Effect of in utero exposure to endocrine disruptors on fetal ...

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The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.40, No.6, 909-916, 2015

Letter

Effect of in utero exposure to endocrine disruptors on fetal steroidogenesis governed by the pituitary-gonad axis: a study in rats using different ways of administration Yudai Kariyazono1, Junki Taura1, Yukiko Hattori1, Yuji Ishii1, Shizuo Narimatsu2, Masatake Fujimura3, Tomoki Takeda1 and Hideyuki Yamada1 Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 2Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushimanaka, Kita-ku, Okayama 700-8530, Japan 3Department of Basic Medical Sciences, National Institute of Minamata Disease, 4058-18 Hama, Minamata 867-0008, Japan 1

(Received June 2, 2015; Accepted October 2, 2015)

ABSTRACT — The effects of endocrine disruptors on testicular steroidogenesis in fetal rats were investigated in a study involving in utero exposure. In the major part of this study, pregnant rats at gestational day (GD)15 were given a single oral administration of the test substance, and then the expression of the following mRNAs in GD20 fetuses was determined: testicular steroidogenic acute-regulatory protein (StAR), a cholesterol transporter mediating the rate-limiting step of steroidogenesis, a ß-subunit of pituitary luteinizing hormone (LH), and a regulator of gonadal steroidogenesis. Among the substances tested, only di(2-ethylhexyl)phthalate (DEHP) reduced the expression of fetal testicular StAR. The others listed below exhibited little effect on fetal StAR: 2,2’,4,4’-tetrabromodiphenylether, tributyltin chloride, atrazine, permethrin, cadmium chloride (Cd), lead acetate (Pb) and methylmercury (CH3HgOH). None of them, including DEHP, lacked the ability to reduce the expression of pituitary LHß mRNA. The present study also examined the potential of metals as modifiers of fetal steroidogenesis by giving them to pregnant dams in drinking water during GD1 and GD20. Under these conditions, Cd and Pb at a low concentration (0.01 ppm) significantly attenuated the fetal testicular expression of StAR mRNA without a concomitant reduction in LHß. No such effect was detected with CH3HgOH even at 1 ppm. These results suggest that: 1) DEHP, Cd and Pb attenuate the fetal production of sex steroids by directly acting on the testis, and 2) chronic treatment during the entire gestational period is more useful than a single administration for determining the hazardous effect of a suspected endocrine disruptor on fetal steroidogenesis. Key words: Endocrine disruptor, Steroidogenic acute-regulatory protein, Luteinizing hormone, Maternal exposure, Fetus, Rats INTRODUCTION It has long been suspected that a number of endocrine disruptors hinder the reproduction and development of humans and wild life, disturbing the transfer of correct phenotypes to the next generations (Gore, 2008; Schug et al., 2011; Frye et al., 2012; Fudvoye et al., 2014). However, the detailed mechanism as well as the exact magnitude of their adverse effects on reproduction is not yet fully understood. A series of our previous studies have revealed

that the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), imprints immaturity in the gender-specific phenotypes such as sexual behavior by transiently lowering gonadal steroidogenesis during perinatal stages, the so-called the 'critical period' (Mutoh et al., 2006; Takeda et al., 2009, 2012 and 2014). More specifically, maternal exposure to TCDD down-regulates many steroidogenic proteins including steroidogenic acute-regulatory protein (StAR), a cholesterol transporter involved in the rate-determining step of steroidogenesis, in pups only at stages

Correspondence: Hideyuki Yamada (E-mail: [email protected]) Vol. 40 No. 6

910 Y. Kariyazono et al.

between the late fetus [gestational day (GD)20] and the early infant [postnatal day 2] (Mutoh et al., 2006; Takeda et al., 2012). The reason why such a transient reduction is serious is that correct masculinization and de-feminization need the stimuli of fetuses/infants by sex-steroids during the critical period (Carlson, 2007). These studies also provided evidence that TCDD initially reduces the expression of pituitary luteinizing hormone (LH) to exert the above effect on peripheral steroidogenesis. Of the two subunits of LH, the ß-subunit is more sensitive to TCDD exposure than LHα (Taura et al., 2014). It would be of great interest to know whether other chemicals suspected as being endocrine disrupters exhibit their effects in a similar manner to TCDD. To address this issue, we examined the potential of the following 8 substances in terms of their effects on gonadal steroidogenesis and its upstream regulators in fetal rats: di(2-ethylhexyl)phthalate (DEHP), 2,2’,4,4’-tetrabromodiphenylether (BDE47), tributyltin chloride (TBT), atrazine, permethrin, cadmium chloride (Cd), lead acetate (Pb) and methylmercury (CH3HgOH). In previous investigations, we gave rats a single oral dose of TCDD at GD15 with the day when pregnancy is confirmed being defined as GD0. Treatment with TCDD at an earlier stage (GD8) produces the same outcome as treatment at GD15 on the steroidogenesis governed by the pituitary-gonad axis (Mutoh et al., 2006). Although the present study examined the effects of test compounds also using a single oral dose at GD15, this schedule of administration might be inappropriate to evaluate their potential. This is because the body persistence of many chemicals examined in this study is shorter than that of TCDD. Also, if the gestational stage sensitive to test substances differs among the chemicals, treatment only at the limited window of pregnancy would miss their hazard to fetal development. Thus, the actual potential of the chemicals suspected for their ability to disrupt fetal steroidogenesis may not be obtained until they are evaluated in a study involving chronic exposure. Regarding this issue, we also examined the effects of Cd, Pb and CH3HgOH on fetal steroidogenesis after giving pregnant rats these substances dissolved in drinking water during the entire gestational period. MATERIALS AND METHODS Materials The following chemicals were obtained from the sources indicated in parentheses: TBT, cadmium chloride (CdCl2 · 2.5H2O) and lead acetate [(CH3COO)2Pb · 3H2O] (Nacalai Tesque Inc., Kyoto, Japan); atrazine and cis-perVol. 40 No. 6

methrin (Wako Pure Chemical Industries, Ltd., Osaka, Japan); DEHP (Kanto Chemical Co., Ltd., Tokyo, Japan); CH3HgOH (Tokyo Chemical Industry, Co., Ltd., Tokyo, Japan); and BDE47 (Accu Standard Inc., New Haven, CT, USA). All other chemicals were of the highest quality commercially available. Animals and treatments All animal experiments were conducted under the approval of the Institutional Animal Care and Experiment Committee of Kyushu University: permission number, A22-152-0 and A24-019-0. Male (9 weeks-old) and female (6-7 weeks-old) Wistar rats were obtained from Kyudo Co., Ltd. (Saga, Japan). They were housed under a photoperiod cycle of 12 hr light/12 hr dark in an air-conditioned specific pathogen-free room, and provided with food and tap water ad libitum. After adaptation for one week, female rats were paired overnight with male rats. When a seminal plug or sperm was observed in the female vagina, the day was designated as GD0 of pregnancy. Pregnant rats at GD15 were given a single oral dose of test chemical or vehicle alone at a dose of 2 mL/kg body weight, and the testis and pituitary were removed from their fetuses at GD20 for analysis. Corn oil, one of the vehicles, was purchased from Ajinomoto Co., Inc. (Tokyo, Japan). The doses of chemicals were as follows: DEHP: 30, 100 and 300 mg/kg/corn oil; TBT: 1, 5 and 10 mg as Sn/kg/corn oil; BDE47: 20, 60 and 200 μg/kg/corn oil; atrazine: 50 mg/kg/corn oil; permethrin: 50 mg/kg/corn oil; CdCl2: 3 mg as Cd/kg/water; CH3HgOH: 8 mg/kg/water; and (CH3COO)2Pb: 3 mg as Pb/kg/water. These doses were set by referring to published works which reported the positive effects of endocrine disruptors (see the first phrase of RESULTS AND DISCUSSION). In a separate experiment, drinking water containing 0.01 and 0.1 ppm CdCl2 was given to pregnant rats during GD1 and GD20. Also in this experiment, the tissues of the GD20 fetuses were removed for analysis of steroidogenesis. Similarly, pregnant rats were treated for 20 days with (CH3COO)2Pb (0.01 and 0.1 ppm drinking water) or CH3HgOH (0.1 and 1 ppm), and GD20 fetuses were examined. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting The expression of mRNAs was quantified by realtime RT-PCR according to the method described previously (Matsumoto et al., 2010). In brief, total RNA was extracted from the testis and pituitary using RNeasy Kits (QIAGEN GmbH, Hilden, Germany). The RNA obtained

911 Effects of endocrine disruptors on fetal steroidogenesis

was treated with gEraser (TaKaRa-bio, Shiga, Japan) to digest contaminated genomic DNA, and reverse-transcribed to its cDNA. Target mRNAs were amplified with Fast SYBR Green Master Mix (Invitrogen, Carlsbad, CA, USA), using a StepOnePlus Real-time PCR system (Invitrogen). The primer designs and the PCR conditions were described elsewhere (Matsumoto et al., 2010; Takeda et al., 2012). The amount of quantified target mRNA was normalized by ß-actin mRNA. Immunoblotting analysis for fetal testicular StAR was carried out according to the method reported previously (Takeda et al., 2009). The target protein was visualized by a reaction involving alkaline phosphatase conjugated with the secondary antibody, and then its image was scanned and quantitated using the ChemiDocTM MP system operated by Image Lab™ software (Bio-RAD Laboratories Inc., Hercules, CA, USA).

Fig. 1.

Statistical analysis Except for immunoblotting, the data for the fetuses of one dam were averaged to become a single analytical unit. In immunoblotting, the testes of all fetuses in one dam were pooled, and the mitochondrial fraction was prepared for analysis. The statistical difference between control and endocrine disrupter-treated group was compared by Student’s t-test. The comparison among multiple groups was conducted by one-way analysis of variance with a post-hoc test (Dunnet’s test), using GraphPad Prism Version 5 software (GraphPad Software, Inc., SanDiego, CA, USA). The statistical significance was set at p < 0.05. RESULTS AND DISCUSSION When pregnant rats at GD15 were given a single oral administration of test substances, only DEHP lowered the expression of testicular StAR mRNA in GD20 fetuses in a dose-dependent fashion (Fig. 1). A downward trend in

Effects of maternal exposure to endocrine disruptors on the fetal expression of testicular StAR mRNA. Pregnant rats at GD15 were given a single oral dose of each endocrine disruptor, and the effect of this treatment on the expression of testicular StAR mRNA in GD20 fetuses was examined. The doses of DEHP, TBT and BDE47 are shown in the figure. The doses of other substances were as follows: atrazine (Atr.), 50 mg/kg; permethrin (Per.), 50 mg/kg; Cd, 3 mg/kg; Pb, 3 mg/kg; and CH3HgOH (MeHg), 8 mg/kg. Each bar represents the mean ± S.E.M. of 6-14 fetuses 2 of which were removed from 3-7 different dams. ** Significantly different from the control (P < 0.01). Vol. 40 No. 6


the fetal expression of testicular mRNA coding for cytochrome P450 17, one of the enzymes necessary for steroidogenesis, was also seen following maternal exposure to DEHP (data not shown). The protein level of StAR in the fetal testis was also attenuated by maternal exposure to DEHP (Fig. 2). These results agree with previous work which showed a DEHP-induced change in the expression of fetal testicular genes (Vo et al., 2009) and the anti-androgenic effect of this substance on the fetal testis (Borch et al., 2006). The other 7 substances examined here did not produce any significant change in the fetal expression of mRNA coding for testicular StAR (Fig. 1). Among them, TBT and BDE47 had no effect at three doses which were within the range causing impaired fetal maturation, and abnormal morphology and altered gene expression in the fetal rat gonads [TBT (chronic treatment); Adeeko et al., 2003; Kishta et al., 2007] and abnormal reproduction in rats (BDE47; Talsness et al., 2008; Suvorov et al.,

Fig. 2.

Effects of maternal exposure to DEHP on the fetal expression of testicular StAR protein. Pregnant rats at GD15 were given a single oral dose of DEHP at the doses indicated, and the expression of testicular StAR in GD20 fetuses was analyzed by immunoblotting. The data for ß-actin is for the loading control. Each bar represents the mean ± S.E.M. of 5-6 dams: the testes of all fetuses in one dam were pooled and a mitochondrial fraction was prepared for the analysis of StAR. * Significantly different from the control (P < 0.05).

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2009). Atrazine, permethrin, Cd, Pb and CH3HgOH were examined only at one dose. However, the doses of these substances were determined in view of their effects on the production of sex steroids and related phenomena reported thus far: i.e., an alteration in the pulsatile expression of LH in adult female rats (atrazine: Foradori et al., 2009a, 2009b); a reduction in StAR expression in adult mice (permethrin: Zhang et al., 2007); impairment of testicular function in adult rats and mice (Cd: Siu et al., 2009; Ji et al., 2010); impairment of testicular function and sexual behaviors in adult rats (Pb: Batra et al., 1998, 2001; Sant’Ana et al., 2001); and impaired behavior and abnormal brain morphology as well as the LD50 for fetuses after in utero exposure (CH3HgOH: Stoltenburg-Didinger and Markwort, 1990; Lee and Han, 1995). It is of great interest whether the substances examined in this study cause abnormality in reproduction and development under the conditions used. Regarding this issue, little abnormality was observed in the litter size, and sex ratio in fetuses and fetal body weight after treating pregnant rats with a single oral dose of DEHP, TBT, BDE47 and CH3HgOH (Supplemental Table 1). As mentioned in the introduction, TCDD lowers gonadal steroidogenesis by initially reducing the pituitary expression of gonadotropins including LH. However, no reducing effect on the pituitary LHß mRNA was observed for all 8 substances examined in this study (Fig. 3). Regarding other pituitary hormones, while TCDD also reduced the fetal expression of growth hormone (Hattori et al., 2014; Taura et al., 2014), neither this hormone nor thyroid-stimulating hormone was attenuated by treatment with DEHP, TBT and BDE47 (data not shown). Instead of a reduction, TBT at the highest dose significantly increased the fetal expression of LHß mRNA (Fig. 3). Therefore, the ability of DEHP to attenuate fetal testicular StAR seems to be due to its direct effect on the regulatory mechanism in the fetal testis. In this regard, DEHP undergoes metabolic activation to a mono(2-ethylhexyl) derivative which can reduce testicular steroidogenesis by increasing active oxygen species (Zhao et al., 2012). It is also known that DEHP easily passes through the blood-placental barrier (BPB) to translocate into fetuses (Stroheker et al., 2006). Therefore, an increase in oxidative stress produced by the DEHP metabolite appears to be one of the mechanisms which provokes a reduction in fetal steroidogenesis. To address the concern whether a single oral administration may be insufficient to cause damage to steroidogenesis in the fetal gonad due to the reasons mentioned in the introduction, we examined the effect of chronic exposure to Cd, Pb and CH3HgOH on fetal steroidogenesis.


Fig. 3.

Effects of maternal exposure to endocrine disruptors on the fetal expression of pituitary LHß mRNA. Refer to the experimental section and the legend to Fig. 1 for the treatment schedule including doses and sample preparation. Each bar represents the mean ± S.E.M. of 3-7 dams: the pituitaries of three fetuses in one dam were pooled. * Significantly different from the control (P < 0.05).

Unlike single oral administration, giving pregnant rats 0.01 ppm Cd or Pb in drinking water from GD1 to GD20 reduced the expression of StAR mRNA in GD20 fetuses (Fig. 4A). This effect was not accompanied by any change in LHß expression (Fig. 4B). As the ten-fold higher dose (0.1 ppm) produced an effect comparable to the 0.01 ppm dose, the concentration of Cd and Pb needed for a reduction in fetal StAR would be less than 0.01 ppm. CH3HgOH had little effect on fetal StAR expression even at 1 ppm. Although chronic treatments with Cd and Pb attenuated the expression of fetal testicular StAR, these treatments had little effect on the litter size, sex ratio and growth of fetuses at GD20 (Supplemental Table 1). Taking both data from single and chronic treatments into consideration, it appears that chemicals with a short duration in the body should be examined for their potential as endocrine disruptors using the dosing schedule for

chronic treatment. However, single administration of Pb, Cd and CH3HgOH had little effect on testicular steroidogenesis in fetuses, although they remained in the body for a long time: i.e., these metals have a biological half-life of one month or more in the whole body, and the half-life is further extended in particular tissues such as the kidney (Cd), bone (Pb) and brain (CH3HgOH) (Amzal et al., 2009; Rice, 1989; Yu et al., 2011). Such failure of these metals to affect fetal steroidogenesis may be due to the poor transfer into fetuses. For instance, the matured BPB has been reported to interfere with the translocation of Cd into the fetus (Boadi et al., 1991; Ji et al., 2011). It is, therefore, an alternative possibility that the failure of Cd given at GD15 to reduce fetal steroidogenesis is due to the inappropriate choice of dosing time when the BPB is already mature. Therefore, treating pregnant dams with chemicals during the whole gestational period is also helpVol. 40 No. 6


Fig. 4.

Effects of chronic treatment of pregnant rats with Cd, Pb and methyl Hg on the fetal expression of mRNAs coding for testicular StAR (A) and pituitary LHß (B). Pregnant rats were given drinking water containing CdCl2, (CH3COO)2Pb and CH3HgOH (MeHg) during GD1 and GD20, and the expression of testicular StAR and pituitary LHß mRNA in GD20 fetuses was analyzed. The concentration of tested metal in the drinking water is shown in the figure. A, Each bar represents the mean ± S.E.M. of 6-12 fetuses 2 of which were removed from different dams. In B, each bar represents the mean ± S.E.M. of 3-7 dams: the pituitaries of three fetuses in one dam were pooled. * Significantly different from the control (P < 0.05).

ful to evaluate their effects on fetal steroidogenesis when transportation of the test compounds into fetuses is rejected by the BPB. In the case of Pb, this metal is known to easily pass through the BPB (Goyer, 1990). Nevertheless, treating pregnant rats with Pb only at GD15 caused little change in the fetal expression of StAR, whereas chronic treatment produced a significant effect. In summary, treatment of pregnant animals with the test chemicals during the entire period of gestation would be a better way to evaluate its potential on fetal steroidogenesis. Similarly, like DEHP discussed before, Cd and Pb are capable of increasing oxidative stress (Cd: Patrick, 2003; Yadav and Khandelwal, 2008) (Pb: Bolin et al., 2006). Thus, an increase in active oxygen species would be one Vol. 40 No. 6

of the mechanisms whereby these metals reduce testicular steroidogenesis in the fetus. The observation that anti-oxidants, i.e., α-lipoic acid and selenium, can alleviate a Cdproduced attenuation in the expression of testicular StAR supports the above view (El-Maraghy and Nassar, 2011). Cd is known to suppress the delivery of Zn to the fetus (Kuriwaki et al., 2005; Kippler et al., 2010). Because Zn deficiency leads to a reduction in testicular steroidogenesis (Hamdi et al., 1997), an alternative possibility is that Cd suppresses the fetal production of sex steroids by lowering the Zn level. Co-administration of Zn alleviates a Cd-induced reduction in the testicular content of testosterone (Gunnarsson et al., 2004). This fact seems to support the importance of a decrease in the level of Zn.


The provisional tolerable weekly intake (PTWI) of Cd has been proposed to be 7 μg/kg/week (WHO, 1993). Approximately 2/3rds of the dietary Cd intake is considered to come from plant products and the remaining amount is derived from animal products (Satarug and Moore, 2004), although some molluscs and crustaceans contain a high level of Cd (Storelli and Marcotrigiano, 2001; Kikuchi et al., 2002). Since, in the present study, the average water consumption was about 13 mL/100 g body weight/day in rats given 0.01 ppm CdCl2, the net intake of Cd in this group was calculated to be 9.1 μg/kg/ week which is close to the PTWI described above. Thus, as far as the damage to gonadal steroidogenesis in fetuses is concerned, the safety level for Cd intake may have to be reconsidered and set at a value lower than the present proposal. The same seems to also apply to Pb exposure: for example, while the PTWI of Pb is set at 25 μg/kg/ week (WHO, 1993), an animal experiment performed in this study indicated that 9.1 μg Pb/kg/week [0.01 ppm (CH3COO)2Pb] given to pregnant dams produces a reduction in gonadal steroidogenesis in their fetuses. As shown in the present study, when Cd and Pb at a single dose of 3,000 μg/kg were given to pregnant rats at GD15 by gavage, these metals failed to affect the fetal pituitary-gonad axis. However, the fetal expression of StAR was reduced by breeding dams for 3 weeks with drinking water containing Cd and Pb (net dose for 3 weeks: 9.1 μg/kg x 3 weeks = 27.3 μg/kg), the level of which was far lower than that of the single oral administration. Taken together, the results of the present study suggest that the effects of endocrine disruptors on the maturation and development in next generations should be assessed by chronic administration to pregnant animals. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; Scientific Research (S) (Project No.: 24221004). Conflict of interest---- The authors declare that there is no conflict of interest. REFERENCES Adeeko, A., Li, D., Forsyth, D.S., Casey, V., Cooke, G.M., Barthelemy, J., Cyr, D.G., Trasler, J.M., Robaire, B. and Hales, B.F. (2003): Effects of in utero tributyltin chloride exposure in the rat on pregnancy outcome. Toxicol. Sci., 74, 407-415. Amzal, B., Julin, B., Vahter, M., Wolk, A., Johanson, G. and Åkesson, A. (2009): Population toxicokinetic modeling of cad-

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