Reproductive Toxicity Study of Bisphenol A, Nonylphenol, and Genistein in Neonatally Exposed Rats

J Toxicol Pathol 2005; 18: 203–207

Short Communication Reproductive Toxicity Study of Bisphenol A, Nonylphenol, and Genistein in Neonatally Exposed Rats

Shuji Noda1, Takako Muroi1, Hideo Mitoma1, Saori Takakura1, Satoko Sakamoto1, Atsushi Minobe1, and Kanji Yamasaki1

1Chemicals Evaluation and Research Institute, 3–822, Ishii, Hita, Oita 877–0061, Japan

Abstract: To investigate the endocrine-mediated effects of neonatal administration of the weak estrogenic compounds, bisphenol A, nonylphenol and genistein, were subcutaneously injected with these compounds at doses of 0.1, 1, and 10 µg/rat/day for 5 days starting on postnatal day 1. Rats were autopsied at 47–50 days. Positive control groups were given diethylstilbestrol (DES) at the same dose levels as the other chemicals. The ano-genital distance, age of vaginal opening and preputial separation, and estrus cyclicity for all living offspring were examined. No abnormalities by the injection of nonylphenol and genistein were detected in this study. In the BPA groups, ano-genital distance in the females in the 1 and 10 µg BPA groups was shorter than in the control group, and the ventral prostate weight was higher in the 10 µg BPA group than in the control group. On the other hand, in the DES groups, delayed preputial separation occurred later in the 0.1 and 1 µg groups than in the control group, and in the 10 µg groups, preputial separation was not completed. Also the estrous stage persisted in female of all DES groups. Underdevelopment of the seminal vesicle and prostate were observed in males of the 1 and 10 µg DES groups, and fewer ovarian corpus lutea, ovarian follicular cysts, and uterine glands, as well as increased uterine epithelial height and squamous metaplasia of the epithelium in the uterus were observed in females of all DES groups. We concluded that the findings observed in the weak estrogenic compound groups were not toxicologically relevant changes and that the present data provide valuable information for the neonatal exposure assay using weak estrogenic compounds. (J Toxicol Pathol 2005; 18: 203–207) Key words: bisphenol A, diethylstilbestrol, endocrine effects, genistein, neonate, nonylphenol, rat

Since a considerable number of chemicals have been assays using the weakly estrogenic compounds, bisphenol A, reported to have possible endocrine-disrupting activity in nonylphenol, and genistein. humans and animals1,2, the Organisation for Economic Co- The test substances were bisphenol A (BPA, CAS No. operation and Development (OECD) has proposed using a 012D2222, Kanto Chemical Co., Tokyo, Japan), p-n- uterotrophic assay as an in vivo screening tests for estrogenic nonylphenol (NP, CAS No. 104-40-5, 98.7% pure, Wako properties of chemicals and the Hershberger assay as an in Pure Chemicals, Tokyo, Japan), and genistein (CAS No. vivo screening test for androgenic properties3–6. In addition, 446-72-0, 98.4% pure, Wako Pure Chemicals). the tests described in the enhanced OECD test guideline no. Diethylstilbestrol (DES, CAS No. 56-53-1, 99.8% pure, 407 (enhanced TG407) have been developed as assays for Wako Pure Chemicals) was used as the positive control for detection of endocrine and other mechanisms in mature rats. the estrogenic compounds. Olive oil (Fujimi Pharmaceutical On the other hand, the neonatal exposure assay has been Company, Osaka, Japan) was used as the vehicle. reported as an assay for detecting endocrine-disrupting The study was conducted in two parts because of the activity in the early life stages of rats and mice, and the small size of the animal room. DES and BPA were tested in endocrine-disrupting activity of estrogenic compounds, such Study 1 and DES, NP and genistein in Study 2. Study 2 was as clomiphene, diethylstilbestrol, ethynylestradiol, 17β- performed 2 months after the completion of Study 1. estradiol, tamoxifen, bisphenol A, and some phytoestrogens Twenty-five pregnant Crj:CD(SD)IGS female rats on 13 have been detected by this assay7–14. Therefore, this assay is days after mating were purchased from Charles River Japan, considered to be a useful method for detecting endocrine- Inc. (Shiga, Japan) for each study. At gestational day 19, the disrupting activity. We performed the neonatal exposure dams were weighed, weight-ranked, and randomly assigned 2 dams per group, to the experimental and control groups, Received: 10 November 2004, Accepted: 21 November 2005 and the remaining dams were used in other studies. The Mailing address: Kanji Yamasaki, Chemicals Evaluation and Research pups that were subsequently born were used in this study. Institute, 3-822, Ishii, Hita, Oita 087–0061, Japan Each chemical was administered to all pups for 5 days TEL: 81-973-24-7211 FAX: 81-973-23-9800 beginning on postnatal day (PND) 1 (day of birth = PND 0), E-mail: [email protected] 204 Neonatal Exposure of Weak Estrogenic Chemicals and on PND 7 the litters were adjusted to four males and four significant difference, the differences between the control females per dam. When the number of pups of either sex group and each of the treatment groups were analyzed by was less than four, the litter size was adjusted to a total of Dunnett’s test. When the variances were not homogeneous, eight pups regardless of sex. The pups were housed with the the Kruskal-Wallis test was used. If it yielded a significant dams in polycarbonate pens until weaning. All pups were difference, the differences between the control group and weaned on PND 21, and then housed in stainless steel, wire- each of the treatment groups were analyzed by a mesh cages for the remainder of the study. Water was nonparametric Dunnett’s test. provided automatically, and the animals were given access No abnormal clinical signs were observed in the rats to a commercial diet (MF, Oriental Yeast Co., Tokyo, Japan) given any of the compounds. The body weight of the rats ad libitum. The animal room was maintained at a given the weakly estrogenic compounds remained normal temperature of 23 ± 2°C and a relative humidity of 55 ± 5%. throughout the study, whereas, the body weight of the female The room was artificially illuminated with fluorescent light rats receiving DES in Study 2 significantly increased in a on a 12-hour light/dark cycle (06:00–18:00 h). All animals dose-dependent manner. were cared for according to the principles outlined in the Ano-genital distance in the females in the 1 and 10 µg guide for animal experimentation prepared by the Japanese BPA groups was shorter than in the control group, and no Association for Laboratory Animal Science. Rats were dose-related changes were observed in rats given other subcutaneously injected with 0, 0.1, 1 or 10 µg/rat/day compounds (Table 2). (approximately 0, 10, 100, and 1000 µg/kg/day body weight) The mean day of vaginal opening did not differ of one of the chemicals or vehicle for 5 days starting on PND significantly between the control group and the groups given 1. We selected the maximum dose of DES as 10 µg/rat/day any of the compounds. There were no differences between based on the data of Medlock et al.11, and the two lower the mean days of preputial separation in the control group doses of DES, 1 and 0.1 µg/rat/day, were set and tested with and any of the groups given the weakly estrogenic the same doses of BPA, NP, and genistein to determine compounds. However, preputial separation occurred later in whether these weakly estrogenic chemicals induce estrogen the 0.1 and 1 µg DES groups than in the control group, and receptor mediated changes. A vehicle control group given complete preputional separation was not detected in the rats olive oil alone was also established. The volume of the olive in the 1 and/or 10 µg DES groups. oil solutions was 0.05 ml for s.c. injection. The physical No abnormal estrous cycling was detected in the BPA, stability of each chemical was assessed with a Fourier NP and genistein groups, whereas the estrous stage persisted transform infrared spectrophotometer (FTS-135, Nippon in all DES groups. Bio-Rad Laboratories K.K., Tokyo, Japan), and the stability, No gross changes were detected in the BPA, NP and homogeneity, and concentration of each chemical genistein groups. However, the seminal vesicle, ventral suspension prepared for administration were confirmed by prostate, and glans penis were smaller in male of the 1 and 10 HPLC. The numbers of pups in each group used to make µg DES groups, and excessive cleavage of the urethral slit, each observation are shown in Table 1. The number of pups occasionally associated with insufficient raphe formation in the 1 µg/rat/day genistein group was smaller than in the between the urethral orifice and vagina in female rats, named other groups because one dam in the group was accidentally cleft phallus, hypospadias and small ovaries were observed sacrificed after gestational day 19. in all of the females of the DES groups. Clinical signs of all animals were recorded daily. Body Relative organ weight changes of male rats are shown weight was measured twice a week throughout the study. in Tables 3 and 4. In this study, only the relative weight was Ano-genital distance was measured on PND 7. Vaginal evaluated for the organ weight change, because the ages at opening and preputial separation were examined. The sacrifice of the rats were different, especially the rats with estrous cycle was evaluated from the day after the first persistent estrus vaginal smears. Seminal vesicle weight was vaginal opening day until the day of necrospy, and rats, significantly lower in all genistein groups without dose- including the DES group, were sacrificed in the diestrous relation. The ventral prostate weight was higher in the 10 µg stage on PND 47–50 in both studies, except for the rats with BPA group. No dose-related changes were detected in male persistent estrus vaginal smears which were sacrificed on rats given NP. However, the epididymis, seminal vesicle, PND 70. The following organs were weighed: pituitary, and ventral prostate weights were significantly lower in the 1 testes, epididymides, ventral prostate, seminal vesicle plus and/or 10 µg DES groups in Study 1, and in Study 2 the testis coagulating gland with fluid, uterus, and ovaries. The and epididymis weights were lower in the 1 and/or 10 µg pituitary was weighed after organ fixation. All weighed DES groups, and the seminal vesicle and ventral prostate organs except the testes were fixed in 10% neutral buffered weights were lower in all DES groups. No dose-related formalin and examined histologically. The testes were fixed organ weight changes were detected in female rats given any in Bouin’s solution and examined. of the weakly estrogenic compounds. On the other hand, the Data were analyzed using Bartlett’s test for ovary weights were significantly lower in all DES groups, homogeneity of variance. When the variances were and the uterine weights were lower in all DES groups in homogeneous at a significance level of 5%, a one-way Study 1 and in the 10 µg DES group in Study 2. analysis of variance was performed. If this test yielded a Histologically, no abnormal findings were detected in Noda, Muroi, Mitoma et al. 205

Table 1. Pup Numbers/Group for Each Chemical

Study Groups Dose (µg/rat/day) 00.1110 MF MF MF MF

1 Vehicle control 8 8 Diethylstilbestrol 8 8 9 7 10 6 Bisphenol A 8 8 6 10 8 8

2 Vehicle control 8 8 Diethylstilbestrol 8 8 8 8 8 8 Nonylphenol 8 8 8 8 8 8 Genistein 884488

M=male, F=female.

Table 2. Ano-genital Distance (Mean ± SD) of Offspring at 7 Days of Age

Study Chemicals Doses (µg/rat/day) Male Female mm AGD/3√ b.w. mm AGD/3√ b.w.

1 Vehicle 0 5.44 ± 0.34 2.17 ± 0.11 2.88 ± 0.21 1.16 ± 0.07 Diethylstilbestrol 0.1 5.45 ± 0.26 2.19 ± 0.12 2.69 ± 0.25 1.10 ± 0.10 1 4.83 ± 0.32 2.07 ± 0.12 2.59 ± 0.26 1.15 ± 0.08 10 4.95 ± 0.21 2.16 ± 0.06 2.86 ± 0.27 1.22 ± 0.12 Bisphenol A 0.1 5.68 ± 0.35 2.18 ± 0.14 2.80 ± 0.27 1.09 ± 0.09 1 5.50 ± 0.57 2.18 ± 0.19 2.61 ± 0.17 1.05 ± 0.06* 10 5.44 ± 0.41 2.09 ± 0.14 2.64 ± 0.20 0.99 ± 0.14*

2 Vehicle 0 6.64 ± 0.55 2.67 ± 0.24 3.35 ± 0.28 1.38 ± 0.10 Diethylstilbestrol 0.1 6.08 ± 0.67 2.39 ± 0.25 3.34 ± 0.34 1.36 ± 0.12 1 5.97 ± 0.40 2.44 ± 0.18 3.10 ± 0.23 1.28 ± 0.09* 10 6.07 ± 0.65 2.54 ± 0.26 3.37 ± 0.20 1.42 ± 0.08 Nonylphenol 0.1 6.71 ± 0.28 2.65 ± 0.08 3.26 ± 0.14 1.31 ± 0.08 1 6.29 ± 0.59 2.54 ± 0.21 3.14 ± 0.13 1.28 ± 0.06 10 6.74 ± 0.60 2.69 ± 0.25 3.16 ± 0.19 1.29 ± 0.07 Genistein 0.1 6.71 ± 0.51 2.77 ± 0.19 3.21 ± 0.16 1.31 ± 0.07 1 6.38 ± 0.31 2.60 ± 0.10 3.06 ± 0.17 1.27 ± 0.09* 10 6.80 ± 0.46 2.68 ± 0.17 3.28 ± 0.20 1.31 ± 0.08

*Significantly different from vehicle control at p<0.05.

the male and female rats given BPA, NP or genistein. gestational days 10–18 resulting in no abnormalities in However, underdevelopment of the seminal vesicle and female offspring, but in which abnormalities such as vaginal prostate was observed in the 1 and 10 µg DES groups, and epithelial stratification and abnormal estrous cycles were fewer ovarian corpus lutea, ovarian follicular cysts and detected in mice given BPA at doses of 15 or 150 µg/mouse uterine glands, as well as increased uterine epithelial height for 5 days starting on PND 113. Thus, the neonatal assay and squamous metaplasia of the glandular epithelium in the method, administered to female neonatal rats for a short uterus were detected in all of the females in the DES groups. time, is considered a useful method for detecting estrogenic The estrogenic compounds DES, ethynylestradiol, activity of chemicals. clomiphen, tamoxifen, BPA and 17β-estradiol, all of which The uterotrophic properties of BPA, NP and genistein except BPA appear to be strongly estrogenic compounds were detected in the rat immature uterotrophic assay19, and based on their receptor binding affinities and the results of abnormal estrous cycles have been observed in rats given uterotrophic assays15–17, were administered to neonatal rats BPA 600 mg/kg in a 28-day repeated toxicity test complying for a short time, and endocrine-mediated effects were with OECD enhanced TG 40720. Thus, some estrogen detected7–9,11–14. There is an interesting study in which BPA receptor mediated changes caused by BPA, NP and genistein was given to pregnant mice at doses of 10 and 100 mg/kg on at high dose levels were already known. Vaginal epithelial 206 Neonatal Exposure of Weak Estrogenic Chemicals

Table 3. Relative Organ Weights (mg/100 g b.w) of Male Rats

Study Chemicals Doses (µg/rat/day) Pituitary Testis Epididymis Seminal vesicle Ventral prostate Body weight (g)

1 Vehicle 0 4.1 ± 0.3 872.7 ± 182.1 125.0 ± 15.7 125.2 ± 24.6 58.9 ± 14.7 279.7 ± 30.8 Diethylstilbestrol 0.1 4.4 ± 0.4 847.3 ± 60.7 111.7 ± 6.9 102.5 ± 18.8 55.0 ± 11.3 283.9 ± 9.6 14.0 ± 0.3 891.6 ± 94.8 115.3 ± 14.6 75.2 ± 30.5 32.0 ± 12.3* 264.2 ± 16.3 10 4.5 ± 0.2 717.9 ± 94.3 91.6 ± 12.6* 26.1 ± 12.1* 31.6 ± 8.6* 231.7 ± 18.1 Bisphenol A 0.1 4.8 ± 0.3* 848.3 ± 46.8 118.4 ± 12.4 130.7 ± 23.6 68.6 ± 19.0 301.8 ± 20.1 14.5 ± 0.4 868.3 ± 38.3 121.5 ± 8.2 147.3 ± 31.6 70.9 ± 12.2 297.6 ± 10.7 10 4.5 ± 0.3 921.0 ± 39.8 128.7 ± 8.0 149.6 ± 25.3 79.4 ± 21.0* 269.7 ± 11.3 2 Vehicle 0 4.3 ± 0.5 901.9 ± 24.0 132.1 ± 10.4 190.5 ± 17.1 78.8 ± 16.4 278.9 ± 28.5 Diethylstilbestrol 0.1 4.4 ± 0.8 901.7 ± 45.7 122.3 ± 8.4 110.0 ± 17.1* 52.1 ± 8.7* 274.5 ± 13.5 14.1 ± 0.4 819.4 ± 92.8 114.1 ± 12.8* 52.6 ± 22.0* 37.9 ± 10.0* 249.3 ± 21.0* 10 4.4 ± 0.3 760.8 ± 97.1* 116.4 ± 16.9* 50.9 ± 20.9* 37.3 ± 8.5* 266.8 ± 13.2 Nonylphenol 0.1 4.3 ± 0.3 866.2 ± 53.8 121.8 ± 8.9 151.3 ± 25.2* 67.2 ± 11.8 291.5 ± 16.4 14.2 ± 0.3 907.2 ± 57.5 129.6 ± 13.1 157.2 ± 30.1* 77.1 ± 15.0 276.5 ± 25.1 10 4.3 ± 0.3 930.0 ± 131.7 128.2 ± 15.9 169.7 ± 18.4 80.9 ± 6.9 277.5 ± 9.6 Genestein 0.1 4.4 ± 0.4 882.0 ± 59.3 125.6 ± 8.6 133.7 ± 26.7* 68.9 ± 15.4 279.4 ± 17.3 14.3 ± 0.2 653.1 ± 177.0* 112.6 ± 7.8* 138.7 ± 16.3* 70.0 ± 6.8 268.5 ± 26.0 10 4.4 ± 0.4 929.5 ± 86.2 128.8 ± 9.7 158.2 ± 30.2* 68.9 ± 10.6 287.9 ± 20.4

*Significantly different from vehicle control at p<0.05.

Table 4. Relative Organ Weights (mg/100 g b.w) of Female Rats

Study Chemicals Doses (µg/rat/day) Pituitary Uterus Ovary Body weight (g)

1 Vehicle 0 5.7 ± 0.5 155.2 ± 13.9 30.7 ± 2.9 237.6 ± 22.3 Diethylstilbestrol 0.1 6.6 ± 0.7* 122.7 ± 8.1** 14.4 ± 2.0** 255.0 ± 13.9 16.6 ± 0.5* 106.1 ± 8.5** 13.1 ± 2.8** 261.3 ± 17.1* 10 6.2 ± 0.7 99.4 ± 16.5** 10.1 ± 1.** 255.0 ± 15.1 Bisphenol A 0.1 6.0 ± 0.8 143.4 ± 12.7 31.1 ± 3.7 252.6 ± 15.8 16.4 ± 0.5** 153.4 ± 14.1 33.6 ± 3.4 244.7 ± 13.0 10 6.1 ± 0.6 160.4 ± 13.8 31.1 ± 2.6 223.4 ± 15.2 2 Vehicle 0 5.7 ± 0.4 143.5 ± 19.4 31.2 ± 2.3 215.7 ± 13.0 Diethylstilbestrol 0.1 6.3 ± 0.9 136.8 ± 18.6 12.2 ± 2.7** 243.4 ± 11.8** 15.7 ± 0.8 115.9 ± 14.0 11.9 ± 3.0** 249.5 ± 14.0** 10 5.6 ± 0.6 103.3 ± 17.4** 9.7 ± 1.6** 252.4 ± 22.5** Nonylphenol 0.1 5.4 ± 0.4 142.0 ± 17.2 25.3 ± 2.2** 230.5 ± 15.6 16.2 ± 0.3 147.3 ± 12.3 31.9 ± 5.6 219.4 ± 23.8 10 5.5 ± 0.7 147.8 ± 9.2 31.8 ± 2.6 211.6 ± 9.3 Genestein 0.1 5.5 ± 0.5 152.6 ± 10.7 29.9 ± 2.1 227.7 ± 16.5 15.0 ± 0.7* 137.1 ± 7.0 31.3 ± 6.7 207.7 ± 14.6 10 5.3 ± 0.3 136.5 ± 14.6 29.7 ± 3.9 221.0 ± 21.8

*Significantly different from vehicle control at p<0.05.

stratification was observed in mice given 150 µg/kg BPA coumestrol and equol, inhibited uterine gland formation but and abnormal estrous cycles in mice given 15 or 150 µg/ reproductive organ structure in male rats was not altered by mouse BPA for 5 days beginning on PND 113, but no neonatal exposure to coumestrol10,23. In the present study, abnormal changes in reproductive function or histological seminal vesicle weight was lower in all genistein groups changes in reproductive organs were observed in rats given without dose-relation and ventral prostate weight was higher 300 µg/kg BPA on the same schedule21. Also, no in the 10 µg BPA group, but no abnormal histological abnormalities were detected in male rats given 8 mg/kg NP findings were detected in these groups. We could not by intraperitoneal injection from PND 1–1022. On the other speculate on the mechanism of the weight changes in the hand, neonatal exposure to phytoestrogens, such as accessory sex organs. 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