Esposizione a livelli ambientali di xenoestrogeni: modelli di studio nel ratto in vivo e rilevanza degli effetti Francesca Farabollini (a), Leonida Fusani (c), Daniele Della Seta (a), Francesco Dessì-Fulgheri (b) a Dip. di Fisiologia, Universita di Siena b Dipartimento di Biologia Evoluzionistica, Universita di Firenze c Dipartimento di Biologia ed Evoluzione, Universita di Ferrara (Gruppo di Ricerca Interuniversitario su Distruttori Endocrini e Sviluppo neurocomportamentale: GRIDES)
Xenoestrogen - Heterogeneous class of Endocrine Disrupting Compounds that mimic the action of estrogen hormones - A number of studies showing experimental alteration of reproductive functions in aquatic wildlife - Plenty of correlational evidence for reproductive and behavioral alterations in wildlife and humans - Experimental evidence for adverse effects in mammals at environmentally relevant concentrations is scarce
In intact animals, xenoestrogens interferes with endogenous estrogen system during development and sensitive phases of adult life Xs: DISRUPTERS or MODULATORS?
Main criticisms to xenoestrogens studies 1.Treatment is not environmental-like Animal model, route, dose, do not mimic exposure from environmental contamination
Environmental-like protocol (I) Sprague-Dawley rats ANIMAL Intact male/female ROUTE oral administration TIME-COURSE to mothers direct 90 day conception birth weaning puberty behavior Early exposure -------- Late effects
Environmental-like protocol (II) SUBSTANCE BPA DOSE Low dose: range of typical human exposure < EPA standards < LOAEL (lowest adverse effect level) Large effects from Small exposure ENDPOINTS BEHAVIOUR (socio-sexual, play, maternal..)
Behaviour - End-point of complex integrated systems - Organized during development - Sexual behavior relevant for reproduction - Fine ethological analysis to reveal sensitive elements
Main criticisms to xenoestrogen studies 2.Compounds have additional, non-estrogenic actions Generalization of xenoestrogens effects is difficult. Tested chemicals may have multiple effects, related to different mechanisms of action and/or different concentration 3. Protocol is not validated for estrogenic activity Sensitivity of the protocol to pure estrogens should be controlled (positive control) Doses of Xs and pure estrogens should be selected on the base of different bioactivity
Environmental-like protocol II: EE SUBSTANCE Ethynyl estradiol (EE) : pure synthetic estrogen estrogenic potency >>BPA environmental contaminant DOSE Low dose : range of environmental exposure High dose : anticonceptional pill ENDPOINTS BEHAVIOUR (reproductive, cognitive..)
Environmental-like protocol III: Pair Model PAIR MODEL -Developmental exposure of both males and females -Adult male and female paired for 15 days END-POINTS - Reproductive Success: fertility and fecundity
WINDOWS OF EXPOSURE PERINATAL (PRE: GD 0-22) (POST: PND 1-21) PERINATAL (GD 0 PND 22) BEHAVIORAL END-POINTS JUVENILE ADULT: SEXUAL AGGR. MATERNAL + (play) + + PUBERAL (PND 23-30) + + ADULT (pregn.+lact.) + BPA Experiments Early exposure to a low dose of bisphenol A affects socio-sexual behaviour of juvenile female rats: Porrini et al. Brain Research Bulletin 65: 261-266 (2005). Altered profiles of spontaneous novelty seeking, impulsive behaviour, and response to D-amphetamine in rats perinatally exposed to bisphenol-a: Adriani et al. Environmental Health Perspectives, 111: 395-401 (2003). Effects of perinatal exposure to bisphenol A on socio-sexual behaviour of female and male rats: Farabollini et al. Environmental Health Perspectives, 110: 409-413 (2002). Pubertal exposure to estrogenic chemicals affects behaviour, in juvenile and adult male rats: Della Seta et al. Hormones and Behavior, 50: 301-307 (2006) Bisphenol-A exposure during pregnancy and lactation affects maternal behaviour in rats: Della Seta et al. Brain Research Bulletin, 65: 255-260 (2005). Bisphenol-A differently affects estrogen receptors-α in cycling and lactating female rats: Aloisi et al. Neuroscience Letters, 310: 49-52 (2001).
PERINATAL EXPOSURE:PRE/POST Genital sniffing Sexual Activity Test: Males Intromission latency Number of Intromissions sec 50 40 30 20 10 0 *# OIL PRE POST sec 200 150 100 50 0 *# OIL PRE POST 20 15 10 5 0 *# OIL PRE POST Ejaculation latency 600 500 400 sec 300 200 100 0 OIL PRE POST OIL: control; PRE: BPA prenatal; POST: BPA postnatal. BPA 40 μg/kg/day p<0.05 * vs oil # vs other treatment.
PUBERTAL EXPOSURE TO Xs conception birth weaning sexual maturity Days - 21 0 21 50 23 30 34 45 90 + Treatment: Xenoestrogen// Control Puberal tests: : novelty seeking, play. Adult tests: sexual orientation, sn sexual behavior. Xs: Bisphenol A 40 μg/kg/day; Ethinylestradiol 0.4 μg/kg/day
PUBERTAL EXPOSURE duration (sec.) 400 350 300 250 200 150 100 50 0 genital sniff Sexual Activity Test: Male * OIL BPA EE frequency 40 35 30 25 20 15 10 5 0 intromission * # OIL BPA EE latency (sec.) 900 800 700 600 500 400 300 200 100 0 intromission * * OIL BPA EE duration (sec) 350 300 250 200 150 100 50 0 BPA 40 μg/kg/day EE 0.4 μg/kg/day refractory period * OIL BPA EE OIL BPA EE * EE or BPA vs OIL p<0.05 # EE vs BPA, p<0.05
PERINATAL EXPOSURE OF MALE RATS: PRENATAL/POSTNATAL Development of sexual behaviour is influenced by perinatal exposure to BPA Impairment in the timing and performance of copulatory activity No clearcut dissociation of effects between PRE and POST (different subsystems involved) PUBERTAL EXPOSURE OF MALE RATS Development of sexual behaviour may still be influenced by pubertal exposure to BPA/EE Sexual behaviour is altered in timing and sequence of copulatory elements Stronger effects with EE and weaker with BPA: Lower sensitivity to BPA in the pubertal phase? Matching of doses? EE< BPA 1:100 On the whole: MALE COPULATORY ELEMENTS ARE SENSITIVE END-POINTS NO CLEARCUT DIFFERENTIATED EFFECTS BETWEEN THE VARIOUS DEVELOPMENTAL PERIODS OF EXPOSURE SENSITIVITY OF THE ANIMAL MODEL IS PROVED BY THE EXPERIMENT WITH EE, AS POSITIVE CONTROL
Environmental-like protocol II: EE SUBSTANCE ANIMAL Ethynyl estradiol (EE) : pure synthetic estrogen estrogenic potency >>BPA environmental contaminant Long- term exposure: from conception to puberty Female rat Pair model: female + male DOSE Low dose : environmental exposure (0.004 ug/kg) High dose : anticonceptional pill (0.4 ug/kg) BEHAVIOUR. ENDPOINTS reproductive behaviour, estrous cycle..
Effects of environmental-like exposure to ethynylestradiol: end-points - Sexual behavior of intact females - Reproductive function in exposed pairs -Development
Effects of EE2 on female sexual behaviour 100 OIL EE4 75 EE400 % females 50 25 0 proceptive lordosis aggressivity Endocrinology 2008
Sexual behaviour of stimulus males paired with EE2 treated females 100 OIL EE4 EE400 75 % males 50 25 0 mount / intromission ejaculation
Effects of EE2 on the estral cycle Estral cycle type Regular 12 Irregular Persistent estrus N 0 OIL EE4 EE400
Effects of EE2 on reproduction of treated pairs 100 OIL EE4 75 EE400 % females 50 25 0 pregnant gave birth
Effects of EE2 on F2 at PND2 N. of pups Mean body mass (g) 12.5 10.0 7.5 5.0 * * OIL EE4 8 7 6 5 4 3 2.5 2 1 0.0 total males females 0 male female Proc R Soc B 2007
Effects of EE2 on development of F1 animals Sex P Number of pups M NS F NS M+F NS Anogenital distance M NS F NS Litter mass M+F NS Body mass PND2 M NS F NS Body mass PND7 M NS F NS Body mass PND14 M NS F NS Body mass PND22 M NS F NS Body mass PND32 M NS F NS
Effects of environmental-like exposure to ethynilestradiol - Development (F1) apparently normal - Adult female sexual behavior disrupted (EE400) or altered (EE4) - In F1 pairs complete reproductive failure (EE400) or altered fecundity (EE4) - Individual body mass of F2 not affected
NTP CONCLUSIONS ON BPA EXPOSURE, April 2008 Some concern for: Neural and behavioral effects In fetuses, infant and children At current human exposure Scientific evidence from animal studies: Low level exposure during development Limitedevidenceof adverseeffects
University of Siena Daniele Della Seta University of Firenze Luca Corrieri Francesco Dessi`-Fulgheri University of Ferrara Leonida Fusani
Critical design components for future research on BPA, National Toxicology Program, guidelines on BPA, April 2008 Appropriate experimental design and statistical analysis, accounting for litter effects Appropriate route (oral) of exposure. Non-oral route should include internal measures of free BPA Multiple doses ranging from low to high Relevant endpoints, biologically plausible outcomes for estrogen-mediated effects on reproduction and behavior
Effects of EE2 on the estral cycle
PERINATAL EXPOSURE TO Xs Mother s status Before mating Pregnancy Lactation Groups PRE Days of treatment -5 POST 0 21 23 42 90+ ADULT BEHAVIOR OIL OIL Pup s status Conception Birth Weaning Cross-fostering Pups Mothers Control (OIL) BPA 40 μg/kg/day