[Show abstract][Hide abstract] ABSTRACT: A 27-day controlled exposure study of adult male African clawed frogs (Xenopus laevis) was conducted to examine the mechanism by which tebuconazole may disrupt steroidogenesis. The fungicide was measured by LC-MS/MS in tank water and in target tissues (adipose, kidney, liver, and brain), and we observed tissue-specific bioconcentration with BCF up to 238. Up to 10 different steroid hormones were quantified in gonads using LC-MS/MS and in plasma using GC-MS/MS and a radioimmunoassay was performed for further measurement of androgens. In order to assess whether effects increased with exposure or animals adapted to the xenobiotic, blood samples were collected 12 days into the study and at termination (day 27). After 12 days of exposure to 100 and 500μgL(-1) tebuconazole, plasma levels of testosterone (T) and dihydrotestosterone (DHT) were increased, while plasma 17β-estradiol (E2) concentrations were greatly reduced. Exposure to 0.1μgL(-1), on the other hand, resulted in decreased levels of T and DHT, with no effects observed for E2. After 27 days of exposure, effects were no longer observed in circulating androgen levels while the suppressive effect on E2 persisted in the two high-exposure groups (100 and 500μgL(-1)). Furthermore, tebuconazole increased gonadal concentrations of T and DHT as well as expression of the enzyme CYP17 (500μgL(-1), 27 days). These results suggest that tebuconazole exposure may supress the action of CYP17 at the lowest exposure (0.1μgL(-1)), while CYP19 suppression dominates at higher exposure concentrations (increased androgens and decreased E2). Increased androgen levels in plasma half-way into the study and in gonads at termination may thus be explained by compensatory mechanisms, mediated through increased enzymatic expression, as prolonged exposure had no effect on circulating androgen levels.
[Show abstract][Hide abstract] ABSTRACT: This paper presents the development, optimization, and validation of a LC-MS/MS methodology to determine the concentration of the antifungal drug and fungicide tebuconazole in a controlled exposure study of African clawed frogs (Xenopus laevis). The method is validated on animal tank water and on tissue from exposed and non-exposed adult X. laevis. Using solid-phase extraction (SPE), the analytical method allows for quantification of tebuconazole at concentrations as low as 3.89 pg mL(-1) in 10 mL water samples. Using bead-beating-assisted matrix solid-phase dispersion (MSPD), it was possible to quantify tebuconazole down to 0.63 pg mg(-1) wet weight liver using 150 mg tissue. The deuterated analogue of tebuconazole was used as internal standard, and ensured method accuracy in the range 80.6-99.7 % for water and 68.1-109 % for tissue samples. The developed method was successfully applied in a 4-week X. laevis repeated-exposure study, revealing high levels of tebuconazole residues in adipose and liver tissue, and with experimental bioconcentration factors up to 18,244 L kg(-1).
[Show abstract][Hide abstract] ABSTRACT: 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 dialogue on their impact on risk assessment.
[Show abstract][Hide abstract] ABSTRACT: The continued presence of gonadotropin-releasing hormone (GnRH) neurons is required for a healthy reproductive lifespan, but factors that maintain postnatal GnRH neurons have not been identified. To begin to understand these factors, we investigated whether (1) fibroblast growth factor (FGF) signaling and (2) interactions with the opposite sex are involved in the maintenance of the postnatal GnRH system. A transgenic mouse model (dnFGFR mouse) with the targeted expression of a dominant-negative FGF receptor (dnFGFR) in GnRH neurons was used to examine the consequence of FGF signaling deficiency on postnatal GnRH neurons. Male dnFGFR mice suffered a significant loss of postnatal GnRH neurons within the first 100 days of life. Interestingly, this loss was reversed after cohabitation with female, but not male, mice for 300-550 days. Further, opposite sex housing in dnFGFR males also increased hypothalamic GnRH peptide levels, promoted a more mature GnRH neuronal morphology, facilitated litter production, and enhanced testicular morphology. Lastly, mice hypomorphic for FGFR3 exhibited a similar pattern of postnatal GnRH neuronal loss as dnFGFR males, suggesting FGF signaling acts, in part, through FGFR3 to enhance the maintenance of the postnatal GnRH system. In summary, we have shown that FGF signaling is required for the continued presence of postnatal GnRH neurons. However, this requirement is not absolute as sexual interactions can compensate for defects in FGFR signaling, thereby rescuing the declining GnRH system. This suggests the postnatal GnRH system is highly plastic and capable of responding to environmental stimuli throughout adult life.
[Show abstract][Hide abstract] ABSTRACT: A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best
achieved when information about a chemical’s potential hazardous effects is obtained as early in the design
process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at “the drawing board.” It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In thispaper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a ‘proof-of-principle’ test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.
Green Chemistry 09/2012; 15(1). DOI:10.1039/c2gc35055f · 8.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.
[Show abstract][Hide abstract] ABSTRACT: The herbicide atrazine is a potent endocrine disruptor, active in fish and amphibians in the low ppb range. Among other effects,
atrazine impairs reproductive development and function including decreased testosterone levels, impaired testicular development,
and low fertility/sperm production in male fish, amphibians, and in some reptiles. Atrazine also feminizes fish, amphibians
and reptiles resulting in the development of oocytes in the testes and complete feminization. In addition to laboratory experiments,
similar effects have been associated with animals in the wild. Although there is some question about how to compare the doses,
adverse effects are also observed in laboratory rodents: including prostate disease, low sperm production, and decreased testosterone
levels in males and mammary cancer, abortion, and impaired mammary development in females. These effects are all consistent
with the induction of aromatase, the enzyme that converts testosterone into estrogen, a mechanism that has been demonstrated
across vertebrate classes. Despite well over 150 publications from at least 50 independent laboratories showing adverse reproductive
effects in all vertebrate classes examined, and recent epidemiological studies associating atrazine exposure with low sperm
counts in men, breast and prostate cancer, and birth defects, the major manufacturer still maintains that “atrazine has been
used safely for 50 years” and the US EPA still struggles with how to evaluate pesticides for endocrine disrupting effects.
[Show abstract][Hide abstract] ABSTRACT: Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".
The Journal of steroid biochemistry and molecular biology 03/2011; 127(1-2):64-73. DOI:10.1016/j.jsbmb.2011.03.015 · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: I am honored to receive the E. E. Just Award for 2010. In my invited essay, I have opted to discuss the state of diversity in the biological sciences with some recommendations for moving forward toward a more positive and inclusive academy. The need to develop cohorts of minority scientists as support groups and to serve as role models within our institutions is stressed, along with the need to ensure that minority scientists are truly included in all aspects of the academy. It is imperative that we increase our efforts to prepare for the unique challenges that we will face as the United States approaches a "majority minority" population in the next 50 years.
Molecular biology of the cell 11/2010; 21(22):3767-9. DOI:10.1091/mbc.E10-05-0414 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Greater than 70% of the world's amphibian species are in decline. We propose that there is probably not a single cause for global amphibian declines and present a three-tiered hierarchical approach that addresses interactions among and between ultimate and proximate factors that contribute to amphibian declines. There are two immediate (proximate) causes of amphibian declines: death and decreased recruitment (reproductive failure). Although much attention has focused on death, few studies have addressed factors that contribute to declines as a result of failed recruitment. Further, a great deal of attention has focused on the role of pathogens in inducing diseases that cause death, but we suggest that pathogen success is profoundly affected by four other ultimate factors: atmospheric change, environmental pollutants, habitat modification and invasive species. Environmental pollutants arise as likely important factors in amphibian declines because they have realized potential to affect recruitment. Further, many studies have documented immunosuppressive effects of pesticides, suggesting a role for environmental contaminants in increased pathogen virulence and disease rates. Increased attention to recruitment and ultimate factors that interact with pathogens is important in addressing this global crisis.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 03/2010; 107(10):4612-7. DOI:10.1073/pnas.0909519107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies from my laboratory, showing the chemical castration (demasculinization) and feminization of amphibians by low but ecologically relevant concentrations of atrazine in the laboratory and in the wild, prompted a critical response from atrazine's manufacturer, Syngenta Crop Protection, and Syngenta-funded scientists. A careful analysis of the published data funded by Syngenta, and of several studies submitted to the US Environmental Protection Agency (EPA) by the Syngenta-funded panel for data evaluation, indicates that the data presented in these studies are not in disagreement with my laboratory's peer-reviewed, published data. Further, the published and unpublished data presented to the EPA by the Syngenta-funded panel (and touted in the popular press) suffer from contaminated laboratory controls; high mortality; inappropriate measure- ments of hormone levels in stressed, sexually immature animals during nonreproductive seasons; and contaminated reference sites. The confound- ing factors in the industry-funded studies severely limit any conclusions about the adverse effects of atrazine on amphibians and prevent meaningful comparisons with my laboratory's published data.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Environmental Health Perspectives 06/2007; 115(5):720-7. DOI:10.1289/ehp.9758 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The popular herbicide atrazine is an endocrine disruptor that demasculinizes and feminizes several species of animals, and co-relates with breast and reproductive disorders in mammalians. We recently reported that atrazine induces human aromatase gene expression via promoter II (ArPII) in a steroidogenic factor 1 (SF-1)-dependent manner. Here, we show that knockdown of SF-1 abolishes ArPII induction by atrazine in H295R cells, which harbor high SF-1 expression and are originally atrazine-responsive. Conversely, exogenous SF-1 enables atrazine to induce ArPII in the otherwise non-responsive KGN cells. Atrazine's effect is independent from protein kinase A and LRH-1, a close relative of SF-1. However, it binds directly to the SF-1, and concomitantly, enhances interactions of SF-1 with co-activator TIF2, and renders more SF-1 binding to ArPII chromatin. Intriguingly, LBD mutations do not alter SF-1's ability to mediate atrazine stimulation, suggesting that atrazine interacts with SF-1 via a region(s) other than the ligand binding pocket. These data suggest that atrazine binds to and activates SF-1 to induce ArPII.
Biochemical and Biophysical Research Communications 05/2007; 355(4):1012-8. DOI:10.1016/j.bbrc.2007.02.062 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 (microg/L). Here, we describe the frequencies at which these malformations occur and compare them with morphologies induced by the estrogen, 17beta-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).