Christiansen, Sofie

Hass, Ulla

Jacobsen, Pernille Rosenskjold

Baggrund: I toksikologisk testning bliver effekter af hormonforstyrrende stoffer ofte undersøgt mere grundigt hos hanner end hos hunner. Hos hanner findes hypotesen om testikulært dysgenese syndrom (TDS), der siger at der er en fælles oprindelse i fostertilværelsen for blandt andet den øgede forekomst af drenge født med hypospadi og unge mænd med lav sædkvalitet, der er set i befolkningen i de senere år. Samtidig kan eksponering for hormonforstyrrende stoffer i fostertilværelsen hos forsøgsdyr medføre lignende effekter på forplantningsevnen og udsættelse for hormonforstyrrende stoffer mistænkes for at medvirke til de ovennævnte effekter på mennesker. Det er blevet foreslået, at et lignende syndrom, kaldet ovarie dysgenese syndrom (ODS), også findes hos hunner. Dette syndrom omfatter ligeledes ændringer af udviklingen af reproduktionssystemet blandt andet som følge af kemisk eksponering, og kan resultere i nedsat frugtbarhed, sygdomme i graviditeten, gynækologiske lidelser og sygdomme senere i livet. Der har imidlertid manglet viden om effekten af eksponering under udviklingen for miljørelevante hormonforstyrrende stoffer i hunner. Da kemisk eksponering kan påvirke den hunlige reproduktive udvikling, er det vigtigt at undersøge effekter hos hunner både tidligt og senere i livet.Metoder: De resultater, som præsenteres i denne afhandling, er fra fem toksikologiske studier, hvor parrede Wistar hunrotter blev doseret peroralt med miljørelevante stoffer alene eller i blandinger i drægtigheds- og laktationsperioden. To studier undersøgte blandinger (miks) af hormonforstyrrende pesticider (Pestimix), to undersøgte en blanding af hormonforstyrrende kemikalier baseret på human eksponering (Contamed), og i et studie blev der testet en positiv kontrol for østrogene effekter, lægemidlet ethinylestradiol (EE2). Projektet med blandingen af de fem pesticider omfattede to pilot studier (samlet kaldet Pestimix RF) og et dosisrespons studie (Pestimix DR). I Contamed projektet blev der testet blandinger af kemikalier, som var modelleret ud fra den høje ende af hvad mennesker kan være udsat for. Projektet omfattede to rottestudier, kaldet Contamed 1 og 2. I disse studier blev 13 kemikalier blandet, hvor data om in vivo hormonforstyrrende effekter og information om menneskelig eksponering var til rådighed. De testede stoffer inkluderede phthalater, pesticider, UV-filtre, bisphenol A, parabener og lægemidlet paracetamol. Sammen repræsenterede kemikalierne flere hormonforstyrrende virkemåder/mekanismer. Endelig er resultaterne fra et dosis-respons forsøg med det østrogene stof EE2 inkluderet. I alle disse undersøgelser blev både han og hun -afkom undersøgt for effekter af eksponeringen, men de endpoints, der blev fokuseret på i denne afhandling, var dem der ses hos hunnerne. De undersøgte endpoints var drægtighedslængde i de drægtige hunner, og i det hunlige afkom anogenital afstand (AGD), antallet af brystvorter, alder for pubertet, målinger af anti -müllerian hormon (AMH) samt østrus cyklus på adskillige tidspunkter i løbet af dyrene levetid.Resultater og diskussion: Forlænget drægtighedslængde blev observeret i Pestimix studierne i grupper doseret med blanding/miks af pesticider, ved doser langt under deres individuelle NOAEL for dette endpoint (”No Observed Adverse Effect Level ”), dvs. den højeste dosis af stoffet, som i dyreforsøg ikke har givet nogen skadelige effekter. Effekterne af miksen blev formentlig forårsaget af azol fungiciderne epoxiconazol og tebuconazol samt prochloraz som tidligere har vist sig at give denne form for effekt. Længere drægtighedslængde blev også observeret i drægtige rottemødre fra EE2 studiet ved den højeste dosis. AGD i hunafkom var øget i både Pestimix RF-studiet og DR studiet. Her blev set effekter i de to højeste miks-grupper, samt hos hunrotter, der modtog epoxiconazol, prochloraz og tebuconazol ved doser svarende til dem, der indgik i den højeste miksdosis. Effekterne i miks-grupperne blev formentlig forårsaget af epoxiconazol, tebuconazol og prochloraz, som tidligere har vist sig at påvirke AGD hos hunrotter. En øget AGD blev også observeret ved den højeste dosis i EE2 studiet. Tidspunktet for pubertetens indtræden blev ikke påvirket i nogen af de udførte forsøg, selv i EE2 studiet, som var en p ositiv kontrol. Det anbefales dog, at registreringen i fremtidige studier påbegyndes i en tidligere alder end ungedag 27, som viste sig at være for sent, fordi nogle dyr allerede havde nået puberteten i den alder. En stigning i antallet af brystvorter blevobserveret i den høje dosisgruppe i EE2 studiet. Mekanismen bag denne virkning er ikke kendt. Østrus cyklus blev ikke påvirket i Pestimix DR, men data var ikke optimale da metodologiske vanskeligheder forårsagede at en stor del af dyrene blev pseudogravide. I de to Contamed studier var der ingen effekt på østrus cyklus i dyrene i 3, 5 eller 9-10 måneders alderen. Men efter 12 måneder havde flere dyr uregelmæssig cyklus i den højeste miksdosis-gruppe sammenlignet med kontroller. Dette indikerer en tidligere reproduktiv ældning (menopause/overgangsalder i mennesker). Dette er et vigtigt resultat, da for tidlig menopause er en alvorlig effekt, og de fleste studier vil overse sådan en effekt, fordi studierne afsluttes for tidligt til at observere den. Der var også et signifikant fald i AMH niveauer i 4 måneder gamle dyr i højeste miksdosis-gruppe og i gruppen behandlet med paracetamol. Der blev hverken observeret effekt på AMH i de ældre dyr eller i de 3 måneder gamle dyr i EE2 studiet. Der skal mere viden til for at afgøre om AMH kan være en god markør for effekter af hormonforstyrrende stoffer på ovariefollikelreserven hos rotter. Power beregninger afslørede at generelt kan man måle omkring en 50% ændring både for effekter på østrus cyklus og AMH.Konklusion: Hunrotter viste sig at være følsomme for eksponering med blandinger af hormonforstyrrende stoffer. I alle 5 studier blev drægtige rotter og /eller hunligt afkom påvirket ved eksponering for de enkelte kemikalier eller miks af hormonforstyrrende kemikalier ved de undersøgte doser. For første gang er der rapporteret mixeffekter på gestationslængden hos drægtige rotter. I afkommet blev der set effekter på AGD, antal brystvorter, østrus cyklus og AMH niveauer, men ikke tidspunkt for pubertet. Effekter på østrus cyklus blev kun set hos de et år gamle rotter og er et tegn på sene effekter af tidlig eksponering. Resultaterne indikerer at hormonforstyrrende stoffer kan være medvirkende faktor til effekter i reproduktionssystemet hos voksne kvinder og ufødte pigebørn. AMH skal undersøges bedre for at vurdere om det er en god markør for effekter af hormonforstyrrende stoffers effekt på ovariefollikelreserven. Mere følsomme endpoints ville være nyttige ved vurderingen af effekter hos hunner og bør være i fokus i fremtidigt arbejde.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

Online Research Database In Technology

Contaminant mixtures and repoductive health: Developmental toxicity effects in rats after mixed exposure to environmentally relevant endocrine disrupting chemicals, with focus on effects in females

http://orbit.dtu.dk/ws/files/74579359/Pernille_Jacobsen_Afhandling..PDF

Contaminant mixtures and repoductive health: Developmental toxicity effects in rats after mixed exposure to environmentally relevant endocrine disrupting chemicals, with focus on effects in females

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