Hayes TB , Stuart AA , Mendoza M , Collins A , Noriega N , Vonk A , Johnston G , Liu R , Kpodzo D .

	Figure 1. Proposed mechanism of atrazine action on amphibians. The induction of aromatase results in a decrease in androgens (aromatase substrate) and a subsequent increase in estrogens (aromatase product). Atrazine thus demasculinizes (chemically castrates) and feminizes amphibians. Molecular and endocrine support for the proposed mechanism has been demonstrated in amphibians as well as all other vertebrate classes examined (see “Discussion”).
	Figure 2. . Experimental design for testing the effects of partial E2 exposure on sex differentiation in X. laevis larvae. The larval period from hatching [NF stage (St.) 48] to the completion of metamorphosis (complete tail reabsorption, NF stage 66) lasts 65 days on average under standard conditions in our laboratory. Sex differentiation occurs between NF stages 52 and 54 (~ 25 days posthatching). Animals were treated with E2 (100 μg/L) for 7 days (beginning at NF stage 50, day 19 posthatching, and ending at NF stage 53, day 26), for 14 days (beginning at NF stage 50, day 19 posthatching, and ending at NF stage 55, day 33), or 49 days (until complete tail reabsorption, NF stage 66). Treatment periods are shown by the solid green horizontal arrows, and termination of treatments, by the vertical dashed lines. In the final experimental group, treatment ended for each individual as they metamorphosed. Black arrows indicate that the animals were reared without E2 exposure after the treatments were completed. Larval pictures adapted from Niewkwoop and Faber (1994).
	Figure 3. Gonads of a control postmetamorphic (NF stage 66) male (A,C) and female (B,D) African clawed frog (X. laevis). Abbreviations: OV, ovarian vesicle; K, kidney. (A,B) The entire kidney–interrenal–gonadal complex. The yellow color is the result of Bouin’s fixative. Arrowheads show the rostral and caudal ends of the animal’s right gonad. (C,D) Transverse cross-sections (8 μm) through the geometric center of each animal’s right gonad. Sections were stained with Mallory’s trichrome stain. Arrow indicates melanophore in the ovary. Scale bar: (A,B) 0.1 mm; (C,D) 10 μm. Figure adapted from Hayes et al. (2002a).
	Figure 4. Gonads of an NF stage 66 male X. laevis showing SSP. The individual shown was exposed to 0.1 ppb atrazine. (A) The entire dissected Bouin’s-fixed kidney–interrenal–gonadal complex. Each of the six testes is numbered. (B,C) Sagittal sections (stained in Mallory’s trichrome stain) through the animal’s right and left gonad, respectively. The six testes are numbered corresponding to numbers in A. Each is a distinct organ based on review of the entire series of sagittal sections. Scale bar: (A,B) 0.1 mm; (C) 40 μm.
	Figure 5. A mixed hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: FB, fatbody; K, kidney; O, ovary; T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B–E) Transverse cross-sections (8 μm) stained in Mallory’s trichrome stain. Sections were taken through areas indicated by the black lines. Note the absence of pigment in the ovaries, a conditional typical in hermaphrodites. Scale bar: (A) 0.1 mm; (B–E) 25 μm.
	Figure 6. A lateral hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: O, ovary, T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B) A transverse cross-section (8 μm) stained in Mallory’s trichrome stain. The section was taken from the center of the gonad indicated by the arrow in A. Scale bar: (A) 0.1 mm; B) 25 μm.
	Figure 7. A rostral-caudal hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: K, kidney, O, ovary, T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B,C) Transverse cross-sections (8 μm) of the animal’s right gonad stained in Mallory’s trichrome stain. The sections were taken from the areas shown by the white arrowheads: right rostral testis in cross-section (B) and right caudal ovary (C). (D–G) Sagittal sections (8 μm) taken from the animal’s left gonad from the areas indicated by the white arrows: sagittal sections of the left rostral testis (D,E) and through the caudal ovary ( F,G). Note absence of pigment in the ovary. Scale bar: (A) 0.1 mm; (B–C) 10 μm; (D–G) 50 μm.
	Figure 8. An animal with nonpigmented ovaries. The individual shown was exposed to 0.1 ppb atrazine. Abbreviations: K, kidney; O, ovary; UO, unpigmented ovaries. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. Although the individual shown has distinct lobes, some animals with this morphology had only very shallow indications of lobes. (B) A transverse cross-section (8 μm) through the geometric center of the animal’s kidney and gonads. The section is stained in Mallory’s trichrome stain. This histology revealed that the animals were in fact females, even though their gonads lacked pigment, typical of normal ovaries. Scale bar: (A) 0.1 mm; (B) 40 μm.
	Figure 9. (A) Frequency of males, females, and specimens with gonadal malformations in controls and atrazine-treated animals (0.1–25 ppb). Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%. (B) Frequency of gonadal malformations only. X-axis is categorical.
	Figure 10. Unpigmented ovaries induced by CPA exposure. Abbreviations: FB, fat body; K, kidneys; UO, unpigmented ovary. Scale bar = 0.1 mm.
	Figure 11. Frequency of males, females, and animals with nonpigmented ovaries in animals exposed to CPA compared with solvent (methanol)-treated controls. Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%.
	Figure 12. SSP (A) and hermaphroditism (B) in animals treated for 7 days (NF stage 50–53) with 100 μg/L E2. Six testes (three on each side) are numbered in A. Abbreviations: O, ovary; T, testis. Scale bar = 0.1 mm for A and B.
	Figure 13. Frequency of males, females, animals with SSP, and hermaphrodites in animals treated for s7, 14, or 49 days with 100 μg/L E2. X-axis is categorical. Control bar shows the control sex ratio. NF stages below the bars indicate the stage at which E2 exposure was terminated. All E2 exposures began at NF stage 50 (Figure 2). Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%.

Babić-Gojmerac, Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence. 1989, Pubmed

Babić-Gojmerac, Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence. 1989, Pubmed
Ball, Hormonal regulation of brain circuits mediating male sexual behavior in birds. 2004, Pubmed
Bulun, Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. 2005, Pubmed
Capel, Effect of scale on the behavior of atrazine in surface waters. 2001, Pubmed
Carr, Response of larval Xenopus laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal morphology. 2003, Pubmed , Xenbase
CHANG, Genic control and hormonal reversal of sex differentiation in Xenopus. 1956, Pubmed , Xenbase
Coady, Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis. 2005, Pubmed , Xenbase
Crain, Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. 1997, Pubmed
Eldridge, Short-term effects of chlorotriazines on estrus in female Sprague-Dawley and Fischer 344 rats. 1994, Pubmed
Eldridge, Estrous cycle patterns of Sprague-Dawley rats during acute and chronic atrazine administration. 1999, Pubmed
Ellem, Local aromatase expression in human prostate is altered in malignancy. 2004, Pubmed
Frank, Pesticide and polychlorinated biphenyl residues in waters at the mouth of the Grand, Saugeen, and Thames Rivers, Ontario, Canada, 1986-1990. 1991, Pubmed
Frank, Survey of farm wells for pesticides, Ontario, Canada, 1986 and 1987. 1990, Pubmed
Friedmann, Atrazine inhibition of testosterone production in rat males following peripubertal exposure. 2002, Pubmed
GALLIEN, [Total inversion of sex in Xenopus laevis Daud, following treatment with estradiol benzoate administered during larval stage]. 1953, Pubmed , Xenbase
Goto-Kazeto, Localization and expression of aromatase mRNA in adult zebrafish. 2004, Pubmed
Hayes, Comment on "Gonadal development of larval male xenopus laevis exposed to atrazine in outdoor microcosms". 2005, Pubmed , Xenbase
Hayes, Atrazine-induced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens): laboratory and field evidence. 2003, Pubmed , Xenbase
Hayes, Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. 2002, Pubmed , Xenbase
Hayes, Metabolism of exogenous steroids by anuran larvae. 1993, Pubmed , Xenbase
Hayes, Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. 1998, Pubmed
Hayes, Herbicides: feminization of male frogs in the wild. 2002, Pubmed
Hayes, Welcome to the revolution: integrative biology and assessing the impact of endocrine disruptors on environmental and public health. 2005, Pubmed
Hecker, Plasma sex steroid concentrations and gonadal aromatase activities in African clawed frogs (Xenopus laevis) from South Africa. 2004, Pubmed , Xenbase
Hecker, Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine. 2005, Pubmed , Xenbase
Holtfreter, [Not Available]. 1931, Pubmed
Ikeda, In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats disrupts brain sexual differentiation. 2005, Pubmed
Jooste, Gonadal development of larval male Xenopus laevis exposed to atrazine in outdoor microcosms. 2005, Pubmed , Xenbase
Keller, Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line. 2004, Pubmed
Kettles, Triazine herbicide exposure and breast cancer incidence: an ecologic study of Kentucky counties. 1997, Pubmed
Kniewald, Effects of s-triazine herbicides on hormone-receptor complex formation, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activity at the anterior pituitary level. 1979, Pubmed
Kniewald, Disorders of male rat reproductive tract under the influence of atrazine. 2000, Pubmed
Kniewald, Effect of s-triazine compounds on testosterone metabolism in the rat prostate. 1995, Pubmed
MacLennan, Cancer incidence among triazine herbicide manufacturing workers. 2002, Pubmed
Merchant-Larios, Ultrastructural events during early gonadal development in Rana pipiens and Xenopus laevis. 1981, Pubmed , Xenbase
Miller, Results from the Lake Michigan Mass Balance study: concentrations and fluxes of atmospheric polychlorinated biphenyls and trans-nonachlor. 2001, Pubmed
Oberdörster, Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones. 2001, Pubmed
Pennington, Analysis of pesticide runoff from mid-Texas estuaries and risk assessment implications for marine phytoplankton. 2001, Pubmed
Reeder, Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs (Acris crepitans). 1998, Pubmed
Roberge, Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor. 2004, Pubmed
Sanderson, Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes. 2001, Pubmed
Sanderson, 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? 2000, Pubmed
Scribner, Changes in herbicide concentrations in Midwestern streams in relation to changes in use, 1989-1998. 2000, Pubmed
Simić, Reversibility of the inhibitory effect of atrazine and lindane on cytosol 5 alpha-dihydrotestosterone receptor complex formation in rat prostate. 1991, Pubmed
Solomon, Ecological risk assessment of atrazine in North American surface waters. 2013, Pubmed
Spanò, Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus). 2004, Pubmed
Stevens, Hypothesis for mammary tumorigenesis in Sprague-Dawley rats exposed to certain triazine herbicides. 1994, Pubmed
Swan, Semen quality in relation to biomarkers of pesticide exposure. 2003, Pubmed
Tavera-Mendoza, Response of the amphibian tadpole (Xenopus laevis) to atrazine during sexual differentiation of the testis. 2002, Pubmed , Xenbase
Tavera-Mendoza, Response of the amphibian tadpole Xenopus laevis to atrazine during sexual differentiation of the ovary. 2002, Pubmed , Xenbase
Trentacoste, Atrazine effects on testosterone levels and androgen-dependent reproductive organs in peripubertal male rats. 2001, Pubmed
Villalpando, Determination of the sensitive stages for gonadal sex-reversal in Xenopus laevis tadpoles. 1990, Pubmed , Xenbase
Wetzel, Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats. 1994, Pubmed