Prenatal Exposure of 2,2,4,4-Tetrabromodiphenyl Ether (PBDE 47) Inducing Sperm Superoxide Anion Generation and Hyperactivation in Male Offspring Mice

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1 Prenatal Exposure of 2,2,4,4-Tetrabromodiphenyl Ether (PBDE 47) Inducing Sperm Superoxide Anion Generation and Hyperactivation in Male Offspring Mice Hsu PC 1, Li CH 1, Chen LW 1, Tseng LH 2, Guo YL 3, Lee CW 1, Tsai SS 4 1 Department of Safety, Health and Environmental Engineering, National Kaohsiung First University of Science and Technology, Kaohsiung, 2 Department of Occupational Safety and Hygiene, Tajen University, Pingtung, Taiwan; 3 Department of Environmental and Occupational Medicine, National Taiwan University (NTU) and NTU Hospital, Taipei; 4 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung Abstract Polybrominated diphenyl ethers (PBDEs) are a class of polyhalogenated aromatic compounds commercially used as fire retardants in consumer products. 2,2,4,4 -Tetrabromodiphenyl ether (PBDE 47) is the predominant congener found in most wildlife and human samples and generally accounts for half of the total PBDEs measured. This study examines the effects of prenatal exposure to PBDE 47 on reproductive function in male offspring mice. The pregnant mice were divided into 4 groups and gavaged with 0, 100, 500 and 2500 mg/kg PBDE 47 in corn oil per day between gestational day (GD) 6 and 17. The male offspring mice were anesthetized and sperm function, testis DNA content, and histopathology were studied on postnatal day 85. We found in the 500- and mg/kg per day groups that prenatal exposure to PBDE 47 induced the damage of sperm chromatin DNA integrity. To our interest, epididymal sperm motility and mitochondrial membrane potential (MMP) were significantly induced and revealed hyperactivation in PBDE 47 treated groups as compared to the controls. Meanwhile, we found the sperm morphological abnormality and superoxide anion (O - 2 ) generation was significantly increased in the 500 mg/kg group. There were no significant changes in DNA content in testis cells and testicular histopathology. In conclusion, there were U-shape dose-response relationships in BDE-47 prenatal exposure and male reproductive outcomes. This is the first study to report the lowest-observed-adverse-effect level (LOAEL) for sperm function to be 500 mg/kg of PBDE 47 in male offspring mice. Future investigations should be performed to study the mechanism of PBDE 47 in utero exposure on oxidative stress and epididymal sperm hyperactivation in pubertal stages of development. Introduction Polybrominated diphenyl ethers (PBDEs) have been commercially used as fire retardants in consumer products such as polyurethane foam, electronics, and textile coating. PBDEs are generally added to products as three commercial mixtures (penta-bde, octa-bde, or deca-bde mixtures), each of which contains different 1 Vol. 71, 2009 / Organohalogen Compounds page

2 proportions of the 209 possible PBDE congeners that vary in their degree of bromination 1. 2,2,4,4 -Tetrabromodiphenyl ether (PBDE 47) is the predominant congener found in most wildlife and human samples and generally accounts for half of the total PBDEs measured 2,3. Although reproductive health risks to humans following PBDE prenatal exposure are unknown, several studies in rodents report potential developmental, reproductive, neurological, and endocrine toxicity 2,4,5,6. Several studies on toxicokinetics of PBDEs in various animal species have been conducted. The results from a number of tissue distribution studies with various tetra- and penta-bdes in rats and mice showed highest affinity to adipose tissue followed by liver, lung, kidney and brain 7,8. A study with tetra-bde showed long terminal half-life, about 23 days, which may indicate high bioaccumulation 7. Materials and Methods The pregnant mice were divided into 4 groups and gavaged with 0, 100, 500 and 2500 mg/kg PBDE 47 in corn oil per day between gestational day (GD) 6 and 17. At postnatal day 85, the animals were anesthetized by CO2 and the reproductive organ were removed and weighed. Epididymal sperm count, motility, sperm chromatin structure analysis (SCSA), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) generation were measured. SCSA, MMP, and ROS generation were analyzed by flow cytometry (FCM). Data were expressed as means ± standard deviation. All statistical analyses were performed on JMP 5.0. A P value of < 0.05 was considered statistically significant. Results and Discussion Body and organ weights. The data showed no significant differences in mean body weight, relative testie, epididymis, and cauda epididymis weights between male offspring prenatally exposed to PBDE 47 and controls (Table 1). The relative total seminal vesicle weight was significantly decreased in the 100- and 500- mg/kg groups as compared with controls (P < 0.05) (Table 1). Sperm function. We found in the 500- and mg/kg per day groups that prenatal exposure to PBDE 47 induced the damage of sperm chromatin DNA integrity (P < 0.05) (Table 2). Epididymal sperm motility and mitochondrial membrane potential (MMP) were significantly induced and revealed hyperactivation in PBDE 47 treated groups as compared to the controls (P < 0.05) (Table 2). The percentage of sperm morphological abnormality and levels of superoxide anion (O - 2 ) generation were significantly increased in the 500 mg/kg group as compared to the control group (P < 0.05) (Table 2). Testicular DNA content and histopathology. There were no significant changes in DNA content in testis cells (data nor shown) and testicular histopathology between PBDE 47-treated and control groups (Figure 1). In this study, this is the first study to report in utero PBDE 47 exposure might cause oxidative stress and epididymal sperm hyperactivation in pubertal stages of development. Other studies found that lead exposure 2 Vol. 71, 2009 / Organohalogen Compounds page

3 induced the produced early onset of capacitation by one of the pathways of ROS generation. These effects consequently result in premature acrosome reaction and reduced zona-intact oocyte-penetrating capability 9. DeLamirande and Gagnon (1993) also found a positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa 10. In conclusion, there were U-shape dose-response relationships in BDE-47 prenatal exposure and male reproductive outcomes. This is the first study to report the lowest-observed-adverse-effect level (LOAEL) for sperm function to be 500 mg/kg of PBDE 47 in male offspring mice. Future investigations should be performed to study the mechanism of PBDE 47 in utero exposure on excessive superoxide anion generation and epididymal sperm hyperactivation in pubertal stages of development. Acknowledgements This study was supported by the National Science Council of Taiwan (NSC B MY3). References 1. Alaee M., Arias, P., Sjodin, A. and Bergman, A. Environ Int 2003;29: Birnbaum L.S. and Staskal D.F. Environ Health Perspect 2004;112:9. 3. Hites R.A. Environ Sci Technol 2004;38: Zhou T., Taylor M.M., DeVito M.J. and Crofton K.M. Toxicol Sci 2002;66: Tseng L.H., Lee C.W., Pan M.H., Tsai S.S., Li M.H., Chen J.R., Lay J.J. and Hsu P.C. Toxicology 2006; Tseng L.H., Li M.H., Tsai S.S., Lee C.W., Pan M.H. and Hsu P.C. Chemosphere 2008;70: Staskal D.F., Diliberto J.J., DeVito M.J. and Birnbaum L.S. Toxicol Sci 2005;83: Darnerud P.O. and Risberg S. Chemosphere 2006;62: Hsu P.C., Hsu C.C., Liu M.Y., Chen L.Y. and Guo Y.L. J Toxicol Environl Health, Part A, 1998;55: DeLamirande E. and Gagnon C. Int J Androl 1993;16:21. 3 Vol. 71, 2009 / Organohalogen Compounds page

4 Table 1. Body and tissue weights of mice prenatally exposed to 2,2,4,4 -tetrabromodiphenyl ether (PBDE 47) (100, 500, and 2500 mg/kg) and unexposed controls Parameters Treatment of PBDE 47 ( g/kg) P value Body weight (g) 37.4± ± ± ± Relative right testis weight (mg/g B.W.) 2.7± ± ± ± Relative left testis weight (mg/g B.W.) 2.9± ± ± ± Relative right epididymis weight (mg/g B.W.) 1.1± ± ± ± Relative left epididymis weight (mg/g B.W.) 1.1± ± ± ± Relative right cauda epididymis weight (mg/g B.W.) 0.4± ± ± ± Relative left cauda epididymis weight (mg/g B.W.) 0.4± ± ± ± Relative total seminal vesicle weight (mg/g B.W.) 7.4± ±0.3 * 6.0±0.3 * 6.7± All data was expressed as means ± S.D.; * p < 0.05 as compared with control group. Table 2. Sperm function of mice prenatally exposed to 2,2,4,4 -tetrabromodiphenyl ether (PBDE 47) (100, 500, and 2500 mg/kg) and unexposed controls Parameters Treatment of PBDE 47 ( g/kg) P value Sperm count (10 6 /ml) 11.4± ± ± ± Sperm motility (%) 40.4±2.9 a 53.8±3.0 * 52.6±2.9 * 55.1±2.7 * MMP (%) a 64.3± ±3.9 * 90.6±3.9 * 85.4±3.7 * <0.001 αt (arbitrary unit) 375.4± ± ± ± COMPαT (%) b 2.4± ± ± ± Sperm H 2 O 2 (arbitrary unit) 64.9± ± ± ± Sperm. O - 2 (%) c 13.0± ± ±7.4 * 29.9± All data was expressed as means ± S.D. * p < 0.05 as compared with control group. a Values represent percentages of sperm population with high mitochondrial membrane potential as assessed by flow cytometric analysis with the JC-1 probe. b COMPαT is the % of sperm with abnormal chromatin DNA structure. c Values represent percentages of sperm population with high. O - 2 generation as assessed by flow cytometric analysis with the HE probe. 4 Vol. 71, 2009 / Organohalogen Compounds page

(A) Control Contr (B) 100 g/kg 100μ (C) 500 g/kg (D) 2500 g/kg

Photomicrographs of H&E stained sections of mouse testis.

of spermatogenesis;(b) 100 g PBDE 47/kg treated group; (C) 500

5 (A) Control Contr (B) 100 g/kg 100μ (C) 500 g/kg (D) 2500 g/kg Figure 1. Photomicrographs of H&E stained sections of mouse testis. (A) Controls showing normal testicular histology with evidence of spermatogenesis;(b) 100 g PBDE 47/kg treated group; (C) 500 g PBDE 47/kg treated group; and (D) 2500 g PBDE 47/kg treated group. Morphology of the testicular tissues appeared normal in all the treated groups as compared with that of the control group. 5 Vol. 71, 2009 / Organohalogen Compounds page

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