I. Glenn Sipes

An Updated Weight of the Evidence Evaluation of Reproductive and Developmental Effects of Low Doses of Bisphenol A

There is controversy over whether low doses of bisphenol A (BPA, CAS no. 80-05-7) cause reproductive and developmental effects in humans. We update the 2004 weight-of-evidence assessment of an expert panel convened by Harvards Center for Risk Analysis by critically evaluating over 50 additional studies published between April 2002 and February 2006 that examine in vivo reproductive and developmental toxicity in mammals at doses ≤5 mg/kg-d. Our findings are consistent with the Harvard study: some statistically significant findings in rats and mice exist but they are generally countered by more numerous studies showing no effect for similar endpoints. No effect is marked or consistent across species, doses, and time points. Some mouse studies report morphological changes in testes and sperm and some non-oral mouse studies report morphological changes in female reproductive organs. Owing to lack of first-pass metabolism, results from non-oral studies are of limited relevance to oral human exposure. Human biomonitoring indicates exposures lower than the “low” doses in the reviewed animal studies. Reports of human health impact are very limited and inconsistent. Taken together, the weight of evidence does not support the hypothesis that low oral doses of BPA adversely affect human reproductive and developmental health.

Mechanisms of acute hepatic toxicity: chloroform, halothane, and glutathione.

The effects of hepatic microsomal enzyme induction with phenobarbital and depletion of hepatic glutathions (CSH) with diethyl maleats on the acute hepatotoxic responses to chloroform and halothane anesthesia were studied in rats. Phenobarbital pretreatment markedly increased the hepatotoxic response to chloroform anesthesia, but had little effect on halothane hepatotoxicity. Hepatic CSH levels were decreased 70–80 per cent by 2 hour* of Chloroform anesthesia in induced rats, but were unchanged in non-induced rats and in animals anesthetized with halothane. Marked destruction of microsomal electron transfer components was observed in the chloroform-anesthetized, induced animals only. Induction caused a large increase in in-vitro covalent binding of 14CHCl3 metabolites to microsomal protein, which could be prevented by CSH. Diethyl maleate pretreatment lowers CSH content approximately 80 per cent. Chloroform anesthesia produced hepatic necrosis and destruction of microsomal enzymes in the absence of induction, but halothane did not. Hepatotoxicity of chloroform appears to be related to two factors: 1) rate of biotransformation; 2) availability of the hepatic antioxidant, CSH. Halothane hepatotoxicity does not proceed by the same sequence of events as does that of chloroform.

The role of epoxidation in 4-vinylcyclohexene-induced ovarian toxicity

4-Vinylcyclohexene (VCH) is present in gases discharged during synthetic rubber production. Chronic treatment of B6C3F1 mice and F-344 rats with VCH by gavage has been shown to induce ovarian tumors in mice but not in rats. Our objective was to understand the mechanism of the species difference in VCH-induced ovarian tumors. Since a critical step in the induction of ovarian tumors is destruction of the small oocyte, small oocyte counts obtained from serially sectioned ovaries were used as an index of toxicity. VCH or its epoxide metabolites [VCH-diepoxide, VCH-1,2-epoxide, and VCH-7,8-epoxide (in mice only)] were given to 28-day-old female mice and rats in corn oil, ip, at doses ranging from 0.07 to 7.4 mmol/kg body wt/day for 30 days. The dose which reduced the small oocyte count to 50% that of control was defined as the ED50. In mice, the ED50 for the reduction in small oocytes by VCH was 2.7 mmol/kg, whereas, no detectable oocyte loss occurred in rats at the highest dose of VCH (7.4 mmol/kg). The potency of the epoxides of VCH was greater than that of VCH in both species. The ED50 for oocyte loss by VCH-1,2-epoxide in mice and rats was 0.5 and 1.4 mmol/kg, respectively. In mice, VCH-7,8-epoxide had comparable potency to VCH-1,2-epoxide (ED50 = 0.7). VCH diepoxide was even more potent with ED50 values of 0.2 and 0.4 mmol/kg, in mice and rats, respectively. The dose response of the blood concentration of VCH-1,2-epoxide in mice after VCH showed that doses of VCH which caused minimal toxicity had the lowest blood level of this ovotoxic epoxide. Pretreatment of mice with the cytochrome P450 inhibitor chloramphenicol (200 mg/kg, ip) inhibited VCH epoxidation in vivo and in vitro and partially protected mice from VCH toxicity. Thus it appears that metabolism of VCH to epoxides and their subsequent destruction of oocytes are critical steps in VCH-induced ovarian tumors. Rats may be resistant to ovarian tumor induction by VCH because the amount of VCH converted to epoxides is insufficient to produce oocyte destruction.

Hypothyroxinemia and hypothermia in rats in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin administration

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment on body temperature and serum and tissue levels of thyroid hormones, glucose, glucagon, insulin, and somatostatin were investigated. Within 7 days following TCDD administration (45 micrograms/kg), rats exhibited hypothyroidism compared to pair-fed controls and rats fed ad libitum. Body temperature was maintained in the pair-fed and ad libitum-fed controls but was significantly decreased in TCDD-treated rats at 2 days. Within 2 weeks of the administration of 90 micrograms TCDD/kg, body temperature was below 35 degrees C with the lowest mean value of 34.5 degrees C recorded on Day 16. Mean body temperatures for control rats ranged from 36.8 to 37.5 degrees C. One week after TCDD administration (45 micrograms/kg), serum thyroxine (T4) declined to 46% of pair-fed controls. The decreased free-thyroxine index indicated that the measured decrease in thyroxine reflected decreased hormone concentrations as opposed to altered protein binding. Hypoglycemia occurred in TCDD-treated rats subsequent to hypothyroxinemia and hypothermia, but it did not develop in the pair-fed controls. At 1 week after administration of 45 micrograms TCDD/kg, serum and pancreatic insulin levels were reduced to 25 and 76% of ad libitum-fed controls, respectively. Hypophagia was determined to be responsible for the decreased growth rate and hypoinsulinemia but did not account for hypothyroxinemia, hypothermia, and hypoglycemia following the administration of TCDD. No significant alterations were detected in serum glucagon or in pancreatic, hepatic, or serum somatostatin levels. Decreased somatostatin in the gastric antrum coincided with a 29% increase in stomach dry weight. The delayed toxicity of TCDD may result, in part, from these hormonal alterations.

Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide

4-vinyl-1-cyclohexene diepoxide (VCD) is known to destroy oocytes in ovaries of immature rats. Since ovaries functionally differ between immature and adult animals, we examined the effect of VCD on oocytes in adult rats. Adult (58 days) and immature (28 days) rats were injected daily (30 days) with vehicle or VCD. Each group contained 10 rats. During this time, cyclicity was determined daily by vaginal cytology. Animals were terminated on day 31 and tissues were collected. Oocytes were counted; livers, spleens, and uteri were weighed. VCD reduced the number of regular estrous cycles/30 days in adults, but not immature rats (n = 20, P < 0.05). VCD reduced the number of oocytes in adult and immature rats (n = 20, P < 0.05). Liver, spleen, or ovarian weights were not affected by VCD in either group. VCD reduced uterine weight in adult (n = 20, P < 0.05) but not in immature rats. These results demonstrate that VCD decreases uterine weight in adult rats and as with immature rats, selectively destroys oocytes in ovaries of adults.

Regulation of substance P by nerve growth factor: disruption by capsaicin

Capsaicin depleted substance P from guinea pig dorsal root ganglia and inhibited the retrograde axoplasmic transport of nerve growth factor (NGF). Doses of capsaicin which depleted substance P also inhibited the retrograde axoplasmic transport of NGF. Inhibition of the retrograde transport of NGF by capsaicin preceded substance P depletion. Supplementation of guinea pigs with mouse NGF completely prevented capsaicin-induced substance P depletion. It is concluded that capsaicin depletes substance P from primary afferent neurons of the adult guinea pig by altering the availability of NGF. The data support a role for NGF in the normal maintenance of neuropeptide levels in some sensory neurons in the adult animal.

Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide

4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P<0.05) the number of preantral follicles by day 30 relative to control. There were no ultrastructural differences in morphology between VCD-treated and control ovaries. Circulating FSH levels in VCD-treated animals were greater (days 120, 240, and 360, P<0.05) than in controls. Cyclicity was disrupted in the VCD-treated group by day 360. These results show that VCD-induced follicular destruction in rats is associated with a sequence of events (loss of preantral follicles, increased plasma FSH, and cyclic disruption) preceding premature ovarian senescence that is similar to events that occur during the onset of menopause in women.

Weight-of-Evidence Evaluation of Reproductive and Developmental Effects of Low Doses of Bisphenol A

Recent public concern has focused on potential reproductive and developmental effects from exposure to low levels of bisphenol A (BPA, CAS number 80-05-7). Two previous published reviews (; ) conducted weight-of-evidence evaluations of in vivo reproductive/developmental toxicity from BPA exposure ≤ 5 mg/kg-d based on studies published through February 2006. Here, an update of those analyses presents additional relevant studies that were published through July 25, 2008, and a weight-of-evidence analysis of the studies evaluated in all three reviews. As with the earlier literature, positive findings: (1) are countered by null findings in more numerous studies; (2) have not been replicated; (3) do not exhibit coherence and plausibility; (4) do not show consistency across species, doses, and time points; and/or (5) were from studies using non-oral exposure routes. Owing to the lack of first-pass metabolism, results from non-oral studies are of limited relevance to human exposure. Exposure levels in most of the low-dose oral and non-oral animal studies are generally much higher than those experienced by even the most exposed people in the general population. The weight of evidence does not support the hypothesis that low oral doses of BPA adversely affect human reproductive and developmental health.

Expression and Redistribution of Cellular Bad, Bax, and Bcl-xL Protein Is Associated with VCD-Induced Ovotoxicity in Rats

Abstract Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25–100 μm) and large (100–250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-xL protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-xL in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-xL protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-xL ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

Weight of the Evidence Evaluation of Low-Dose Reproductive and Developmental Effects of Bisphenol A

ABSTRACT A panel convened by the Harvard Center for Risk Analysis (HCRA) evaluated the weight of evidence for potential developmental and reproductive toxicity of bisphenol A (BPA, CASRN 80-05-7) in animals at doses well below the Lowest Observed Adverse Effect Level (LOAEL) of 50 mg/kg-day previously identified by the U.S. Environmental Protection Agency (US EPA) and even US EPAs reference dose (RfD) of 0.05 mg/kg-day. The effects are hypothesized to occur through an endocrine-modulating mode of action, specifically through estrogen receptors. The panel focused on potential male reproductive effects but also examined other endpoints possibly associated with hormone-like effects. The review considered studies published through April 2002. A formal deliberation framework focused on consistency, generalizability, and biological plausibility. The panel found no consistent affirmative evidence of low-dose BPA effects for any endpoint. Inconsistent responses across rodent species and strains made generalizability of low-dose BPA effects questionable. Lack of adverse effects in two multiple-generation reproductive and developmental studies casts doubt on suggestions of significant physiological or functional impairment. The panel was concerned about generalization of non-oral administration results to oral exposures. Differences in the pattern of BPA responses compared to estradiol or diethylstilbestrol (DES) cast doubt on estrogenicity as a low-dose mechanism of action for BPA. Finally, there is indirect evidence that humans may be less sensitive to possible estrogenic effects from BPA exposure due to pharmacodynamic factors. The panel recommended replication of existing studies under carefully controlled conditions and further study of BPAs pharmacokinetics and pharmacodynamics. The study was funded by a grant from the American Plastics Council.