Effect of bisphenol A and co-administration of bisphenol A and vitamin C on epididymis of adult rats: A histological and biochemical study

K. C. Chitra¹, K. Ramachandra Rao², P. P. Mathur¹

Keywords: bisphenol A; epididymis; spermatozoa; ascorbic acid; antioxidant enzymes; lipid peroxidation; reactive oxygen species; oxidative stress

Abstract

Aim: To study the effect of bisphenol A on the epididymis and epididymal sperm of rats and the possible amelioration action of co-administration with vitamin C. Methods: Male Wistar rats were orally administered bisphenol A (0.2 µgkg^-1day^-1, 2 µgkg^-1day^-1 and 20 µgkg^-1day^-1) and 0.2 µg, 2 µg and 20 µg bisphenol A + 40 mg vitamin Ckg^-1day^-1 for 60 days. On day 61, rats were killed with anesthetic ether and sperm collected from epididymis were used for assessment of sperm count, motility and viability and biochemical studies. A 1 % homogenate of epididymis was prepared and used for biochemical estimations. Caput, corpus and cauda epididymis were fixed in Bouin's fixative for histological studies. Results: Administration of bisphenol A caused a reduction in the epididymal sperm motility and count and the sperm viability remained unchanged. The activities of superoxide dismutase and glutathione peroxidase decreased, while the levels of lipid peroxidation increased in epididymal sperm and epididymis at all doses. Co-administration with vitamin C reversed the effect of bisphenol A-induced oxidative stress in epididymal sperm and epididymis. A complete degeneration of epididymal epithelium in caput, corpus and cauda regions with reduction in the number of sperms were observed at all doses of bisphenol A-treated rats. Conclusion: Bisphenol A induced oxidative stress in epididymis and caused degeneration of the epididymal epithelium of rats. Co-administration with vitamin C had a protective effect against the bisphenol A-induced toxicity in epididymal sperm and epididymis.

1 Introduction

Bisphenol A, an environmental contaminant, enters into humans as it leaches from the lining of tin cans into foods, from dental sealants into saliva and from polycarbonate bottles into their contents. In 1993, about 109 tons of bisphenol A was released into the air or surface water [1]. Recently, in India the usage of plastic beverage containers and the release of bisphenol A-containing industrial wastewater has been increased, resulting in considerable human exposure. Bisphenol A has been shown to decompose into many kinds of metabolites, probably including bisphenol A radical by a reaction of radical oxygen in the oral environment and thus could occur in many biological systems other than dental resin and plastic containers [2]. Bisphenol A glycidyl methacrylate has been shown to act neither as activators nor inhibitors of CYP3A4 and CYP3A7, the major forms of carcinogen-activating enzymes found in human liver [3]. There is also a concern that the estrogenicity of bisphenol A may elicit toxicity to mammalian developmental and reproductive processes.

Accumulation of bisphenol A in male reproductive organs has certain clinical implications since exposure during fetal life has been shown to decrease the efficiency of sperm production in mice. There are some reports suggesting the testicular toxicity of bisphenol A in both rats and mice [4, 5]. However, neonatal treatment of bisphenol A at 2.2 mg/kg to 300 mg/kg caused no histopathological changes to male reproductive organs [6]. Previous studies have reported that low doses (0.2 µgkg^-1day^-1 to 20 µgkg^-1day^-1) of bisphenol A induced oxidative stress in epididymal sperm after 45 days of treatment [7] and in liver of rats after 30 days of treatment [8]. Other environmental contaminants such as lindane[9, 10], TCDD [11, 12], methoxychlor [13], nonylphenol [14], HCH [15], iron [16] have also been shown to induce oxidative stress in testis, epididymis and epididymal sperm of rats.

Vitamin C, an endogenous water-soluble natural antioxidant, protects the tissues from reactive oxygen species (ROS), and cyclophosphamide induced testicular oxidative stress [17]. Co-administration of ascorbic acid along with vitamin E has been shown to improve functional parameters of sperm such as semen volume, sperm motility, sperm count, sperm concentration and viability in rats [18]. In the present study a therapeutic dose of vitamin C was co-administered with 0.2 µg/kg to 20 µg/kg of bisphenol A to see if vitamin C could reverse the effect of bisphenol A-induced oxidative stress in epididymal sperm and epididymis of rats. The experiments done in this study comply with the current laws of India.

2 Materials and methods

Male rats of Wistar strain (45 days) were purchased from the Central Animal House, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India. Animals were housed in plastic cages under a well-regulated light and dark (12 h: 12 h) schedule at 24 3 and provided with standard commercial laboratory chow and tap water ad libitum.

Bisphenol A [2, 2-Di (4-hydroxyphenyl) propane] of 97 % purity was obtained from S.D. Fine-Chem, India. Malondialdehyde was obtained from Merck-Schuchardt, Germany. NADPH and glutathione oxidized were obtained from SISCO Research Laboratories, Mumbai, India. Horseradish peroxidase (HRP), bovine serum albumin (BSA), deoxyribonucleic acid (DNA), thiobarbituric acid and pyrogallol were obtained from Himedia Laboratories, Mumbai, India. All other chemicals were of analytical grade and obtained from local commercial sources.

Animals were divided into three groups of four rats each. Group I was administered orally with bisphenol A dissolved in olive oil at 0.2 µgkg^-1day^-1, 2 µgkg^-1day^-1 and 20 µgkg^-1day^-1 separately for 60 days. Group II received the similar doses of bisphenol A along with 40 mg of vitamin C for 60 days and Group III only received the vehicle and served as the control. The animals were fasted overnight and killed 24 h after the last treatment using an overdose of anesthetic ether.

The caput, corpus and cauda epididymidis from the rats were cleared from adhering tissues and fixed in Bouin's fixative for histological study. After dehydration in alcoholic series and cleaning in xylol, the tissues were embedded in paraffin wax and 5 µm sections were cut. The sections were stained with hematoxylin-eosin and examined under a light microscope. Photomicrographs were taken using Trinocular Research Microscope with automatic exposurometer (Nikon Optihot Model, Japan).

Sperm viability test was done by the method as described in the WHO Laboratory Manual [19]. Briefly, an aliquot of 100 µL epididymal sperm was mixed with 100 µL 0.5 % eosin solution on a microscopic slide covered with a cover slip and examined after 30 sec under light microscope at 200. Two hundred spermatozoa were counted and the percentage of unstained (viable) sperm was recorded.

Epididymal sperm motility was evaluated by the method as described by Linder et al. [20]. Briefly, cauda epididymal fluid was collected using a pipette tip and diluted to 2 mL with Ham's F-12 medium at 32 . Approximately 10 µL of the mixture was placed in a Neubauer hemocytometer and the percentage of motile sperm were counted.

Epididymal sperm were collected by slicing the epididymis in 5 mL of Ham's F-12 medium, and then incubated for 5 min at 32 . Spermatozoa were counted by the method as previously described [13]. Briefly, 5 µL of epididymal sperm were diluted with 95 µL of Ham's F-12 medium and approximately 10 µL of the diluted sperm suspension were transferred to a counting chamber. To prevent dehydration the sample was placed in a humid chamber. After standing for 5 min, the intact spermatozoa that settled down were counted under a light microscope at 200.

The epididymal sperm suspension was centrifuged at 225 g for 10 min at 4 and the pellet was resuspended in normal saline. The sperm were homogenized for ten seconds and centrifuged at 800 g for 10 min at 4 . The supernatant was used for biochemical studies. The epididymides were washed several times in cold saline to remove as far as possible the sperm attached to the tissue. A 1 % homogenate of cauda epididymis was prepared in cold saline. The homogenates were centrifuged at 800 g for 20 min at 4 and the supernatant used for various biochemical assays. Protein was estimated by the method of Lowry et al. [21] and DNA by the method of Burton [22]. Superoxide dismutase [23], glutathione peroxidase [24], and lipid peroxidation [25] were estimated both in epididymal sperm and in epididy-mis. Since there was a positive correlation (r = 0.90, n=24) between the sperm count and DNA contents in the epididymal sperm [14], the DNA content was routinely used as an indicator of sperm count and the results of biochemical estimations in epididymal sperm were expressed in terms of mg DNA.

Data were presented as meanSD. Statistical analyses were performed using a one-way analysis of variance (ANOVA) followed by unpaired Student's t-test. Differences were considered significant at P<0.05.

3 Results

Bisphenol A did not significantly change epididymal sperm viability, while sperm motility and count decreased in a dose-dependent manner compared with the corresponding control groups (Figure 1~3). Co-administration of bisphenol A and vitamin C did not significantly change the epididymal sperm motility and count (Figure 1~3).

Bisphenol A at all the dose levels caused a complete degeneration of the epithelia of the caput, corpus and cauda epididymidis (Figure 4).

Figure 4a. Photomicrograph showing histological changes in caput epididymis of rats after 60 days of bisphenol A treatment (50). A: Control; B-D: 0.2 µgkg^-1day^-1, 2 µgkg^-1day^-1 and 20 µgkg^-1day^-1, respectively. In bisphenol A-treated rats epididymal epithelium degeneration (~) can be seen. (L) denotes lumen of the epididymis without sperm.

Figure 4c. Photomicrograph showing histological changes in the cauda epididymis of rats after 60 days of bisphenol A treatment (50). A: Control; B-D: 0.2 µgkg^-1day^-1, 2 µgkg^-1day^-1 and 20 µgkg^-1day^-1, respectively. (~) denotes degeneration of epididymal epithelium, (h) indicates occlusion of epididymal epithelium, (L) denotes epididymal lumen of without sperm.

In bisphenol A-treated rats, the activities of superoxide dismutase and glutathione peroxidase decreased significantly (P<0.05) in epididymal sperm compared with the controls (Table 1) with a dose-dependent increase in the levels of lipid peroxidation in epididymal sperm (Table 1). Co-administration of bisphenol A and vitamin C did not alter the activities of antioxidant enzymes and the levels of lipid peroxidation in the epididymal sperm of rats (Table 1).

Table 1. Effect of bisphenol A and co-administration of bisphenol A and vitamin C on antioxidant enzymes and levels of lipid peroxidation in epididymal sperm of rats (n=4). ^bP<0.05,compared with the controls. dnmol pyrogallol oxidized/ min/mg DNA at 32 ; ^enmol NADPH oxidized/ min/mg DNA at 32 ; ^fmmol malondialdehyde produced/15 min/mg DNA at 32 .

The activities of superoxide dismutase and glutathione peroxidase decreased significantly (P<0.05) in the cauda epididymidis compared with the controls (Table 2). A dose-dependent increase in the levels of lipid peroxi-dation was observed in the epididymis (Table 2). Co-administration of bisphenol A and vitamin C did not alter the activities of antioxidant enzymes and the levels of lipid peroxidation in the epididymis (Table 2).

Table 2. Effect of bisphenol A and co-administration of bisphenol A and vitamin C on antioxidant enzymes and levels of lipid peroxidation in cauda epididymis (n=4). ^bP<0.05, compared with the controls. ^dnmol pyrogallol oxidized/ min/mg protein at 32 ; ^enmol NADPH oxidized/ min/ mg protein at 32 ; ^fµmol malondialdehyde produced/15 min/ mg protein at 32 .

Administration of bisphenol A at doses of 0.2 µg/kg, 2 µg/kg and 20 µg/kg decreased epididymal sperm count and sperm motility which may be due to increased lipid peroxidation [26]. Co-administration of bisphenol A and vitamin C could impart protective effect against bisphenol A-induced toxicity on the epididymal sperm motility and sperm count.

In the present study bisphenol A induced a complete degeneration of epididymal epithelium with reduction in the number of spermatozoa in the caput, corpus and cauda epididymidis, which may be due to a decrease in serum testosterone levels or a reduction in 5a-reductase activity in the epididymis.

The epididymis and spermatozoa are highly rich in polyunsaturated fatty acids and thus susceptible to damages induced by ROS. To counteract the effects of ROS, the epididymis and spermatozoa are equipped with antioxidant defence systems, namely, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Increased lipid peroxidation may indicate an increased generation of free oxygen radicals in the epididymis, which has been associated with sperm mid-piece abnormalities and count decline [26].

Lipid peroxidation is highly toxic to spermatozoa and causes an irreversible arrest of sperm motility, damage of sperm integrity and other sperm functions [27]. Oxidative destruction of polyunsaturated fatty acids has also been shown to be extremely damaging to epididymal membranes since it proceeds as a self-perpetuating chain reaction [28]. Incubation of ascorbic acid with the environmental contaminant, methoxychlor, protects epididymal sperm of goats from oxidative damage [29]. In the present study co-administration of vitamin C reversed the oxidative stress induced by bisphenol A in epididymal sperm and epididymis of rats.

The authors thank the staff of Bioinformatics Centre, Pondicherry University, Pondicherry for providing various facilities. KCC acknowledges the Lady Tata Memorial Trust, Mumbai, India for a Junior Scholarship. PPM acknowledges the receipt of financial support from the Population Council, New York, USA (Grant Nos. B99.047P-9/ ICMC and B99.048 R/ ICMC).

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Correspondence to: Dr. P. P. Mathur, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India.
Tel: +91-413-265 5212, Fax: +91-413-265 5211
E-mail: ppmathur@hotmail.com
Received 2003-06-27   Accepted 2003-07-28

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