Tyrone B. Hayes

Regulatory decisions on endocrine disrupting chemicals should be based on the principles of endocrinology

For years, scientists from various disciplines have studied the effects of endocrine disrupting chemicals (EDCs) on the health and wellbeing of humans and wildlife. Some studies have specifically focused on the effects of low doses, i.e. those in the range that are thought to be safe for humans and/or animals. Others have focused on the existence of non-monotonic dose-response curves. These concepts challenge the way that chemical risk assessment is performed for EDCs. Continued discussions have clarified exactly what controversies and challenges remain. We address several of these issues, including why the study and regulation of EDCs should incorporate endocrine principles; what level of consensus there is for low dose effects; challenges to our understanding of non-monotonicity; and whether EDCs have been demonstrated to produce adverse effects. This discussion should result in a better understanding of these issues, and allow for additional dialog on their impact on risk assessment.

Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses

Atrazine is the most commonly used herbicide in the U.S. and probably the world. It can be present at several parts per million in agricultural runoff and can reach 40 parts per billion (ppb) in precipitation. We examined the effects of atrazine on sexual development in African clawed frogs (Xenopus laevis). Larvae were exposed to atrazine (0.01–200 ppb) by immersion throughout larval development, and we examined gonadal histology and laryngeal size at metamorphosis. Atrazine (≥0.1 ppb) induced hermaphroditism and demasculinized the larynges of exposed males (≥1.0 ppb). In addition, we examined plasma testosterone levels in sexually mature males. Male X. laevis suffered a 10-fold decrease in testosterone levels when exposed to 25 ppb atrazine. We hypothesize that atrazine induces aromatase and promotes the conversion of testosterone to estrogen. This disruption in steroidogenesis likely explains the demasculinization of the male larynx and the production of hermaphrodites. The effective levels reported in the current study are realistic exposures that suggest that other amphibian species exposed to atrazine in the wild could be at risk of impaired sexual development. This widespread compound and other environmental endocrine disruptors may be a factor in global amphibian declines.

Pesticide Mixtures, Endocrine Disruption, and Amphibian Declines: Are We Underestimating the Impact?

Amphibian populations are declining globally at an alarming rate. Pesticides are among a number of proposed causes for these declines. Although a sizable database examining effects of pesticides on amphibians exists, the vast majority of these studies focus on toxicological effects (lethality, external malformations, etc.) at relatively high doses (parts per million). Very few studies focus on effects such as endocrine disruption at low concentrations. Further, most studies examine exposures to single chemicals only. The present study examined nine pesticides (four herbicides, two fungicides, and three insecticides) used on cornfields in the midwestern United States. Effects of each pesticide alone (0.1 ppb) or in combination were examined. In addition, we also examined atrazine and S-metolachlor combined (0.1 or 10 ppb each) and the commercial formulation Bicep II Magnum, which contains both of these herbicides. These two pesticides were examined in combination because they are persistent throughout the year in the wild. We examined larval growth and development, sex differentiation, and immune function in leopard frogs (Rana pipiens). In a follow-up study, we also examined the effects of the nine-compound mixture on plasma corticosterone levels in male African clawed frogs (Xenopus laevis). Although some of the pesticides individually inhibited larval growth and development, the pesticide mixtures had much greater effects. Larval growth and development were retarded, but most significantly, pesticide mixtures negated or reversed the typically positive correlation between time to metamorphosis and size at metamorphosis observed in controls: exposed larvae that took longer to metamorphose were smaller than their counterparts that metamorphosed earlier. The nine-pesticide mixture also induced damage to the thymus, resulting in immunosuppression and contraction of flavobacterial meningitis. The study in X. laevis revealed that these adverse effects may be due to an increase in plasma levels of the stress hormone corticosterone. Although it cannot be determined whether all the pesticides in the mixture contribute to these adverse effects or whether some pesticides are effectors, some are enhancers, and some are neutral, the present study revealed that estimating ecological risk and the impact of pesticides on amphibians using studies that examine only single pesticides at high concentrations may lead to gross underestimations of the role of pesticides in amphibian declines.

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis)

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

Herbicides: Feminization of male frogs in the wild

Atrazine is the most commonly used herbicide in the United States and probably in the world. Here we investigate the effects of exposure to water-borne atrazine contamination on wild leopard frogs (Rana pipiens) in different regions of the United States and find that 10–92% of males show gonadal abnormalities such as retarded development and hermaphroditism. These results are supported by laboratory observations, which together highlight concerns over the biological effects of environmental atrazine on amphibians.

Sex determination and primary sex differentiation in amphibians: Genetic and developmental mechanisms

Most amphibians lack morphologically distinguishable sex chromosomes, but a number of experimental techniques have shown that amphibian sex determination is controlled genetically. The few studies suggesting that environment influences sex determination in amphibians have all been conducted at temperatures outside of the range normally experienced by the species under study, and these effects probably do not occur under natural conditions. No sex-determining genes have been described in amphibians, and sex differentiation can be altered by treatment with exogenous steroid hormones. The effects of sex steroids vary extensively between species, and a variety of steroids can alter the sex ratios of treated larvae. The role of endogenous sex steroids in gonadal differentiation has not been fully explored; thus the natural role of steroids in amphibian gonadal differentiation is unknown. Sex steroid receptors have not been examined in amphibian gonads, and the mechanism of steroid action on the gonad is unclear. In addition to steroids, the thyroid hormones may play a role in gonadal differentiation. Pituitary gonadotrop(h)ins affect gonadal growth, but not differentiation or maturation of gonads. In addition to the issue of resolving the mechanisms underlying hormone action in gonadal differentiation, other debates concerning interactions between the developing gonads and the invading germ cells, and even the origin of the medullary and cortical portions of the developing gonads, remain unresolved. Studies examining links between sex determination and gonadal differentiation are needed. In addition, examinations of variation in steroidal effects on gonadal development in a phylogenetic context are lacking.

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans.

Background Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP. Methods We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1’s role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1–dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII. Results Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this “orphan” receptor. Conclusion The current findings are consistent with atrazine’s endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.

Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17beta-estradiol): Support for the demasculinization/feminization hypothesis.

Atrazine is a potent endocrine disruptor that both chemically castrates and feminizes male amphibians. It depletes androgens in adult frogs and reduces androgen-dependent growth of the larynx in developing male larvae. It also disrupts normal gonadal development and feminizes the gonads of developing males. Gonadal malformations induced by atrazine include hermaphrodites and males with multiple testes [single sex polygonadism (SSP)], and effects occur at concentrations as low as 0.1 ppb (μg/L). Here, we describe the frequencies at which these malformations occur and compare them with morphologies induced by the estrogen, 17β-estradiol (E2), and the antiandrogen cyproterone acetate, as a first step in testing the hypothesis that the effects of atrazine are a combination of demasculinization and feminization. The various forms of hermaphroditism did not occur in controls. Nonpigmented ovaries, which occurred at relatively high frequencies in atrazine-treated larvae, were found in four individuals out of more than 400 controls examined (1%). Further, we show that several types of gonadal malformations (SSP and three forms of hermaphroditism) are produced by E2 exposure during gonadal differentiation, whereas a final morphology (nonpigmented ovaries) appears to be the result of chemical castration (disruption of androgen synthesis and/or activity) by atrazine. These experimental findings suggest that atrazine-induced gonadal malformations result from the depletion of androgens and production of estrogens, perhaps subsequent to the induction of aromatase by atrazine, a mechanism established in fish, amphibians, reptiles, and mammals (rodents and humans).

Variation in thyroid hormone action and tissue content underlies species differences in the timing of metamorphosis in desert frogs

Summary Hormonal control of post‐embryonic morphogenesis is well established, but it is not clear how differences in developmental endocrinology between species may underlie animal diversity. We studied this issue by comparing metamorphic thyroid hormone (TH) physiology and gonad development across spadefoot toad species divergent in metamorphic rate. Tissue TH content, in vitro tail tip sensitivity to TH, and rates of TH‐induced tail tip shrinkage correlated with species differences in larval period duration. Gonad differentiation occurred before metamorphosis in species with long larval periods and after metamorphosis in the species with short larval periods. These differences in TH physiology and gonad development, informed by phylogeny and ecology of spadefoot metamorphosis, provide evidence that selection for the short larval periods in spadefoot toads acted via TH physiology and led to dramatic heterochronic shifts in metamorphic climax relative to gonad development.

Evolutionary Patterns of Diversity in Spadefoot Toad Metamorphosis (Anura: Pelobatidae)

Abstract The larvae of spadefoot toads exhibit extreme developmental/endocrinological diversity. For example, New World spadefoot toads (Scaphiopus and Spea) have the shortest larval periods known among anurans, and the tadpoles of Old World spadefoot taxa (Pelobates) are among the largest known. To analyze the patterns of this diversity in an evolutionary context, we generated comparable larval growth and development data from 10 of the 11 taxa of spadefoot toads and from one taxon of parsley frog (Pelodytes), the nearest spadefoot toad relative. We found dramatic differences in growth and development among taxa, which indicated that taxon-specific physiology, rather than phenotypic plasticity, underlies larval period diversity. For all eight response variables (development rate, three growth rates, time to forelimb emergence, time to tail resorption, mass at tail resorption, and body length at tail resorption), taxa within genera were similar to each other and were different from taxa in other genera. Larvae of Scaphiopus were small with short larval periods, larvae of Spea were large with short larval periods, larvae of Pelobates were large with long larval periods, and larvae of Pelodytes were small with long larval periods. Even though taxa within the same genus live in different environments, larval growth and development correlated with phylogenetic groupings rather than breeding habitat. Mapping larval data onto a molecular phylogeny indicated that short larval periods, as well as rapid embryonic development and high temperature tolerance, originated within the spadefoot toad family.