Exposure to Bisphenol AF During Embryonic Development Causes Feminization in Male Chickens
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Exposure to Bisphenol AF during embryonic development causes feminization in male chickens Mimmi Wänn Degree project in biology, Bachelor of science, 2019 Examensarbete i biologi 15 hp till kandidatexamen, 2019 Biology Education Centre and Department of Environmental Toxicology, Uppsala University Supervisor: Anna Mattsson Contents Abstract .............................................................................................................................. 3 Svensk sammanfattning ...................................................................................................... 4 Introduction ....................................................................................................................... 5 Background ........................................................................................................................ 5 Bisphenol A in our everyday life ................................................................................................ 5 Why hazardous? ........................................................................................................................ 6 Effects in humans ....................................................................................................................... 7 Survey and analysis of alternatives to Bisphenol A ................................................................... 7 Bisphenol AF .............................................................................................................................. 7 The domestic fowl as a model organism ..................................................................................... 8 Sexual differentiation ................................................................................................................. 9 Gene expression in sex differentiation and the importance of estrogen ..................................... 9 The impact of BPA exposure on sexual differentiation and molecular mechanism ................. 10 Ovarian histology ..................................................................................................................... 10 Testicular histology .................................................................................................................. 10 Histology of ovotestis ............................................................................................................... 11 Aim and objective ............................................................................................................. 11 Materials and methods ...................................................................................................... 11 Ethics ....................................................................................................................................... 11 Incubation ................................................................................................................................ 11 Treatment ................................................................................................................................ 12 Dissection, gross morphology and sampling ............................................................................ 13 Image analysis of gonadal histology ......................................................................................... 13 Statistics ................................................................................................................................... 14 Results .............................................................................................................................. 14 Body weight and mortality rate ............................................................................................... 14 Liver weight and HSI ............................................................................................................... 15 Female right Müllerian duct length ......................................................................................... 16 Male right:left testes area ratio ................................................................................................ 16 Frequencies of ovotestis in males ............................................................................................. 16 1 Histology .................................................................................................................................. 18 Discussion ........................................................................................................................ 19 Author contribution .......................................................................................................... 22 Acknowledgements ........................................................................................................... 22 References ........................................................................................................................ 23 2 Abstract The exposure to endocrine disrupting chemicals exerts an imminent threat to wildlife and mankind, causing adverse effects, as for instance malformations and functional impairment of the reproductive system. Bisphenol A (BPA) has been extensively used in the plastic industry for decades and is known to hold appreciated characters as great flexibility and resistance. Leakage of BPA has unfortunately been detected and both human and wildlife get continuously exposed to low concentrations of this xenoestrogen in our everyday life. Studies investigating the estrogen-like influence of BPA exposure in birds, demonstrated altered development in the reproductive organs during sexual differentiation. Today there are numerous bisphenols on the market, serving as BPA alternatives due to their similar structure and function. Bisphenol AF (BPAF) is one of many BPA analogues that was chosen for further evaluation in a survey by Kemikalieinspektionen in 2011. The major aim of this study was to examine the impact of BPAF during the embryonic development of the chicken embryo and focused on gonadal differentiation endpoints. Injection into the air chamber of the incubated eggs was executed during embryonic development and examination performed prior to anticipated hatching. Causation of increased mortality induced by BPAF was statistically significant, established at 70 μg/g egg and 175 μg/g egg. Male right:left testes area ratio was statistically significant, affected by treatment dose 70 μg/g egg, as well as liver weight and liver:body weight ratio, hepatosomatic index. No effects of treatment were found in females. Gross morphology revealed an increasing frequency of feminization in male testes, which was statistically significant at 175 μg/g egg. Feminization was characterised by ovarian-like appearance of the left testis. Histological analysis only confirmed ovotestis in one individual, the same individual showing pronounced characteristics of ovotestis in the gross morphology assessment. This individual displayed features of ovarian-like cortex with abundant clusters of oocyte-like germ cells, and lacunae in the medulla. The results from our study indicate a similar estrogenic treatment effect of BPAF, as for BPA, during early-life exposure to the developing chicken embryo. Hence human and wildlife health concerns should be further addressed regarding potential exposure of BPA alternatives in our everyday life. Keywords: Bisphenol AF; ovotestis; feminization; sex differentiation; endocrine disruptors; estrogenic properties; chicken embryo. 3 Svensk sammanfattning Exponeringen för hormonstörande ämnen utgör ett överhängande hot mot mänskligheten och djurlivet. Dessa ämnen orsakar skadliga effekter, som exempelvis missbildningar och funktionell nedsättning av reproduktionsorganen. Bisfenol A (BPA) har sedan flera årtionden använts i stor utsträckning av plastindustrin och är känt för egenskaper så som hög flexibilitet och tålighet. Olyckligtvis har läckage av BPA upptäckts i vår vardag och både människor och djur blir kontinuerligt utsatta för låga koncentrationer av denna xenoöstrogen. Studier, som har undersökt östrogenlik inverkan vid exponering av BPA hos fåglar, uppvisade förändrad utveckling av reproduktionsorganen under könsdifferentieringen. Idag finns det ett flertal bisfenoler på marknaden, vilka tjänar som alternativ till BPA tack vare liknande funktion och strukturell uppbyggnad. Bisfenol AF (BPAF) är en av många BPA-analoger som blev utvalda för vidare undersökning om dess toxikologiska effekter i en kartläggning av Kemikalieinspektionen 2011. Syftet med denna studie var att undersöka inverkan av BPAF under embryonalutvecklingen hos kycklingembryo och fokuserade på gonaddifferentiering som process och slutpunkt. De befruktade hönsäggen injicerades in i luftkammaren och embryot undersöktes före förväntad kläckning. BPAF inducerade ökad mortalitet, som var statistiskt signifikant vid 70 μg/g ägg och 175 μg/g ägg. Förhållandet mellan hanlig höger:vänster testikelarea, levervikt och förhållandet mellan lever:kroppsvikt (hepatosomatiskt index) påverkades vid 70 μg/g ägg. Inga behandlingseffekter återfanns hos honor. Morfologisk undersökning avslöjade en ökande feminiseringsfrekvens av testiklar hos hanar, vilket var statistiskt signifikant vid 175 μg/g ägg. Feminiseringen karakteriserades av ovarielikt utseende hos den vänstra testikeln. Histologisk analys konfirmerade endast ovotestis hos en individ, samma individ som uppvisade markanta karaktärsdrag av ovotestis vid den morfologiska bedömningen. Denna individ visade egenskaper