1999 Progress Report: Metabolic Androgenization of Invertebrates by Endocrine-Disrupting ChemicalsEPA Grant Number: R826129
Title: Metabolic Androgenization of Invertebrates by Endocrine-Disrupting Chemicals
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Carleton, James N
Project Period: October 1, 1997 through September 30, 2000 (Extended to September 30, 2002)
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $406,155
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Health , Safer Chemicals , Endocrine Disruptors
Objective:The overall objective of this program is to definitively characterize the phenomenon of metabolic androgenization in invertebrates and to elucidate the putative relationships among chemical exposure, metabolic androgenization, and toxicological consequences such as pseudohermaphrodism, developmental abnormalities, and reproductive impairment. The studies are being conducted in the model crustacean, the water flea (Daphnia magna) and the model gastropod, the mud snail (Illyanassa obsoleta).
The first objective of the program is to definitively characterize the biotransformation of testosterone by daphnids and mud snails and evaluate the effects of specific environmental chemicals on these biochemical processes. We hypothesize that effects on testosterone biotransformation are responsible for various effects of the chemicals on reproductive processes in these invertebrate species. Experiments with daphnids were completed during the first year of this project. These results demonstrated that daphnids extensively biotransform testosterone into various hydroxylated, oxido-reduced, and conjugated derivatives. The putative endocrine-disrupting chemicals (4-nonylphenol, propiconazole) acted similarly by inhibiting the metabolic elimination of testosterone as the glucose-conjugated derivative. Tributyltin, another compound under study, had somewhat different effects on testosterone biotransformation by daphnids. This compound increased overall testosterone biotransformation rates but decreased the relative contribution of glucose conjugation to the total rate of biotransformation. Taken together, these results indicate that these compounds function similarly to inhibit the metabolic elimination of testosterone as the glucose conjugate. Glucose conjugation is the major metabolic process leading to the inactivation and elimination of testosterone in this species.
Metabolism of testosterone by the mud snail was investigated during the past year. Surprisingly, no significant conversion of testosterone to polar hydroxylated or conjugated derivatives by the mud snail was detected. Rather, testosterone was rapidly converted to highly apolar derivatives that are sequestered within the organism. The identification of these testosterone derivatives is underway. Preliminary indications are that they are fatty acid conjugates of testosterone. The discovery that testosterone is not significantly eliminated from the mud snail as polar conjugates indicates that interference with such processes by the chemicals under study, as originally hypothesized, is unlikely. Once the testosterone derivatives have been identified, experiments will be undertaken to determine whether the chemicals under study either interfere with the formation of the nonpolar derivatives or facilitate the de-conjugation of the derivatives. Either process would increase the amount of free testosterone in the snails that could be responsible for putative masculinizing effects of the chemicals.
The second objective of the program is to identify physiological perturbations associated with metabolic androgenization. Using the crustacean model, we have determined that chemicals that elicit metabolic androgenization also interfere with normal embryo development. During the past year, methods were developed for monitoring daphnid embryo development in vitro and these methods were used to characterize normal embryo development. Six stages of embryo development were readily discerned: (1) cleavage, (2) gastrulation, (3) early organogenesis, (4) mid-organogenesis, (5) late organogenesis, and (6) neonatal. Comparative studies were then undertaken with testosterone and the chemicals under study (4-nonylphenol and propiconazole) to determine whether the developmental toxicity of the environmental chemicals is consistent with that observed with testosterone. Testosterone and the environmental chemicals induced the same types of embryonic abnormalities and exhibited parallel concentration-response curves. Further, testosterone was shown to directly elicit toxicity to the developing embryos. In contrast, embryo toxicity of the environmental chemicals was greatest when maternal organisms were exposed to the chemicals. These results support the hypothesis that the environmental chemicals elevate testosterone levels in the maternal daphnids and these elevated testosterone levels are responsible for the embryo toxicity.
Experiments were conducted with the mud snail to determine whether imposex (male sex characteristics in females) could be induced by testosterone, tributyltin (positive control), and 4-nonylphenol. Thus far, aqueous exposure to the materials has not resulted in a significant incidence of imposex. The experimental design was therefore modified, whereby the test materials are administered to the snails by daily injection. A significant incidence of imposex was demonstrated among snails injected with 20 ng tributyltin per day for 14 days. Similar experiments are underway with testosterone and 4-nonylphenol. Preliminary results suggest that both of these compounds also will cause imposex among the injected snails. Once the appropriate experimental design has been developed for the induction of imposex, the effects of these treatments on testosterone metabolism and homeostasis will be determined. We will test the hypothesis that the chemicals under study cause imposex by inhibiting testosterone conjugation in increasing levels of free testosterone within the organisms. Elevated testosterone levels have previously been associated with imposex.
The third objective of the program is to evaluate cause-effect relationships between androgenization and the observed physiological responses associated with androgenization. We have demonstrated that direct exposure of daphnids to steroidal androgens, but not estrogens, a progestogen, a corticosteroid, or an insect juvenoid, causes developmental abnormalities. These results directly implicate the increase in steroidal androgen levels resulting from chemically induced metabolic androgenization as the cause of the developmental abnormalities. We have hypothesized that this effect is due to alterations in lipid homeostasis resulting in improper nutrient provision to the embryos. To test this hypothesis, studies have been initiated to characterize normal levels of various lipids classes in daphnids and to evaluate the effects of chemical exposure on the transfer of lipids to the embryos. Initial studies have indicated that maternal exposure to testosterone is not a prerequisite for the embryo toxicity elicited by this steroid. Therefore, interference with the maternal transfer of lipids to the embryos by testosterone is not likely to be responsible for the developmental toxicity. Furthermore, experiments conducted with propiconazole have demonstrated that triglyceride levels are not significantly altered in daphnids by exposure to this compound. Triglyceride is the major form of stored energy provided to the embryos by the maternal organisms. Thus, the embryo toxicity caused by testosterone appears not to be due to effects of testosterone on the provision of energy to the embryo.
Experiments conducted with the insect juvenoid methoprene demonstrated that this compound did not elicit embryo toxicity as observed with testosterone. However, experimental results did indicate that this compound can interfere with the development of sex characteristics in males and can influence daphnid parthenogenetic reproduction at nanomolar exposure concentrations. These interesting results prompted us to continue experiments with this compound aimed at: (1) establishing whether juvenoids function as hormones in crustaceans as they do in insects, and (2) determining the susceptibility of juvenoid-regulated processes to endocrine disruption. Results thus far indicate that methoprene elicits two modes of toxicity to daphnids. At concentrations above 30 nM, methoprene severely reduced fecundity and increased the time required to attain reproductive maturation. A threshold effect concentration (~30 nM) was clearly evident for these effects. At concentrations below 30 nM, methoprene reduced fecundity in a concentration-dependent manner. However, the concentration-response curve for this effect was significantly different from that observed for this same parameter at concentrations above 30 nM. Furthermore, concentrations as low as 5 nM methoprene, and perhaps lower, significantly reduced juvenile molt frequency. A threshold effect concentration was not clearly evident for these effects at concentrations as low as 2.0 nM. Finally, preliminary data suggest that low nanomolar concentrations of methoprene may actually stimulate reproduction of daphnids. Taken together, these results suggest that juvenoid hormones function in regulating daphnid maturation and reproduction, and methoprene may interfere with the function of endogenous juvenoids.
Future Activities:Experiments will continue in daphnids aimed at evaluating the cause-effect relationships between chemically induced metabolic androgenization and developmental toxicity. Endogenous testosterone levels will be determined in daphnids by radioimmunoassay and confirmed by GC-MS. The effects of chemically induced metabolic androgenization on endogenous testosterone levels will then be determined. These experiments will establish whether exposure to the endocrine-disrupting chemicals elevates endogenous testosterone levels sufficiently to cause developmental toxicity. The production of daphnid vitellogenin antibodies is underway. These antibodies will be used in immunoassays to determine whether steroidal androgens or metabolic androgenization interfere with vitellogenin transfer to or utilization by embryos. Methods also will be developed to assess whether daphnids and mud snails possess high-affinity testosterone-binding proteins (i.e., receptors or other targets of toxicity). If such binding activity is measured, then experiments will be conducted during which the organisms will be co-exposed to testosterone and other experimentally identified ligands to the binding site. Agonist/antagonist interactions between testosterone and the ligands will then be evaluated in vivo with respect to the induction of developmental toxicity (daphnids) or imposex (mud snails). Identity of the testosterone derivatives produced by mud snails will be established. Effects of chemicals that induce imposex on the generation of these testosterone metabolites will then be established. We hypothesize that these chemicals will inhibit the formation of these testosterone conjugates just as they inhibit glucose conjugation resulting in increased levels of bioavailable testosterone within the organisms. This would provide a mechanism by which these chemicals cause imposex.
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||LeBlanc GA, McLachlan JB. Molt-independent growth inhibition of Daphnia magna by an anti-androgen. Environmental Toxicology and Chemistry 1999;18:1450?1455.||