uncertainty about T1S results. Descriptions include the in vivo uterotropic assay, which detects estrogens and antiestrogens; the pubertal female assay, which assesses steroidogenesis, antithyroid activity, antiestrogenicity, and HPG function; and the Hershberger assay, which detects the weight of androgen-dependent tissues in castrate-immature-male rats (antiandrogens). Of the several alternative mammalian in vivo assays proposed, a short-term pubertal male rat assay appears most promising for inclusion in T1 or T1.5. An additional in utero-lactational screening protocol is being evaluated, but appears to be better suited for T1.5 or T2 due to the size, complexity, and duration of the assay. The adult intact male assay, also proposed as an alternative for T1, attempts to identify EDCs in a hormonal battery, but has limited value as a screen due to lack of sensitivity and specificity. For Tier 2 testing, the number of endocrine-sensitive endpoints and offspring (F1) examined in multigenerational tests must be thoughtfully expanded for EDCs on a mode-of-action-specific basis, with consideration given to tailoring T2 based on the results of T1S.

Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate.

Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.

Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues.

Male offspring exposed in utero to antiandrogens often display alterations in androgen-dependent developmental markers (e.g., anogenital distance [AGD], nipple retention) together with clearly adverse responses such as genital malformations and reproductive tract lesions. The objectives of this study were to determine whether in utero exposure to flutamide results in permanent changes in male AGD and nipple retention, characterize the dose-response relationship between flutamide-mediated alterations in these landmarks and clearly adverse antiandrogenic effects, and establish the predictive value and relationship between AGD and nipple retention, and other adverse manifestations. Male offspring were exposed in utero to 0, 6.25, 12.5, 25, or 50 mg/kg/day (po) of flutamide from gestation days 12 to 21. Offspring were uniquely identified at birth, and various androgen-mediated end points (AGD, areola/nipple retention, cryptorchidism, reproductive tract weights, and malformation incidence) were examined throughout life. In utero flutamide exposure significantly decreased the AGD on postnatal day (PND) 1 and increased areola/nipple retention in male rats on PND 13. Flutamide-induced alterations in AGD and areolae/nipples in early postnatal life correlated with a reduction in AGD and retained nipples observed in the adult. Prenatal flutamide exposure resulted in dose-responsive increases in cryptorchidism. Hypospadias were observed in all flutamide-exposed offspring. In utero flutamide exposure induced partial or complete prostate agenesis and decreased the weights of the seminal vesicles, levator ani bulbocavernosus (LABC) muscle, testes, and epididymides in a dose-dependent manner. Epididymal malformations were observed mainly in the 50 mg/kg/day flutamide dose group. In general, flutamide-induced alterations in dihydrotestosterone (DHT)- and testosterone (T)-dependent development each had similar respective dose-response curves. DHT-mediated development was more sensitive to in utero flutamide exposure than T-dependent processes. However, the dose-response curves for flutamide-induced changes in cryptorchidism and seminal vesicle weight were intermediate between the dose-response curves for DHT- and T-mediated development, indicating that proper development of these tissues may require both androgens. The LABC also displayed a dose-dependent decrease in weight that was similar to dose-response observed with seminal vesicle weight and was the most sensitive T-dependent end point measured. Flutamide-induced decreases in AGD predicted subsequent malformations as evidenced by logistic regression and receiver operator characteristic analysis of malformations versus AGD. However, the AGD that would predict a 10% incidence of seminal vesicle malformations is equivalent to a female AGD. An almost fully feminized phenotype of 10-12 nipples was observed in animals that had malformations in T-dependent tissues, whereas 6 or more nipples were observed in animals with malformation in DHT-dependent tissues. These data suggest that flutamide-mediated changes in AGD and nipple retention are not sensitive predictors of altered T-mediated development.

Effects of phthalate esters on the developing reproductive tract of male rats.

Phthalate esters are a large group of chemical agents used predominantly as plasticizers and solvents. Certain members of this chemical class have been shown to cause reproductive and developmental toxicity. Recent attention has focused on the potential of these agents to interfere with male reproductive development through a postulated antiandrogenic mechanism. Observations have focused on di-n-butyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP) and butyl benzylphthalate, with most information relating to dose-response relationships obtained for DBP. Neither DBP, DEHP nor their major metabolites interacted with human or rodent androgen receptors (AR) in transcriptional activation assays. DBP was administered during the critical window of development of the male reproductive system, after which the resulting offspring were examined until adulthood. DBP elicited marked effects on the developing male reproductive tract, including malformations of the epididymis and vas deferens, and hypospadias. Retention of thoracic nipples/areolae and reductions in anogenital distance were also noted. Surprisingly, Leydig cell adenomas were induced in some male offspring at 100 days of age. All these events occurred in the absence of any toxicity in the pregnant dam. Examination of testes from fetal rats indicated markedly reduced testosterone levels and increased Leydig cell numbers after DBP administration to the dams. Leydig cells were positive for AR and 3-betahydroxysteroid dehydrogenase.

Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat.

Linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) is a herbicide that blocks androgen action in the male rat. Studies were undertaken to characterize the ability of linuron to activate transcription through the human androgen receptor (AR) in vitro and to determine whether in utero linuron exposure induces dose-responsive alterations in androgen-dependent reproductive development in the male rat. In vitro, linuron competitively antagonized transcriptional activity of the AR induced by dihydrotestosterone (DHT) in a dose-responsive manner with an equilibrium dissociation constant (K(B)) of 75.8 x 10(-8) M. Pregnant rats were administered linuron by gavage at 0, 12.5, 25, or 50 mg/kg/day (n = 11/group) from gestation day 12 to 21. Anogenital distance of resulting offspring was unaffected, whereas male areola/nipple retention was increased in a dose-responsive manner. Hypoplastic testes in adult offspring were seen in 2/56 rats (2/10 litters), 8/69 rats (4/11 litters), and 5/44 rats (3/8 litters), while hypoplastic epididymides occurred in 1/56 rats (1/10 litters), 8/69 rats (4/11 litters), and 2/44 rats (1/8 litters) in the 12.5, 25, and 50 mg/kg/day dose groups, respectively. Partial agenesis of the epididymides was observed in 3/44 rats (2/8 litters) only in the 50 mg/kg/day group. These data indicate that in utero exposure to linuron preferentially impairs testosterone-mediated, rather than DHT-mediated, reproductive development. This effect is distinctly different from the effects induced by flutamide, an AR antagonist that shares structural similarities with linuron. Furthermore, these data suggest that dose-response studies utilizing late gestational exposure to endocrine-active compounds may be more robust than the traditional or EPA-modified multigeneration protocols in identifying adverse effects.

Prioritization of NTP reproductive toxicants for field studies.

Population studies that evaluate human reproductive impairment are time consuming, expensive, logistically difficult, and with limited resources must be prioritized to effectively prevent the adverse health effects in humans. Interactions among health scientists, unions, and industry can serve to identify populations exposed to potential hazards and develop strategies to evaluate and apply appropriate controls. This report describes a systematic method for prioritizing chemicals that may need human reproductive health field studies. Rodent reproductive toxicants identified from the National Toxicology Program (NTP) Reproductive Assessment by Continuous Breeding (RACB) protocol were prioritized on the basis of potency of toxic effect and population at risk. This model for prioritization links NTP findings with data from the National Occupational Exposure Survey (NOES) and the Hazardous Substance Data Base (HSDB) or the High Production Volume Chemical Database (HPVC) to prioritize chemicals for their potential impact on worker populations. The chemicals with the highest priority for field study were: dibutyl phthalate, boric acid, tricresyl phosphate, and N, N-dimethylformamide.

Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation.

Di(n-butyl) phthalate (DBP) is a commercially important plasticizer and ubiquitous environmental contaminant. Since previous, limited dose-response studies with DBP that reported alterations in male reproductive development and function failed to establish a NOAEL (no-observed-adverse-effect level), an extensive dose-response study was conducted. Pregnant CD rats were given DBP by gavage at 0, 0.5, 5, 50, or 100 mg/kg/day (n = 19-20) or 500 mg/kg/day (n = 11) from gestation day 12 to 21. In male offspring, anogenital distance was decreased at 500 mg DBP/kg/day. Retained areolas or nipples were present in 31 and 90% of male pups at 100 and 500 mg/kg/day, respectively. Preputial separation was not delayed by DBP treatment in males with normal external genitalia, but cleft penis (hypospadias) was observed in 5/58 rats (4/11 litters) at 500 mg/kg/day. Absent or partially developed epididymis (23/58 rats in 9/11 litters), vas deferens (16/58 animals in 9/11 litters), seminal vesicles (4/58 rats in 4/11 litters), and ventral prostate (1/58 animals) occurred at 500 mg/kg/day. In 110-day-old F(1) males, the weights of the testis, epididymis, dorsolateral and ventral prostates, seminal vesicles, and levator ani-bulbocavernosus muscle were decreased at 500 mg/kg/day. At 500 mg/kg/day, widespread seminiferous tubule degeneration was seen in 25/58 rats (in 9/11 litters), focal interstitial cell hyperplasia in 14/58 rats (in 5/11 litters), and interstitial cell adenoma in 1/58 rats (in 1/11 litters). For this 10-day prenatal (embryonic and fetal) exposure to DBP, the NOAEL and LOAEL (lowest-observed-adverse-effect level) were 50 and 100 mg/kg/day, respectively. This is currently the lowest NOAEL described for the toxicity of DBP.

Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment.

The National Toxicology Program (NTP) conducted a continuous breeding study in SD rats with di-n-butyl phthalate (DBP) given via the diet at dose levels of up to 650 mg/kg/day. In the parental generation effects on reproduction were modest (small decreases in litter size and pup weight following treatment). However, the F(1) male offspring had marked decreases in fertility (at 650 mg/kg/day), with reduced sperm counts and reproductive tract malformations on reaching adulthood. A no-observed-adverse-effect level (NOAEL) was not established for the study [lowest-observed-adverse-effect level (LOAEL) 66 mg/kg/day]. In a study conducted at CIIT, the majority of these adverse changes could be reproduced over a similar dose range, but with a much shorter dosing regimen covering a critical window of development (gestation days 12-20). A default risk assessment for DBP indicates a reference dose (RfD) of 66 microg/kg/day, based on a LOAEL of 66 mg/kg/day and default factors of 10 for inter-species and inter-individual differences and the lack of a NOAEL. Human exposure data would indicate worst-case scenarios to infants (via formula) in the dose range of the RfD. A default risk assessment appears to be inappropriate since rodents, unlike primates, metabolize phthalate diesters (including DBP) to monoesters extensively in the gut following oral administration. It is believed that the monoester is the active principle for induction of reproductive and developmental toxicity of specific phthalate esters. Thus, if humans produce very low levels of the monoester from an environmental exposure to the diester, the likelihood of any reproductive or developmental toxicity via the oral route appears extremely remote.

Fertility and developmental neurotoxicity effects of inhaled hydrogen sulfide in Sprague-Dawley rats.

In this study, we examined whether perinatal exposure by inhalation to hydrogen sulfide (H2S) had an adverse impact on pregnancy outcomes, offspring prenatal and postnatal development, or offspring behavior. Virgin male and female Sprague-Dawley rats (12 rats/sex/concentration) were exposed (0, 10, 30, or 80 ppm H2S; 6 h/day, 7 days/week) for 2 weeks prior to breeding. Exposures continued during a 2-week mating period (evidence of copulation = gestation day 0 = GD 0) and then from GD 0 through GD 19. Exposure of dams and their pups (eight rats/litter after culling) resumed between postnatal day (PND) 5 and 18. Adult male rats were exposed for 70 consecutive days. Offspring were evaluated using motor activity (PND 13, 17, 21, and 60+/-2), passive avoidance (PND 22+/-1 and 62+/-3), functional observation battery (PND 60+/-2), acoustic startle response (PND 21 and 62+/-3), and neuropathology (PND 23+/-2 and 61+/-2). There were no deaths and no adverse physical signs observed in F0 male or female rats during the study. A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation, and the average number of implants per pregnant female. Exposure to H2S did not affect pup growth, development, or performance on any of the behavioral tests. The results of our study suggest that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat at occupationally relevant exposure concentrations (< or =10 ppm).

Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide.

Gestational and lactational exposure to di(n-butyl) phthalate (DBP) at >/=250 mg/kg/day disrupts male rat reproductive development and function. Although this indicates an antiandrogenic mechanism, DBP and its biologically active metabolite do not interact with the androgen receptor (AR) in vitro. In the present study, we compared the effects of DBP and the antiandrogen flutamide using a shorter exposure during the prenatal period of male sexual differentiation in rats. Pregnant CD rats received DBP at 0, 100, 250, or 500 mg/kg/day po (n = 10) or flutamide at 100 mg/kg/day po (n = 5) from Gestation Days 12 to 21. In F1 males, DBP (500 mg/kg/day) and flutamide caused hypospadias; cryptorchidism; agenesis of the prostate, epididymis, and vas deferens; degeneration of the seminiferous epithelium; and interstitial cell hyperplasia of the testis. Flutamide and DBP (250 and 500 mg/kg/day) also produced retained thoracic nipples and decreased anogenital distance. Interstitial cell adenoma occurred at 500 mg DBP/kg/day in two males. The only effect seen at 100 mg DBP/kg/day was delayed preputial separation. In contrast to flutamide, DBP caused a low incidence of prostate agenesis and hypospadias with no vaginal pouch. The low incidence of DBP-induced intraabdominal testes contrasted with the high incidence of inguinal testes seen with flutamide. Thus prenatal male sexual differentiation is a sensitive period for the reproductive toxicity of DBP. A no observed adverse effect level was not established and the lowest observed (adverse) effect level was 100 mg/kg/day. Flutamide and DBP disrupted the androgen signaling necessary for male sexual differentiation but with a different pattern of antiandrogenic effects. DBP is an example of an environmental antiandrogen that disrupts androgen-regulated male sexual differentiation without interacting directly with the AR, as does flutamide.

Rodent Leydig cell tumorigenesis: a review of the physiology, pathology, mechanisms, and relevance to humans.

Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years because of its common occurrence in rodent bioassays and the importance in assessing the relevance of this tumor type to humans. To date, there have been no comprehensive reviews to identify all the compounds that have been shown to induce LCTs in rodents or has any review systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. This review attempts to fill these deficiencies in the literature by comparing the cytology and ontogeny of the LC, as well as the endocrine and paracrine regulation of both normal and tumorigenic LCs. In addition, the pathology of LCTs in rodents and humans is compared, compounds that induce LC hyperplasia or tumors are enumerated, and the human relevance of chemical-induced LCTs is discussed. There are plausible mechanisms for the chemical induction of LCTs, as typified by agonists of estrogen, gonadotropin releasing hormone (GnRH), and dopamine receptors, androgen receptor antagonists, and inhibitors of 5alpha-reductase, testosterone biosynthesis, and aromatase. Most of these ultimately involve elevation in serum luteinizing hormone (LH) and/or LC responsiveness to LH as proximate mediators. It is expected that further work will uncover additional mechanisms by which LCTs may arise, especially the role of growth factors in modulating LC tumorigenesis. Regarding human relevance, the pathways for regulation of the hypothalamo-pituitary-testis (HPT) axis of rats and humans are similar, such that compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because GnRH and prolactin receptors are either not expressed or are expressed at very low levels in the testes in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear not to be relevant to humans; however, the potential relevance to humans of the remaining five pathways of LCT induction cannot be ruled out. Therefore, the central issue becomes what is the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. This evidence includes the following: (1) the human incidence of LCTs is much lower than in rodents even when corrected for detection bias; (2) several comparative differences exist between rat and human LCs that may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs; (3) endocrine disease states in man (such as androgen-insensitivity syndrome and familial male precocious puberty) underscore the marked comparative differences that exist between rats and man in the responsiveness of their LC's to proliferative stimuli; and (4) several human epidemiology studies are available on a number of compounds that induce LCTs in rats (1,3-butadiene, cadmium, ethanol, lactose, lead, nicotine) that demonstrate no association between human exposure to these compounds and induction of LC hyperplasia or adenomas. (ABSTRACT TRUNCATED)

Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism?

Di(n-butyl) phthalate (DBP), a widely used plasticizer suspected of having estrogenic properties, was investigated for its effects on the prenatal and early neonatal development of the reproductive tract. Pregnant CD rats (n = 10) were given DBP at 0, 250, 500, or 750 mg/kg/day (p.o.) throughout pregnancy and lactation until their offspring were at postnatal day 20. Maternal body weights throughout the dosing period were comparable in all groups. At 750 mg/kg/day, the number of live pups per litter at birth was decreased and maternal effects on pregnancy and postimplantation loss are likely to have occurred. Anogenital distance was decreased at birth in the male offspring at 500 and 750 mg/kg/day. The epididymis was absent or underdeveloped in 9, 50, and 71% of adult offspring (100 days old) at 250, 500, and 750 mg/kg/day, respectively, and was associated with testicular atrophy and widespread germ cell loss. Hypospadias occurred in 3, 21, and 43% of males and ectopic or absent testes in 3, 6, and 29% of males at 250, 500, and 750 mg/kg/day, respectively. Absence of prostate gland and seminal vesicles as well as small testes and seminal vesicles were noted at 500 and 750 mg/kg/day. Vaginal opening and estrous cyclicity, both estrogen-dependent events, were not affected in the female offspring, although low incidences of reproductive tract malformations were observed at 500 and 750 mg/kg/day. In the male offspring, DBP produced the same spectrum of effects elicited by the antiandrogen flutamide. Thus, DBP specifically impaired the androgen-dependent development of the male reproductive tract, suggesting that DBP is not estrogenic but antiandrogenic in the rat at these high dose levels. For human risk assessment, determining if this toxicity is metabolite-mediated will be critical, since marked species differences in metabolism exist.

Expression cloning of a rat testicular transcript abundant in germ cells, which contains two leucine zipper motifs.

The aim of the present study was to identify specific, novel germ cell markers that could be used to monitor normal and abnormal spermatogenesis. Of several cloned cDNAs isolated from an adult rat testis cDNA library using an expression screening strategy, clone 813B4 (700 base pairs) hybridized exclusively to three mRNA transcripts in samples isolated from rat testes on and after Day 21 of life and to epididymides from some, but not all, adult rats. After further screening, two identical clones encoding a 2.2-kilobase cDNA (KTT4) were isolated and found to contain an open reading frame of 578 amino acids including two leucine zipper motifs. On Northern blots, KTT4 mRNA was abundant in samples from round spermatids, and homologous mRNAs were present in testes from mice and marmosets. A zoo blot revealed that the KTT4 gene is conserved in humans, monkeys, mice, dogs, and cattle. On sections of rat testes, KTT4 mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter was abundant in round and elongating spermatids until step 15. Expression of KTT4 was not altered by ethane dimethane sulphonate-induced androgen withdrawal, but in rats treated 14 days previously with methoxyacetic acid, a marked reduction in KTT4 was noted associated with the depletion of round spermatids. In conclusion, the present study identified a conserved gene expressed in meiotic and post-meiotic germ cells; database searches have shown it to be homologous to recently published sequences for an outer dense fiber protein of the sperm tail (Odf2/Odf84).

Leydig cell hyperplasia and adenoma formation: mechanisms and relevance to humans.

Leydig cell adenomas are observed frequently in studies evaluating the chronic toxicity of chemical agents in laboratory animals. Doubts have been raised about the relevance of such responses for human risk assessment, but the question of relevance has not been evaluated and presented in a comprehensive manner by a broad group of experts. This article reports the consensus conclusions from a workshop on rodent Leydig cell adenomas and human relevance. Five aspects of Leydig cell biology and toxicology were discussed: 1) control of Leydig cell proliferation; 2) mechanisms of toxicant-induced Leydig cell hyperplasia and tumorigenesis; 3) pathology of Leydig cell adenomas; 4) epidemiology of Leydig cell adenomas; and 5) risk assessment for Leydig cell tumorigens. Important research needs also were identified. Uncertainty exists about the true incidence of Leydig cell adenomas in men, although apparent incidence is rare and restricted primarily to white males. Also, surveillance databases for specific therapeutic agents as well as nicotine and lactose that have induced Leydig cell hyperplasia or adenoma in test species have detected no increased incidence in humans. Because uncertainties exist about the true incidence in humans, induction of Leydig cell adenomas in test species may be of concern under some conditions. Occurrence of Leydig cell hyperplasia alone in test species after lifetime exposure to a chemical does not constitute a cause for concern in a risk assessment for carcinogenic potential, but early occurrence may indicate a need for additional testing. Occurrence of Leydig cell adenomas in test species is of potential concern as both a carcinogenic and reproductive effect if the mode of induction and potential exposures cannot be ruled out as relevant for humans. The workgroup focused on seven hormonal modes of induction of which two, GnRH agonism and dopamine agonism, were considered not relevant to humans. Androgen receptor antagonism, 5 alpha-reductase inhibition, testosterone biosynthesis inhibition, aromatase inhibition, and estrogen agonism were considered to be relevant or potentially relevant, but quantitative differences may exist across species, with rodents being more sensitive. A margin of exposure (MOE; the ratio of the lowest exposure associated with toxicity to the human exposure level) approach should be used for compounds causing Leydig cell adenoma by a hormonal mode that is relevant to humans. For agents that are positive for mutagenicity, the decision regarding a MOE or linear extrapolation approach should be made on a case-by-case basis. In the absence of information about mode of induction, it is necessary to utilize default assumptions, including linear behavior below the observable range. All of the evidence should be weighed in the decision-making process.

Detection of germ cell-derived proteins in testicular interstitial fluid: potential for monitoring spermatogenesis in vivo.

The aim of the present study was to assess whether proteins secreted by the seminiferous tubules (ST) can be detected in testicular interstitial fluid (IF) and testicular (TV), spermatic (SV), and peripheral venous (PV) plasma from adult rats. An antiserum was raised against seminiferous tubule-conditioned medium (STCM) prepared from adult rats and used in conjunction with Western blot analysis to screen IF and blood samples resolved by one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of IF and PV were analyzed from control adult rats and rats exposed to scrotal heating (43 degrees C for 30 minutes) 24 hours earlier to ascertain whether damage to spermatogenesis would affect 'leakage' of proteins from the seminiferous tubules. In all control rats, the STCM antiserum specifically detected three proteins in testicular IF with molecular weights of 24, 16, and 14 kDa, respectively. Heat treatment increased the abundance of these proteins and induced the appearance of several other less-abundant proteins, all with molecular masses below 25 kDa. Two of the proteins present in IF were identified, the 24-kDa protein as phosphatidylethanolamine-binding protein (PEBP), and the 14-kDa protein as an androgen-regulated protein (ARP-2). Both of these proteins have been shown in previous studies to be secreted by round spermatids. Our results suggest that germ cell secretory products can gain access to the interstitium under both normal physiological conditions and more easily after induction of damage to spermatogenesis. The antiserum was unable to detect any ST-derived proteins in blood, although it is likely that this result may be due to insensitivity of the presently used techniques. The development of specific immunoassays for germ cell-secreted proteins (e.g., PEBP and ARP-2) should enable more definitive assessment of whether proteins secreted by the seminiferous epithellum can be measured in blood and thus provide a potential means of monitoring spermatogenesis.

Application of benchmark dose risk assessment methodology to developmental toxicity: an industrial view.

The U.S. EPA first signalled its intention to use benchmark dose risk techniques in 1991. Subsequently, publication of draft Guidelines for the Risk Assessment of Reproductive Toxicity data indicated the Agency's intention for wide use of the technique. In developmental toxicity experiments, a number of factors need to be considered before attempting benchmark dose calculations, as compared to the conventional NOAEL approach. For example, care in the assessment of potential litter effects (the litter is the unit of such a study) on the data and whether the data are continuous (e.g. foetal body weight) or discontinuous (e.g. specific or grouped developmental defects where the abnormality is present or absent). Two examples of the use of the benchmark dose approach will be made. First, in the analysis of foetal body weight, where a benchmark dose estimate for an agent producing a 5% decrease in mean foetal weight may be calculated from a shift in the distribution of foetal weights between groups, or by conversion of data to reflect changes in the incidence of 'small' pups (i.e. those towards the extreme of the normal range). The second example involves studies conducted on the developmental toxicity of a triazole antifungal. In the first study, the agent was clearly teratogenic, but a NOAEL was not established and thus necessitated a second study. Analysis of benchmark does estimates (e.g. for % foetuses malformed) from the first study indicated that these were not significantly changed when the data from the second study were combined (i.e. the second study did not aid the risk assessment). The benchmark dose approach has significant scientific and practical advantages over the conventional NOAEL methodology in risk assessments derived from developmental toxicity studies.

The effect of implantation of osmotic pumps on rat thyroid hormone and testosterone levels in the plasma, an implication for tissue 'S' phase studies.

In order to monitor the effect of the procedures required to s.c. implant osmotic pumps into rats on plasma thyroid and testosterone hormone levels, male Fischer 344 rats (8-10 weeks old) were divided into six groups of 10 rats and the groups treated in the following manner: (1) controls housed 5 per cage; (2) controls housed individually; (3) animals anaesthetised for surgery and individually housed; (4) anaesthetised, sham operated and individually housed; (5) anaesthetised, s.c. implanted with osmotic pumps containing saline and individually housed; (6) anaesthetised, s.c. implanted with osmotic pumps containing 5-bromo 2-deoxyuridine (BRDU) and individually housed. Four days after performing the surgery the study was terminated and the level of hormones in the plasma determined by radio immunoassay (RIA). Tri-iodothyronine (T3) and thyroxine (T4) plasma levels (free and total) were significantly decreased with each additional step in the procedure used for the s.c. implantation of an osmotic pump containing BRDU, when compared with the individually housed controls. Similarly, testosterone plasma levels were significantly decreased by the s.c. implantation of osmotic pumps, implying a 'stress' response might occur following implantation. These observations might need to be considered by investigators when performing toxicological research which, as part of the study, uses osmotic pumps for the delivery of the nucleotide precursor required for monitoring cells in 'S' phase.