Rochelle W. Tyl

Environmental Factors and Puberty Timing: Expert Panel Research Needs

Serono Symposia International convened an expert panel to review the impact of environmental influences on the regulation of pubertal onset and progression while identifying critical data gaps and future research priorities. An expert panel reviewed the literature on endocrine-disrupting chemicals, body size, and puberty. The panel concluded that available experimental animal and human data support a possible role of endocrine-disrupting chemicals and body size in relation to alterations in pubertal onset and progression in boys and girls. Critical data gaps prioritized for future research initiatives include (1) etiologic research that focus on environmentally relevant levels of endocrine-disrupting chemicals and body size in relation to normal puberty as well as its variants, (2) exposure assessment of relevant endocrine-disrupting chemicals during critical windows of human development, and (3) basic research to identify the primary signal(s) for the onset of gonadotropin-releasing hormone–dependent/central puberty and gonadotropin-releasing hormone–independent/peripheral puberty. Prospective studies of couples who are planning pregnancies or pregnant women are needed to capture the continuum of exposures at critical windows while assessing a spectrum of pubertal markers as outcomes. Coupled with comparative species studies, such research may provide insight regarding the causal ordering of events that underlie pubertal onset and progression and their role in the pathway of adult-onset disease.

Two-Generation Reproductive Toxicity Study of Dietary Bisphenol A in CD-1 (Swiss) Mice

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

General background on the hypothalamic-pituitary-thyroid (HPT) axis.

This article reviews the thyroid system, mainly from a mammalian standpoint. However, the thyroid system is highly conserved among vertebrate species, so the general information on thyroid hormone production and feedback through the hypothalamic-pituitary-thyroid (HPT) axis should be considered for all vertebrates, while species-specific differences are highlighted in the individual articles. This background article begins by outlining the HPT axis with its components and functions. For example, it describes the thyroid gland, its structure and development, how thyroid hormones are synthesized and regulated, the role of iodine in thyroid hormone synthesis, and finally how the thyroid hormones are released from the thyroid gland. It then progresses to detail areas within the thyroid system where disruption could occur or is already known to occur. It describes how thyroid hormone is transported in the serum and into the tissues on a cellular level, and how thyroid hormone is metabolized. There is an in-depth description of the alpha and beta thyroid hormone receptors and their functions, including how they are regulated, and what has been learned from the receptor knockout mouse models. The nongenomic actions of thyroid hormone are also described, such as in glucose uptake, mitochondrial effects, and its role in actin polymerization and vesicular recycling. The article discusses the concept of compensation within the HPT axis and how this fits into the paradigms that exist in thyroid toxicology/endocrinology. There is a section on thyroid hormone and its role in mammalian development: specifically, how it affects brain development when there is disruption to the maternal, the fetal, the newborn (congenital), or the infant thyroid system. Thyroid function during pregnancy is critical to normal development of the fetus, and several spontaneous mutant mouse lines are described that provide research tools to understand the mechanisms of thyroid hormone during mammalian brain development. Overall this article provides a basic understanding of the thyroid system and its components. The complexity of the thyroid system is clearly demonstrated, as are new areas of research on thyroid hormone physiology and thyroid hormone action developing within the field of thyroid endocrinology. This review provides the background necessary to review the current assays and endpoints described in the following articles for rodents, fishes, amphibians, and birds.

The Developmental Toxicity of Bisphenol A in Rats and Mice

Bisphenol A (BPA) was evaluated for developmental toxicity in CD rats (0, 160, 320, or 640 mg/kg/day) and CD-1 mice (0, 500, 750, 1000, or 1250 mg/kg/day) dosed daily by gastric intubation on Gestational Days 6 through 15. Timed-pregnant dams were sacrificed 1 day prior to parturition, the uterine contents were examined, and all fetuses were examined for external, visceral, and skeletal malformations. In rats, maternal weight gain during gestation, weight gain corrected for gravid uterine weight, and weight gain during treatment were significantly reduced at all BPA doses. Gravid uterine weight and average fetal body weight per litter were not affected by BPA. No increase in percentage resorptions per litter or percentage fetuses malformed per litter was detected. In mice, maternal mortality occurred at all BPA doses, reaching 18% at the high dose, which also produced a significant decrease in maternal body weight gain during gestation and treatment. Weight gain corrected for gravid uterine weight was not affected by BPA. Reductions in gravid uterine weight and average fetal body weight were observed with the 1250 mg/kg dose of BPA. Relative maternal liver weight was increased at all doses of BPA. There was a significant increase in the percentage of resorptions per litter with 1250 mg BPA/kg/day. Malformation incidence was not altered by BPA. Thus, BPA treatment at maternally toxic dose levels during organogenesis produced fetal toxicity in mice but not in rats and did not alter fetal morphologic development in either species.

Effects of acrylamide on rodent reproductive performance.

Acrylamide monomer causes peripheral neurotoxicity, mutagenicity, clastogenicity, male reproductive toxicity, prenatal lethality, and endocrine-related tumors in rodents. Acrylamide (and/or its metabolite glycidamide) binds to dopamine receptors and spermatid protamines and inhibits activity of kinesin and dyneine, resulting in interference with neuronal intracellular transport and sperm motility. Glycidamide binds to various proteins and DNA. Acrylamide at low doses decreases litter size, with rats more sensitive than mice. At higher doses, sperm morphology and motility and neurotoxicity are affected, which decreases mating frequency. Acrylamide does not affect female reproduction (females exhibit neurotoxicity). Dominant lethal mutations cause decreased newborn litter size. The mechanisms of action appear to be: (1) acrylamide/glycidamide binding to spermatid protamines, causing dominant lethality and effects on sperm morphology; and (2) acrylamide binding to motor proteins, causing distal axonopathy, including hindlimb weakness/paresis, and effects on mounting, sperm motility, and intromission. Glycidamide-induced mutations appear to play no role in reproductive or neurologic toxicity.

The developmental toxicity of ethylene glycol in rats and mice

Timed-pregnant CD rats and CD-1 mice were dosed by gavage with ethylene glycol (EG) in distilled water on gestational days (gd) 6 through 15 (0, 1250, 2500, or 5000 mg kg-1 day-1 for rats; and 0, 750, 1500, or 3000 mg kg-1 day-1 for mice). Females were observed daily during treatment, but no maternal deaths or distinctive clinical signs were noted. Dose-related decreases in maternal weight gain during treatment were significant at all doses in rats and at the mid and high doses in mice. Gravid uterine weight was reduced in both species at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. At termination (gd 20, rats; gd 17, mice), the status of uterine implantation sites was recorded, and live fetuses were weighed and examined for external, visceral, and skeletal malformations. Dose-related increases in postimplantation loss per litter were observed in both species with the high dose significantly above controls only in rats. Fetal body weight per litter was significantly reduced at the mid and high doses in rats and at all doses in mice. The percentage of malformed live fetuses per litter and/or the percentage of litters with malformed fetuses was significantly elevated in all EG dose groups and greater than 95% of litters were affected at the high dose for each species. A wide variety of malformations were observed; the most common in both species were craniofacial and neural tube closure defects and axial skeletal dysplasia. EG produced severe developmental toxicity in two rodent species at doses that apparently failed to produce any serious maternal effects.

Developmental Toxicity Evaluation of Dietary Di(2-ethylhexyl) phthalate in Fischer 344 Rats and CD-1 Mice

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizing agent, was evaluated for developmental toxicity in timed-pregnant Fischer 344 rats (22-25 dams/dose) and CD-1 mice (24-30 dams/dose). DEHP was administered in the diet on gestational Days (gd) 0 through 20 at 0.0, 0.5, 1.0, 1.5, or 2.0% (rats) and on gd 0 through 17 at 0.00, 0.025, 0.05, 0.10, or 0.15% (mice). At termination (gd 20, rats; gd 17 mice), all fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal toxicity and reduced fetal body weight per litter were observed at 1.0, 1.5, and 2.0%. Increased resorptions and decreased number of live fetuses/litter were observed at 2.0%. Maternal food consumption was reduced and water consumption was increased in all DEHP groups. The number and percentage of fetuses malformed per litter were unaffected by treatment. In mice, maternal toxicity, increased resorptions and late fetal deaths, decreased number of live fetuses, and reduced fetal body weight per litter were observed at 0.10 and 0.15%. Maternal food and water consumption exhibited a dose-related upward trend with food consumption significantly increased at 0.15%. The number and percentage of fetuses malformed per litter (open eye, exophthalmia, exencephaly, short, constricted, or no tail, major vessel malformations, fused or branched ribs, and fused or misaligned thoracic vertebral centra) were elevated at 0.05, 0.10, and 0.15% DEHP. In conclusion, DEHP was not teratogenic at any dose tested in Fischer 344 rats when administered in the feed throughout gestation but did produce maternal and other embryofetal toxicity at 1.0, 1.5, and 2.0%. In contrast, DEHP administration throughout gestation in CD-1 mice resulted in an increased incidence of malformations at doses which produced maternal and other embryofetal toxicity (0.10 and 0.15%) and at a dose (0.05%) which did not produce significant maternal toxicity. No treatment-related embryofetal toxicity including teratogenicity was observed in mice at 0.025% or in rats at 0.5% DEHP.

Relationship between acrylamide reproductive and neurotoxicity in male rats

To determine whether there is a relationship between the reproductive and neurotoxic effects of acrylamide monomer (AM), the first week of the study design of Sublet et al. ¿14 was duplicated: Long-Evans male rats were gavaged with AM in water, 25/group, at 0, 5, 15, 30, 45, or 60 mg/kg/day for 5 days (days 1 through 5). On Day 8, males were paired overnight with untreated virgin females (1 : 1) in proestrus/estrus. On day 9, males were evaluated for forelimb and hindlimb grip strength. Five males/group were perfusion fixed, 20/group were used for andrologic assessment, and all were necropsied. Perfusion-fixed sciatic nerves were examined histologically. Sperm-positive females were examined for preimplantation and postimplantation loss at midpregnancy. At 15 to 60 mg/kg/day, males exhibited significantly reduced weight gain, reduced mating, fertility, and pregnancy indices by trend analysis (significant at 60 mg/kg/d by pairwise comparison), and increased postimplantation loss and dominant lethal factor, F(L)%, at 45 and 60 mg/kg/day. At 60 mg/kg/day, the sperm beat cross frequency was increased, with no significant effects on epididymal sperm motility or concentration, and hindlimb grip strength was decreased, with no pathologic lesions in sciatic nerves. Therefore, epididymal sperm, mating, and neurotoxic effects were observed at AM doses that also resulted in increased postimplantation loss, possibly by different mechanisms.

The induction of bladder stones by terephthalic acid, dimethyl terephthalate, and melamine (2,4,6-triamino-s-triazine) and its relevance to risk assessment

Terephthalic acid (TPA), dimethyl terephthalate (DMT), and melamine (MA) induced calculi and transitional cell hyperplasia in urinary bladders of rats. A high incidence of calculi was induced in weanling rats, but the incidence was much lower in adult rats ingesting the same dietary concentration of the chemical. The dose-response curves for the induction of urolithiasis in weanling rats were extremely steep, consistent with the fact that the formation calculi can occur in urine that is supersaturated, but not in urine that is undersaturated with respect to the stone components. In the cases of TPA and DMT, stones were composed primarily of calcium terephthalate (CaTPA). By determining the solubility of CaTPA, the concentration of TPA that would be required to achieve urinary saturation was calculated, and a conservative estimate of the amount of TPA or DMT that would have to be absorbed in order to induce calculi was derived. TPA and MA induced bladder tumors in rats in chronic feeding studies. However, it is likely that these tumors were secondary to the development of calculi. TPA and MA are apparently nongenotoxic, and they do not appear to be metabolized. Increased cell replication in the urothelium of the bladder caused by chronic physical injury was probably a major factor in the mechanism of induction of bladder tumors by bladder stones. Bladder neoplasms occurred primarily in the high dose groups, and they were usually, although not invariably, associated with stones. The possibility that stones were passed or were lost during processing of tissues for histopathologic examination could explain the absence of calculi from some of the neoplastic bladders. The formation of bladder calculi is an example of a threshold effect. Although there is strong evidence linking bladder stones with the induction of tumors, the existence of thresholds in chemical carcinogenesis continues to be controversial. A decision by the U.S. Environmental Protection Agency concerning the levels of MA allowed to occur in the food chain indicates that data regarding thresholds, even in the case of urolithiasis, are not being utilized in the risk assessment process.

Basic exploratory research versus guideline-compliant studies used for hazard evaluation and risk assessment: bisphenol A as a case study.

Background Myers et al. [Environ Health Perspect 117:309–315 (2009)] argued that Good Laboratory Practices (GLPs) cannot be used as a criterion for selecting data for risk assessment, using bisphenol A (BPA) as a case study. They did not discuss the role(s) of guideline-compliant studies versus basic/exploratory research studies, and they criticized both GLPs and guideline-compliant studies and their roles in formal hazard evaluation and risk assessment. They also specifically criticized our published guideline-compliant dietary studies on BPA in rats and mice and 17β-estradiol (E2) in mice. Objectives As the study director/first author of the criticized E2 and BPA studies, I discuss the uses of basic research versus guideline-compliant studies, how testing guidelines are developed and revised, how new end points are validated, and the role of GLPs. I also provide an overview of the BPA guideline-compliant and exploratory research animal studies and describe BPA pharmacokinetics in rats and humans. I present responses to specific criticisms by Myers et al. Discussion and conclusions Weight-of-evidence evaluations have consistently concluded that low-level BPA oral exposures do not adversely affect human developmental or reproductive health, and I encourage increased validation efforts for “new” end points for inclusion in guideline studies, as well as performance of robust long-term studies to follow early effects (observed in small exploratory studies) to any adverse consequences.