Abstract
The effects of bisphenol A (BPA) were examined on sex hormones of F1 generation male rats during weaning period. Female rats were exposed to BPA from day 0 after pregnancy to the weaning period at doses of 50, 100, or 200 mg kg−1. The sex hormone levels of F1 generation male rats were determined. This study shows that F0 generation female rats fed with 200 mg kg−1 BPA had a significantly higher serum prolactin (PRL) levels at the end of weaning. Significantly higher levels of serum estradiol (E2) were also found in female rats fed 100 or 200 mg kg−1 BPA. Serum levels of E2 in F1 male generation rats were higher in treatment groups compared to control groups while serum testosterone (T) levels were lower. Follicle stimulating hormone (FSH) in F1 generation rats fed 200 mg kg−1 was markedly decreased. The relative testicular weights were significantly less in 100 and 200 mg kg−1 BPA groups. BPA was found to alter the sex hormone levels in F1 male rats during weaning period and thus disrupted endocrine functions.
Introduction
Bisphenol A (BPA), a known environmental estrogen (Owens and Koeter Citation2003; Roy et al. Citation1997; Toppari et al. Citation1996), is used largely in the manufacture of polycarbonate plastics, polystyrene resins, and dental sealants. The halogenated derivatives of BPA, such as tetrabromobisphenol A, are widely used as flame retardants for building material, paints, plastic products including epoxy resin, electronic circuit boards, and other electronic equipments. Depolymerization of these products results in BPA and its derivatives, leaching into foods (Brotons et al. Citation1995), infant formula from plastic bottles (Biles et al. Citation1999), saliva of patients treated with dental sealants (Nicolas et al. Citation1996; Olea et al. Citation1996; Pulgar et al. Citation2000), and in fresh food at the µg to mg kg−1 levels (Vivacqua et al. Citation2003). BPA has been detected in the concentration range of 0.1–10 ppb in human blood, urine, and fetal tissues, and related BPA levels in blood and fat tissues have also been reported (Ikezuki et al. Citation2002; Schonfelder et al. Citation2002; Thomsen, Lundanes, and Becher Citation2001; Vom Saal and Hughes Citation2005). Recently Willhite, Ball, and McLellan (Citation2008) derived an oral reference dosage of 0.016 mg kg−1-day−1.
BPA exerts low toxicity (Staples et al. Citation1998) with a rat oral median lethal dose (LD50) of 3.25 g kg−1, inhalation exposure of LD50 is 0.02%, and mouse oral LD50 is 2.4 g kg−1. Exposure can occur through contact with skin, or via inhalation or ingestion. Blot, Janning, and Michna (Citation2001) estimated that the maximum amount of daily human intake of BPA is 1 µg kg−1. Various studies demonstrated that BPA exerts adverse effects on endocrine, reproductive, immune, and nervous systems (Roy et al. Citation1997; Willhite, Ball, and McLellan Citation2008). Several investigators found that BPA interacted with estrogen receptors and interfered with endocrine metabolism leading to harmful effects (Owens and Koeter Citation2003; Roy et al. Citation1997). In this study, the effects of BPA were examined on sex hormone levels in F1 generation male rats during weaning period by radioimmunoassay.
Materials and methods
Animals
Approximately 20-day-old SD male rats obtained from animal experiment center of Chongqing University of Medical sciences were used.
Chemicals and treatments
BPA was purchased from Sigma Corporation, United States, and luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T), and estradiol (E2) radioimmunoassay kits were purchased from Tianjin Concord Medical Technology Co., Ltd. After male and female SD rats were acclimatized for 1 week, two female rats were mated with a male rat (female rats and male rats were mated at a ratio of 2 : 1) overnight and the day when copulatory plugs were found was designated as GD-0. The GD-0 rats were randomly assigned to either three treatment or control, five rats/group. Rats in the treatment groups were orally gavaged with BPA at the dose of 50, 100, or 200 mL kg−1, while control groups received an equivalent volume of corn oil. The rats were orally gavaged from GD-0 to day 20 in male offspring. All pregnant mothers and male offspring were sacrificed by femoral artery bleeding following anesthesia. Blood samples were put in a 37○C water bath for 30 min, followed by centrifugation at 1200 g for 10 min. Serum samples were stored at −20○C until analysis. Both testes were excised and weighed.
Sex hormone assay
Serum of pregnant rats was used to measure LH, FSH, E2, and PRL concentrations by radioimmunoassay. Male rat serum was used to determine LH, FSH, E2, and T concentrations.
Statistical analyses
One-way ANOVA was performed using the SPSS 10.0 software, with p < 0.05 taken as statistical significance.
Results
Body weight and serum sex hormone levels in lactating rats
Serum concentration of sex hormones and body weight of lactating rats in control and treatment groups’ data were not shown. There was no significant difference between treatment and control groups in body weight. However, PRL levels of 200 mg kg−1 BPA group were significantly higher control, and E2 levels of 100 and 200 mg kg−1 BPA groups were also significantly increased.
Serum level of sex hormones in 20-day-old F1 male rats
Serum concentration of sex hormones of F1 male rats in control and treatment groups are shown in . The levels of FSH of 200 mg kg−1 BPA group blood serum were markedly reduced. However, the levels of E2 of 50, 100, and 200 mg kg−1 BPA groups were significantly elevated accompanied by markedly lower T levels in BPA groups.
Table 1. Concentration of serum LH, FSH, E2, and T in 20-day-old F1 male rats.
Testicular parameters in 20-day-old F1 male rats
Data of the body weight and the testicular weights of F1 male generation are shown in . Body weight of 50, 100, and 200 mg kg−1 BPA groups was significantly lower than control group, and relative testicular weight in 100 and 200 mg kg−1 BPA group were markedly lower than the control group.
Table 2. Hormone parameters in 20-day-old F1 male rats.
Discussion
This study showed that the blood serum hormone levels of F0 parental female rats were altered by BPA exposure. The PRL and E2 levels of blood serum of 200 mg kg−1 BPA group were markedly increased. In this study, BPA may simulate estrogen to increase the level of serum PRL, because estrogen has a direct role in stimulating PRL gene expression (Wu et al. Citation1999). In addition, in vitro studies showed that E2 can promote PRL synthesis and release (DeMaria, Livingstone, and Freeman Citation2000). As there are estrogen receptors in the pituitary gland PRL cell, E2 can activate PRL gene through the estrogen response element near the PRL gene 5′ end of enhancer after E2 bound with the receptor.
In this study, the serum hormone levels were significantly altered by BPA in F1 rats during weaning. The levels of FSH and LH were increased at low BPA doses, but FSH declined at 200 mg kg−1 BPA indicative of hormesis. In contrast, BPA produced the elevation in the levels of blood serum E2, but a fall in blood serum T. In the early stages of male gonadal development, the levels of LH and FSH directly affect germ cells differentiation and replication; in the neonatal period, FSH secreted by the prehypophysis initiated spermatogenesis by promoting the role of T on Sertoli cell growth and function, thus FSH indirectly influences spermatogenesis. However, spermatogenesis in rat testis is not determined by FSH to a considerable extent, and LH, which is the main endocrine-regulating factor involved in T synthesis, is secreted and synthesized by pituitary gland. LH affects the spermatogenesis in the seminiferous tubules of testis because of a direct impact on the level of T. Atanassova et al. (Citation2000) reported the exposure of neonatal rats to estrogens or estrogen-like substances altered hypothalamic–pituitary–testicular axis function. It would appear that the exposure to BPA in the F0 generation affected F1 male generation sex hormone levels indicative of PPA-induced alterations in reproductive functions.
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