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The current study examined the effects on the red-eared slider turtle of extremely low doses of three endocrine-disrupting compounds (EDCs)--trans-Nonachlor, chlordane, and p,p'-DDE-singly and in mixtures. Previous studies using the red-eared slider turtle have proven its value as an organism for obtaining information about the effects of endocrine-disrupting compounds. The sex of the turtle, easily manipulated by exposure during embryogenesis, continues to be a marker of effects. When red-eared slider turtle embryos incubating at a temperature that normally produces a male-biased sex ratio are exposed to these compounds singly, the sex ratio of the resulting hatchlings shifts significantly to a female bias. The current work offers further evidence that the red-eared slider turtle provides a way to quantify the effects of mixtures and has the potential as a model for evaluating additivity and synergy. The results provide information about how very low doses (parts per billion) of these compounds behave in mixtures. The dose ranges were 0.125-0.5 ng/egg for trans-Nonachlor and chlordane and 7-28 ng/egg for p,p'-DDE. Results suggest the possibility that two of the compounds may exert effects in mixtures via complementary pathways; when applied singly, the effects of chlordane and p,p'-DDE were inversely related to dose, but in mixtures, which were essentially a greater dose of EDC, the compounds had an increased effect. In all cases, mixtures resulted in 100% females.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider., (Copyright 2001 Wiley-Liss, Inc.)

In the red-eared slider turtle, pesticides can alter expected sex outcomes, a major step in the inferred pathway of sex determination, and hatchling steroid physiology. Changes such as these can profoundly affect an organism's fitness. Other potential markers for effects on fitness include hatchling mass, hatchling use of maternal stores (residual yolk), and especially early hatchling growth rates. In the current study, red-eared slider turtles were exposed during embryogenesis to one of three compounds-chlordane, trans-Nonachlor, or p,p'-DDE-all of which affect sex determination in this species. Turtles were weighed at hatching, after a 28-d fasting period, and after 14 d of ad libitum feeding. All three compounds had some population-wide effects on changes in mass from time point to time point when compared to controls. From hatching to the end of the 28-d fast, turtles exposed in the egg to the mid-range doses of trans-Nonachlor and of p,p'-DDE lost mass and underwent a change in mass significantly different from controls. Additionally, turtles exposed to the two higher doses of trans-Nonachlor and the mid-range dose of chlordane grew significantly more than controls after 14 d of ad libitum feeding. These results point to a role for pesticides in endocrine disruption that extends beyond sex determination and sex development.

Gonadal sex in the red-eared slider turtle is determined by the incubation temperature that the embryo experiences during the mid-trimester of development. High temperatures result in female-biased sex ratios, and low temperatures produce male-biased sex ratios. The physiological equivalent of temperature appears to be a combination of the nature and abundance of steroidogenic enzymes and their products-including estradiol and its precursor, testosterone-and aromatase, the enzyme that converts testosterone to estradiol. Aromatase has been hypothesized to play a major role in the female developmental pathway in this species, and research in other species with temperature-dependent sex determination points to the brain as an organ that transduces the temperature signal into an aromatase response. In this study, we used a tritiated water assay to compare the pattern of estradiol biosynthesis at male- and female-producing temperatures in the brain and adrenal-kidney-gonad (AKG) through development. The pattern for both sexes in the AKG was one of increased activity after the temperature-sensitive period (TSP), but with no significant difference between sexes. In the brain, however, putative females exhibited a significantly higher level of aromatase activity than putative males at the beginning of the TSP, after which activity in both male and female brains decreased, dropping below detection in females before hatch. These results point to the brain as a site of aromatase response to temperature in this species, and they suggest that the product of aromatase activity, estradiol, may induce alterations in the neuroendocrine axis controlling gonadal sex steroid hormone production., (Copyright 2000 Academic Press.)

Many compounds in the environment capable of acting as endocrine disruptors have been assayed for their developmental effects on morphogenesis; however, few studies have addressed how such xenobiotics affect physiology. In the current study we examine the effects of three endocrine-disrupting compounds, chlordane, trans-nonachlor, and the polychlorinated biphenyl (PCB) mixture Aroclor 1242, on the steroid hormone concentrations of red-eared slider turtle (Trachemys scripta elegans) hatchlings treated in ovo. Basal steroid concentrations and steroid concentrations in response to follicle-stimulating hormone were examined in both male and female turtles treated with each of the three compounds. Treated male turtles exposed to Aroclor 1242 or chlordane exhibited significantly lower testosterone concentrations than controls, whereas chlordane-treated females had significantly lower progesterone, testosterone, and 5[alpha]-dihydrotestosterone concentrations relative to controls. The effects of these endocrine disruptors extend beyond embryonic development, altering sex-steroid physiology in exposed animals.

Incubation temperature determines gonadal sex in the red-eared slider turtle, Trachemys scripta. However, little is known about the long-term effects of incubation temperature on traits other than gonadal sex in this species. To investigate the hypothesis that incubation temperature (independent of gonadal sex) influences sex steroid levels after hatching, we incubated eggs of the red-eared slider turtle at three temperatures (26, 28.6, and 31 degrees C). We then measured plasma levels of dihydrotestosterone, estradiol, progesterone, and testosterone in 6-wk-old males from 26 degrees C and 28.6 degrees C eggs, and in 6-wk-old females from 28.6 degrees C and 31 degrees C eggs. We found that dihydrotestosterone levels were not influenced by incubation temperature or gonadal sex. However, progesterone levels were significantly higher in males from 26 degrees C eggs than in males from 28.6 degrees C eggs. In contrast, testosterone levels did not differ between males from 26 degrees C versus males from 28.6 degrees C eggs, but they were significantly higher in females from 28.6 degrees C than in females from 31 degrees C eggs. Progesterone and testosterone levels did not differ between males and females from 28.6 degrees C eggs. Temperature also influenced estradiol levels in both sexes, but the effects were enigmatic. We conclude that incubation temperature has lasting effects on sex steroid levels even after hatching.

Xenobiotics suspected of being estrogenic-the PCB aroclor 1242 and the pesticides toxaphene, dieldrin, p,p'-DDD, cis-Nonachlor, trans-Nonachlor, p,p'-DDE, and chlordane-were examined for their ability to override a male-producing incubation temperature and result in female hatchlings in the red-eared slider, a turtle with temperature-dependent sex determination. Compounds were assayed in the environmentally relevant concentrations detected in alligator eggs from Lake Apopka, Florida, singly, in concert with one another, and with estradiol. Compounds assayed alone and resulting in significant sex reversal were trans-Nonachlor, cis-Nonachlor, aroclor 1242, p,p'-DDE, and chlordane. When administered with estradiol, only one of the compounds, chlordane, caused sex reversal at significant levels. When applied together, however, the eight compounds assayed resulted in significant sex reversal., (Copyright 1999 Academic Press.)

Gonadal sex in the red-eared slider turtle, Trachemys scripta, is determined by incubation temperature during embryonic development. Evidence suggests that temperature determines sex by influencing steroid hormone metabolism and/or sensitivity: steroidogenic enzyme inhibitors or exogenous sex steroid hormones and their man-made analogs override (or enhance) temperature effects on sex determination. Specifically, nonaromatizable androgens and aromatase inhibitors induce testis differentiation at female-producing temperatures, whereas aromatizable androgens and estrogens induce ovary differentiation at male-producing temperatures. Moreover, natural estrogens and temperature synergize to produce more females than would be expected if estrogens and temperature had purely additive effects on sex determination. In this study, we use sex reversal of turtle embryos incubated at a male-producing temperature to examine synergism among steroidal estrogens: estrone, 17ss-estradiol, and estriol. A low dose of 17ss-estradiol (200 ng) showed significant synergism when administered with a single low dose of estriol (10 ng). Likewise, a single low dose of estrone (250 ng) had a synergistic effect when combined with the same low dose of estriol (10 ng). We conclude that the weak natural estrogens estrone and 17ss-estradiol synergize with a low dose of the more potent estriol to reverse gonadal sex during the critical period of sexual differentiation. These results suggest that weak environmental estrogens may also synergize with stronger natural estrogens.

Risk assessments for nongenotoxic chemicals assume a threshold below which no adverse outcomes are seen. However, when an endogenous chemical, such as 17ss-estradiol (E2), occurs at a concentration sufficient to cause an effect, the threshold is already exceeded. Under these circumstances, exogenous estradiol is not expected to provide a threshold dose. This principle is demonstrated for E2 in the red-eared slider, a turtle with temperature-dependent sex determination. In this species, gonadal sex is determined by egg incubation temperature; female development requires endogenous estrogen produced by elevated temperature. While normal production of females by endogenous estrogens is not an adverse effect, exogenous estrogens can sex reverse presumptive males, which can be an adverse effect. A large dose-response study was conducted using seven doses and a vehicle control (starting n = 300/group); a single E2 dose was applied to the eggshell of recently laid eggs. Animals were sexed after hatching. The incubation temperature chosen, 28.6 degrees C, generates a minority of females. Thus, the criteria for testing the threshold hypothesis were met, i.e., there is evidence that there is endogenous estrogen and that it generates an irreversible response. The lowest E2 dose tested, 400 pg/egg (40 ng/kg), sex reversed 14.4% of the animals, demonstrating very low dose sensitivity. The data were fit with a modified Michaelis-Menten equation, which provided an estimate of 1.7 ng/egg for endogenous estradiol. The median effective dose (ED50) was 5.0 +/- 2.0 ng/egg (95% confidence limits), of which 1.7 ng/egg was endogenous estradiol and 3.3 ng/egg came from the applied estradiol. There was no apparent threshold dose for E2. A smaller replication confirmed these results. These results provide a simple biologically based dose-response model and suggest that chemicals which act mechanistically like E2 may also show no threshold dose. If so, even low environmental concentrations of such chemicals may carry risk for sex reversal.