Tammy E. Stoker

Triclosan Exposure Modulates Estrogen-Dependent Responses in the Female Wistar Rat

Triclosan is an antimicrobial found in personal care and sanitizing products, such as soaps, toothpaste, and hair products. There have been recent concerns for the possible effects on human health, as triclosan has been detected in human breast milk, blood, and urine samples. In a previous study, we found that triclosan alters serum thyroid hormone and testosterone concentrations in male rats. In the current study, we evaluated the effects of triclosan in the female Wistar rat following exposure for 21 days in the Endocrine Disruptor Screening Program pubertal protocol and the weanling uterotrophic assay (3-day exposure). In the pubertal study, triclosan advanced the age of onset of vaginal opening and increased uterine weight at 150 mg/kg, indicative of an estrogenic effect. In the uterotrophic assay, rats received oral doses of triclosan (1.18, 2.35, 4.69, 9.37, 18.75, 37.5, 75, 150, and 300 mg/kg) alone, 3 microg/kg ethinyl estradiol (EE), or triclosan (same doses as above) plus 3 microg/kg EE. Uterine weight was increased in the EE group (positive control) as compared with the control but was not affected by triclosan alone. However, there was a significant dose-dependent increase in the group cotreated with EE and triclosan (>or= 4.69 mg/kg) as compared with EE alone, indicating a potentiation of the estrogen response on uterine weight. This result was well correlated with potentiated estrogen-induced changes in uterine histology. Serum thyroid hormone concentrations were also suppressed by triclosan in this study, similar to other studies in the male and female rat. In conclusion, triclosan affected estrogen-mediated responses in the pubertal and weanling female rat and also suppressed thyroid hormone in both studies. The lowest effective concentrations in the rodent model are approximately 10 (for estrogen) and 40 (for thyroid hormone) times higher than the highest concentrations reported in human plasma.

THE EFFECTS OF TRICLOSAN ON PUBERTY AND THYROID HORMONES IN MALE WISTAR RATS

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a potent antibacterial and antifungal compound that is widely used in personal care products, plastics, and fabrics. Recently triclosan has been shown to alter endocrine function in a variety of species. The purpose of this study was to determine effects of triclosan on pubertal development and thyroid hormone concentrations in the male rat. Weanling rats were exposed to 0, 3, 30, 100, 200, or 300 mg/kg of triclosan by oral gavage from postnatal day (PND) 23 to 53. Preputial separation (PPS) was examined beginning on PND 33. Rats were killed on PND 53, organ weights were recorded and serum was collected for subsequent analysis. Triclosan did not affect growth or the onset of PPS. Serum testosterone was significantly decreased at 200 mg/kg, however no effects were observed on androgen-dependent reproductive tissue weights. Triclosan significantly decreased total serum thyroxine (T4) in a dose-dependent manner at 30 mg/kg and higher (no observed effect level of 3 mg/kg). Triiodothyronine (T3) was significantly decreased only at 200 mg/kg, but thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly increased at 100 mg/kg triclosan and above suggesting that the induction of hepatic enzymes may have contributed to the altered T4 and T3 concentrations, but it does not appear to correlate with the T4 dose-response. This study demonstrates that triclosan exposure does not alter androgen-dependent tissue weights or onset of PPS; however, triclosan exposure significantly impacts thyroid hormone concentrations in the male juvenile rat.

Effects of Castration and Chronic Steroid Treatments on Hypothalamic Gonadotropin-Releasing Hormone Content and Pituitary Gonadotropins in Male Wild-Type and Estrogen Receptor-α Knockout Mice

Testicular androgens are integral components of the hormonal feedback loops that regulate circulating levels of LH and FSH. The sites of feedback include hypothalamic areas regulating GnRH neurons and pituitary gonadotropes. To better define the roles of androgen receptor (AR), estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) in mediating feedback effects of sex steroids on reproductive neuroendocrine function, we have determined the effects of castration and steroid replacement therapy on hypothalamic GnRH content, pituitary LHβ and FSHβ messenger RNA (mRNA) levels, and serum gonadotropins in male wild-type (WT) and estrogen receptor-α knockout (ERKO) mice. Hypothalami from intact WT and ERKO males contained similar amounts of GnRH, whereas castration significantly reduced GnRH contents in both genotypes. Replacement therapy with estradiol (E2), testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic GnRH content in castrated (CAST) WT mice; only the androgens were effective in CAST ...

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.

Developmental Exposure to a Commercial PBDE Mixture, DE-71: Neurobehavioral, Hormonal, and Reproductive Effects

Developmental effects of polybrominated diphenyl ethers (PBDEs) have been suspected due to their structural similarities to polychlorinated biphenyls (PCBs). This study evaluated neurobehavioral, hormonal, and reproductive effects in rat offspring perinatally exposed to a widely used pentabrominated commercial mixture, DE-71. Pregnant Long-Evans rats were exposed to 0, 1.7, 10.2, or 30.6 mg/kg/day DE-71 in corn oil by oral gavage from gestational day 6 to weaning. DE-71 did not alter maternal or male offspring body weights. However, female offspring were smaller compared with controls from postnatal days (PNDs) 35-60. Although several neurobehavioral endpoints were assessed, the only statistically significant behavioral finding was a dose-by-age interaction in the number of rears in an open-field test. Developmental exposure to DE-71 caused severe hypothyroxinemia in the dams and early postnatal offspring. DE-71 also affected anogenital distance and preputial separation in male pups. Body weight gain over time, reproductive tissue weights, and serum testosterone concentrations at PND 60 were not altered. Mammary gland development of female offspring was significantly affected at PND 21. Congener-specific analysis of PBDEs indicated accumulation in all tissues examined. Highest PBDE concentrations were found in fat including milk, whereas blood had the lowest concentrations on a wet weight basis. PBDE concentrations were comparable among various brain regions. Thus, perinatal exposure to DE-71 leads to accumulation of PBDE congeners in various tissues crossing blood-placenta and blood-brain barriers, causing subtle changes in some parameters of neurobehavior and dramatic changes in circulating thyroid hormone levels, as well as changes in both male and female reproductive endpoints. Some of these effects are similar to those seen with PCBs, and the persistence of these changes requires further investigation.

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.

The dithiocarbamate fungicide thiram disrupts the hormonal control of ovulation in the female rat

Thiram has been reported to inhibit dopamine-beta-hydroxylase (D beta H), thereby affecting norepinephrine (NE) synthesis. Because NE is a neurotransmitter that is known to play an important role in the hypothalamic regulation of pituitary function, the acute effects of the thiram on the hormonal control of ovulation in the rat were investigated. Ovariectomized, estrogen-primed female rats were given a single injection of thiram (0, 6, 12, 25, 50, and 100 mg/kg, i.p.) at 1100 h and serum LH was measured in serial bleeds. Thiram at 100 and 50 mg/kg completely blocked the LH surge in all rats tested, while 12 and 25 mg/kg blocked the surge in 40 and 75% of the treated animals, respectively. Six mg/kg had no effect. Ovulation was then assessed in intact, proestrous females in response to thiram administration (0, 12, 25, or 50 mg/kg) at 0900, 1100, 1300, or 1800 h. Ovulation was blocked by 25 and 50 mg/kg at 1300 h in all rats, but when injected at 1100 h only the 50 mg/kg dose was effective. No such blockade was found with 50 mg/kg injected at 0900 and 1800 h. To assess the influence of thiram on the LH surge in intact rats, additional females were dosed at 1300 h on the day of proestrus and blood collected over that same day. Thiram at 50 mg/kg blocked the LH surge in all rats, while 25 mg/kg blocked the surge in 60% of the females tested. No effect occurred with 12 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorotriazine Herbicides and Metabolites Activate an ACTH-dependent Release of Corticosterone in Male Wistar Rats

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

Blockade of ovulation in the rat by the fungicide sodium N-methyldithiocarbamate: Relationship between effects on the luteinizing hormone surge and alterations in hypothalamic catecholamines

Sodium N-methyldithiocarbamate (SMD), also known as metam sodium, is a commonly employed soil fungicide and nematocide. Structurally related dithiocarbamates have been found to decrease norepinephrine (NE) synthesis by suppressing the activity of dopamine-beta-hydroxylase. Because brain hypothalamic catecholamine (CA) activity is involved in generating the proestrus afternoon surge in blood luteinizing hormone (LH) which stimulates the final stages of ovulation, this study explored the effect of SMD on this hormonal trigger and its relationship to changes in hypothalamic CAs. Ovariectomized, steroid-primed Long-Evans rats showed a dose-related (25-100 mg/kg, IP) suppression of the surge and a drop in NE when SMD was given at 1100 h, a few h prior to the expected LH rise. The surge effect was reversed by the alpha-adrenergic agonist clonidine. With cycling rats, a decline with dose (50-300 mg/kg, 1300 h, proestrus) was seen in the percentage of ovulating females, with earlier injections (0900 h) being less effective at the highest dose. At all doses, low circulating levels of LH and prolactin at 1600 h suggested either a blockade in the proestrus surges of each hormone or a displacement in their time of occurrence. Anterior and posterior hypothalamic NE fell by 3 h postinjection and was accompanied by a rise in dopamine, while serotonin was unchanged. Although there was a distinct parallel between the alterations in regional CAs and the incidence of ovulation at the high doses of SMD, the relationship did not hold as the dose decreased. A similar dissociation between ovulation and CAs was seen when equimolar doses of SMD or methylisothiocyanate, a principal metabolite, were given by gavage. At the regional level of analysis employed, the data indicate that while IP injections of SMD are able to block the LH surge and ovulation in these rats, the dose-response relationship suggests that, along with induced alterations in CA metabolism, an additional factor may be involved in the observed effects.

The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat.

Triclosan (TCS), an antibacterial, has been shown to be an endocrine disruptor in the rat. We reported previously that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth in rats exposed to EE and TCS in the uterotrophic assay, whereas TCS alone had no effect. To further characterize this potentiation, we evaluated the effect of co-exposure with lower doses of EE that are comparable to the concentrations in hormone replacement regimens and began to assess the mechanisms by which this potentiation occurs. Changes in uterine weight, epithelial cell growth, and estrogen-sensitive gene expression were assessed. TCS expectedly enhanced the uterotrophic response to EE, however at significantly lower doses of EE. Similarly, TCS increased the EE-induced stimulation of epithelial cell height following cotreatment. Cotreatment also enhanced the estrogen-induced change in gene expression, which was reversed with an ER antagonist. Furthermore, the TCS-induced potentiation was independent of ER activation, as no effects were observed in the ER TA assay.