Authored by Christopher Edet Ekpenyong*
Abstract
Background: Biological factors affecting the therapeutic doses of ascorbic acid (AA) against xenobiotic-induced oxidative stress (OS) and reproductive toxicity have been established, however, the effect of gender is yet to be thoroughly researched and ascertained. The present study aimed to assess gender disparities in the effect of AA against gasoline vapor (GV)-induced reproductive toxicity in rats.
Methods: Thirty-five matured male and female Wistar Albino rats weighing between 200 and 250g were divided into 5 groups (n=7per group). Group 1 served as unexposed control, groups 2, 3, 4, and 5 were exposed to GV for 6 weeks. Groups 3, 4, and 5 in addition to being exposed to GV were treated with low, medium, and high doses of AA for 2 weeks of the 6 weeks of exposure and treatment. Animals were sacrificed and blood samples and reproductive organs were obtained for analysis and histopathological examination respectively.
Results: Exposure to GV alone significantly P<0.05 decreased serum estrogen, progesterone, and testosterone levels. Serum levels of estrogen and progesterone were significantly (P<0.05) higher in the low-dose AA-treated female animals, whereas the highest serum level of testosterone was found in the high-dose AA treated male animals. A corresponding significant decrease in serum FSH and LH levels were also found in the low and high doses of AA treated female and male groups respectively.
Conclusion: There is a gender difference in the effect of AA against GV-induced OS and reproductive toxicity. Therefore, gender-related dose adjustment should be considered when using AA to manage OS-related male or female reproductive disorders.
Keywords:Petroleum fume; Vitamin C; Gender; Oxidative stress; Reproduction; Toxicity
Introduction
Gasoline consists of several hydrocarbons and additives that constitute significant environmental pollutants. Exposure to hydrocarbon fumes by humans is common and widespread due to the extensive domestic and industrial applications. Exposure by humans can be through dermal, inhalation, or ingestion routes, with inhalation being the most common exposure route and can occur at any point along the production and distribution chain. Many people are exposed to petroleum fumes daily, especially those whose residence or workplace is close to petrochemical industries, refineries, oil fields, gasoline refueling stations, trafficcongested areas and gasoline combustion stations. About 100 million people are exposed to hydrocarbon constituents of gasoline per week when refueling at a self-service gasoline station [1]. Also, gasoline service station attendants are at a higher risk of exposure to gasoline inhalation for many hours a week and about 8086 minutes per year. Available data indicate that >3.6 billion gallons of gasoline vaporize into the air as gasoline vapor (GV) [2]. Following inhalation, GV is absorbed and distributed in the body. Within the body, it undergoes further toxicokinetic processes leading to the generation of reactive oxygen species (ROS), oxidative stress (OS), Gasoline consists of several hydrocarbons and additives that constitute significant environmental pollutants. Exposure to hydrocarbon fumes by humans is common and widespread due to the extensive domestic and industrial applications. Exposure by humans can be through dermal, inhalation, or ingestion routes, with inhalation being the most common exposure route and can occur at any point along the production and distribution chain. Many people are exposed to petroleum fumes daily, especially those whose residence or workplace is close to petrochemical industries, refineries, oil fields, gasoline refueling stations, trafficcongested areas and gasoline combustion stations. About 100 million people are exposed to hydrocarbon constituents of gasoline per week when refueling at a self-service gasoline station [1]. Also, gasoline service station attendants are at a higher risk of exposure to gasoline inhalation for many hours a week and about 8086 minutes per year. Available data indicate that >3.6 billion gallons of gasoline vaporize into the air as gasoline vapor (GV) [2]. Following inhalation, GV is absorbed and distributed in the body. Within the body, it undergoes further toxicokinetic processes leading to the generation of reactive oxygen species (ROS), oxidative stress (OS),
Materials and Methods
Experimental Animals
Thirty-five mature Wistar Albino rats weighing between 200 and 250g were obtained from the animal house of the Department of Pharmacology, Faculty of Pharmacy, University of Uyo, Akwa Ibom State, Nigeria. They were kept in well-ventilated cages for 7 days to acclimatize. They were allowed access to food and water ad libitum. All animals were fed rat chow (Vital Feeds, Grand Cereal Ltd, Jos).
Segregation of Animals
The animals were randomly divided into 5 groups (n=7 per group). They were exposed to GV in the exposure chambers (60 x 80x 100cm3)
Group 1 served as unexposed control and was orally gavaged 2ml of normal saline for 6wks.
Group 2 was exposed to GV alone for 6wks and maintained on normal animal feed.
Group 3 was exposed to GV for 6wks, fed a normal diet, and orally gavaged 100mg/kg of AA for 2 wks of the 6wks.
Group 4 was exposed to GV for 6wks, fed a normal diet, and orally gavaged 200mg/kg of AA for 2 wks of the 6wks.
Group 5 was exposed to GV for 6wks, fed a normal diet, and orally gavaged 300mg/kg of AA for 2 wks of the 6wks.
Collection of experimental samples for analysis
After 2 weeks of AA administration, the animals were weighed and anesthetized with chloroform soaked in a swab of cotton wool in a desiccator. The blood sample was collected by cardiac puncture and emptied into labeled specimen bottles, for biochemical evaluation including determination of estrogen, progesterone, follicle-stimulating hormone (FSH), Luteinizing hormone (LH), testosterone level, catalase (CAT), and malondialdehyde (MDA) levels. Animals were sacrificed by cervical dislocation and reproductive organs testis and ovary were carefully removed and fixed in a suitably treated formalin reagent and thereafter, subjected to normal routine histological procedures/examination.
Biochemical Analysis
Estimation of CAT and MDA activities
CAT activity was determined by the Titrimetric method. Tissue lipid peroxidation was quantified by estimating the plasma concentration of MDA using the thiobarbiturate acid reactive substance (TBARS) method and measured spectrophotometrically at 532nm. Serum estrogen, progesterone, testosterone, LH, and FSH levels were determined by Enzyme-Linked Immuno-sorbent assay (ELISA) as described by Tietz [7].
Histopathological tissue processing
The fixed tissues were dehydrated in different grades of alcohol as follows; two changes of 70% and 95% alcohol for a period of 2hrs each, two changes of 100% also known as absolute alcohol for a period of 2hrs. Dehydrated tissues were cleared using xylene. Tissues were impregnated with two changes of paraffin wax in the oven at the temperature of 60°C for 1hr 30mins) each to ensure they were fully embedded. Tissues were transferred from the final wax bath to molds filled with molten wax, allowed to solidify and thereafter, properly oriented for sectioning. The paraffin block was sectioned at 5μm after cooling the surface of the tissues with an ice bar. Ribbons were gently picked with Carmel brush and dropped in a water bath containing water at 60°C to enable ribbons float, expand and flatten out. Slides were rubbed with thymol containing egg albumen and gently dipped into the bath to pick up the flattened out tissue ribbons [8]. Haematoxylin and Eosin (H&E) staining techniques [8] were applied in staining the tissue sections.
Haematoxylin and eosin staining procedures
Tissue sections were deparaffinized in two changes of xylene and hydrated through graded series of alcohols in descending order and were rinsed in water and stained with Haematoxylin for 10mins. Tissue sections were rinsed and differentiated in one percent (1%) acidic alcohol and blued in running water using saturated lithium carbonate solution until sections appear sky blue. The blued section was counterstained in the Eosin solution for 3mins. Tissues were washed in water and dehydrated in ascending grades of alcohol, cleared in xylene, and mounted in DPX covered with coverslips and observed under the microscope.
Microscopy
Processed slides were viewed under a light microscope at magnification (X400), and photomicrographs obtained were linked to the computer using the microscope’s camera.
Statistical Analysis
Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS), version 20.0, and M. S. Excel. The one-way analysis of variance (ANOVA) and posthoc Tukey least significant difference (LSD) test was used to analyze the data and to determine the significance respectively. Data are expressed as Mean + Standard Error of Mean (S.E.M) and tables were used to illustrate the variations in the numerical values across the experimental groups. The P. values <0.05 were considered statistically significant.
Results
Antioxidant activity
Serum MDA increased significantly in GV alone group and decreased significantly (P<0.05) in a dose-independent manner in both male and female animals. In female animals, serum MDA significantly (P<0.05) increased in GV alone group, and significantly (P<0.05) decreased in GV plus low dose AA compared to the gasoline alone, group. Interestingly, serum CAT decreased significantly (P<0.05) in GV plus a medium and high dose of AA compared with the GV alone group (Figures 1-4).
Serum estradiol level
Means serum level of estradiol significantly (P<0.05) decreased in the GV-alone group compared with the control group. Estradiol significantly increased in GV plus low, medium, and high doses compared with the GV-alone group. The highest increment occurred in the group treated with low-dose of AA (Figure 5).
Serum follicle-stimulating hormones (FSH) level
Exposure of the female animals to GV-alone caused a significantly (P<0.05) decreased in serum FSH compared with normal control, whereas in male animals exposed to GV had a nonsignificant (P<0.05) changes in serum FSH compared with normal control. Also, the highest ameliorative effect of AA was obtained from the highest dose in female whereas, in the male animals, it was the medium dose that produced the highest ameliorative effect (Figure 6,7).
Serum luteinizing hormone (LH) level
In female animals, there was no significant (P>0.05) difference between the serum level of LH between the GV alone and the normal control. In the male animals, a significant (P<0.05) decrease in serum LH compared with the normal control was observed. In both sexes, medium doses of AA produced the highest protective effect on serum LH (Figure 8,9).
Serum progesterone level
Exposure to GV significantly (P<0.05) decreased in serum progesterone low dose of AA produced a significantly increased in serum progesterone level compared with normal control. Medium and high doses caused a significant (P<0.05) decrease in serum progesterone (Figure 10).
Exposure to GV caused a non-significant decrease in serum testosterone levels compared to levels in the normal control group. The highest dose of AA produced the highest ameliorative effect on the GV-induced decrease in serum testosterone levels (Figure 11).
Figures 12 to 26 show the changes in the histomorphology of the ovaries (Figures 12-16), testis (Figures 17-21), and epididymis (Figures 22-25) following the exposure to GV, and concomitantly treated with AA. Similar to the changes in the biochemical markers of ovarian and testicular endpoints, a greater improvement in the ovarian histomorphology was found in the low dose AA-treated group, whereas the testicular and epididymal histomorphology showed greater improvement in the high dose AA-treated groups (Figure 12-26).
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