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.

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.

Atrazine is the most commonly used herbicide in the United States and probably the world. Atrazine contamination is widespread and can be present in excess of 1.0 ppb even in precipitation and in areas where it is not used. In the current study, we showed that atrazine exposure (≥ 0.1 ppb) resulted in retarded gonadal development (gonadal dysgenesis) and testicular oogenesis (hermaphroditism) in leopard frogs (Rana pipiens). Slower developing males even experienced oocyte growth (vitellogenesis). Furthermore, we observed gonadal dysgenesis and hermaphroditism in animals collected from atrazine-contaminated sites across the United States. These coordinated laboratory and field studies revealed the potential biological impact of atrazine contamination in the environment. Combined with reported similar effects in Xenopus laevis, the current data raise concern about the effects of atrazine on amphibians in general and the potential role of atrazine and other endocrine-disrupting pesticides in amphibian declines.

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.

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. Atrazineexposed 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. Atrazineexposed 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.A trazine is one of the most widely used pesticides in the world. Approximately 80 million pounds are applied annually in the United States alone, and atrazine is the most common pesticide contaminant of ground and surface water (1). Atrazine can be transported more than 1,000 km from the point of application via rainfall and, as a result, contaminates otherwise pristine habitats, even in remote areas where it is not used (2, 3). In fact, more than a half million pounds of atrazine are precipitated in rainfall each year in the United States (2).In addition to its persistence, mobility, and widespread contamination of water, atrazine is also a concern because several studies have shown that atrazine is a potent endocrine disruptor active in the ppb (parts per billion) range in fish (4, 5), amphibians (6-12), reptiles, and human cell lines (5, 13-15), and at higher doses (ppm) in reptiles (16-18), birds (19), and laboratory rodents (20-28). Atrazine seems to be most potent in amphibians, where it is active at levels as low as 0.1 ppb (6-10). Although a few studies suggest that atrazine has no effect on amphibians under certain laboratory conditions (29,30), in other studies, atrazine reduces testicular volume; reduces germ cell and Sertoli cell numbers (11); induces hermaphroditism (6, 8, 10); reduces testosterone (10); and induces testicular oogenesis (7-9, 31). Furthermore, atrazine contamination is associated with demasculinization and feminization of amphibians in agricultural areas where atrazine is used (32) and directly correlated with atrazine contamination in the wild (7,9,33,34).Despite the wealth of data from larvae and newly metamorphosed amphibians, the ultimate impacts of atrazine's developmental effects on reproductive function and fitness at sexual maturity, which relate more closely to population level effects and amphibian declines, have b...

SummaryGreater 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.

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The jojoba (Simmondsia chinensis) plant produces esters of longchain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.