Evaluation of the diphenyl herbicide, oxyfluorfen, for effects on thyroid hormones in the juvenile rat.

Recently, oxyfluorfen, a pre- and post-emergent diphenyl ether herbicide, was identified in our laboratory as an inhibitor of iodide uptake by the sodium iodide symporter (NIS), the first key step in the synthesis of thyroid hormones (THs). This inhibition was observed in vitro, using both a human NIS engineered cell line (hNIS-HEK293T-EPA) and a rat thyroid follicular cell line (FRTL-5). Oxyfluorfen was found to be a potent inhibitor of NIS activity with an EC50 of approximately 2 µM in both cell lines with no observed cytotoxicity at any concentration tested up to 100 µM. The current research tested the hypothesis that oxyfluorfen alters circulating concentrations of THs. This hypothesis was first tested in a pilot study with both juvenile male and female rats exposed to oxyfluorfen for 4 days at 0, 125, 250 and 500 mg/kg/day. Once we identified that this short-term 4-day oxyfluorfen exposure suppressed both total serum thyroxine (T4) and triiodothyronine (T3) at all doses, we tested seven lower concentrations of oxyfluorfen (0.8125 to 62.5 mg/kg day) in an 8-day exposure paradigm to more closely evaluate the dose-response. We found that oxyfluorfen suppressed serum T4 with a LOEL of 3.25 mg/kg/day and T3 with a LOEL 62.5 mg/kg/day. Analytical chemistry of the serum showed an accumulation over time following oral exposure to oxyfluorfen in both the 4- and 8-day groups. Analytical chemistry of the thyroid glands in the 8-day study revealed higher accumulation in the thyroid as compared to the serum (2 to 3- fold at 62.5 mg/kg). No changes in thyroid weight or serum TSH were observed following the 8-day exposure. This study is the first to demonstrate an effect of oxyfluorfen on serum thyroid hormones in the rat. Additional studies are needed to further evaluate the effects on thyroid homeostasis with extended exposures and the potential implications of the observed effects.

Evaluation of triclosan in the Hershberger and H295R steroidogenesis assays.

Triclosan (TCS) is an antibacterial widely used in personal care products that exhibits endocrine disrupting activity in several species, with reports of altered thyroid, estrogen and androgen signaling pathways. To evaluate the androgenic mode of action, TCS was evaluated for androgen receptor mediated effects in the Hershberger assay and for altered androgen synthesis in the H295R steroidogenesis assay. In the Hershberger assay, castrated males were dosed by oral gavage for 10 days with corn oil (vehicle) or TCS (50 or 200 mg/kg/day) in the presence or absence of testosterone proprionate (TP, 0.2 mg/kg/day) prior to assessing accessory sex tissues (ASTs) weights. TCS alone or in combination with TP did not alter androgen dependent AST weights. Assessment of serum thyroxine (T4) demonstrated a significant dose-dependent decrease by TCS (50 or 200 mg/kg/day) co-administered with TP and TCS (200 mg/kg) without TP, but no differences in liver or thyroid weights. In the H295R assay, TCS from 0.01 to 10 µM had no effect on testosterone production but TCS at 3 µM and above did induce a significant increase in estrogen production. At 10 µM, TCS produced significant cytotoxicity which confounded the interpretation of the estrogenic effect at that concentration. Thus, TCS had no effect on androgen synthesis or activity in the models used, but did enhance estrogen production and suppress serum T4.

Reproductive effects of maternal and pre-weaning undernutrition in rat offspring: age at puberty, onset of female reproductive senescence and intergenerational pup growth and viability.

Maternal and/or postnatal undernutrition are widespread in human populations and are components of many experimental developmental and reproductive toxicology bio-assays. This study investigated in utero and/or pre-weaning undernutrition effects on reproductive maturation and senescence in the Sprague-Dawley rat as well as potential intergenerational effects. Pregnant rats were given food ad libitum or at 50% of normal dietary intake throughout pregnancy. Their offspring (control or IUGR) were cross-fostered to control dams with litter sizes of 8 or 16 pups (control and undernourished). Offspring body weights were reduced and onset of male puberty slightly delayed in animals from large postnatal litters. Similar body weight effects were observed in females but there was no difference in the age of vaginal opening. Female reproductive senescence as measured by onset of estrus acyclicity occurred at a younger age in IUGR-8-pup and Control-16-pup groups compared to Control-8-pup or IUGR-16-pup groups. Females were bred to control males and no evidence of adverse reproductive effects was found in any F2 groups. The offspring of the F1 generation did not show an intergenerational effect as documented in humans.

Regional differences in the pituitary distribution of luteinizing hormone in the gonadectomized and proestrous female rat.

Previous data have shown that regional differences in the presence of anterior pituitary luteinizing hormone (LH) generally correlate with the comparable disparities in distribution of gonadotropes throughout the gland. In female rats, the differences are apparent over the estrous cycle, but are more prominent during the hours preceding the proestrus surge of LH. The current experiments examined (1) if such regional disparities are present throughout the surge window, (2) if differences are mirrored by release of LH in vitro and (3) if the appearance of regional differences is altered in ovariectomized females. Results showed that a comparative elevation in the rostral portion of the pituitary during the pre-surge period diminishes and finally disappears concurrent with the rise in circulating LH. This increase in rostral LH concentrations is reflected in this region by a comparable effect in vitro on stimulated LH secretion from pituitary fragments, although the effect is somewhat diminished by referencing release against tissue concentrations of LH present in a contralateral rostral fragment. Ovariectomies conducted at 1500h on proestrus, at a time when a significant regional difference has faded, resulted in a prompt increase in LH across all areas of the pituitary, and the emergence of a marked augmentation in rostral concentrations over the ensuing 72h. The effect was not seen when ovariectomies were performed on estrus. These data show that, while a regional disparity in anterior pituitary LH is present as circulating concentrations of estradiol rise prior to the LH surge, the removal of this steroid feedback at a time when LH synthesis is normally amplified accentuates the difference between the rostral region and other areas of the pituitary.

Evaluation of ammonium perchlorate in the endocrine disruptor screening and testing program's male pubertal protocol: ability to detect effects on thyroid endpoints.

The U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 male pubertal protocol was designed as a screen to detect endocrine-disrupting chemicals which may alter reproductive development or thyroid function. One purpose of this in vivo screening protocol is to detect thyrotoxicants via a number of different mechanisms of action, such as thyroid hormone synthesis or clearance. Here we evaluate the ability of this EDSP male pubertal protocol to detect the known thyrotoxicant ammonium perchlorate as an endocrine disruptor. Ammonium perchlorate is a primary ingredient in rocket fuel, fertilizers, paints, and lubricants. Over the past 50 years, potassium perchlorate has been used to treat hyperthyroidism in humans. Perchlorate alters thyroid hormone secretion by competitively inhibiting iodide uptake by the thyroid gland. In this study, ammonium perchlorate was administered at 62.5, 125, 250, and 500 mg/kg to male Wistar rats based on a pilot study of oral dosing. Doses of 125-500 mg/kg perchlorate decreased T4 in a dose-dependent manner. TSH was significantly increased in a dose-responsive manner at the same doses, while T3 was unchanged at any dose. Thyroid histology was significantly altered at all doses, even at the 62.5 mg/kg, with a clear dose-dependent decrease in colloid area and increase in follicular cell height. No effects on preputial separation, a marker of pubertal progression, or reproductive tract development were observed at any dose. These results demonstrate that the male pubertal protocol is useful for detecting thyrotoxicants which target the thyroid axis by this mechanism (altered uptake of iodide). This study also found that perchlorate exposure during this period did not alter any of the reproductive developmental endpoints.

In vivo and in vitro anti-androgenic effects of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture.

PBDEs have been synthesized in large quantities as flame retardants for commercial products, such as electronic equipment and textiles. The rising in levels of PBDEs in tissues in wildlife species and in human milk and plasma samples over the past several years have raised concerns about possible health effects. Recently, we showed that the PBDE mixture, DE-71, delayed puberty and suppressed the growth of androgen-dependent tissues in male Wistar rat following a peri-pubertal exposure. These effects suggested that DE-71 may be either inducing steroid hormone metabolism or acting as an androgen receptor (AR) antagonist. To elucidate the potential anti-androgenic effects of this mixture, we evaluated DE-71 in several in vivo assays, which are responsive to alterations in androgen activity. In a pubertal exposure study designed to further evaluate the delay in preputial separation (PPS), we observed a dose-dependent delay in PPS with 60 and 120 mg/kg/day of DE-71 (4 and 5 days) and a corresponding suppression of ventral prostate (VP) and seminal vesicle growth at both doses. Adult males exposed to 60 mg/kg DE-71 for 3 days resulted in a significant increase in luteinizing hormone and a non-significant increase in testosterone, androstenedione and estrone. DE-71 also tested positive for anti-androgenic activity in an immature rat Hershberger assay, with decreases in mean VP and seminal vesicle weight following doses of 30-240 mg/kg. DE-71 and the individual BDE congeners which comprise the mixture (BDE-47, -99, -100, -153, -154) were also evaluated in vitro. First, AR binding was evaluated in a competitive binding assay using rat VP cytosol. In addition, we evaluated gene activation in a transcriptional activation assay using the MDA-kb2 cell line which contains an endogenous human AR and a transfected luciferase reporter. DE-71 and BDE-100 (2, 4, 6-pentaBDE) both inhibited AR binding, with IC50s of approximately 5 microM. In addition, DE-71 and two of the congeners (BDE-100 and BDE-47) inhibited DHT-induced transcriptional activation. The pattern of inhibition shown in the double-reciprocal plot for BDE-100 and the linear slope replot confirmed that the in vitro mechanism is pure competitive inhibition, with a inhibition constant (Ki) of 1 microM. The delay in puberty in the male rat and decreased growth of androgen-dependent tissues observed previously following exposure to DE-71 were likely due to this inhibition of AR binding by several of the congeners which make up this mixture.

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.

Xenoendocrine disrupters-tiered screening and testing: filling key data gaps.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1). In vivo, the uterotropic assay detects estrogens and antiestrogens, while steroidogenesis, antithyroid activity, (anti)estrogenicity and HPG function are assessed in a 'Pubertal Female Assay'. (Anti-) androgens are detected in the Hershberger Assay (weight of AR-dependent tissues in castrate-immature-male rats). Fish and amphibian assays also are being developed. The fathead minnow assay can identify EDCs displaying several mechanisms of concern, including AR and ER receptor agonists and antagonists and inhibitors of steroid hormone synthesis. An amphibian metamorphosis assay is being developed to detect thyroid-active substances. Several alternative mammalian in vivo assays have been proposed. Of these, a short-term pubertal male rat assay appears most promising. An in utero-lactational screening protocol also is being evaluated. For Tier 2, the numbers of endocrine sensitive endpoints and offspring (F1) examined in multigenerational tests need to be expanded for EDCs. Consideration should be given to tailoring T2, based on the results of T1S. Tier 1 and 2 also should examine relevant mixtures of EDCs. Toxicants that induce malformations in AR-dependent tissues produce cumulative effects even when two chemicals act via different mechanisms of action.

Delayed ovulation and pregnancy outcome: effect of environmental toxicants on the neuroendocrine control of the ovary(1).

In the female rat, we have shown that a burst exposure to environmental toxicants known to alter noradrenergic function will block the ovulatory surge of LH when administered during a sensitive period on the day of vaginal proestrus. Such treatments will delay ovulation by 24 h and affect embryo survival. These results demonstrate clearly that brief, appropriately timed, toxicant exposure can initiate a cascade of changes that can alter reproductive outcome. However, we also found that continued exposure to the same compound is without an apparent influence on the reproductive capacity of the female, indicating that the female can become tolerant to such adverse reproductive effects. These observations raise a number of questions concerning the approaches currently used to examine potential reproductive toxicants. In this review, we describe the consequences of appropriately timed exposures to chlordimeform and dithiocarbamates on the timing of ovulation and subsequent alterations in pregnancy outcome. We also review the available literature on phenobarbital delays in ovulation and oocyte function in the rodent and the relevance to ovulatory delays in the human.

The effects of atrazine on female wistar rats: an evaluation of the protocol for assessing pubertal development and thyroid function.

The effects of atrazine (ATR), a chlorotriazine herbicide, on the onset of puberty were evaluated in Wistar rats. Female rats were dosed by oral gavage from postnatal day(s) (PND) 22 through PND 41 with 0, 12.5, 25, 50, 100, or 200 mg ATR/kg. Vaginal opening (VO) was significantly delayed 3.4, 4.5, or greater than 6.8 days by 50, 100, and 200 mg/kg, respectively. VO had not occurred in 4 of 15 females in the 200 mg/kg group by the time of necropsies (PND 41). Body weight (bw) at necropsy was reduced in the 200 mg/kg group by 11.6%, but was not different from the control (0) in the 50 and 100 mg/kg groups. To examine the influence of reduced bw on pubertal development, a group of pair-fed controls was included whose daily food intake was dependent upon the amount consumed by their counterpart in the 200 mg/kg group. Although necropsy bw was reduced to the same extent as the ATR females, VO in the pair-fed controls was not significantly delayed. Adrenal, kidney, pituitary, ovary, and uterine weights were reduced by 200 mg/kg ATR. Serum T(3), T(4), and TSH were unaltered by ATR, which was consistent with no histopathologic/morphologic changes in the thyroid. Estrous cyclicity was monitored in a second group of females from VO to PND 149. The number of females displaying regular 4- or 5-day estrous cycles during the first 15-day interval after VO was lower in the 100 and 200 mg/kg ATR and pair-fed controls. Irregular cycles were characterized by extended periods of diestrus. By the end of the second 15-day interval (PND 57-71), no effects on estrous cyclicity were observed. These data show that ATR can delay the onset of puberty and alter estrous cyclicity in the female Wistar rat ( NOAEL of 25 mg/kg). Reduced food consumption and bw did not account for the delay in VO, because this effect was not observed in the pair-fed controls. In addition, the effect on estrous cyclicity was observed in the 100 mg/kg ATR group where no significant reduction in bw was observed.

The effect of atrazine on puberty in male wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function.

Since atrazine (ATR), a chlorotriazine herbicide, has been shown previously to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) through a direct effect on the central nervous system (CNS), we hypothesized that exposure to ATR in the EDSTAC male pubertal protocol (juvenile to peripubertal) would alter the development of the male rat reproductive system. We dosed intact male Wistar rats from postnatal day (PND) 23 to 53 and examined several reproductive endpoints. ATR (0, 12.5, 25, 50, 100, 150, or 200 mg/kg) was administered by gavage and an additional pair-fed group was added to compare the effects of any decreased food consumption in the high dose group. Preputial separation (PPS) was significantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg ATR dose groups. PPS was also delayed in the pair-fed group, although significantly less than in the high dose-ATR group. The males were killed on PND 53 or 54, and pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were removed. ATR (50 to 200 mg/kg) treatment resulted in a significant reduction in ventral prostate weights, as did the reduced food consumption of the pair-fed group. Testes weights were unaffected by atrazine treatment. Seminal vesicle and epididymal weights were decreased in the high dose-ATR group and the control pair-fed group. However, the difference in epididymal weights was no longer significantly different when body weight was entered as a covariable. Intratesticular testosterone was significantly decreased in the high dose-ATR group on PND 45, but apparent decreases in serum testosterone were not statistically significantly on PND 53. There was a trend for a decrease in luteinizing hormone (LH) as the dose of ATR increased; however, dose group mean LH was not different from controls. Due to the variability of serum prolactin concentrations on PND 53, no significant difference was identified. Although prolactin is involved in the maintenance of LH receptors prior to puberty, we observed no difference in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed dose-related increases that were significant only in the 200 mg/kg-ATR group. No differences were observed in thyroid stimulating hormone (TSH) and thyroxine (T4) between the ATR groups and the control; however triiodothyronine (T3) was elevated in the high dose-ATR group. No differences in hormone levels were observed in the pair-fed animals. These results indicate that ATR delays puberty in the male rat and its mode of action appears to be altering the secretion of steroids and having subsequent effects on the development of the reproductive tract, which appear to be due to ATR's effects on the CNS. Thus, ATR tested positive in the pubertal male screen that the Endocrine-Disrupter Screening and Testing Advisory Committee (EDSTAC) is considering as an optional screen for endocrine disrupters.

Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Endocrine Disrupter Screening and Testing Advisory Committee.

Puberty in mammalian species is a period of rapid interactive endocrine and morphological changes. Therefore, it is not surprising that exposure to a variety of pharmaceutical and environmental compounds has been shown to dramatically alter pubertal development. This concern was recognized by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) that acknowledged the need for the development and standardization of a protocol for the assessment of the impact of endocrine-disrupting compounds (EDC) in the pubertal male and recommended inclusion of an assay of this type as an alternative test in the EDSTAC tier one screen (EPA, 98). The pubertal male protocol was designed to detect alterations of pubertal development, thyroid function, and hypothalamic-pituitary-gonadal (HPG) system peripubertal maturation. In this protocol, intact 23-day-old weanling male rats are exposed to the test substance for 30 days during which pubertal indices are measured. After necropsy, reproductive and thyroid tissues are weighed and evaluated histologically and serum taken for hormone analysis. The purpose of this review was to examine the available literature on pubertal development in the male rat and evaluate the efficacy of the proposed protocol for identifying endocrine-disrupting chemicals. The existing data indicate that this assessment of puberty in the male rat is a simple and effective method to detect the EDC activity of pesticides and toxic substances.

Atrazine disrupts the hypothalamic control of pituitary-ovarian function.

The chloro-S-triazine herbicides (i.e., atrazine, simazine, cyanazine) constitute the largest group of herbicides sold in the United States. Despite their extensive usage, relatively little is known about the possible human-health effects and mechanism(s) of action of these compounds. Previous studies in our laboratory have shown that the chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these studies led us to hypothesize that these herbicides disrupt endocrine function primarily through their action on the central nervous system. To evaluate this hypothesis, we examined the estrogen-induced surges of luteinizing hormone (LH) and prolactin in ovariectomized Sprague-Dawley (SD) and Long-Evans hooded (LE) rats treated with atrazine (50-300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 mg/kg) suppressed the LH and prolactin surge in ovariectomized LE, but not SD female rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 females. Three daily doses of atrazine suppressed the estrogen-induced LH and prolactin surges in ovariectomized LE females in a dose-dependent manner, but this same treatment was without effect on serum LH and prolactin in SD females. The estrogen-induced surges of both pituitary hormones were suppressed by atrazine (75-300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated after 21 days of treatment. Three experiments were then performed to determine whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. These included examination of the ability of (1) the pituitary lactotrophs to secrete prolactin, using hypophyosectomized females bearing pituitary autotransplants (ectopic pituitaries); (2) the synthetic gonadotropin-releasing hormone (GnRH) to induce LH secretion in females treated with high concentrations of atrazine for 3 days; and (3) atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from pituitaries, using a flow-through perifusion procedure. In conclusion, the results of these studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD female rats by altering the hypothalamic control of these hormones. In this regard, the LE female appeared to be more sensitive to the hormone suppressive effects of atrazine, as indicated by the decreases observed on treatment-day 3. These experiments support the hypothesis that the effect of atrazine on LH and prolactin secretion is mediated via a hypothalamic site of action.

Perinatal exposure to estrogenic compounds and the subsequent effects on the prostate of the adult rat: evaluation of inflammation in the ventral and lateral lobes.

Although the effects of estrogenic compounds administered during the perinatal period on the size and morphology of the prostate have been well documented, the effects of such exposures on inflammatory changes in the prostate have not been well characterized. Since neonatal estradiol exposure has been shown to cause periods of hyperprolactinemia later in life and a relationship exists between high prolactin levels and rat lateral prostate inflammation, we hypothesized that an exposure to environmental compounds with estrogenic activity could result in an increase in lateral prostate inflammation in adulthood. To investigate this possibility and compare differences between estrogen agonists and antagonists, we examined the effect of a perinatal exposure to 17beta-estradiol, the insecticide methoxychlor, the partial estrogen agonist tamoxifen, and the pure antiestrogen ICI 182,780. Dams were dosed from gestation day (GD)18 to parturition and then the pups were dosed from postnatal day (PND) 1 to 5 with 0.1 mL of a solution of 0.355 mM and .0178 mM by sc injection, respectively, of all compounds in sesame oil, except for methoxychlor, which was administered only to the dam by gavage from GD 18 through PND 5 at a dose of 50 mg/kg in sesame oil. At 90 d of age, the weight of the lateral and ventral prostate in the estradiol group was significantly decreased. Tamoxifen caused a decrease in the weight of the lateral prostate, whereas the ventral lobe was not affected. ICI 182,780 did not alter prostate weight. The methoxychlor exposure increased the lateral lobe weight, but the ventral lobe weight was not affected. In the estradiol and tamoxifen groups, an inflammatory infiltrate was observed in the ventral prostates in 45.0 and 27.8% of the animals, respectively. There was a significant increase in the percent and severity of inflammation in the lateral prostate (as determined by a myeloperoxidase or neutrophil quantification assay) in the estradiol, tamoxifen, and methoxychlor groups as compared to controls. The ICI group was comparable to the controls in both ventral and lateral lobes. This study demonstrates that perinatal exposure to estrogenic compounds can result in alterations in the size of the adult prostate and increase the incidence of prostatitis.

Prepubertal exposure to compounds that increase prolactin secretion in the male rat: effects on the adult prostate.

To test the hypothesis that a transient increase in prolactin (PRL) secretion prior to puberty can result in an alteration of the adult prostate, male rats were exposed from postnatal Days (PND) 22 to 32 to compounds that increase PRL secretion. These compounds included pimozide (a dopamine antagonist), estradiol-17beta, and bisphenol A (a monomer of polycarbonate plastics reported to have weak estrogenic activity). During dosing, pimozide (PIM), bisphenol A (BPA), and estradiol-17beta (E(2)) stimulated an increased secretion of PRL. At 120 days of age, the lateral prostate weight was increased in the PIM and BPA groups as compared to the vehicle-injected controls. Examination of the prostates revealed inflammation in the lateral lobes of all treated groups. Results of a myeloperoxidase assay, a quantitative assay to assess acute inflammation, indicated an increase in the percentage of males with neutrophil infiltrate in the lateral prostates of the PIM and E(2) treatment groups compared to their respective controls. The histological evaluations of these tissues confirmed an increase in luminal polymorphonuclear cells and interstitial mononuclear cells of the lateral prostates in all treatment groups. Administration of the dopamine agonist, bromocriptine, to the estradiol-implanted males from PND 22 to 32 reversed the induction of lateral prostate inflammation by estradiol, suggesting that PRL was necessary for the inflammatory effect. This study demonstrates that prepubertal exposures to compounds that increase PRL secretion, albeit through different mechanisms, can increase the incidence of lateral prostate inflammation in the adult.

Maternal exposure to atrazine during lactation suppresses suckling-induced prolactin release and results in prostatitis in the adult offspring.

The availability of prolactin (PRL) to the neonatal brain is known to affect the development of the tuberoinfundibular (TIDA) neurons and, as a consequence, lead to alterations in subsequent PRL regulation. Without early lactational exposure to PRL (derived from the dam's milk), TIDA neuronal growth is impaired and elevated PRL levels are present in the prepubertal male. These observations, combined with the finding that alterations in PRL secretion (i.e., hyperprolactinemia) in the adult male rat have been implicated in the development of prostatitis, led us to hypothesize that early lactational exposure to agents that suppress suckling-induced PRL release would lead to a disruption in TIDA development, altered PRL regulation, and subsequent prostatitis in the male offspring. To test this hypothesis, suckling-induced PRL release was measured in Wistar dams treated twice daily with the herbicide atrazine (ATR, by gavage, on PND 1-4 at 0, 6.25, 12.5, 25, and 50 mg/kg body weight), or twice daily with the dopamine receptor agonist bromocriptine (BROM, sc, at 0.052, 0.104, 0.208, and 0.417 mg/kg); BROM is known to suppress PRL release. Similarly, atrazine has also been reported to suppress PRL in adult females. Serum PRL was measured on PND 3 using a serial sampling technique and indwelling cardiac catheters. A significant rise in serum PRL release was noted in all control females within 10 min of the initiation of suckling. Fifty-mg/kg ATR inhibited suckling-induced PRL release in all females, whereas 25 and 12.5 mg/kg ATR inhibited this measure in some dams and had no discernible effect in others. The 6.25 mg/kg dose of ATR was without effect. BROM, used here as a positive control, also inhibited suckling-induced PRL release at doses of 0.104 to 0.417 mg/kg, with no effect at 0.052 mg/kg. To examine the effect of postnatal ATR and BROM on the incidence and severity of inflammation (INF) of the lateral prostate of the offspring, adult males were examined at 90 and 120 days. While no effect was noted at 90 days of age, at 120 days, both the incidence and severity of prostate inflammation was increased in those offspring of ATR-treated dams (25 and 50 mg/kg). The 12.5 mg/kg ATR and the two highest doses of BROM increased the incidence, but not the severity, of prostatitis. Combined treatment of ovine prolactin (oPRL) and 25 or 50 mg/kg ATR on PND 1-4 reduced the incidence of inflammation observed at 120 days, indicating that this increase in INF, seen after ATR alone, resulted from the suppression of PRL in the dam. To determine whether or not there is a critical period for these effects, dams were dosed with 25 and 50 mg/kg on PND 6-9 and PND 11-14. Inflammation was increased in those offspring from dams treated on PND 6-9, but this increase was not significant. Dosing on PND 11-14 was without effect. These data demonstrate that ATR suppresses suckling-induced PRL release and that this suppression results in lateral prostate inflammation in the offspring. The critical period for this effect is PND 1-9.

Neuroendocrine and reproductive effects of contemporary-use pesticides.

Work in our laboratory has focused on the hypothesis that certain environmental contaminants will interfere with reproductive function because they disrupt the neuroendocrine regulation of gonadal function. In this article, we review the evidence that certain classes of contemporary-use pesticides alter gonadotropin secretion through a disruption of hypothalamic mechanisms. Specifically, we discuss the effect of formamidine and dithiocarbamate pesticides on the noradrenergic control of pituitary hormone secretion, ovarian function, and pregnancy in the rat. This is followed by a review of studies evaluating the effect of a chlorotriazine herbicide, atrazine, on the hormonal control of ovulation and lactation. We also discuss the physiological consequences of these endocrine alterations in the female, how toxicant-induced endocrine alterations may differ in physiological outcome in the male and female, and the fact that the reproductive risk assessment of some pesticides that act on the central nervous system (CNS) may be influenced by the development of tolerance.

Effect of atrazine on ovarian function in the rat.

The effect of the chlorotriazine herbicide, atrazine, on ovarian function was studied in Long-Evans hooded (LE-hooded) and Sprague-Dawley (SD) rats. Atrazine was administered by gavage for 21 d to females displaying regular 4-d estrous cycles. In both strains, 75 mg/kg/d disrupted the 4-d ovarian cycle; however, no distinct alteration (i.e., irregular cycles but not persistent estrus or diestrus) was apparent at this dose. At 150 mg/kg/d, atrazine induced repetitive pseudopregnancies in females of both strains. The highest dose tested (300 mg/kg/d) also induced repetitive pseudopregnancies in the SD females, while the ovaries of the LE-hooded female appeared regressed and the smear cytology was indicative of the anestrous condition. Although a NOAEL was not established, the doses employed in this experiment were in excess of those used in chronic feeding studies in which an early onset of mammary gland tumors was noted. These data demonstrate that atrazine can disrupt ovarian function and bring about major changes in the endocrine profile of the female.

Characterization of pregnancy outcome following thiram-induced ovulatory delay in the female rat.

A single injection of the dithiocarbamate fungicide, thiram, suppresses the proestrous surge of LH and delays ovulation for 24 h. In this study, we examined fertility after a thiram-induced delayed ovulation. Females were injected with thiram (50 mg/kg, IP) on proestrus (1300 h) and mated on the following evening. Control and thiram-treated, but nondelayed, females were injected and mated on the same day. The number of females in the thiram-delayed group that became pregnant was reduced and litter size on GD 20 was reduced: however, no obvious morphological anomalies were seen. The number of pregnant females and litter size was not altered in the thiram-nondelayed rats, indicating that it is the thiram-induced delay in ovulation and not the exposure to thiram per se that was responsible for altered pregnancy outcome. On GD 7 and 11, the number of live fetuses per litter was reduced in the delayed females, but the number of implantation sites was not different from controls. On GD 11 the mean developmental score, head length, crown-rump length, and somite number in the delayed group were also reduced, indicating retarded development of live embryos. These results demonstrate that delayed ovulation induced by a single thiram exposure does not alter the number of oocytes released or the number that implant. However, the concept from these females are compromised during midgestation.

Suppression of oocyte release in rats by local administration of the noradrenergic neurotoxin DSP4.

The presence of noradrenergic neuronal innervation in the ovaries and cyclic alterations in ovarian noradrenaline suggest a role for such innervation in oocyte release. The current experiments evaluated the relationship between ovulation and alterations in ovarian concentrations of noradrenaline induced by unilateral, intrabursal administration of the specific noradrenergic neurotoxin DSP4. Intrabursal injections of DSP4 (0-10 mumoles per ovary) given at 19:00 h at pro-oestrus induced a prompt, dose-related reduction in ovarian noradrenaline on the injected and non-injected sides. Although this result suggests that injected material was reaching the contralateral ovary, ovulation was suppressed only on the injected side. This suppression was persistent, and lasted through at least the next two cycles following either unilateral or bilateral treatment. The reductions in noradrenaline could be mostly, if not entirely, attenuated by prior administration of desipramine which blocks re-uptake of noradrenaline, while the ipsilateral ovulatory effects remained unchanged. Although it has been reported that DSP4 binds the opiate receptor, intrabursal co-administration of the antagonist naloxone was ineffective in altering ovulatory suppression. These results suggest that while decreases in ovarian noradrenaline in response to local exposure to a noradrenergic neurotoxin may accompany a reduction in oocyte release or a block in ovulation, the anti-ovulatory effect of DSP4 is independent of the changes in noradrenaline concentrations and may be due to some other ovarian response.