Effects of altered food intake during pubertal development in male and female wistar rats.

The U.S. Environmental Protection Agency is currently validating assays that will be used in a Tier I Screening Battery to detect endocrine disrupting chemicals. A primary concern with the Protocols for the Assessment of Pubertal Development and Thyroid Function in Juvenile Male and Female Rats is that a nonspecific reduction in body weight (BWT) during the exposure period may potentially confound the interpretation of effects on the endocrine endpoints. Wistar rats were underfed 10, 20, 30, or 40% less than the ad libitum food consumed by controls from postnatal days (PNDs) 22 to 42 (females) or PNDs 23 to 53 (males). Terminal BWT of females and males were 2, 4, 12, and 19% and 2, 6, 9, and 19% lower than controls, respectively. In the females, neither the age of pubertal onset nor any of the thyroid hormone endpoints were affected by food restriction (FR) that led to a 12% decrease in BWT. Similarly, none of the male reproductive endpoints examined were altered by FR that led to a 9% BWT decrease. However, decreased triiodothyronine and thyroxin was observed in FR males with a 9% reduced BWT. While these data support the use of the maximum tolerated dose for BWT (10%) for the female protocol, effects on the male thyroid endpoints indicate that a slightly lower limit (

Moderating influence of the drinking water disinfection by-product dibromoacetic acid on a dithiocarbamate-induced suppression of the luteinizing hormone surge in female rats.

The disinfection by-product dibromoacetic acid (DBA) has been found in female rats to increase circulating concentrations of both estradiol (E2) and estrone (E1). This effect is apparently due, at least in part, to a suppression in hepatic catabolism. The present study investigated whether DBA, by increasing sex steroid levels, is able either to augment the hypothalamic up-regulation involved in triggering a luteinizing hormone (LH) surge, or to affect the ability of the neurotoxicant sodium dimethyldithiocarbamate (DMDC) to block the surge. Sprague-Dawley rats were gavaged for 14 days with DBA (0-150mg/kg) and ovariectomized on dosing day 11, and at the same time implanted with an estradiol capsule to generate daily LH surges. An injection of 0.1mM/kg DMDC was administered at 13:00h on day 14 and blood was sampled over the afternoon. DBA induced a dose-related increase in total estrogens. For identified surges, areas under the LH curve partitioned into two groups, comprising the two lower (0 and 37.5mg/kg DBA) and the two higher (75 and 150mg/kg) treatment groups. Consequently, low and high DBA groups were compared and found to be significantly different. At 150mg DBA/0.1mM DMDC, the timing of an identifiable LH peak was comparable to non-DMDC females, unlike the 37.5mg DBA/0.1mM DMDC group in which the appearance of peak concentrations was delayed. A significant effect with DBA treatment alone was not present. Results indicated that this exposure to DBA induced a dose-related increase in total estrogen concentrations that paralleled a diminished DMDC blockade of the LH surge. The effect appeared to be attributable to an augmentation in the estrogen-associated up-regulation in brain mechanisms stimulating the surge.

Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

Pubertal development in female Wistar rats following exposure to propazine and atrazine biotransformation by-products, diamino-S-chlorotriazine and hydroxyatrazine.

We showed previously that the chlorotriazine herbicide, atrazine (ATR), delays the onset of pubertal development in female rats. ATR and its biotransformation by-products are present in soil and groundwater. Since current maximum contaminant levels are set only for ATR, it is important to determine whether these by-products can also alter pubertal development and possibly pose a cumulative exposure hazard. We evaluated the effects of two ATR by-products, diamino-s-chlorotriazine (DACT) and hydroxyatrazine (OH-ATR), and a structurally similar chlorotriazine, propazine (PRO), on female pubertal development. Rats were gavaged from postnatal days (PNDs) 22 through PND 41 with DACT (16.7, 33.8, 67.5, 135 mg/kg), OH-ATR (22.8, 45.7, 91.5, 183 mg/kg), or PRO (13, 26.7, 53, 106.7, 213 mg/kg). The dose range for each chemical was selected as the molar equivalent of ATR (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal opening (VO) and killed on PND 41. DACT, a by-product of ATR that occurs in the environment and is also the primary chlorinated metabolite of ATR in animal tissue, delayed VO by 3.2, 4.8, and 7.6 days compared to the controls (33.1 +/- 0.4 (SE) days of age) following exposure to 33.8, 67.5, and 135 mg/kg, respectively. The no effect level (NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for ATR (25 mg/kg). Although the body weight (BW) on PND 41 was reduced by the high dose of DACT (8.4% reduction), this reduction did not exceed the criteria for selecting the maximum tolerated dose (e.g., a dose that causes >10% decrease in BW at necropsy). None of the lower doses of DACT caused a significant difference in BW gain. Additionally, 33.8, 67.5, and 135 mg/kg of DACT significantly increased the BW on the day of VO. PRO (107 or 213 mg/kg) delayed VO by 4 days but did not alter the BW on PND 41. While no significant delays in pubertal development were observed in two separate dose-response studies with doses ranging up to 183 mg/kg (OH-ATR), a minor but statistically significant delay in the onset of puberty in a pilot study using OH-ATR raises the possibility that an effect might occur following exposure to higher doses. However, it is clear from these data that OH-ATR has a much lower potency when compared with equimolar doses of DACT and PRO. Together, these data demonstrate that PRO and DACT can delay the onset of puberty in the female rat at doses equimolar to ATR and provide the scientific basis for the use of additivity in the upcoming risk assessments.