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Advances in Environmental Biology, 7(14) December 2013, Pages: 4823-4827
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Advances in Environmental Biology
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Investigating Effect of Vitamins E and C on Sexual Cell Lineages in Adult Male Rats
under Oxidative Stress due to Sulfasalazine
1,2
Keshavarz Mojtaba, 1,3Kargar Jahromi Hossein, 4Khodaparast Zahra, 5Bathaee Seyed Hamid, 6Saberi
Roza, 7Azhdari Sara, 7Farzam Mohammad
1
Jahrom University of Medical Sciences, Jahrom, Iran.
Young Researchers Club Elite, Shiraz Branch, Islamic Azad University, Shiraz, Iran.
3
Young Researchers Club Elite, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
4
Developmental Biology, Islamic Azad University, Arsanjan, Fars, Iran
5
Institution of Supreme Education and Industry of Maragheh, Iran.
6
Department of Biology, Islamic Azad University, Jahrom, Fars, Iran.
7
Department of Anatomy and Embryology, International Branch, Shiraz University, Shiraz, Iran.
2
ARTICLE INFO
Article history:
Received 28 October 2013
Received in revised form 24
January 2014
Accepted 28 January 2014
Available online 25 February 2014
Key words:
Sulfasalazine, Vitamins E Vitamins
EC, Rat, Spermatogenesis, Oxidative
Stress.
ABSTRACT
Background & Objective: Sulfasalazine is a drug used for treatment of inflammatory
diseases such as inflammatory bowel disease and is prescribed long term for patients.
Consumption of this drug causes a range of side effects such as creating sterility in
men. Therefore, in this study the effect of vitamins E and C in the prevention of the
effects of sulfasalazine on spermatogenesis of male rats was studied. Materials &
methods: Adult male rats (150-200 gr), Wistar race, were divided into 5 groups of 8
(control group and four experimental groups). Rats in experimental groups 1, 2, 3 and 4
received sulfasalazine (600 mg/kg) daily for 14 consecutive days via gavage. Also,
during this period rats of experimental group 2 in addition to sulfasalazine received
vitamin C (20 mg/kg) daily, experimental group 3 received vitamin E (200 mg/kg)
daily, and experimental group 4 received vitamin C (20 mg/kg) plus vitamin E (200
mg/kg daily) via gavage. Rats of control group just received regular food and water. At
the end of the period, counting sexual cells was performed by light microscopy on testis
sections. At the end of experiment, the results were analyzed statistically by ANOVA
test and Duncan's test, P≤0.05 was considered significan t. Results: Results show that
sulfasalazine consumption has an effect on number of sex cells and created significant
reduction in the P≤0.05 level, and the combination of vitamins E and C prevents this
reduction. Discussion: Results of this study suggest that sulfasalazine with increased
oxidative stress causes impaired spermatogenesis, and vitamins E and C by decreasing
oxidative stress prevents the damaging effects of sulfasalazine on spermatogenesis.
© 2013 AENSI Publisher All rights reserved.
To Cite This Article: Keshavarz Mojtaba, Kargar Jahromi Hossein, Khodaparast Zahra, Bathaee Seyed Hamid, Saberi Roza, Azhdari Sara,
Farzam Mohammad., Investigating Effect of Vitamins E and C on Sexual Cell Lineages in Adult Male Rats under Oxidative Stress due to
Sulfasalazine. Adv. Environ. Biol., 7(14), 4823-4827, 2013
INTRODUCTION
Investigating the causes of infertility in men and how to prevent the creation of infertility in a community is
a valuable aid to patient who is suffering infertility. Sulfasalazine is a drug used for the treatment of
inflammatory bowel disease and is also used for treating diseases such as ulcerative colitis, Crohn’s disease and
also rheumatoid arthritis. These diseases are chronic and relapsing and patients should use this medication their
entire life which will lead to dangerous side effects, one of which is sterility in men. Construction of
sulfasalazine is composed of two parts, sulfa pyridine and 5-aminosalicylate which are connected by a bond.
Active part of the medicine is 5-aminosalicylate and side effects of medicine are related to sulfa pyridine [1].
Several studies have shown that taking sulfasalazine may cause sterility in men. For the first time Liu and
colleagues showed this effect in a patient with ulcerative colitis [2]. In another study Tat and colleagues showed
abnormal sperm in the semen of patients treated with sulfasalazine [3]. Fukushima and colleagues examined the
mechanism of sterility induced by sulfasalazine [4]. They showed that sperm motility and acrosome reaction are
important for fertilization and will be reduced by sulfasalazine. This indicates sulfasalazine's effect is in
spermatid and epididymal stage. In a recent study it has been shown that sulfasalazine decreases the number of
spermatids and percentage of motile cells in testicular. Moreover, it has been shown that abnormalities exist in
Corresponding Author: Hossein Kargar Jahromi, Jahrom University of Medical Sciences, Jahrom, Iran.
Tel: +989361554955 E-mail: [email protected]
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Hossein Kargar Jahromi et al, 2013
Advances in Environmental Biology, 7(14) December 2013, Pages: 4823-4827
sperm morphology, like sperm without tails at dose of 600 mg per day. The mechanism of sulfasalazine's effect
on infertilityhas not been identified. Several mechanisms have been proposed regarding the effect of
sulfasalazine on spermatogenesis. One mechanism that has been proposed is induced oxidative stress;
medications that cause the production of reactive oxygen species may play an important role in reducing the
quality and quantity of the semen [4]. Active oxygen causes lipid peroxidation in fatty acids of the cell
membrane and so it increases cell membrane permeability which causes immobility and sperm death [5,6]. In a
recent study, it has been shown that sulfasalazine decreases the body’s antioxidant defense enzymes such as
superoxide dismutase and glutathione reductase [4]. Due to chronic use of sulfasalazine and its effect on
sterility, the use of materials that can help prevent side effects of the drug could be one way to solve this
problem. Due to increased oxidative stress by sulfasalazine, it appears that antioxidant compounds can be
effective in relieving the side effects of the drug. Vitamin E or alpha-tocopherol are antioxidant compounds [7],
vitamin E is a fat-soluble vitamin that contains detergent free radicals. One tocopherol molecule breaking
antioxidant molecule chains can inhibit two lipid proxils radical, and so inhibit two chains reaction of
proxidation [8]. Vitamin C is an antioxidant compound which is soluble in water and it has a role in
neutralization of free radicals and removing oxidative stress. In addition to cleaning free radicals, it causes other
antioxidants such as vitamin E and to enter the cycle [9]. Regarding the subject of this study, the goal of this
research is to investigate the protective effect of vitamin E and C on the effects of sulfasalazine on
spermatogenesis in laboratory rats.
MATERIALS AND METHODS
In the present experimental study, 40 adult male Wistar rats weighting 150 to 200 gr were used. Animals
were maintained in Jahrom Animal house in the Jahrom Medical Sciences University in standard conditions (12
hours light/12 hours dark and at temperature 2 ± 22°C). The animals were randomly divided into 5 groups as
follows:
The Control group (C): did not receive any medicine and maintenance and nutritional conditions were
similar to other groups.
The First experimental group (E1): received sulfasalazine at the dose of 600 mg per kg of body weight by
gavage for 14 days. Dose selection was based on drug consumption dose and also previous studies on the effects
of induced sterility oxidative stress.
The Second experimental group (E2): In addition to sulfasalazine at a dose of 600 mg per kg of body weight
per day received vitamin C at a dose of 20 mg per kg of body weight by gavage 15 minutes prior to receiving
sulfasalazine.
The Third experimental group (E3): Once daily received sulfasalazine at a dose of 600 mg per kg of body
weight, plus vitamin E at a dose of 200 mg per kg of body weight by gavage 15 minutes prior to receiving
sulfasalazine.
The Fourth experimental group (E4): Received sulfasalazine at a dose of 600 mg per kg of body weight per
day plus vitamin C at a dose of 20 mg per kg of body weight and 200 mg of vitamin E in the form of gavage.
This group was given vitamins E and C 15 minutes before receiving sulfasalazine. It should be noted that during
the study, all animals were cared for according to the ethical principles of the animal protection law (SPCA)
passed in 2006 in USA (10 , 2). Fifteenth days after the start of the study, in sterile conditions, the left and right
testes were removed by creating a gap in the lower abdomen, and the left testis was placed in Bouin’s fixative
solution for histological examination.
Review of histological testis:
The left testis was removed from Bouin’s fixative solution and tissue molding with paraffin and by
microtome cut sections with a thickness of 5 microns were produced and stained with hematoxylin-eosin
method. Stained slides were assessed by light microscope by a person who had no knowledge of the category
and with the following characteristics: Number of spermatogonia cells, primary spermatocytes, sertoli, leydig,
spermatids, and spermatozoa. In each sample 12 semen tubes were examined by light microscope with
magnification of ×100. Obtained values were expressed as mean number of cells in each tube.
Statistical analysis:
For data analysis SPSS software version 16 was used, and to compare groups one way ANOVA and
Duncan’s test were used. Based on Duncan’s test, if there is a common target in each group there is no
significant difference. The P≤0.05 was considered as statistically significant. The means and standard deviations
were calculated.
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4823-4827
Results:
Histological examination of the testis:
According to Table 1 it can be seen that sulfasalazine caused a significant reduction in the number of
spermatogonia, primary spermatocytes but Vitamin E and C combined together have significantly increased in
all experimental groups compared to the control group (Figures 1 to 5).
Table 1: The values (mean ± SEM, n=8 each) of testes cells of control rats, and rats treated with sulfasalazine, sulfasalazine +vitamin C,
sulfasalazine +vitamin E, or sulfasalazine +vitamin C and vitamin E.
Treatment
Spermatogonia
Primary
Spermatozoa
Leydig
Sertoli
Spermatids
spermatocytes
Control
81.4 + 1.3a
b
160 + 6.1a
118.4 + 1.7a
100.6 + 2.1b
63 + 1.1
Sulfa+vit C
80.9 + 105a
170.7 + 2.1c
29.7 + 1.2c
130.3 + 3.5e
Sulfa + vit E
81.6 + 1.6a
178.5 + 2.1d
35.3 + 0.67a
161.5 + 3.5d
c
e
d
259.6 + 3.8c
90.8 + 1.2
226.7 + 7.5
26.5 + 0.95
b
Sulfa
Sulfa + vit E &vit C
69.6 + 2.8
36.6 + 2.5a
b
40.7 + 0.99
79.2 ± 2.1 a
± 1.5
100.5 ± 1.7 c
116 ± 1.1 d
170.1 ± 2.1 e
64.4
b
33.4 ± 1.2 a
± 0.70 b
34.5 ± 0.88 a
34.3 ± 0.73 a
38 ± 0.86 c
22.3
Sulfa: sulfasalazine; vit C: vitamin C; vit E: vitamin E. If in each group, there is at least one common letter (a,b,c,d,e), there is no significant
difference in this case. Data is given based on the average standard error of the mean. Significant difference is P ≤ 0.05
Fig. 1: Photo micrograph of the seminiferous tubular in control group, staining with Hemotoxylin eosin,
magnification of ×100, wave mode and the reproductive corrugated epithelium of the tubes 4 to 8 cell
layer.
Fig. 2: Photo micrograph seminiferous tubular in the sulfasalazine group (experiment 1), Hematoxylin and eosin
staining, magnification ×100, spermatogenesis is stopped at the stage of primary spermatocytes,
thickening of the interstitial space is visible due to fluid retention (edema).
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4823-4827
Fig. 3: Photo micrograph seminiferous tubular in sulfasalazine +vitamin C (experiment 2), stained with
Hematoxylin and eosin, magnification of ×100, most of the tubes are rich in reproductive epithelium
and sperm cells.
Fig. 4: Photo micrograph of seminiferous tubular in sufasalazine group + vitamin E (experiment 3),
Hematoxylin and eosin staining, magnification of ×100, most tubes contains multiple layers of
reproductive epithelium, maturity until the final stage of sperm production continues so that all tubes
containing the slides of the lumen are filled by these cells.
Fig. 5: Photo micrograph seminiferous tubular in sufasalazine group + vitamin C + vitamin E (experimental 4),
Hematoxylin and eosin staining, magnification ×100 , about of 7 tubes seminiferous, perfectly normal
epithelium in about 6 to 8 layers.
Discussion and conclusion:
According to the studies, sulfasalazine consumption causes increased oxidative stress and so increases
MDA enzyme (which is the final product of lipid peroxidation by reactive oxygen species (ROS)). Subsequently
Ros generate causes stop cell cycle and increasing apoptosis process, thus it will reducing the overall number of
sperm [2,11,12]. According to this research, sulfasalazine can cause free radical production (ROS) in the cell. It
can be argued that production of free radicals in testicular germ cells which are quite sensitive causes the loss of
them. Other studies found that Ros was produced from two different sources in a sperm liquid called damaged
spermatozoa cells and activated leukocytes that in high amounts, with disorder in the structure of DNA, reduce
the percentage of live sperm and non-sperm binding to the ovum, causing infertility in men [13]. According to
this research and also publications by the center of “control and disease protection” it is stated that chemical
materials disrupt endocrine system (EDC) with the production of free radicals (ROS) and they are able to reduce
oxidative damage to biology molecules such as DNA and protein [14]. It is possible that sulfasalazine by
creating free radical (Ros) and mutant in testis tissue, especially the sensitive cells of spermatogonia, primary
spermatocyte, spermatid and spermatozoa, cause serious damage and loss of these cells. So there is a risk of
damage and loss of interstitial cells and sertoli in this way. Also, the function of interstitial cells is affected by
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4823-4827
sertoli cells [15]. Vitamin E or alpha-tocopherol due to lipid solubility can prevent destructive effects of Ros on
sperm parameters [15]. Because a-tocopherol molecule as a chain breaking antioxidant can inhibit two lipid
proxils radical and so inhibit chain potential reaction of high oxidation. VitaminC or ascorbic acid is an
antioxidant soluble in water that also eliminate free radicals Ros cause the entrance of other antioxidant such as
vitamin E to the cycle, thus it is possible that both of these antioxidants can prevent the side effects of
sulfasalazine [16].
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the staf of the Research department of Medical Science
University of Jahrom.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Fukushima, T., M. Kato, T. Adachi, Y. Hamada, M. Horimoto, M. Komiyama, et al., 2005. Effects of
sulfasalazine on sperm acrosome reaction and gene expression in the male reproductive organs of rats.
Toxicol Sci., 85: 675-682.
Levi, A.J., A.M. Fisher, L. Hughes, W.F. Hendry, 1979. Male infertility due to sulphasalazine. Lancet, 2:
276-278.
Toth, A., 1979. Male infertility due to sulphasalazine. Lancet, 2: 904.
Fukushima, T., Y. Hamada, M. Komiyama, Y. Matsuno, C. Mori, I. Horii, 2007. Early changes in sperm
motility, acrosome reaction, and gene expression of reproductive organs in rats treated with sulfasalazine.
Reprod Toxicol., 23: 153-157.
Chitra, K.C., C. Latchoumycandane, P.P. Mathur, 2003. Induction of oxidative stress by bisphenol A in
the epididymal sperm of rats. Toxicology, 185: 119-127.
Fujii, J., Y. Iuchi, S. Matsuki, T. Ishii, 2003. Cooperative function of antioxidant and redox systems
against oxidative stress in male reproductive tissues. Asian J Androl., 5: 231-242.
Pascoe, G., F. Olafs dottier, D. Read, 1987. Vitamin E protection against chemical induced cell injury.
Maintenance of cellular protein thiols as a cytoprotective mechanism. Arch. Biochem. Biophys., 256: 150158.
John, S., M. Kale, N. Rathore, D. Bhatnagar, 2001. Protective effect of vitamin E in dimethoate and
malathion induced oxidative stress in rat erythrocytes. J. Nutr. Biochem., 12: 500-504.
Aldana, L., V. Tsutsumi, A. Craigmill, M.I. Silveira, E.G. De Mejia, 2001. Tocopherol modulates liver
toxicity of the prethroidcypermethrin. Toxicol. Lett., 125: 107-116.
Chan, A.C., 1993. Partners in defense, vitamin E and vitamin C. Can J. Physiol. Pharmacol., 1: 25-31.
Draper, H.H., M. Hadley, 1990. Malondialdehyde determination as index of lipid peroxidation. Meth.
Enzymol., 186: 421-31.
Storey, B.T., 1997. Biochemistry of the induction and prevention of lipoperoxidative damage in human
spermatozoa. Mol Hum Reprod, 3: 203-13.
Wolf, R., D. Wolf, V. Ruocco, 1998. Vitamin E: the radical protector. J. Eur. Acad Dermatol Venereol.,
10: 103-117.
Choi, S.M., S.D. Yoo, B.M. Lee, 2004. Toxocological characteristics of endocrine disrupting chemicals:
Developed mental toxicity, Carcinogenicity and mutagenicity: Journal of toxicology and environmental
health, Part B., 7: 1-32.
Kobayashi, H., E. Gill-Guzman, A.M. Mahran, 2001. Quality control of reactive oxygen species
measurement by Iuminal-dependent chemiluminescenceassay. J. Androl., 22: 568-574.
Acharya, U.R., M. Mishra, J. Patro, et al., 2008. Effect of vitamins C and E on spermatogenesis in mice
exposed to cadmium. Reproductive Toxicology, 25: 84-8.