IN VITRO AND IN VIVO TOXICOLOGICAL EFFECTS OF SODIUM ARSENITE ON PREPUBERTAL AND ADULT MALE MICE REPRODUCTIVE SYSTEM

Abstract

The metal element namely “Arsenic” is abundantly found on earth. It exists in several trivalent and pentavalent oxidation states like -3, +3 and +5. In both human body and atmosphere, it occurs as inorganic and organic compounds. Sodium arsenite (NaAsO2) is considered more toxic and biologically active than sodium arsenate. Arsenic is a renowned carcinogen an an integral part of around 245 minerals. Its has also been used in industrial, agricultural, and medicinal fields. Arsenic accumulates preferentially in the skin, hair and nails of mammals. Drinking water is a common source through which living organisms are exposed to arsenic. Arsenic levels < 10 µg/L are declared safe for drinking purposes by the World Health Organization (WHO) whereas, the maximum contamination levels are 50 µg/L. In recent years, arsenic contamination through drinking water has immensely increased in the sub-continent, especially in Pakistan with some most populated cities having arsenic concentration up to 200 ug/L. Adult male reproductive toxicity caused by arsenic has been extensively reported in adult males, however no data are present as regards to the in vitro gonadotoxicity due to NaAsO2 in prepubertal and adult male mice. Therefore, the present study was designed to (i) evaluate possible in vitro toxic effects of NaAsO2 in prepubertal and adult male mice reproductive organs (ii) and to assess the in vivo reproductive organs development and function after sub-chronic and chronic exposures of NaAsO2. Three experiments (six replicates for each) were conducted using post-natal day (PND) 5 Balb-c mice testis in an in vitro culture system. Prepubertal (infantile) mice testis pieces approximately 1 mm3 were placed on nucleopore membrane, floating on 1ml of α-MEM and 10% KSR. Testicular cultures were incubated at 34°C in a 5% CO2 incubator. Cultures were left untreated for 24 h (Day1). Media were changed after 24 h to provide fresh nutrients to the growing culture. In the first experiment, cultures were treated with 0.01, 0.05, 0.1, 0.5, and 1 µM NaAsO2 exposure for 1 day. Whereas, in second (0.1, 0.5, and 1 µM) and third experiment (10, 50, and 100 µM) NaAsO2 concentrations were administered for six days. Cultures were fixed on day seven and proceeded for hematoxylin and eosin staining and double immunohistochemistry, Cleaved caspase-3 (CC3) and mouse vasa homologue (MVH) for determining apoptosis and germ cells, respectively. Adult mice testicular fragments were incubated at 37°C, while the caudal part of epididymes were incubated at 33°C, each for 2 and 24 h, with NaAsO2 at 1, 10, 50, and 100 µM concentrations. Levels of Reactive oxygen species (ROS), TBARS, antioxidant enzymes, testosterone concentrations, and the extent of sperm DNA damage were estimated. Sperms were incubated with Human tubal fluid at 37°C in 5% CO2 incubator, and sperm motility, viability, and morphology were assessed after 2 h. For evaluating the reproductive toxicity of arsenic in vivo following the chronic and sub-chronic exposure, NaAsO2 (0.01, 5, and 10 mg/L) was orally administered in male mice from the PND 25 to PND 53 and 114. Histological, hormonal, sperm parameters, oxidative stress, antioxidant enzymes, and DNA damage were assessed. Results demonstrated that in vitro exposure of prepubertal (infantile) testicular cultures to NaAsO2 for 1 and 6 days with 1µM NaAsO2 as the highest dose in experiment 1 and 2 respectively, did not cause any change in the testicular morphology, germ cell density, or CC3 expression. However, exposure of prepubertal (infantile) testis to arsenic concentrations in the third experiment (10, 50, and 100 µM NaAsO2) induced pronounced changes, whereby germ cell number revealed reduction in a dose dependent manner, while CC3 expression increased a hundred-fold after 50 µM arsenic exposure. Two hours incubation of adult mice testicular and epididymal tissues with NaAsO2 demonstrated significant increases in the ROS, lipid peroxidation, and sperm DNA damage. However, decreases occurred in catalase, peroxidase, and superoxide dismutase levels at 50 and 100 µM concentrations. Whereas, following 24 h incubation, groups treated with 10, 50 and 100 µM NaAsO2 concentrations, revealed significantly greater toxic effects by elevating oxidative stress markers and sperm DNA damage while decreasing levels of antioxidant enzymes, As for testosterone concentration, a non-significant decrease occurred at 2 h of incubation while significant reduction was noticeable after 24 h of incubation with NaAsO2. Low dose of 1 µM NaAsO2 remained unable to induce any toxic effects. In vitro sperm motility and viability were substantially reduced with increasing concentrations of NaAsO2. Similarly, sperm morphology was affected at higher tested doses (50, and 100 µM NaAsO2). On the other hand, in vivo sub-chronic (PND 25-53) NaAsO2 exposure at high doses (5 and 10 mg/L) produced significant decrease in the antioxidant enzymes, sperm parameters (sperm count, viability, and motility), and hormones (LH, FSH, testosterone), while ROS and TBARS levels were substantially increased. Morphological changes in the testis as well as sperm DNA damage were evident only at 10 mg/L dose. In contrast, in vivo chronic exposure (PND 25-114) of NaAsO2 even at low dose (0.01 mg/L) induced severe harmful effects on mouse reproductive organs. Considerable changes in mouse testis, and body weight, as well as morphological changes in testis and epididymes were noticed in arsenite exposed mouse. Testicular oxidative stress was induced along with reduction in hormonal, and sperm parameters. Sperm DNA damage was observed at 5 and 10 mg/L arsenic concentrations. In vivo experiment results showed that higher doses of NaAsO2, after both sub-chronic and chronic exposure, initiated adverse effects on mouse reproductive system. Overall, the present study concludes that in vitro and in vivo NaAsO2 treatment induces both time and dose-dependent toxic effects on prepubertal and adult male mouse reproductive organs.