Effects of combined exposure to anti-androgens on development and sexual dimorphic behaviour in rats

Summary Background: Androgens are key regulators of male sexual differentiation during the in utero and early postnatal development. Exposure to endocrine disrupting chemicals (EDCs) that counteract androgen action at some stage in these periods can permanently demasculinise male foetuses and lead to malformations of the reproductive tract. The incidence of hypospadias (where the opening of the urethra is on the underside of the penis) in young boys has increased over the last decades but it is still unclear whether human exposure to endocrine disrupting chemicals may be responsible for this increase. It is well-known that humans are exposed to a mixture of endocrine disrupting chemicals but risk assessment is currently based on the no observed adverse effect levels (NOAELs) for effects of one chemical at a time. The NOAEL is the highest tested dose at which no statistically or biologically adverse effects can be identified and is used in regulatory toxicology as a point of departure for establishing “acceptable” exposures for humans. Purpose and methods: The overall purpose of this thesis is to explore the need for improving the future risk assessments of mixtures of EDCs. This is done by examining the following questions:  Are there combined effects at NOAEL levels for individual anti-androgens based on the effects on anogenital distance, nipple retention and external malformations in male rats?  Can the combined effects be predicted based on the model approaches; dose addition or independent action?  Is sexually dimorphic behaviour in rats affected at lower dose levels of anti-androgens and thereby a more sensitive endpoint than morphological effects on the male external reproductive organs? The thesis is based on the results of in vivo studies where mated female Wistar rats were exposed to anti-androgens either alone or in mixtures during pregnancy and lactation. The endpoints examined for anti-androgenic effects in the offspring were: Anogenital distance (AGD), nipple retention (NR), and external (morphological) malformations in pups and sexually mature male rats. Furthermore, the effects of the anti-androgens were studied in the offspring at different age period using several behavioural tests. Additionally, the development and use of a new test for mating behaviour was a part of this project. Results and discussion: Results from the single chemical dose response studies showed that the NOAEL values found were very close to the NOAELs used by various regulatory bodies. It was also clear that DEHP (di-(2-ethylhexyl) phthalate) at a relatively low dose of 10 mg/kg bw/day caused adverse anti-androgenic effects on male rat development. The drug, finasteride was by far the most potent chemical, and it exhibited dose-response relationships with very shallow gradients for all endpoints. In the mixture study with 3 similarly acting anti-androgens (vinclozolin, flutamide and procymidone), a combination of doses of each chemical, which on their own did not change the AGD statistically significantly, induced clear combination effects. Furthermore, exposure to low doses of the individual chemicals showed only modest effects on NR, while the mixture induced NR in the males that clearly approached the female values. Severe malformations of external genitalia (hypospadias) were observed in male rats with the mixture while the individual compounds did not cause any such effects. Increased frequencies (56%) of external malformations (hypospadias) were observed after exposure to a mixture of the three chemicals compared to administration of the three chemicals alone (0%). AGD was a good early biomarker, as a 25% reduction in mean AGD measured on postnatal day 1 was likely to result in clear malformations in approximately 50% and marked malformations in approximately 25% of the adult male rats. These combined effects could be predicted fairly accurately on the basis of information about the potency of the individual chemical components by using the dose addition concept. Behaviour was as sensitive an endpoint as the morphological parameters (AGD, NR and malformations) in the mixture study with 3 similarly acting anti-androgens (vinclozolin, flutamide and procymidone). Results showed that learning was impaired in mixture exposed males when tested in the Morris water maze. In the mixture study with 4 dissimilarly acting anti-androgens (vinclozolin, finasteride, DEHP and prochloraz) AGD, NR, and reproductive organ weights at PND (postnatal day) 16 were clearly affected in the mixture groups at dose levels where the individual chemicals caused no or only minor effects. The combined effects were equally well predicted by dose addition or independent action. The parameter ‘retained nipples’ was the most sensitive endpoint, with effects becoming noticeable at the lowest doses. Changes in AGD were almost as sensitive an endpoint, followed by reductions in prostate and LABC (levator ani/bulbocavernosus muscles) weights, and genital malformations. In addition, the experimentally observed responses for external malformations clearly exceeded the predictions, suggesting that the combined effect of DEHP, vinclozolin,prochloraz and finasteride is synergistic with respect to malformations of external sex organs. To clarify, this thesis refers to effects which exceed expectations as synergism and those which meet expectations as additivity. Behaviour was a less sensitive endpoint than the morphological parameters (AGD, NR and malformations) in the mixture study with 4 dissimilarly acting anti-androgens (vinclozolin, finasteride, DEHP and prochloraz). Conclusion and perspectives: The data from the extended developmental toxicity studies with rats presented in this thesis suggest that dose addition models can in most cases predict the combined effects of anti-androgens and demonstrate that marked effects can occur at mixture doses below the NOAELs for the single chemicals. Since unhindered androgen action is essential for human male development in foetal life, these findings are highly relevant to human risk assessment. This must be emphasised because the results clearly indicate that risk assessment based on NOAELs for single anti-androgens may underestimate the risk for hypospadias and other disruptions of male sexual differentiation. Generally, behaviour was not as sensitive an endpoint as the as the development of the reproductive system but male behaviour may be just as sensitive an endpoint after exposure to mixtures of 3 androgen receptor (AR) antagonists (vinclozolin, flutamide, and procymidone). The learning behaviour in Morris water maze was in this study impaired at the same dose levels at which effects on NR and weights of epididymides, ventral prostate and bulbouretral glands were observed. Behavioural testing could therefore provide useful complementary information and contribute to a broader picture of the toxicity of the EDCs alone or in mixtures than studies that only take the development of reproductive organs into account.