Leon Earl Gray

Antifertility effect of methoxychlor in female rats: dose- and time-dependent blockade of pregnancy.

Long-term exposure to methoxychlor (MXC), an estrogenic pesticide, produces infertility in rats, and short-term exposure blocks the decidual cell response (DCR). To address the short-term effects of MXC on fertility, the differential effects of MXC dosage and timing of administration (relative to implantation) on several gestational parameters were investigated. When MXC was administered during early pregnancy (Days 1-8), a dose-dependent decline in implantations and uterine weight was seen with no effect on ovarian weight or corpora lutea; MXC reduced serum progesterone at all doses. Preimplantation administration of MXC (Days 1-3 of pregnancy) produced a decline in implantations and uterine weight, while postimplantation dosing (Days 4-8 of pregnancy) increased resorptions to 100%, decreased uterine weight, and reduced serum progesterone without affecting the number of implantations, ovarian weight, or number of corpora lutea. The DCR of pseudopregnancy was inhibited by 500 mg/kg/day MXC when administered either pre- or postimplantation, but 200 mg/kg/day was without effect in either regimen. When hormonally primed, long-term ovariectomized rats were exposed to doses of 500 mg/kg/day, MXC blocked the induced DCR seen in controls. The data show that short-term MXC dosing during early pregnancy produces a dose-related infertility. The bloickade of pregnancy by the preimplantation administration of MXC may be mediated by a direct effect on preimplantation uterine development. The fetal resorption seen following postimplantation dosing is considered a manifestation of both reduced serum progesterone and the direct disruption of normal decidual development by MXC.

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.

A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion.

Abstract Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

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.

Dose-Response Assessment of Fetal Testosterone Production and Gene Expression Levels in Rat Testes Following In Utero Exposure to Diethylhexyl Phthalate, Diisobutyl Phthalate, Diisoheptyl Phthalate, and Diisononyl Phthalate

Several phthalate esters have been linked to the Phthalate Syndrome, affecting male reproductive development when administered to pregnant rats during in utero sexual differentiation. The goal of the current study was to enhance understanding of this class of compounds in the Sprague Dawley (SD) fetal rat following exposure on gestational days (GDs) 14-18 by determining the relative potency factors for several phthalates on fetal testes endpoints, the effects of a nine phthalate mixture on fetal testosterone (T) production, and differences in SD and Wistar (W) strain responses of fetal T production and testicular gene expression to di(2-ethylhexyl) phthalate (DEHP). We determined that diisobutyl phthalate (DIBP) and diisoheptyl phthalate (DIHP) reduced fetal testicular T production with similar potency to DEHP, whereas diisononyl phthalate (DINP) was 2.3-fold less potent. DINP was also less potent at reducing StAR and Cyp11a gene expression levels, whereas DIBP was slightly more potent than DEHP. We observed that administration of dilutions of a mixture of nine phthalates (DEHP, DIHP, DIBP, dibutyl-, benzyl butyl-, dicyclohexyl-, diheptyl-, dihexyl-, and dipentyl phthalate) reduced fetal T production in a dose-dependent manner best predicted by dose addition. Finally, we found that the differential effects of in utero DEHP treatment on epididymal and gubernacular differentiation in male SD and W rats (0, 100, 300, 500, 625, 750, or 875 mg DEHP/kg/day) are likely due to tissue-specific strain differences in the androgen and insl3 signaling pathways rather than differential effects of DEHP on fetal testis T and insl3 production.

Effects of low subchronic doses of methoxychlor on the rat hypothalamic-pituitary reproductive axis☆

The pesticide methoxychlor (MXC) is known to possess a weak estrogenic action and has been found to have a number of toxic effects on the rodent reproductive system, primarily at the gonadal level. The purpose of this study was to explore the influence of MXC on the pituitary and hypothalamic components of the male reproductive system at dose levels that were without detectable testicular effects. At 21 days, male Long-Evans rats were gavaged daily with 25 or 50 mg/kg MXC in corn oil. Controls received vehicle only. After 8 weeks of dosing, no significant changes were seen in serum LH, FSH, or prolactin, nor in the pituitary concentrations of LH or FSH. Pituitary prolactin was elevated for both doses, and pituitary fragments perifused in vitro released more prolactin than did controls. The concentration of gonadotropin-releasing hormone (GnRH) was higher in the mediobasal hypothalamus, but only for the 50-mg/kg group. At this dose, there was a corresponding increase in the KCl-stimulated release of GnRH. The data suggest that previously reported reproductive effects of MXC may be mediated, at least in part, through an elevation in prolactin concentration and release, which in turn is able to influence hypothalamic levels of GnRH. This prolactinemic effect may well represent an early component of the adverse action of MXC on the reproductive system.

Methoxychlor induces estrogen-like alterations of behavior and the reproductive tract in the female rat and hamster: Effects on sex behavior, running wheel activity, and uterine morphology

The current investigation was designed to determine if the pesticide methoxychlor (M) mimicked the effects of estrogen in the brain and on behavior. Running wheel activity (RWA) and sex behaviors were evaluated in this study because the role of estrogen in the regulation of these behaviors has been thoroughly established. M exposure at 400 mg/kg/day (90% pure) induced high levels of acyclic RWA and persistent vaginal estrus in the female rats. Following ovariectomy (ovx), RWA declined precipitously in controls but remained at high levels in M-treated-ovx females. M also produced estrogen-like alterations of the uterine endometrial epithelium, the ovary, and growth after ovx. In another study, ovx female rats were dosed with M at 200 mg/kg/day and then with progesterone (P). P acts as an antiestrogen and specifically suppresses estrogen-induced RWA. P blocks the synthesis of estrogen receptors in the CNS and reproductive tract but does not lower RWA induced by nonestrogenic mechanisms. After 14 days of M administration RWA was increased fourfold over the ovx-oil-treated females. Subsequently, P injections reduced RWA levels far below those seen when the ovx-M-treated rats were injected with oil. The P-induced decline represents a 95% inhibition of the M-induced increase in RWA. Subsequently, M-treated-ovx rats and hamsters were injected with P and tested for their ability to display reproductive behaviors when paired with a stud male. Female sexual behaviors are induced by the administration of estrogen followed by progesterone. In this study the M-treated females displayed reproductive behaviors, in contrast to the oil-treated rats and hamsters. The observation that the high levels of RWA induced by methoxychlor treatment in ovx rats can be suppressed by concurrent progesterone injections demonstrates that the increase in RWA is due to the estrogenic effects of methoxychlor on the CNS. The fact that methoxychlor, followed by P injections, induces behavioral estrus in the rat and hamster extends this estrogenicity to other areas in the CNS.

Methoxychlor affects the decidual cell response of the uterus but not other progestational parameters in female rats

The pesticide methoxychlor (MXC) is a proestrogen which is metabolized to a compound that has been shown to exhibit estrogenic activity in vivo and in vitro. Following long-term exposure of female rats to MXC, fertility is reduced and fetotoxicity is evident. However, the effects of MXC on several aspects of maternal reproductive physiology, including the decidual cell response (DCR), ovarian weight, serum progesterone levels, and corpora lutea maintenance, have not been previously described. In the present study, the ability of MXC to interfere with progestational events essential for implantation and the maintenance of pregnancy in the rat was investigated. MXC was administered to pseudopregnant rats, decidualization was induced on Day 4, and the DCR and related parameters were evaluated following euthanasia on Day 9. DCR induction during estrone administration served as a positive control. MXC inhibited decidualization in a dose-dependent manner: while 100 mg/kg/day had no effect, doses of 200, 300, 400, and 500 mg/kg/day produced incremental inhibition of the response. No effects of MXC on ovarian weight, serum progesterone levels, or number of corpora lutea were observed. Estrone also inhibited the DCR in a dose-dependent manner. The data suggest that the anti-fertility effect of MXC is mediated by a suppression of decidualization and that such suppression, which is characteristic of estrogenic compounds, is a direct uterine effect. In the DCR model system, the estrogenic activity of MXC is 1/20,000 times the activity of estrone.

Transgenerational Effects of Di (2-Ethylhexyl) Phthalate in the Male CRL:CD(SD) Rat: Added Value of Assessing Multiple Offspring per Litter

In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis, and other androgen-dependent tissues. In order to define the dose-response relationship between di(2-ethylhexyl) phthalate (DEHP) and the Phthalate Syndrome of reproductive alterations in F1 male rats, Sprague-Dawley (SD) rat dams were dosed by gavage from gestational day 8 to day 17 of lactation with 0, 11, 33, 100, or 300 mg/kg/day DEHP (71-93 males per dose from 12 to 14 litters per dose). Some of the male offspring continued to be exposed to DEHP via gavage from 18 days of age to necropsy at 63-65 days of age (PUB cohort; 16-20/dose). Remaining males were not exposed after postnatal day 17 (in utero-lactational [IUL] cohort) and were necropsied after reaching full maturity. Anogenital distance, sperm counts and reproductive organ weights were reduced in F1 males in the 300 mg/kg/day group and they displayed retained nipples. In the IUL cohort, seminal vesicle weight also was reduced at 100 mg/kg/day. In contrast, serum testosterone and estradiol levels were unaffected in either the PUB or IUL cohorts at necropsy. A significant percentage of F1 males displayed one or more Phthalate Syndrome lesions at 11 mg/kg/day DEHP and above. We were able to detect effects in the lower dose groups only because we examined all the males in each litter rather than only one male per litter. Power calculations demonstrate how using multiple males versus one male/litter enhances the detection of the effects of DEHP. The results at 11 mg/kg/day confirm those reported from a National Toxicology Program multigenerational study which reported no observed adverse effect levels-lowest observed adverse effect levels of 5 and 10 mg/kg/day DEHP, respectively, via the diet.