|Year : 2014 | Volume
| Issue : 1 | Page : 5-8
Harmful effects of ascorbic acid and α-tocopherol on male reproductive organs of rats chronically exposed to sodium nitrate
Isyaku U. Yarube1, Joseph O. Ayo2, Muhammad Y. Fatihu3
1 Department of Physiology, Faculty of Medicine, Bayero University, Kano, Nigeria
2 Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
3 Department of Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
|Date of Web Publication||15-May-2014|
Dr. Isyaku U. Yarube
P. O. Box 492, Kano
Source of Support: None, Conflict of Interest: None
Background: Humans and animals are exposed to sodium nitrate through the air, food and drinking water. Because nitrates induce oxidative stress, the effects of antioxidant vitamins ascorbic acid and α-tocopherol on oxidative damage induced by sodium nitrate on reproductive organs of male Wistar rats were studied.
Methodology: Five rats each in four groups were treated for 60 days. They received distilled water (Group I), 3 mg/100 g body weight NaNO 3 (Group II), 3 mg/100 g body weight NaNO 3 + 50 mg/100 g body weight ascorbic acid (Group III) and 3 mg/100 g body weight NaNO 3 + 30 mg/100 g body weight α-tocopherol (Group IV). Live weights of the animals were obtained at the beginning of the experiment and every 2 weeks subsequently. At the end of the study, the left testis, epididymis and seminal vesicle of each rat was carefully removed and weighed. These organs were also examined for histopathological lesions.
Results: Testis:body weight ratio and seminal vesicle:body weight ratio of Group II animals were less than (P < 0.05) those of the control. Epididymis:body weight and seminal vesicle:body weight ratios of Group III animals were significantly lower than those of Group II. Testis:body weight ratio of Group IV animals was significantly less than that of Group II. There was testicular degeneration with absence of spermatids and spermatogenic cells in the semeniferous tubules and absence of epididymal sperm reserve in Group IV animals.
Conclusions: The results indicate that intake of ascorbic acid and α-tocopherol may enhance oxidative damage in the testis, epididymis and seminal vesicle during chronic sodium nitrate exposure.
Keywords: Ascorbic acid, male reproductive organs, oxidative stress, rats, sodium nitrate, α-tocopherol
|How to cite this article:|
Yarube IU, Ayo JO, Fatihu MY. Harmful effects of ascorbic acid and α-tocopherol on male reproductive organs of rats chronically exposed to sodium nitrate. J Med Trop 2014;16:5-8
|How to cite this URL:|
Yarube IU, Ayo JO, Fatihu MY. Harmful effects of ascorbic acid and α-tocopherol on male reproductive organs of rats chronically exposed to sodium nitrate. J Med Trop [serial online] 2014 [cited 2020 Jan 17];16:5-8. Available from: http://www.jmedtropics.org/text.asp?2014/16/1/5/132567
| Introduction|| |
Humans and animals are inevitably exposed to sodium nitrate due to its widespread presence in the air,  food and drinking water,  as well as usage in agriculture,  medicine  and industry.  Nitrates are known to induce oxidative stress through the release of nitrite and nitric oxide (NO),  as well as blockage of cytochrome oxidase due to inhibition of electron transport in the mitochondrial respiratory chain.  Oxidative stress is involved in a vast array of physiological and pathological processes. ,,,, Antioxidant vitamins, ascorbic acid (vitamin C) and α-tocopherol (vitamin E) have been shown to modify the effects of oxidant substances in various biological systems. , They possess anti-oxidant ,,, as well as pro-oxidant activities. 
The aim of this study was to examine the effects of ascorbic acid and α-tocopherol on sodium nitrate-induced oxidative stress on the testis, epididymis and seminal vesicles of adult male Wistar rats.
| Methodology|| |
A total of 20 adult male Wistar rats aged between 4.5 and 7 months and weighing 134-199 g, were used for the study. They were kept in large cages and given access to distilled water and Pelletized Growers Feed™ (Vital Feeds, Jos, Nigeria) ad libitum.
Preparation of Administered Substances
50 g of sodium nitrate (BDH Chemicals Limited, Poole, England) was dissolved in 2 L of distilled water to make a stock solution containing 25 mg/ml NaNO 3 , from which the animals were fed during the experiment. Five hundred tablets (50,000 mg) of vitamin C (Em-vit C™ 100 mg tablets) (Emzor Pharmaceutical Industries, Lagos, Nigeria) were crushed into powder and dissolved in distilled water to prepare a solution containing 250 mg/ml of ascorbic acid. Similarly, 85 capsules of vitamin E (Efishal 200™, Shalina Laboratories, Pvt, Mumbai, India) were cut open and emptied into a clean container. Vegetable oil was added up to 50 ml to prepare a suspension containing 340 mg/ml of the α-tocopherol. The solutions were kept at room temperature and α-tocopherol was protected from direct contact with air and sunlight to avoid degradation, by stocking in a dark and air-tight jar. Appropriate amounts of NaNO 3 solution and the vitamins were collected using 1 ml syringe for administration.
The animals were divided into four groups of five rats each. Drugs (or distilled water) were administered orally using a metallic cannula at 8.00 am daily for 8 weeks. Group I (Control) received distilled water. Group II (NaNO 3 -treated group) received 3 mg/100 g body weight NaNO 3 .  Group III (NaNO 3 + ascorbic acid-treated group) received 3 mg/100 g body weight NaNO 3 + 50 mg/100 g body weight ascorbic acid.  Group IV (NaNO 3 + α-tocopherol-treated group) received 3 mg/100 g body weight NaNO 3 + 30 mg/100 g body weight α-tocopherol. 
Samples and Data Collection
Live weights of the animals were obtained using a triple beam balance (Model OHAUS, 700 Series, Floram Park, NJ, U. S. A) at the beginning of the experiment and every 2 weeks subsequently. Percentage weight gain for each group was determined from mean body weight of that group at the beginning and end of the experiment. Similarly, the ratio of weight of organs (testis, epididymis and seminal vesicle) to live weight of animal before sacrifice was determined.
At the termination of the experiment, the animals were weighed, euthanized by chloroform inhalation in a closed chamber and subsequently sacrificed. The left testis, left epididymis and left seminal vesicle of each rat were carefully removed and immediately dried with blotting paper and weighed using Mettler H80 balance (England). One animal from each group was selected at random for histopathological examination. Testis, epididymis and seminal vesicle were removed from each animal and fixed in 10% neutral buffered formalin. Tissue slides were prepared by paraffin embedding and read using HM - Lux microscope (Germany) at the magnification of ×200.
Descriptive statistics and t-test of independent samples using SPSS version 15.0 (SPSS, Chicago, IL, USA) was used to process the data obtained, which were expressed as mean ± standard error of the mean. P values < 0.05 were considered to be significant.
| Results and Discussion|| |
Effects on Body Weight Gain
Group I had an initial and final weight of 134.1 ± 42.7 g and 195.5 ± 27.7 g, respectively, equivalent to 46% weight gain [Figure 1]. Initial, final and percentage weight gains for Group II was 148.2 ± 8.3 g, 213.8 ± 43.4 g and 44%, respectively. Group III had 172.3 ± 13.6 g, 228.4 ± 19.9 g and 33%; whereas Group IV had 199.1 ± 16.8 g, 219.7 ± 25.5 g and 10%, respectively. Within the 2 months period of the study, weight gain was recorded in both the control and the experimental groups [Figure 1]. Carlström et al.,  reported that dietary nitrates reduced weight gain through release of NO. In the current study, percentage weight gain in the nitrate-treated group was less than that of the control, though the difference was not statistically significant. The ascorbic acid and α-tocopherol-treated groups recorded less weight gain (α-tocopherol gained less than ascorbic acid group) when compared with the nitrate and control groups. This was, apparently, because the two groups had higher initial weight (α-tocopherol group more than ascorbic acid), hence less potential for further gain. However, this result is consistent with the finding that α-tocopherol induced weight loss in normal and diabetic rats. 
|Figure 1: Change of weight of the animals during the experiment. Values are expressed as mean ± standard error of the mean; n = number of animals per group|
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Effects on Organ: Body Weight Ratio
The nitrate-treated animals had significantly lower weights of testis and seminal vesicle [Figure 2]. This suggests that treatment with sodium nitrate significantly reduced testicular and seminal vesicle weight, apparently due to its ability to induce oxidative stress through increase levels of NO and peroxynitrite (ONOO−). ,
|Figure 2: Organ:body weight ratios of animals at the termination of the experiment. Values are expressed as mean ± standard error of the mean; n = number of animals in a group aP < 0.05 compared to Group I, bP < 0.05 compared to Group II|
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Testicular weight of the α-tocopherol-treated animals was significantly less than that of the control and nitrate-treated animals [Figure 2], indicating that α-tocopherol enhanced the testicular weight loss caused by sodium nitrate in these rats. Sahinturk et al. (2007)  reported that α-tocopherol and ascorbic acid co-treatment showed only partial protective effects on testicular weight and histology in rats that received ethane diamethane sulfonate. Again, α-tocopherol did not prevent testicular weight loss after 7 days as it did after 3 days, indicating the heterogeneous effects of α-tocopherol. This is consistent with other findings that antioxidant vitamins cause beneficial and harmful effects in different conditions. ,,, This result, however, contradicts previous findings that α-tocopherol reversed the toxic effects of oxidant substances. ,
Similarly, the weights of testes, epididymis and seminal vesicle of the ascorbic acid-treated animals were significantly lower than those of the nitrate-treated rats [Figure 2]. This indicates that ascorbic acid enhanced the effects of sodium nitrate on these organs. The pro-oxidant effects of ascorbic acid had been demonstrated. , Hemilä and Kaprio, , have suggested that the effects of ascorbic acid and α-tocopherol in human subjects can be beneficial as well as deleterious, depending on many biological factors, such as duration of exposure, age, physical exercise, body mass index. Further research is, therefore, needed not only to define the appropriate doses of the vitamins, but also to ascertain the characteristics of individuals who may benefit from their administration.
Histopathological examination showed testicular degeneration with absence of spermatids and spermatogenic cells in semeniferous tubules [Plate 1] and absence of epididymal sperm reserve [Plate 2], in α-tocopherol-treated rats. This is consistent with organ weight reduction observed in this study. No significant lesions in the nitrate and ascorbic acid treated groups and is in line with findings elsewhere. ,
A limitation of this study is the fact that the total antioxidant capacity, which could serve to confirm at the molecular level the involvement and the amount of oxidative stress as the major mechanism of the observed systemic effects, was not determined. However, there is sufficient evidence in the literature supporting the induction of oxidative stress by nitrates,  as well as the anti-oxidant and pro-oxidant effects of ascorbic acid and α-tocopherol. ,, Nitrates, through the release of nitrites and NO may be involved in many physiological and pathological processes in the body and this represents an avenue for further researches.
| Conclusion|| |
Sodium nitrate administration negatively affected the testes, epididymis and seminal vesicle by reducing the organ:body weight ratios. Ascorbic acid and α-tocopherol enhanced the negative effects of sodium nitrate on these organs, apparently through their pro-oxidant properties. It is recommended that the vitamins be avoided during prolonged exposure to sodium nitrate by fertilizer factory workers and farmers.
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[Figure 1], [Figure 2]