EK
Potential chemical obesogens
Animal studies
Bisphenol A (BPA) (found in some plastics carbonless copy paper and can linings)
Perfluoroalkyl compounds (used in nonstick cookware and water-repellent and stain-resistant fabrics)
Organotins (used in agriculture and industry, used as wood preservatives in marine areas)
Dithiocarbamates (found in cosmetics and agricultural products)
Nonylphenol (found in cosmetics and household cleaners)
Fine particulate matter (air pollutant from burning fuels and wood, from road dust, aerosols and other sources)
Organophosphate pesticides (used for termite control, in home garden products, and in some pet collars)
Atrazine (pesticide used in agriculture that can contaminate drinking water)
Nicotine
Human studies
DDE (a breakdown product of DDT, a persistent pesticide that is now banned)
PCBs (persistent chemicals used as lubricants and flame retardants, now banned)
HCB (a persistent fungicide, now banned)
Oxychlordane (a persistent pesticide, now banned)
Beta-hexachlorocyclohexane (a persistent insecticide, now banned)
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Contaminants that cause Obesegons
Potential chemical obesogens
Animal studies
Bisphenol A (BPA) (found in some plastics carbonless copy paper and can linings)
Perfluoroalkyl compounds (used in nonstick cookware and water-repellent and stain-resistant fabrics)
Organotins (used in agriculture and industry, used as wood preservatives in marine areas)
Dithiocarbamates (found in cosmetics and agricultural products)
Nonylphenol (found in cosmetics and household cleaners)
Fine particulate matter (air pollutant from burning fuels and wood, from road dust, aerosols and other sources)
Organophosphate pesticides (used for termite control, in home garden products, and in some pet collars)
Atrazine (pesticide used in agriculture that can contaminate drinking water)
Nicotine
Human studies
DDE (a breakdown product of DDT, a persistent pesticide that is now banned)
PCBs (persistent chemicals used as lubricants and flame retardants, now banned)
HCB (a persistent fungicide, now banned)
Oxychlordane (a persistent pesticide, now banned)
Beta-hexachlorocyclohexane (a persistent insecticide, now banned) Dioxins and furans (persistent chemicals formed by incineration of PVC plastic and other substances)
Maternal smoking during pregnancy
Both animal and human studies
PBDEs (flame retardants that are still used in consumer products)
Phthalates (found in some plastics)
Chemical pesticides in food and water, particularly atrazine and DDE (dichlorodiphenyldichloroethylene—a DDT breakdown product), have been linked to increased BMI in children and insulin resistance in rodents.26,27 Certain pharmaceuticals, such as the diabetes drug Avandia® (rosiglitazone), have been linked to weight gain in humans and animals,9,17 as have a handful of dietary obesogens, including the soy phytoestrogen genistein28 and monosodium glutamate.15—Most known or suspected obesogens are endocrine disruptors. Many are widespread,29 and exposures are suspected or confirmed to be quite common. In one 2010 study, Kurunthachalam Kannan, a professor of environmental sciences at the University at Albany, State University of New York, documented organotins in a designer handbag, wallpaper, vinyl blinds, tile, and vacuum cleaner dust collected from 24 houses.30 Phthalates, plasticizers that also have been related to obesity in humans,31 occur in many PVC items as well as in scented items such as air fresheners, laundry products, and personal care products.
One of the earliest links between human fetal development and obesity arose from studies of exposure to cigarette smoke in utero.32,33 Although secondhand-smoke exposure has decreased by more than half over the past 20 years, an estimated 40% of nonsmoking Americans still have nicotine by-products in their blood, suggesting exposure remains widespread.34 Babies born to smoking mothers are frequently underweight, but these same infants tend to make up for it by putting on more weight during infancy and childhood.35 “If a baby is born relatively small for its gestational age, it tries to ‘play catch-up’ as it develops and grows,” explains Retha Newbold, a developmental biologist now retired from the NTP.
This pattern of catch-up growth is often observed with developmental exposure to chemicals now thought to be obesogens, including diethylstilbestrol (DES), which Newbold spent the last 30 years studying, using mice as an experimental model. Doctors prescribed DES, a synthetic estrogen, to millions of pregnant women from the late 1930s through the 1970s to prevent miscarriage. The drug caused adverse effects in these women’s children, who often experienced reproductive tract abnormalities; “DES daughters” also had a higher risk of reproductive problems, vaginal cancer in adolescence, and breast cancer in adulthood.36 Newbold discovered that low doses of DES administered to mice pre- or neonatally also were associated with weight gain,37 altered expression of obesity-related genes,38,39 and modified hormone levels.38,39
“What we’re seeing is there’s not a difference in the number of fat cells, but the cell itself is larger after exposure to DES,” Newbold says. “There was also a difference in how [fat cells] were distributed—where they went, how they lined up, and their orientation with each other. The mechanism for fat distribution and making fat cells are set up during fetal and neonatal life.”
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Atrazine –Endocrine Damaging –and Disorienteering Sexual Development
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.
amphibian decline
endocrine disruption
pesticide
sex reversal
Atrazine 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 been unexplored. In the present study, we examined the long-term effects of atrazine exposure on reproductive development and function in an all-male population of African clawed frogs (Xenopus laevis), generated by crossing ZZ females (sex-reversed genetic males) to ZZ males (SI Materials and Methods). The advantage of using this population is that 100% of the animals tested were genetic males. As a result, all hermaphrodites and females observed are ensured to be genetic males that have been altered by endocrine disruption. We examined sex ratios, testosterone levels, sexual dimorphism, reproductive behaviors, and fertility in males exposed to 2.5 ppb atrazine throughout the larval period and for up to 3 years after metamorphosis.
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Results
Feminization.
All of the control animals reared to sexual maturity (n = 40) were males, on the basis of external morphology, whereas only 90% of the atrazine-treated animals (36 of 40) appeared male at sexual maturity (on the basis of the presence of keratinized nuptial pads on the forearms and the absence of cloacal labia). The other 10% of atrazine-exposed animals (n = 4) lacked visible nuptial pads on the forearms and had protruding cloacal labia, typical of females (Fig. 1). Upon dissection of two of the apparent females and laparotomy in another two, we confirmed that animals with cloacal labia were indeed females from the present study, on the basis of the presence of ovaries (Fig. 1F). To date, two atrazine-induced females have been maintained, mated with control males (Fig. 1G), and produced viable eggs (Fig. 1H). The resulting larvae were all male when raised to metamorphosis and sampled (n = 100), confirming that atrazine-induced females were, in fact, chromosomal males. Furthermore, atrazine-induced females lacked the DM-W further confirming that these atrazine-induced females were indeed chromosomal males (Fig. 2). These ZZ females expressed gonadal aromatase, as did true ZW females (n = 4, from our stock colony), but ZZ males (n = 8, control or treated) did not (Fig. 2).
Testes.
Atrazine exposure resulted in a significant reduction in the relative number of testicular tubules with mature sperm bundles in 2007 (n = 18; ANOVA: F = 8.65, df = 1, P < 0.01); that is, atrazine decreased the frequency of tubules with mature spermatozoa (G test: GH = 13545.2, df = 15, P < 0.001). Similar effects were not observed (P > 0.05) in animals (n = 10) 1 year later at 3 years after metamorphosis, in 2008. Other features of the gonads that were examined were not significantly different (P > 0.05).
Atrazine decreased androgen-dependent sperm production, mating behavior, and fertility. (A and C) Largest testicular cross-sections for representative control (A) and atrazine-exposed males (C) from 2007. (B and D) Magnification of individual tubules for control (B) and atrazine-exposed (D) males. Arrowheads in B and D show outline of tubules. Control tubules are typically filled with mature spermatozoa bundles, whereas the majority of tubules in atrazine-exposed males lack mature sperm bundles and are nearly empty, with only secondary spermatocytes (SS) along the periphery of the tubule. (E) Fertility for control (Con) and atrazine-exposed (Atr) males. Pooled data from both 2007 and 2008 study are shown. *P < 0.005 (ANOVA). (F) Fertility plotted against sperm content (percentage of tubules with mature sperm bundles) for control males (black symbols) and atrazine-exposed males (red symbols) for the 2007 (circles) and the 2008 (squares) studies. Dashed lines indicate the lower limit for controls for fertility and sperm content. Sample size differs from the number of trials because no data are available from females that did not lay eggs. (Bar in A applies to A and C; in B applies to B and D.) —Previous studies showed that atrazine demasculinizes (chemically castrates) and feminizes exposed amphibian larvae, resulting in hermaphrodites (8, 10) or males with testicular oocytes (7, 9) at metamorphosis. Since our initial publications (7, 9, 10), the effects of atrazine on amphibian development and the significance of these effects to amphibian declines have been a subject of debate (30, 35, 36). Although some investigators, including Carr et al. (6), reported statistically significant effects of atrazine on gonadal morphology in X. laevis (P < 0.0003 for multiple testes and P = 0.0042 for hermaphrodites), others, using different experimental conditions and different populations of the same species, suggested that atrazine had no effect (29). Essential to this debate, however, is (i) the terminology used to describe gonadal abnormalities; (ii) the expertise and ability of other researchers to recognize abnormalities; (iii) the possibility of natural variation in sex differentiation processes between species and even between populations (or strains) within a species (37); and (iv) the long-term consequences and significance of the observed abnormalities to amphibian reproductive fitness. Here we describe complete and functional female development in genetic (ZZ) males exposed to atrazine, not the production of hermaphrodites or males with testicular oocytes. Thus, there is no confusion in the present study regarding proper terminology or proper identification. Furthermore, because we used an all genetic (ZZ) male colony and genotyped the atrazine-induced ZZ females, there is no question that atrazine completely sex-reversed genetic (ZZ) males, resulting in reproductively functional females. — suggest that sex-reversal by atrazine (complete feminization of genetic males) is not a species-specific effect but rather one that occurs across nonamniote vertebrate classes
The present study thoroughly examines the long-term effects of atrazine on reproductive function in amphibians. Although a single published study attempted to examine long-term reproductive effects of atrazine in amphibians (38), the authors did not report examinations of morphology. Furthermore, their examination of fertility and breeding of atrazine-exposed males was conducted after animals were injected with reproductive hormones (human chorionic gonadotropin, hCG), effectively providing “hormone replacement therapy” and reversing the effects of atrazine. The present study represents a more thorough examination of the effects of atrazine on sex hormone production, testosterone-dependent development and morphology, male reproductive behavior, and fertility