1 National Food Institute, Technical University of Denmark2 Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark
Background: In toxicological testing, effects of endocrine disrupters are in most cases more thoroughly investigated in males than in females. In males the hypothesis of testicu lar dysgenesis syndrome (TDS) proposes that there is a common origin in fetal life of the increase in frequency observed in later years of for example incidence of boys born with hypospadias and young men with low semen quality in the human male population. Furthermore, it has been observed in animal studies that exposure during fetal life to endocrine disrupters may lead to similar adverse reproductive effects. It has been proposed that a similar syndrome, called the ovarian dysgenesis syndrome (ODS), exists for females. This syndrome encompasses alterations in reproductive development caused by chemical exposure in sensitive windows of development that may result in fecundity impairments, gravid diseases, gynecological disorders or later onset adult diseases. However, experimental evidence on the effects of developmental exposure to environmentally relevant endocrine disrupting chemicals in females has been missing attention. Since chemical exposure can affect female reproductive development it is important to investigate effects in females both early and later in life. Methods: The results, presented in this thesis, were obtained in five developmental toxicology studies. Two studies investigated mixtures of endocrine disrupting pesticides (Pestimix), two investigated mixtures of endocrine disrupting chemicals based on human exposures (Contamed), and one study tested a positive control for estrogenic effects, ethinyl estradiol (EE2). The project with the mixture of the five pesticides included two range-finding studies (collectively called Pestimix RF) and a dose response study (Pestimix DR). In the Contamed project, mixtures were modeled based on high end human intakes, and the project involved two developmental mixture studies in rats, called Contamed 1 and 2. In these studies 13 chemicals where data on in vivo endocrine disrupting effects and information on human exposures was available, were selected. The tested chemicals included phthalates, pesticides, UV-filters, bisphenol A, parabens and the drug paracetamol. Together the chemicals represented several modes of action with regard to endocrine disrupting mode of action. Finally, results from a dose response study on the estrogenic drug EE2 were included. In all of these studies, both male and female offspring were investigated for adverse effect of the exposure, however, the endpoints which were focused on in this thesis, were only in females. One endpoint was assessed in the pregnant dams i.e. gestational length and several endpoints were investigated in the female offspring including anogenital distance (AGD), number of nipples, onset of puberty, measurements of Anti-Müllerian hormone (AMH) and estrous cyclicity at several time point during the animals life span. Results and discussion: Prolonged gestational length was observed in the Pestimix studies at mixture doses consisting of the pesticides at doses far below their individual no observed adverse effect level (NOAELs)for this endpoint. The effects in the mixture groups were probably caused by the azole fungicides and prochloraz which have earlier been shown to cause this effect. A longer gestational length was also observed in dams from the EE2 study at the highest dose. AGD in female offspring was increased in both the Pestimix RF study and the DR study. Here effects were seen in the two highest mixture groups, and a similar effect was seen in females receiving epoxiconazole, prochloraz and tebuconazole, at doses equal to those included in the highest mixture dose. The effects in the mixture groups were probably caused by the azole fungicides and prochloraz, which have earlier been shown to affect AGD in females. An increased AGD was also observed in the highest dose group in the EE2 study. Puberty onset was not affected in any of the studies even in EE2 study. It is, however, recommended that in future studies recording of this endpoint is commenced at an earlier age than at pup day 27, which turned out to be too late because some of the animals had already reached puberty at that age. An increase in number of nipples was observed in the high dose group in the EE2 study. The mechanism behind this effect is not known. Estrous cyclicity was not affected in Pestimix DR but the data was not optimal as methodological difficulties caused a large proportion of the animals to become pseudopregnant. In the two Contamed studies there were no effects on estrous cyclicity in the animals at 3, 5 or 9 months of age. However, at 12 months, more animals were irregularly cycling in the highest mixture group compared with controls indicating an advance in timing of reproductive senescence. This is an important finding as premature reproductive aging is a serious effect and most studies will miss such an effect because the studies are terminated too early to observe it. There was also a significant decrease in AMH levels in 4 months old animals in the highest mixture group and the group treated with paracetamol. No effects on AMH were observed in the older animals or in the 3 months old animals in EE2 study. More works need to be done investigating if AMH can be a good marker of effects on ovarian reserve in rats. Power calculations revealed that generally the effect had to be around or above a 50% change both for effects on estrous cycle and AMH in order to be able to detect them with. Conclusion: Female rats were affected by treatment with single EDCs or mixtures of EDCs. In all 5 studies pregnant rats and/or female offspring were affected by exposure to single chemicals or the mixture of endocrine disrupting chemicals at the investigated doses. In dams mixture effects on gestational length were reported for the first time. The affected endpoints in the female offspring were AGD, number of nipples, estrous cyclicity and AMH levels but not onset of puberty. Estrous cycle was affected only in the one-yearold female offspring and not at a younger age indicating early reproductive aging, a late effect of early exposure. The results indicate endocrine disrupters may be a contributing factor to effects of the reproductive system in women.AMH needs to be investigated more before it can be determined whether it is a marker useful in assessing effects in the ovaries after exposure to EDCs. More sensitive endpoints would be helpful when assessing effects in females and should be the focus of further studies.