Joseph Ostby

Late Gestational Exposure to the Fungicide Prochloraz Delays the Onset of Parturition and Causes Reproductive Malformations in Male but Not Female Rat Offspring

Abstract Prochloraz (PZ) is an imidazole fungicide that displays multiple endocrine activities. It inhibits steroid synthesis via P450 modulation and acts as an androgen receptor (AR) antagonist, but its effects on male sexual differentiation have not been described. The purpose of the current study was to expand in vitro observations and to determine whether PZ affected sexual differentiation. PZ effects on AR-mediated gene expression were tested using a cell line (MDA-kb2) containing endogenous AR and stably transfected with an MMTV-luc reporter. PZ concentrations greater than 1 μM caused a dose-dependent inhibition of dihydrotestosterone-induced gene expression. PZ also inhibited R1881 binding to the rat AR (IC50 ∼60 μM). In vivo, pregnant rats received PZ by gavage from Gestational Day 14 to 18 at doses of 31.25, 62.5, 125, and 250 mg/kg of body weight per day. PZ delayed delivery in a dose-dependent manner and resulted in pup mortalities at the two highest doses. In male offspring, anogenital distance and body weight were slightly reduced at 3 days of age. Additionally, female-like areolas were observed at 13 days of age at frequencies of 31%, 43%, 41%, and 71% in the lowest-dose to highest-dose groups, respectively. Weights of androgen-dependent tissues showed dose-dependent reductions. Hypospadias and vaginal pouches were noted in all males treated with 250 mg/kg, whereas those defects were observed in 12.5% and 6.25%, respectively, of males treated with 125 mg/kg. Treatment did not affect age of preputial separation in animals without penile malformations. Despite severe malformations in males, no malformations were noted in females. Together, these results indicate that PZ alters sexual differentiation in an antiandrogenic manner.

Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p′-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat:

Antiandrogenic chemicals alter sexual differentiation by a variety of mechanisms, and as a consequence, they induce different profiles of effects. For example, in utero treatment with the androgen receptor (AR) antagonist, flutamide, produces ventral prostate agenesis and testicular nondescent, while in contrast, finasteride, an inhibitor of 5α-dihydrotestosterone (DHT) synthesis, rarely, if ever, induces such malformations. In this regard, it was recently proposed that dibutyl phthalate (DBP) alters reproductive development by a different mechanism of action than flutamide or vinclozolin (V), which are AR antagonists, because the male offsprings display an unusually high incidence of testicular and epididymal alterations—effects rarely seen after in utero flutamide or V treatment. In this study, we present original data describing the reproductive effects of 10 known or suspected anti-androgens, including a Leydig cell toxicant ethane dimethane sulphonate (EDS, 50 mg kg−1 day−1), linuron (L, 100 mg kg−1 day−1), p,p′-DDE (100 mg kg−1 day−1), ketoconazole (12-50 mg kg−1 day−1), procymidone (P, 100 mg kg−1 day−1), chlozolinate (100 mg kg−1 day−1), iprodione (100 mg kg−1 day−1), DBP (500 mg kg−1 day−1), diethylhexyl phthalate (DEHP, 750 mg kg−1 day−1), and polychlorinated biphenyl (PCB) congener no. 169 (single dose of 1.8 mg kg−1). Our analysis indicates that the chemicals discussed here can be clustered into three or four separate groups, based on the resulting profiles of reproductive effects. Vinclozolin, P, and DDE, known AR ligands, produce similar profiles of toxicity. However, p,p′-DDE is less potent in this regard. DBP and DEHP produce a profile distinct from the above AR ligands. Male offsprings display a higher incidence of epididymal and testicular lesions than generally seen with flutamide, P, or V even at high dosage levels. Linuron treatment induced a level of external effects consistent with its low affinity for AR [reduced anogenital distance (AGD), retained nipples, and a low incidence of hypospadias]. However, L treatment also induced an unanticipated degree of malformed epididymides and testis atrophy. In fact, the profile of effects induced by L was similar to that seen with DBP. These results suggest that L may display several mechanisms of endocrine toxicity, one of which involves AR binding. Chlozolinate and iprodione did not produce any signs of maternal or fetal endocrine toxicity at 100 mg kg−1 day−1. EDS produced severe maternal toxicity and a 45% reduction in size at birth, which resulted in the death of all neonates by 5 days of age. However, EDS only reduced AGD in male pups by 15%. Ketoconazole did not demasculinize or feminize males but rather displayed anti-hormonal activities, apparently by inhibiting ovarian hormone synthesis, which resulted in delayed delivery and whole litter loss. In summary, the above in vivo data suggest that the chemicals we studied alter male sexual differentiation via different mechanisms. The anti-androgens V, P, and p,p′-DDE produce flutamide-like profiles that are distinct from those seen with DBP, DEHP, and L. The effects of PCB 169 bear little resemblance to those of any known anti-androgen. Only in depth in vitro studies will reveal the degree to which one can rely upon in vivo studies, like those presented here, to predict the cellular and molecular mechanisms of developmental toxicity.

Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat:

In humans and rodents, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of 100 or 200 mg vinclozolin (V) kg−1 day−1 during sexual differentiation in rats induces female-like anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, suprainguinal ectopic scrota/testes, a vaginal pouch, epididymal granulomas, and small to absent sex accessory glands in male offspring. Vinclozolin is metabolized to at least two active forms, M1 and M2, that display antiandrogenic activity by binding the androgen receptor (AR). Here, we present information on the reproductive effects of oral treatment with low dosage levels of V during sexual differentiation of the male rat. Vinclozolin was administered to the dam at 0, 3.125, 6.25, 12.5, 25, 50, or 100 mg kg−1 day−1 from gestational day 14 to postnatal day 3 (the period of fetal/neonatal testicular testosterone synthesis and sexual differentiation). At doses of 3.125 mg V kg−1 and above, AGD was significantly reduced in newborn male offspring and the incidence of areolas was increased. These effects were associated with permanent alterations in other androgen-dependent tissues. Ventral prostate weight in one year old male offspring was reduced in all treatment groups (significant at 6.25, 25, 50, and 100 mg kg−1 day−1), and permanent nipples were detected in males at 3.125 (1.4%), 6.25 (3.6%), 12.5 (3.9%), 25 (8.5%), 50 (91%), and 100 (100%) mg V kg−1 day−1. To date, permanent nipples have not been observed in a control male from any study in our laboratory. Vinclozolin treatment at 50 and 100 mg kg−1 day−1 induced reproductive tract malformations and reduced ejaculated sperm numbers and fertility. Even though all of the effects of V likely result from the same initial event (AR binding), the different endpoints displayed a wide variety of dose-response curves and ED50s. The dose-response data for several of the functional endpoints failed to display an obvious threshold. These data demonstrate that V produces subtle alterations in sexual differentiation of the external genitalia, ventral prostate, and nipple tissue in male rat offspring at dosage levels below the previously described no-observed-effect-level (NOEL). These effects occur at a dosage level an order of magnitude below that required to induce malformations and reduce fertility. Hence, multigenerational reproduction studies of antiandrogenic chemicals that were not conducted under the Environmental Protection Agencys new Harmonized Multigenerational Test Guidelines, which include endpoints sensitive to antiandrogens at low dosage levels, could yield a NOEL that is at least an order of magnitude too high.

The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro

Procymidone is a dicarboximide fungicide structurally related to the well-characterized fungicide vinclozolin. Vinclozolin metabolites bind to mammalian androgen receptors (AR) and act as AR antagonists, inhibiting androgen-dependent gene expression in vivo and in vitro by inhibiting AR-binding to DNA. The current study was designed to determine if procymidone acted as an AR antagonist in vitro and to describe the dosage levels of procymidone that alter sexual differentiation in vivo. In vitro, procymidone inhibited androgen from binding the human AR (hAR) in COS (monkey kidney) cells transfected with hAR at 3.16 μM. In vitro, procymidone acted as an androgen antagonist, inhibiting dihydrotestosterone (DHT)-induced transcriptional activation at 0.2 μM in CV-1 cells (cotransfected with the hAR and a MMTV-luciferase reporter gene). In vivo, maternal procymidone exposure at 0, 25, 50, 100, or 200 mg kg−1 day−1 during gestation and early lactation (gestational day 14 to postnatal day 3) altered reproductive development of male offspring at all dosage levels tested. Male offspring exhibited shortened anogenital distance (at 25 mg kg−1 day−1 and above), permanent nipples, reduced weight of several androgen-dependent tissues (levator ani and bulbocavernosus muscles, prostate, seminal vesicles, Cowpers gland and glans penis), and malformations (hypospadias, cleft phallus, exposed os penis, vaginal pouch, hydronephrosis, occasional hydroureter, epididymal granulomas, and ectopic, undescended testes). In addition, perinatal procymidone treatment had a marked effect on the histology of the lateral and ventral prostatic and seminal vesicular tissues of the offspring (at 50 mg kg−1 day−1 and above). These effects consisted of fibrosis, cellular infiltration, and epithelial hyperplasia. This constellation of effects is similar to that produced by perinatal exposure to vinclozolin. However, procymidone appears to be slightly less potent in inducing malformations than vinclozolin by a factor of about two. In summary, the antiandrogenic activity of procymidone was demonstrated in vivo and in vitro in cell lines transfected with hAR. Since the role of androgens in mammalian sexual differentiation is highly conserved, it is likely that humans would be adversely affected by procymidone in a predictable manner if the human fetus was exposed to sufficient levels during critical stages of intrauterine and neonatal life.

Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat

Vinclozolin is a well-characterized antiandrogenic fungicide. It produces adverse effects when administered during sexual differentiation, and it alters reproductive function in adult male rats by acting as an androgen-antagonist. Two active metabolites of vinclozolin, M1 and M2, compete with natural androgens for the rat and human androgen receptors (ARs), an effect that blocks androgen-induced gene expression in vivo and in vitro. In addition to their effects during perinatal life, androgens play a key role in pubertal maturation in young males. In this regard, the present study was designed to examine the effects of peripubertal oral administration of vinclozolin (0, 10, 30, or 100 mg kg−1 day−1) on morphological landmarks of puberty, hormone levels, and sex accessory gland development in male rats. In addition, as binding of the M1 and M2 to AR alter the subcellular distribution of AR by inhibiting AR-DNA binding, we examined the effects of vinclozolin on AR distribution in the target cells after in vivo treatment. We also examined serum levels of vinclozolin, M1, and M2 in the treated males so that these could be related to the effects on the reproductive tract and AR distribution. Vinclozolin treatment delayed pubertal maturation (at 30 and 100 mg kg−1 day−1) and retarded sex accessory gland and epididymal growth. Serum luteinizing hormone (LH; significant at all dosage levels) and testosterone and 5α-androstane,3α,17β-diol (at 100 mg kg−1 day−1) levels were increased. Testis size and sperm production, however, were unaffected. It was apparent that these effects were concurrent with subtle alterations in the subcellular distribution of AR. In control animals, most AR were in the high salt cell fraction, apparently bound to the natural ligand and DNA. Vinclozolin treatment reduced the amount of AR in the high salt (bound to DNA) fraction and it increased AR levels in the low salt (inactive, not bound to DNA) fraction. M1 and M2 were found in the serum of animals from the two highest dosage groups, but they were present at levels well below their K i values. In summary, these results suggest that when the vinclozolin metabolites occupy a small percentage of AR in the cell, this prevents maximal AR-DNA binding and alters in vivo androgen-dependent gene expression and protein synthesis, which in turn results in obvious alterations of morphological development and serum hormone levels. It is noteworthy that similar exposures during prenatal life result in a high incidence of malformations in male rats.

The estrogenic and antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats

This study was designed to determine if long-term exposure to high doses of methoxychlor (M) would alter pituitary or testicular endocrine functions in either an estrogenic or antiandrogenic manner. Weanling male Long-Evans hooded rats were dosed daily with M (po) at 0, 200, 300, or 400 mg kg−1 day−1 for 10 months. Methoxychlor treatment delayed puberty by as much as 10 weeks and reduced fertility and copulatory plug formation in a dose-related manner at the initial mating. During mating, M-treated males exhibited shorter latencies to mount and ejaculate versus control males, but the number of intromissions prior to ejaculation was unaffected, indicating that M enhanced the arousal level in the males in an estrogen-dependent manner. Most treated males eventually mated but time-to-pregnancy was lengthened. Very low sperm counts were associated with infertility, while prolonged delays in puberty reduced fecundity. Methoxychlor treatment with 200 to 400 mg kg−1 day−1 failed to mimic the chronic effects of a sustained (8 months) low dose of estradiol-17β (3-mm silastic implants) on pituitary or testicular hormone levels. Estradiol administration increased pituitary weight 4-fold, serum levels of luteinizing hormone (LH) were reduced by almost 50%, and serum prolactin was increased 40-fold, while M did not affect any of these measures. These data demonstrate that M affects the CNS, epididymal sperm numbers, and the accessory sex glands and delays mating without significantly affecting the secretion of LH, prolactin, or testosterone. These data indicate that M did not alter pituitary endocrine function in either an estrogenic or antiandrogenic manner. To our knowledge, these data provide the first in vivo example of such a pronounced degree of target tissue selectivity to an environmental endocrine-disrupting chemical.

An environmental antiandrogen, vinclozolin, alters the organization of play behavior.

During mammalian sexual differentiation, the androgens, testosterone and dihydrotestosterone are critical for the organization of the male phenotype. In rats, play behavior is sexually dimorphic. Administration of exogenous androgens during the perinatal period results in masculine-like play behavior of juveniles. Recently, there has been increasing concern about the potential for environmental endocrine-disrupting chemicals (EDCs) to alter sexual differentiation in mammals. One such EDC is the fungicide and androgen receptor (AR) antagonist, vinclozolin. We tested whether developmental exposure to an EDC could alter androgen-dependent behaviors such as play. To examine this possibility, neonatal male rats were injected from Postnatal Days (PND) 2 to 3 with corn oil, pharmacological antiandrogen flutamide (50 mg/kg/day) or vinclozolin (200 mg/kg/day); whereas neonatal females were treated either with corn oil or testosterone propionate (TP, 250 microg/kg/day). At PNDs 36-37, animals were observed for social play. Behaviors associated with general social activity, such as sniffing and dorsal contact, were unaffected by treatment or sex. However, play behavior in males treated with flutamide or vinclozolin was significantly reduced to near-female levels when compared to control males. Play behavior in females exposed to TP during the neonatal period was significantly increased when compared with control females. Hence, this study suggests that perinatal exposure to vinclozolin, an environmental antiandrogen, can alter androgen-dependent behavior, such as play, in the male rat.

Prenatal dinocap exposure alters swimming behavior in mice due to complete otolith agenesis in the inner ear.

Exposure to the fungicide dinocap during gestation produces behavioral abnormalities in the house mouse that are not apparent at birth but become obvious at weaning. Pregnant mice (CD-1) were exposed on Days 7 to 16 of gestation to dinocap at 0, 6, 12, or 25 mg/kg/day and the postnatal behavioral development of the offspring was assessed. Torticollis (head-tilting) appears in the treated offspring at 3 weeks of age (4.4% at 12 and 25.3% at 25 mg/kg/day) and, during a test of swimming ability, many of the mice (6.8% at 12 and 47.2% at 25 mg/kg/day) sink below the surface or are unstable and swim on their side in the water. These behavioral abnormalities are the result of agenesis of the otoliths in the inner ears. These were the only developmental defects noted in the 12 mg/kg/day dosage group. In this group 4.4% of the mice displayed torticollis, 9.2% did not swim normally, 19% were missing one or more whole otoliths (7.7% were missing all four otoliths), and partial agenesis of the crystalline material was seen in an additional 11.6% of the mice. The frequency of behavioral and inner ear defects increased in the higher dosage group, but the order of sensitivity of the effects did not change.

TOXICANT-INDUCED HYPOSPADIAS IN THE MALE RAT

Prenatal exposure to endocrine disrupting chemicals that interfere with the androgen or insulin like factor 3 signaling pathways during sexual differentiation can induce malformations of the reproductive tract of the male rodent offspring. The pattern of malformations in the male depends upon the specific mechanism of action of the toxicant, the dosage level administered and the timing of administration during pregnancy. Hypospadias occurs in male rats or mice after maternal treatment with 1). potent estrogens or estrogenic drugs, 2). drugs that inhibit 5 alpha reductase, 3). drugs, herbicides and dicarboximide and conazole fungicides that act as androgen receptor (AR) antagonists, and 4). drugs, herbicides and conazole fungicides that inhibit cytochrome P450 enzymes involved in steroid hormone synthesis. In addition, 5). several phthalate diesters including di-n-butyl phthalate (DBP), di-n-ethylhexyl phthalate (DEHP), and benzylbutyl phthalate(BBP) also induce hypospadias and by altering fetal testis Leydig cell differentiation, resulting in reduced steroid and peptide hormone production.

The postnatal effects of prenatal exposure to low doses of nitrogen (2,4-dichlorophenyl-p-nitrophenyl ether) in Sprague-Dawley rats☆

Nitrofen was administered to pregnant Sprague-Dawley rats by gavage on days 8-16 of gestation at 5 different dose levels--0, 0.46, 1.39, 4.17 and 12.5 mg/kg/day. Diaphragmatic hernias were found in pups that died immediately after birth at the 3 highest dose levels. At the 1.39-mg/kg dose level 3 of the 4 pups examined had diaphragmatic hernias, at the 4.17-mg/kg dose level 2 out of 3 pups had diaphragmatic hernias, and at the 12.5-mg/kg dose level all 5 pups found dead had diaphragmatic hernias. Locomotor activity of the offspring was measured on postnatal days 17 and 24, and hyperactivity was evident at the 3 highest dose levels. However, when the rats were later tested at 45, 49 and 90 days of age they had apparently recovered from this earlier hyperactivity. In the female rat, nitrofen did not delay the onset of puberty as measured by the age of vaginal opening or the age at first estrus. At necropsy of the offspring which began on postnatal day 133, Harderian gland weight reduction and hydronephrosis were seen at the 4.17- and 12.5-mg/kg dose levels, while no effects were found in body, liver, testes, seminal vesicle, kidney, or lung weights. Results of the present study and earlier studies demonstrate that rats are more sensitive than mice to the teratogenic effect of nitrofen (Gray et al., Science, 215 (1982) 293 and Gray et al., Toxicol. Appl. Pharmacol., 67 (1983) 1). In general, nitrofen affects the same organ systems in rats as it does in mice, but the rank order of sensitivity of these effects differs from those described earlier in the mouse by Gray et al. (Toxicol. Appl. Pharmacol., 67 (1983) 1).