Low-Dose Effects of Bisphenol a on Early Sexual Development in Male and Female Rats

REPRODUCTIONRESEARCH

Low-dose effects of bisphenol A on early sexual development in male and female rats

Soﬁe Christiansen1, Marta Axelstad1, Julie Boberg1, Anne Marie Vinggaard1, Gitte Alsing Pedersen2 and Ulla Hass1 1Division of Toxicology and Risk Assessment and 2Division of Food Chemistry, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark Correspondence should be addressed to S Christiansen; Email: [email protected]

Abstract

Bisphenol A (BPA) is widely detected in human urine and blood. BPA has been reported to impair many endpoints for reproductive and neurological development; however, it is controversial whether BPA has effects in the microgram per kilogram dose range. The aim of the current study was to examine the influence of BPA on early sexual development in male and female rats at dose levels covering both regulatory no observed adverse effect levels (NOAELs) (5 and 50 mg/kg bw per day) as well as doses in the microgram per kilogram dose range (0.025 and 0.25 mg/kg bw per day). Time-mated Wistar rats (nZ22) were gavaged during pregnancy and lactation from gestation day 7 to pup day 22 with 0, 0.025, 0.25, 5 or 50 mg/kg bw per day BPA. From 0.250 mg/kg and above, male anogenital distance (AGD) was significantly decreased, whereas decreased female AGD was seen from 0.025 mg/kg bw per day and above. Moreover, the incidence of nipple retention in males appeared to increase dose relatedly and the increase was statistically significant at 50 mg/kg per day. No significant changes in reproductive organ weights in the 16-day-old males and females and no signs of maternal toxicity were seen. The decreased AGD at birth in both sexes indicates effects on prenatal sexual development and provides new evidence of low-dose adverse effects of BPA in rats in the microgram per kilogram dose range. The NOAEL in this study is clearly below 5 mg/kg for BPA, which is used as the basis for establishment of the current tolerable daily intake (TDI) by EFSA; thus a reconsideration of the current TDI of BPA appears warranted. Reproduction (2014) 147 477–487

Introduction (Salian et al. 2011) or from thermal and recycled paper (Vinggaard et al. 2000, Liao & Kannan 2011). Bisphenol A (BPA) is a high-production volume chemical The toxicity of BPA has been extensively investigated used mainly as a monomer in the manufacturing of by industrial, governmental and academic research numerous chemical products, including polycarbonate groups in short- as well as long-term animal studies, plastics and epoxy resins. BPA is present in many including several reproductive toxicity and multi- products, and consumer exposure may arise from generational exposure studies. Health concerns regard- migration of BPA from polycarbonate or epoxy-lined ing human exposures to BPA have in the past arisen food and drink containers, from dental sealants, from its well-known oestrogenic properties (Salian et al. from microwave containers into food during heating 2011). BPA has afﬁnity for nuclear oestrogen receptors (ERa and ERb) and oestrogenic activity in vivo giving rise This paper forms part of a special issue of Reproduction on Endocrine to increased uterine wet weights in the uterotrophic Disrupters. This article was presented at the 7th Copenhagen Workshop assay (Kanno et al. 2001). Furthermore, BPA exposure on Endocrine Disrupters, 28–31 May 2013. The meeting was supported during development has been shown to affect semen by the Danish Ministry of the Environment – Environmental Protection Agency as an activity under the Danish Centre on Endocrine Disrupters. quality (Sakaue et al. 2001, Salian et al. 2009), alter Publication of this special issue has been supported by the Society for prostate weights (Vom et al. 1998, Timms et al. 2005, Reproduction and Fertility. The opinions or views expressed in this Prins & Korach 2008) and increase the incidence of special issue are those of the authors, and do not necessarily reﬂect the prostate intraepithelial neoplasia (PIN) lesions in the opinions or recommendations of the Danish Ministry of the Environment prostate of adult rodents (Prins et al. 2011). Moreover, – Environmental Protection Agency or the Society for Reproduction and low-dose effects have been observed for a wide range of Fertility. The Guest Editors for this special issue were Anna-Maria Andersson, Hanne Frederiksen, Niels Erik Skakkebæk, Rigshospitalet, endpoints. In rodent studies, adverse effects of BPA doses Denmark, Kenneth M Grigor, Western General Hospital, Edinburgh, UK at or below 50 mg/kg bw per day have been reported and Jorma Toppari, University of Turku, Finland. for reproductive organ weights (Ashby et al. 1999,

q 2014 Society for Reproduction and Fertility DOI: 10.1530/REP-13-0377 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/30/2021 01:01:34PM via free access 478 S Christiansen and others

Chitra et al.2003), mammary gland development estimated to be in the range of 0.335–0.388 mg/kg bw per (Munoz-de-Toro et al. 2005) and behaviour, e.g. anxious day (95% percentile) (EFSA 2013). A total high exposure behaviour and impaired learning (Della Seta et al. 2005, (95% percentile) from food and non-food sources is Ryan & Vandenbergh 2006, Gioiosa et al.2007, estimated to be in the range of 0.500–0.642 mg/kg bw per Gonçalves et al.2010, Ayyanan et al. 2011). The day in teenagers and adults (EFSA 2013). majority of these low-dose effects have been observed after exposure during the developmental period when both the reproductive system and the brain are most Materials and methods susceptible to endocrine disturbances. Chemicals From multi-generation reproductive toxicity studies in rats, conducted according to standardised toxicity test BPA (purity O99.5%, CAS no. 80-05-7) was purchased from guidelines (Tyl et al. 2002, 2008), a no observed adverse Sigma–Aldrich. Corn oil used both as negative control and as effect level (NOAEL) of 5 mg/kg bw per day has been vehicle was purchased from Sigma–Aldrich. The corn oil was established. In these studies, the critical effects were provided to the laboratory in glass bottles. The dosing solutions changes in body and organ weights in adult and offspring were kept in glass bottles in the dark, at room temperature, and rats, and liver effects in adult mice. A tolerable daily continuously stirred during the dosing period. The levels of BPA intake (TDI) of 50 mg/kg bw per day has been obtained by in the solutions were verified by chemical analysis. dividing this NOAEL by an uncertainty factor of 100. The TDI was evaluated by EFSA in 2006 and has been Animals and treatment reaffirmed in 2008 and 2010 (EFSA 2006, 2008, 2010). However, an increasing number of research studies A total of 110 time-mated nulliparous, young adult Wistar rats conducted during the last decade have reported the (HanTac:WH, SPF, Taconic Europe, Ejby, Denmark) were effects of BPA in animals at doses at or below this NOAEL supplied at gestation day (GD) 3 of pregnancy. The day when (Richter et al. 2007). So far these data have not been a vaginal plug was detectable was designated as GD 1 and considered adequately relevant or reliable by regulatory independently of actual day of delivery, the expected day of delivery, GD 23 was designated as pup day (PD) 1. Thereby, the bodies because of either small animal number per group age of the pups was related to the time of conception, but was and/or a limited number of dose levels, and thus rather similar to postnatal age. these studies have not affected the establishment of the The animals were housed in pairs until GD 17 and alone TDI or other health-based guidance values for BPA (US thereafter under standard conditions in semi-transparent poly- FDA 2008, EFSA 2010). However, in 2008 an assessment sulfone (PSU) type III cages (PSU 80-1291HOOSU Type III, by NTP-CERHR Expert Panel (National Toxicology Tecniplast, Buguggiate, Italy) (15!27!43 cm) with Aspen Program – Center for the Evaluation of Risks to Human wood chip bedding (Tapvei, Gentofte, Denmark), Enviro Dri Reproduction) concluded that rodent studies suggest that nesting material (Brogaarden, Lynge, Denmark) and Tapvei BPA causes neural and behavioural alterations related to Arcade 17 (Aspen wood) shelters (Brogaarden). They were disruptions in normal sex differences in rats and mice at placed in an animal room with controlled environmental doses below NOAEL (0.01–0.2 mg/kg per day) (Chapin conditions with a 12 h light:12 h darkness cycle with light et al. 2008). In 2010, the FDA concurred with the NTP’s intensity 500 lux starting at 2100 h, humidity 55%G5, assessment on these endpoints (US FDA 2010). temperature at 21G1 8C and ventilation changing air ten times The aim of this study was to investigate early sexual per hour. All animals were fed on a standard diet with Altromin development in male and female Wistar rats after 1314 (soy- and alfalfa-free, Altromin GmbH, Lage, Germany). perinatal exposure to BPA using both a sufficient number Acidified tap water (to prevent microbial growth) in PSU bottles of litters per group and including several dose levels. The (84-ACBTO702SU Tecniplast) were provided ad libitum.The investigated endpoints included anogenital distance PSU bottles and cages as well as the aspen wood shelters (instead (AGD), nipple retention (NR) and prepubertal reproduc- of plastic) were used to reduce the risk of migration of BPA that tive organ weights, endpoints which previously have potentially could confound the study results. proven sensitive for detecting the effects of endocrine- The study was performed using three blocks (separated by 1 week), and all dose groups were equally represented in the disrupting chemicals (Gray et al. 1999, Hass et al. 2007, blocks, i.e. the 22 time-mated rats per dose group were Metzdorff et al. 2007, Christiansen et al. 2008). Also, this allocated among blocks. Dams not giving birth were elimi- study aimed at examining low doses in the microgram nated from the experiment. On the day after arrival (GD 4), the per kilogram dose range (25 and 250 mg/kg bw per day) time-mated dams were pseudo-randomly distributed into the as well as dose levels similar to those that form the basis groups with similar body weight distributions. The dams were for establishment of the current TDI. distributed into five dose groups (0, 0.025, 0.250, 5 or In adult humans, the daily mean exposure in previous 50 mg/kg bw per day respectively), and gavaged by qualified assessments is estimated to be within the range of 0.4–1.5 mg animal technicians with a stainless steel probe 1.2!80 mm BPA/kg bw per day (EFSA 2006, WHO 2011, ANSES 2011). (Scanbur, Karlslunde, Denmark) once daily from GD 7 to PD In a novel EFSA draft report on exposure, dietary exposure 22 (day of delivery excluded), at a constant volume of 2 ml/kg for adults (including women of childbearing age) is bw per day. The exposure period was chosen to cover the most

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Downloaded from Bioscientifica.com at 09/30/2021 01:01:34PM via free access Developmental bisphenol A exposure in rats 479 sensitive periods of the development of the reproductive system. The individual doses were based on the body weight of the animal on the day of dosing. The dams were inspected twice a day for general toxicity including changes in clinical appearance. Body weights were recorded on GD 4 and daily during the dosing period to monitor changes in weight gain, to follow pregnancy status and to adjust dose according to weight. Animal experiments were carried out at the DTU National Food Institute (Mørkhøj, Denmark) facilities. Ethical approval was given by the Danish Animal Experiments Inspectorate. The authorization number given is 2012-15-2934-00089 C4. The experiments were overseen by the National Food Institutes in-house Animal Welfare Committee for animal care and use.

AGD and NR Figure 2 This picture shows retained nipples in an exposed male rat pup on PD 14. Picture from DTU FOOD, 2013. Female rats have nipples, The day after delivery the pups were counted, sexed, weighed whereas male rats possess only rudimentary mammary glands but no and checked for anomalies. Pups found dead were macro- nipples. This is because locally produced DHT causes regression or scopically investigated for changes when possible. AGD (Fig. 1) apoptosis of the nipple anlagen in male rats. However, foetal exposure was measured the day after delivery in all offspring using an to anti-androgens can block this process, causing the male offspring to ocular stereomicroscope. All offspring were weighed on PD 6. display nipples similarly to their female littermates. On PD 14, all male and female pups were weighed and examined for number of areolas/nipples (NR), described as a prostate, seminal vesicles, levator ani/bulbocavernosus muscles dark focal area (with or without a nipple bud) located where (LABC), bulbouretral glands, adrenals, thyroid, retroperitoneal fat nipples are normally present in female offspring (Fig. 2). Female pad, liver and stomach were excised and weighed. From females, rats normally have 12–13 nipples whereas male rats usually the ovaries, thyroid, retroperitoneal fat pad, liver and stomach have none. The same technician, who is skilled in these were excised and weighed. measurements and was blinded with respect to exposure groups, recorded both AGD measurements and NR counts. Chemical analysis

Section of male and female offspring PD 16/17 The BPA concentrations in the dosing solutions (0, 0.0125, 0.125, 2.5 and 25 mg/ml) were analysed, blinded with respect to On PD 16 and 17, one male and one female pup from each litter exposure groups with an accredited method using liquid (nZ18–21) were weighed and decapitated, blinded with respect chromatography negative electrospray ionisation tandem mass to exposure groups. From males, the testes, epididymides, ventral spectrometry LC–ESI–MS/MS (Acquity-Quattro Ultima, Waters, Milford, MA, USA) monitoring the transition reactions m/z 227O212 and m/z 227O133. The samples were extracted with (1:1) n-heptane (HPLC-grade) and methanol:water (3:1) and purified by C18 solid-phase extraction. BPA concentrations in the samples (duplicate) were quantified by external standard cali- bration (r2O0.99) with the addition of internal standard of BPA-d16 (from Sigma–Aldrich) into samples, standards and blanks. The relative S.D. was below 11% and the recovery of BPA in the samples was 86%. The limit of detection was 15 mg/l. The analysis confirmed that the BPA concentrations in the dosing solutions were within the range (95% CI) of what was added.

Statistical analysis For all analyses, the a level was set at 0.05 and the litter was the statistical unit. When more than one pup from each litter was examined, statistical analyses were adjusted using litter as an independent, random and nested factor. AGD and organ weights were analysed using body weight as a covariate. Birth weights were analysed using the number of offspring per litter as covariate. Both data on birth weight and data on the dams (pregnancy and lactation data) were analysed using Figure 1 Anogenital distance (AGD) in male and female rats. AGD is the ANOVA, followed by Dunnett’s post hoc test in SAS (SAS distance from the anus to the developing genital bud. The AGD is Enterprise Guide 4.3 Statistical Software (SAS Institute, Inc., normally about twice as long in male as in female rats (Farris 1949). Cary, NC, USA)). www.reproduction-online.org Reproduction (2014) 147 477–487

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Table 1 Pregnancy and litter data from dams and offspring exposed to 0, 0.025, 0.250, 5 and 50 mg BPA/kg bw per day from GD 7 to PD 22. Control 0.025 mg/kg per day 0.250 mg/kg per day 5 mg/kg per day 50 mg/kg per day Dams and litters nZ18 nZ21 nZ17 nZ18 nZ18 Dam BW gain, GD 7–GD 21 82.4G26.4 91.2G14 82.9G22.5 81.1G20.8 78.8G17.1 Gestation length (days) 23.0G0.6 22.9G0.36 22.9G0.6 22.9G0.2 23.0G0.3 % postimplantation loss 7.9G11.5 8.0G10.2 11.3G22.5 5.9G4.9 13.0G21.8 % perinatal loss 8.5G8.0 10.8G12.4 9.2G7.9 6.8G5.4 17.9G26.5 Litter size 11.8G3.3 11.7G2.3 12.1G3.5 11.4G2.6 11.2G4.1 % perinatal deaths 3.2G5.4 3.3G7.2 2.5G4.9 1.0G2.8 8.6G23.4 % males 50.5G18.5 49.8G14.7 45.1G22.5 49.7G15.3 56.2G10.4 Offspring Male birth weight 6.2G0.5 6.3G0.5 6.1G0.5 6.3G0.5 5.9G0.9 AGD males (mm) 4.1G0.07 3.9G0.1 3.8G0.2 3.8G0.1 3.8G0.2 AGD females (mm) 2.2G0.1 2.1G0.1 2.0G0.1 2.0G0.1 2.0G0.1 Nipples males 0.1G0.2 0.2G0.3 0.2G0.2 0.3G0.4 0.4G0.6

Data represent group means based on litter meansGS.D. nZ17–21 l (22 time-mated in each group). Statistically signiﬁcant ﬁndings (P!0.05) are written in bold.

The number of nipple/areolas (NR) was assumed to follow NR in males a binomial distribution with a response range between 0 and qmax, with qmax being equal to the biologically The number of nipples in female rats is usually 12 vs zero possible maximal number of nipples in rats, either 12 or 13. in males. Perinatal BPA exposure induced NR in male The choice of qmax was decided by considering the global offspring in a dose-related manner and the increase in fit (information criterion of Schwarz). Litter effects on NR and NR was statistically significant at the highest dose of over-dispersion in the data were accounted for by using 50 mg/kg (Fig. 6). There were no changes in number of generalised estimating equation as reported in Christiansen nipples in females (data not shown). et al. (2012). The effect in the males was small, as the mean value at the highest dose of 50 mg/kg was below 0.6 nipples. Results Pregnancy data, postnatal growth and general toxicity Autopsy and organ weights at PD 16 No general maternal toxicity was observed at any of the Weight of the testes, uterus, ovaries, epididymides, doses administered in the study. BPA exposure had no ventral prostate, seminal vesicles, LABC, bulbouretral significant effect on maternal weight gain during glands, adrenals, thyroid, liver, and stomach was pregnancy (GD 7 to GD 21), or on maternal weight on unaffected by BPA exposure (Table 2). However, the PND 1, and the mean gestational length was also weight of the retroperitoneal fat pad was significantly unaffected (Table 1). Moreover, post-implantation loss, increased in male offspring from the highest dose group litter size, birth weights, sex ratio and perinatal loss of 50 mg/kg, when data were analysed with body weight were unaffected after in utero and lactational exposure as a covariate and also when the relative weight of the fat to BPA and so were maternal body weight gains, neonatal deaths and offspring body weights in the Anogenital distance, males postnatal period (Table 1). 27

24 Anogenital distance * * * The prenatal exposure to BPA significantly decreased 21 AGD in both male and female offspring (Figs 3 and 4). The decrease was statistically significant at all doses 18 in the females (P!0.0001 for all groups except for 15 the 0.025 mg/kg group for which PZ0.002) and from 0.25 mg/kg bw per day and above in the 3 males (P!0.0001). 0 In order to evaluate whether the observed effects could be due to unusually high control values, the AGD in both BPA-5 Control BPA-50 BPA-0.25 male and female controls was compared with historic BPA-0.025 control data from two recent similar studies from our Figure 3 Effects of BPA on AGD in prenatally exposed male rats on the laboratory. There were no statistically significant day after delivery. Results are shown as mean units GS.E.M., nZ17–21 l differences between control levels of AGD in the present (1 unitZ0.164 mm). Statistical significance is indicated by *, with and in previous studies (Fig. 5). P!0.01. AGD was reduced in males from 0.25 mg/kg bw per day.

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Anogenital distance, females inhibitor finasteride (Clark et al. 1990, Bowman et al. 15 * 2003, Christiansen et al. 2009). Interestingly, a new *** study that examines the effects of short-term exposure to 12 BPA on mRNA levels of 5a-reductase isozymes in prostate of adult castrated rats show that BPA signi- 9 ficantly decreased the mRNA levels of both 5a-reductase isozymes (Sańchez et al. 2013). This indicates that BPA is 6 a5a-reductase inhibitor and could be one explanation for the shallow dose–response similarly as for finasteride. 3 Another example of such a shallow dose–response curve is the effect on AGD in male rats after in utero exposure 0 to the oestrogenic antimicrobial preservative butylparaben (Boberg J, Axelstad M, Christiansen S, Isling LK, BPA-5 Control BPA-50 Geyic G, Mandrup K & Hass U 2013. Butylparaben BPA-0.25 BPA-0.025 alters anogenital distance, mammary development, and Figure 4 Effects of BPA on AGD in prenatally exposed female rats on the reproductive organ weights in perinatally exposed rats. day after delivery. Results are shown as meanGS.E.M., nZ17–21 l Personal communication). In female rats, AGDs can in (1 unitZ0.164 mm). Statistical significance is indicated by *, with some cases also be affected by exposure to endocrine ! P 0.01. AGD was reduced in females from 0.025 mg/kg bw per day. disruptors. In most studies of anti-androgenic exposure, female AGDs have not been affected (Hass et al. 2007, pad was compared with controls. No effect was seen in Christiansen et al. 2009); however, androgen exposure the female offspring (Table 2). has been shown to increase female AGD (Ostby & Gray 2001) and exposure to oestrogenic agents like ethinyl estradiol (EE2) and genistein have been shown to Discussion increase or decrease female AGDs, depending on study design (Levy et al. 1995, Casanova et al. 1999, Delclos In this study, clear effects of BPA exposure in the et al. 2009, Golub et al. 2010, Mandrup et al. 2013). microgram per kilogram dose range were detected on Due to the shallow dose–response curves observed in the AGD of male and female offspring, an effect which is this study, it was evaluated whether unusually high control a well-known predictor for adverse effects on reproduc- values could have caused the observed statistically tive health in male offspring. The study was designed as a significant differences. However, both male and female high-quality study, in order to be applicable by control AGD values in this study were similar to control regulatory authorities. This means that the study values in recent studies from the same laboratory. The fact included a large number of rats per treatment group that the AGDs were evaluated blindly to exposure group, and four dose levels. Moreover, an effort was made to avoid potential confounding of the results, such as water bottles and animal cages were made of PSU to reduce 30 the risk that BPA or related compounds would leach from Males the plastic and the animal diet was soy- and alfalfa-free. Females S.D. 20 AGD was significantly decreased in both male and ± female offspring at most of the tested BPA doses; however, for both sexes the dose–response curves were very shallow. No clear increase in response was seen 10 with increasing dose, i.e. the AGDs were almost equally AGD, mean lowered in males exposed to 250 mg/kg and 50 mg/kg and in females exposed to 25 mg/kg and 50 mg/kg. 0 Reduced AGD in male rats is usually a sign of anti- androgenic exposure during sexual development. When exposed to potent anti-androgens, AGDs in the male rodents are usually reduced in a dose-related manner, and for some chemicals, such as certain drugs or Control previousControl BPA, study this study Control previousControl BPA, study this study pesticides, perinatal exposure can even lead to males Control subsequent study Control subsequent study having female-like AGDs (Ostby et al. 1999, Parks et al. 2000, Hass et al. 2007, Christiansen et al. 2010). Figure 5 Historic control values for AGD males and females. Data from the control group in the present BPA study and in two not yet published However, other anti-androgenic chemicals show more studies from the same period (previous and subsequent) are shown as shallow dose–response curves. One example of such a mean units GS.D. nZ15–19 l. No statistically significant differences substance is the anti-androgenic drug and 5a-reductase were seen. www.reproduction-online.org Reproduction (2014) 147 477–487

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Z 0.6 * toxicity study, Long–Evans rats (n 6–9 in exposed groups) were exposed orally to either EE2 or 2, 20, 200 mg BPA/kg bw per day from GD 7 to PND 18 0.4 (Howdeshell et al. 2008, Ryan et al. 2010). In females, no effect on AGD was seen (Howdeshell et al. 2008, Ryan et al. 2010), whereas AGDs appeared reduced in male 0.2 offspring at the highest dose level (7% shorter than control values). However, no statistically significant No. of nipples/areolas change was detected (Howdeshell et al. 2008). In this 0.0 study, male AGDs were very similarly decreased, i.e. by 6% at 250 mg/kg bw per day. The lack of a statistically BPA-5 Control BPA-50 significant effect in the Howdeshell et al. (2008) study BPA-0.25 BPA-0.025 could be due to the relatively small group size of 6–9 l, Figure 6 Effects of BPA on nipple retention in perinatally exposed male compared with the 17–22 l per group in this study. In rats on PD 14. Results are shown as meanGS.E.M., nZ17–21 l. another study, pregnant Sprague–Dawley rats (nZ10) Statistical significance is indicated by *, with P!0.01. were fed a diet containing BPA from GD 6 to PND 21, at doses of 0.017, 0.17 or 1.7 mg/kg bw per day, and AGD the large numbers of litters included in the study and the was examined in 5 week-old and 3 month-old males and low P values, consolidate that the observed AGD effects females. No effect was seen on male AGDs, whereas were unlikely to be random findings. AGDs were significantly shorter in 1-month-old females A number of studies have investigated the effects of exposed to 0.17 and 1.7 mg/kg, but not at 3 months of perinatal BPA exposure on AGDs in rat offspring. Some of age. As the effects were only seen in the younger females, them corroborate the present findings, whereas others the authors did not consider the results to be biologically contradict them. In a developmental toxicity study, SD significant (Kobayashi et al.2012). However, these rats (nZ18–20) were administered by gavage to doses of reductions in female AGDs corroborate the findings 0.1 and 50 mg BPA/kg bw per day, from GD 6 to GD 21, from this study, and it is possible that measurement of and AGDs were assessed in male offspring only (Talsness AGD in newborn male and female rats, instead of 1- and et al. 2000). Significantly shorter AGDs were seen in rats 3-month-old rats, would have yielded some even more exposed to 50 mg/kg bw per day, whereas no effects were similar results to the present ones. This study has only seen at 100 mg/kg; however, this dose was lower than the examined AGD at the time of birth and not at a later age. lowest effective dose found in this study, so these results However, even if such a measurement would have shown fit well with the present ones. In another developmental that the decreases in AGD were transient we do not find

Table 2 Body and organ weights for BPA-exposed male and female pups at PD 16/17. nZ15–20 (litter). Control 0.025 mg BPA 0.250 mg BPA 5 mg BPA 50 mg BPA Males Number of malesa 18 20 14 16 17 Body weights (g) 28.6G4.6 29.9G4.3 28.3G2.3 28.6G3.0 28.7G5.3 Right testis (mg) 51.3G7.6 51.4G7.8 51.0G4.6 50.5G7.0 51.0G9.8 Left testis (mg) 50.9G7.4 51.3G7.6 50.3G4.3 49.9G6.6 50.7G9.2 Ventral prostate (mg) 11.7G3.6 11.2G2.8 11.1G2.4 11.5G2.6 10.8G2.9 Epididymides (mg) 22.8G3.8 21.7G3.1 21.1G2.7 21.0G5.7 21.9G3.4 Seminal vesicles (mg) 7.1G2.0 7.5G2.3 7.1G1.8 7.5G2.8 7.1G1.8 LABCb (mg) 26.0G4.4 25.7G5.6 23.9G3.8 27.2G6.5 24.3G5.0 Bulbourethral glands (mg) 1.7G0.4 1.6G0.3 1.5G0.4 1.6G0.4 1.6G0.4 Liver (mg) 753.7G114.7 778.2G136.4 722.5G67.4 739.2G75.3 746.6G150.1 Adrenals (mg) 7.5G2.1 7.3G1.8 7.2G1.5 7.9G1.4 7.6G1.6 Retroperitoneal fat (mg) 38.0G11.2 42.5G13.2 38.7G11.6 36.6G12.3 46.4G12.5c Females Number of femalesa 15 20 16 18 16 Body weights (g) 29.1G2.6 31.2G4.0 29.4G4.7 30.9G3.0 29.6G5.3 Right ovary (mg) 2.9G0.77 2.8G0.73 2.9G0.81 2.8G0.66 2.9G0.74 Left ovary (mg) 3.2G1.04 3.1G0.7 3.2G0.77 3.1G0.59 3.3G1.22 Liver (mg) 798.8G66.7 847.8G141.0 780.4G127.1 822.8G100.6 846.2G178.6 Thyroidea (mg) 4.7G0.9 5.2G1.2 4.7G0.9 5.0G1.3 4.8G0.9 Retroperitoneal fat (mg) 29.5G12.3 31.1G9.7 31.0G12.5 32.3G10.3 30.3G10.0

Data represent meansGS.D. Statistically significant findings (P!0.05) are written in bold. aOne male/female pup per litter. bLevator ani/bulbocavernosus muscles abbreviated LABC. cThe retroperitoneal fat pad was significantly increased in the males from 50 mg BPA when data were analysed with body weight as a covariate and also when the relative weight of this fat pad was compared with the controls.

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Downloaded from Bioscientifica.com at 09/30/2021 01:01:34PM via free access Developmental bisphenol A exposure in rats 483 that this would weaken the findings seen at birth. studies are difficult to compare directly due to differences An altered AGD in male or female offspring at the time in tested species and exposure scheme. of birth indicates that the endocrine signalling, which is Overall, it can be concluded that AGD effects in necessary for normal reproductive development, has rodents after BPA exposure are conflicting, probably due been altered (Bowman et al. 2003, Hotchkiss et al. 2004). to varying study designs, tested species and strains, Such altered programming can have adverse conse- routes of exposure, dose levels and statistical power for quences on reproductive functions later in life (Hotchkiss detecting effects. However, the overall weight of et al. 2004, Christiansen et al. 2008, Welsh et al. 2008), evidence precludes dismissing the findings of decreased even if AGD is no longer affected. AGD at low doses in this study. In a small toxicity study treating Sprague–Dawley rats In this study, BPA also caused retention of areolae or (nZ4) with BPA in drinking water during gestation and nipples in male offspring in a dose-related manner; lactation (estimated dose from 0.1 to 1.2 mg/kg bw per however, the effect was small and only statistically day), no difference in AGD during the neonatal period significant in the highest dose group (50 mg/kg). Previous was seen in neither male nor female pups (Rubin et al. studies have examined areolas/nipples in rats and found 2001); however, the low sensitivity of this study means no effects (Tyl et al. 2002, Howdeshell et al. 2008). As that no clear conclusion should be drawn from it. In an with the effects on AGD, differences in study design may effort to set a reliable NOAEL, Tyl et al. (2002) performed explain why an effect was seen in this study but not in a multi-generational study in Sprague–Dawley rats other studies. It is, however, important to note, that even (nZ26–30), using dietary BPA exposure at doses of though the observed effects on NR was quite small, the 0.001, 0.02, 0.3, 5, 50 and 500 mg/kg bw per day. AGD increased number of areolas/nipples seen in the BPA- was assessed in the F2 and F3 generations, and in male exposed male offspring supports that the perinatal BPA offspring no significant effects were seen. In females, seems to adversely affect sexual differentiation. increased neonatal AGD was seen at some doses in the During the last decade, it has become evident that F2, but not in the F3, and therefore the findings were assessment of AGD and NR in rodent offspring can be used as markers of impaired androgen action within the considered to be not biologically relevant by the authors. critical programming windows of sexual differentiation As this study had high statistical power, the discrepancies (Welsh et al. 2008, 2010). These endpoints have been in AGD result between the study by Tyl et al. (2002) and shown to be highly predictive of increased risk of adverse the present one are difficult to explain, but they could be reproductive toxicity effects later in life, including caused by differences in rat strain (Sprague–Dawley vs increased incidence of hypospadias and cryptorchidism, Wistar), exposure (dietary vs gavage) and/or the decreased penile length and seminal vesicle weight generation of studied offspring (F2 vs F1). (Bowman et al. 2003, Christiansen et al. 2008, Welsh Effects of developmental BPA exposure on AGD have et al. 2008), and assessment of both AGD and NR has also been evaluated in mice. Honma et al. (2002) Z been recognised for regulatory purposes. Both measure- subcutaneously dosed pregnant mice (n 10) with 2 or ments are mandatory in the newly accepted OECD Test m 20 g/kg bw BPA and found increased AGD (measured guideline (Extended one-generation reproductive m on PND 21) in female offspring exposed to 2 g/kg, but toxicity study (OECD 2012)), and statistically significant not to 20 mg/kg. AGDs were no longer significantly effects are considered adverse and useful for setting a increased at day 60. In males, increased AGD was seen in NOAEL (OECD 2013). both dose groups; however, this was only significant at In humans, recent studies have reported shorter AGD in PND 60. These altered AGDs were reported at lower boys with hypospadias or cryptorchidism as compared doses than those observed in this study; however, the with boys with normal genitalia (Hsieh et al. 2008), and different species and dosing scheme make it difficult to decreased AGD in adult men has been correlated to compare the results directly. BPA exposure was also changes in semen parameters and decreased testosterone evaluated in a murine two-generation study. Tyl et al. level (Eisenberg et al. 2011, 2012a, 2012b, Mendiola (2008) found that in male offspring, absolute AGDs were et al. 2011). The reproductive effects of prenatal BPA significantly reduced at the highest tested dose of exposure in humans was evaluated by Miao et al. (2011), 600 mg/kg per day on PND 21, and at both 600 mg/kg who compared AGD in sons of workers who had or had per day and the second highest dose of 50 mg/kg, when not been occupationally exposed to BPA during preg- adjusted for terminal body weight. However, no nancy. The results showed that parental occupational significant reductions in male AGD were seen at the exposure to BPA during pregnancy was associated with lower doses of 3, 30, 300 or 5000 mg/kg. According to the shortened AGD in male offspring. This data indicates authors, these effects were not considered to be treatment that BPA can potentially disturb the development of the related because of the absence of effects on PND 0 for foetal reproductive systems in humans (Miao et al. 2011). F1/F2 males and on PND 21 for F2 females. However, the An inverse association between shorter male AGD in findings of reduced male AGDs after BPA exposure may human infants and prenatal phthalate exposure (particu- corroborate the results from this study, even though the larly di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl www.reproduction-online.org Reproduction (2014) 147 477–487

Downloaded from Bioscientifica.com at 09/30/2021 01:01:34PM via free access 484 S Christiansen and others phthalate (DBP)) has also been reported (Swan et al. BPA-induced effects on AGD and nipples in rat offspring 2005, Swan 2008). Similarly, girls androgenised due are caused by an interference of BPA with the to congenital adrenal hyperplasia have longer AGD effect, synthesis and/or metabolism of oestradiol and compared with healthy girls (Bongiovanni & Kellenbenz testosterone. According to the available in vitro data, 1962). This suggests that AGD can also be a predictor of BPA has the potential to affect all these pathways. persistent disturbances in human sexual differentiation. The reduced AGD in males is a sign of demasculinisation Another marker for disruption of sexual development and may be caused by inhibited foetal testosterone in rodents is reproductive organ weights. When levels and/or an AR antagonistic effect. In females, the measured in prepuberty, male reproductive organ reduced AGD is a sign of super-feminisation and can be weights have in numerous studies been shown to be hypothesised to be linked to BPA-induced ER agonism reduced by anti-androgenic compounds (Metzdorff et al. and/or metabolism. However, further studies would be 2007, Christiansen et al. 2009, Jacobsen et al. 2012). In needed to elucidate this. this study, no significant changes in reproductive organ In adult humans, the daily mean exposure in previous weights were seen in offspring at PD 16/17. This is in assessments is estimated to be within the range of contrast to some studies showing increased prostate 0.4–1.5 mg BPA/kg bw per day (EFSA 2006, WHO 2011, weights after perinatal BPA exposure; however, most ANSES 2011). In a novel EFSA draft report on exposure, other studies have looked at the prostate weights in adult dietary exposure for adults (including women of offspring, which is a big difference to this study (Nagel childbearing age) is estimated to be in the range of et al. 1997, Elswick et al. 2000, Gupta 2000). However, 0.335–0.388 mg/kg bw per day (95% percentile) (EFSA the multi-generational studies by Tyl et al. (2002, 2008), 2013). A total high exposure (95% percentile) from food in mice and rats, did not find any significant effects on and non-food sources is estimated to be in the range of adult prostate weight, so the BPA effects on adult prostate 0.500–0.642 mg/kg bw per day in teenagers and adults weights are still controversial. (EFSA 2013). These estimated exposure levels to BPA are An increased weight of the retroperitoneal fat pad was only somewhat lower than the dose that caused adverse seen in males at the highest dose level. Finding of enlarged effects, which leaves little margin of exposure. fat deposits is in agreement with many studies showing The presented data are the first results from this large study BPA-induced effects on obesity-related parameters and on BPA, which will be extended to include assessment adipocyte differentiation, thereby indicating a link of sexually dimorphic behaviours, mammary gland between BPA exposure and obesity development (vom development, semen quality and reproductive organ weight Saal et al.2012). and histopathology in adolescent and adult offspring. The mode of action of BPA leading to the effects in this study is not clarified and may involve several differential modes of actions. In the ToxCast programme, launched Conclusions by US-EPA to develop ways to predict potential toxicity of chemicals and to develop a cost-effective approach for Perinatal BPA exposure caused adverse effects on both prioritising the thousands of chemicals that need toxicity male and female rat sexual development at low doses. At testing, BPA was shown to have an effect in 101 out of doses from 250 mg/kg and above, male AGD at birth was 467 in vitro toxicity assays (Reif et al. 2010). Thus, BPA’s significantly decreased, whereas decreased female AGD toxicological effects are not limited to the classical was seen from 25 mg/kg bw per day and above. Moreover, oestrogenic mechanism (Shelby 2008) and comprise the incidence of NR in males appeared to increase dose several other signalling pathways. In addition to being an relatedly and the increase was statistically significant at ER agonist, BPA also show activity on the androgen 50 mg/kg per day. The decreased AGD in both sexes receptor (AR), other nuclear receptors and xenobiotic indicates endocrine effects on sexual development and metabolising enzymes (including aromatase) (Reif et al. provides new evidence of adverse effects of BPA in rats in 2010). In the literature, BPA has shown oestrogenic the microgram per kilogram dose range. properties, the ability to antagonise AR activation with The NOAEL in this study is clearly below the an IC50 in the micromolar range (Paris et al. 2002, NOAEL of 5 mg/kg for BPA that is used as the Bonefeld-Jorgensen et al. 2007) and the ability to basis for establishment of the current TDI by EFSA, inhibit aromatase (Bonefeld-Jorgensen et al. 2007). In thus a reconsideration of the current TDI of BPA addition, BPA has been found to inhibit testosterone appears warranted. production and E2 metabolism in H295R cells (Zhang et al. 2011). Consequently, interpretation of BPA effects only in terms of an oestrogen-mimicking effect would be Declaration of interest too simplistic. The involvement of several of these The authors declare that there is no conflict of interest that mechanisms could explain the effects observed in this could be perceived as prejudicing the impartiality of the study at low doses of BPA. It is conceivable that the research reported.

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