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Effects of boric acid feeding on the oxidative stress parameters in testes, sperm parameters and DNA damage in mice
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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Effects of boric acid feeding on the oxidative stress parameters in testes, sperm parameters and DNA damage in mice
T
Serdar Aktasa,*, Cavit Kuma, Melih Aksoyb a b
Adnan Menderes University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Aydın, Turkey Adnan Menderes University, Faculty of Veterinary Medicine Department of Reproduction and AI, Aydin, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Boric acid Oxidative stress Sperm parameters DNA damage
This study was aimed to determine the effects of boric acid on oxidative stress, testicular tissue and spermatozoon DNA. Experiments were performed with Swiss Albino mice divided equally into two groups based on the tratment period: one for 4 and the other for 6-week duration. These groups were further divided into subgroups as Control and those administered daily at oral doses of 115 mg/kg, 250 mg/kg and 450 mg/kg of boric acid. Then, testicular tissue were examined postmortem and analyzed using ex-vivo biochemical tools for oxidative stress, spermatozoon membrane integrity, sperm motility and live cell rate (%). In both 4 and 6-week groups, v. seminalis weight, membrane integrity, motility, live cells and GSH levels exhibited a decreasing trent compared to the controls. In addition, 6-week group had a decrease in SOD level. MDA level was higher in controls in both 4 and 6-week groups. Spermatozoon DNA was intact in the 4-week group, but damaged in the 6-week group, and the degree of the damage dependent on the administered dose. Boric acid induces oxidative stress in testicular tissue, and its long-term application (only 6 weeks) caused damage in spermatozoon DNA.
1. Introduction Boron is an essential trace element for plants [1], but, both humans and animals also need certain amounts per day to maintain normal vital functions [2]. Average daily intake of boron is reported to be between total 0.5–1 mg [3]. Although the role of boron in metabolic activities is not fully understood, it is effective in membrane function and stability, hormone receptors and transmembrane signaling in the cell [4]. Boron is not present as a single element in nature, and is always found together with either hydrogen, oxygen, sodium or calcium. The most common boron minerals in nature are borax and kernite. Boric acid (BA; H3BO3) is the most popular boron compound used in medicine, cosmetics and other industries. BA is an inorganic compound and contains about 17.48 % elemental boron in its structure. It is weakly acidic and easily soluble in water. Albeit it is not absorbed by skin, but can easly enter the body from damaged skin or via dust particles. BA taken orally is absorbed 90 % through the digestive system. The half plasma rate (t½) is about 21 h and is excreted unchanged from the kidneys without metabolism in the body with a detection time up to 96 h in urine [5,6]. Existing data on the toxic level of BA acid in humans is not clear. Information about dosage and duration in humans is limited only to accidental high ingestion and suicide cases. In rats, Weir and Fisher [7] ⁎
reported that 1170 ppm dose was harmful to the reproductive system. In another study, BA administered to F344 rats at a dose of 9000 ppm for 28 days resulted in testicular tissue lesions, decreased spermiation, and extended period of BA application caused testicular atrophy [8]. Linder et al. [9] reported acute, subchronic and chronic effects of BA in rats, and sperm samples were collected by applying 2000 mg/kg/day BA for 2, 14, 28, 57 days. In the sperm samples taken on day 28; spermatozoon maturation disorders and morphological abnormalities were comparatively higher in BA treated rats compared to the control group. There is limited information about toxicity and cytodestructive nature of BA in the body except reproductive system. Neverthless boron contents alone can be toxic. Major toxicity of BA appears during reproductive and developmental stages [8–10] and it also reported to potentiate the mutagenicity of thermal neutron irradiation in Chinese hamster ovary cells [40]. The studies dealing with the effects of BA feeding on sperm cell DNA integrity in mice and rats are scarce. Furthermore, in mice, wide range of dose applications of BA and its effects on the oxidative stress parameters in testicular tissue are not avialable. Therefore; this study was aimed to evaluate the effects of different dose levels of BA fed by oral gavage on the oxidative stress markers and DNA damage in testicular tissue, and the sperm quality parameters in mice.
Corresponding author. E-mail address: [email protected] (S. Aktas).
https://doi.org/10.1016/j.jtemb.2019.126447 Received 12 September 2019; Received in revised form 6 December 2019; Accepted 16 December 2019 0946-672X/ © 2019 Published by Elsevier GmbH.
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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2. Materials and methods
2.3. Assessment of sperm quality parameters
2.1. Animals and treatments
2.3.1. Sperm cell motilty and viability Sperm samples were taken from cauda epididymis and were diluted with PBS (10 μl). A 3 μl sperm suspension was covered with a coverslip. The ratio of motile spermatozoa was determined by evaluating at least five different microscopic fields observed under phase contrast microscope attached to a heating table, pre-set to 37 °C. The motility and velocity parameters of neat sperm samples were quantified by using computer assisted semen analyzer (CASA, Sperm Class Analyser, Microptic, Barcelona, Spain), and the values were given as percentage (%) for each sample. The rate of the dead-alive spermatozoon was charterized by supravital staining technique. Briefly, 10 μl of semen suspension was carefully mixed with 10 μl of eosin-nigrosin dye (3 % sodium citrate, 3 % nigrosin and 1 % eosin) and smear was prepared using coverslip and observed under light microscope at 40X magnification. The total number of unstained spermatozoa were counted out of 200 cells and the percentage of the live spermatozoon was calculated for each mouse.
In this present study, 80 sexually mature Swiss Albino male mice (12 weeks of age, and weighed between, 35–40 g) were used as sperm donors. Animals were purchased from Veterinary Control and Research Institute in Bornova/Izmir, Turkey. The experiments were performed at Adnan Menderes University, Faculty of Medicine, Laboratory of Experimental The study was approved by local animal ethics committee of Aydın Adnan Menderes University (Approval number: B.30.2.ADU.0.00.00.00./50.04/2012/084). The animals were fed regular diet and provided water ad libitum. Throughout the study duration, 12/12 h light dark cycles with temperature 22 ± 2 °C were applied. Mice divided equally into two groups (n = 40) based on the duration of the experiment: either 4 or 6-week. Each group was further divided into equal subgroups as Control (n = 10) and those administered with BA dailly at doses of 115 mg/kg (n = 10), 250 mg/kg (n = 10) and 450 mg/kg (n = 10). Daily doses were determined based on the previous publications [10–12,39]. Animal weights were recorded at the beginning of the study and weekly. The amounts of boric acid to be given daily to each mouse were dosed. Boric Acid (Sigma-B0252, USA) dissolved in 0.5 ml of water was given orally by gavage (22 G, Harvard Apparatus). At the end of either 4 or 6 weeks, the mice were fasted overnight and euthanized for postmortem analysis.
2.3.2. Sperm membrane integrity To evaluate the membrane integrity of spermatozoa, hypoosmotic swelling test (HE-test) was performed in combination with eosin nigrosin staining technique [18]. For this purpose, 20 μl of sperm sample was incubated in 180 μl fructose solution with an osmotic pressure of 80 mOsm/L for 15 min at 37 °C. After incubation, sperm cells were well mixed and eosin nigrosin staining was performed. A minimum of 200 spermatozoon were counted under the light microscope at 40× magnification, and the percentage of the spermatozoon with curved tails and white heads (maintaining membrane integrity) was determined.
2.2. Measurement of antioxidant enzyme activity 2.2.1. Sampling Mice were euthanized by cervical dislocation under anaesthesia with Ketamine (Ketasol %10 Ricterpharma/Austria) and Xylazine (Xylazınbio %2 Bioveta/Czech Republic). Epididymal sperm samples, v seminalis and testicular tissues were isolated. The testicular tissues were homogenized in PBS (150 mM, pH 7.4) in an ice cube for 2000 cycles for 1 min. Later, the homogenates were centrifuged at 7000 rpm in a cooled centrifuge for 10 min at 4 °C and the supernatants were stored at −80 °C (NU 9668E, Nuaire, Japan) until the further analysis. Tissue homogenized (80) testes samples were prepared and examined for measuring total protein (TP), malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT) values. To determine the amount of TP, a commercial test kit was utilized (A2300, Archem Health Ind. Co., Turkey) and the results were expressed per mg of soluable protein.
2.3.3. Sperm cell DNA integrity Alkaline comet assay method was used to determine the percentage of DNA damaged spermatozoa. In this method, intact (negative control) and DNA damaged (positive control) sperm samples were observed and compared for morphology of the sperm DNA mass after electrophoresis. In the positive control, DNA damage was induced by 3 h incubation at 44 °C in water bath. Afterwards, comet and round shaped DNA with different tail length in the positive control and negative control groups were observed respectively. A number of replicates were performed with to find the consistent results for validation of the method before primary experiments. After the pretreatment, preparations were dried, stained with 70 μl DAPI (40,6-diamidino-2-phenyindole-dilactate), and covered with coverslip [19]. Randomly drawn images containing at least 100 sperm cells were recorded for evaluation using a QuickcamPro camera of fluorescence microscope. The rates of DNA damaged spermatozoa were determined by the assessment of the images using, “Image J software” (Version 1.47v; NIH. USA) program [20]. The degree of DNA damage was ranged between rating scale of 0–4.
2.2.2. Malondialdehyde activity To determine the level of lipid peroxidation in testicular tissue MDA (end product of lipid peroxidation) content was measured using thiobarbituric acid method as described by Yoshioka et al. [13], Draper and Hadley [14]. MDA forms a pink complex by incubating with TBA at 90 °C in an aerobic environment. The complex was read by spectrophotometer to measure the MDA concentration by the absorbance at a wavelength of 532 nm.
2.4. Statisical analysis The data was evaluated using SPSS (version 11.5), and after determining the normal distribution of the parameters by Shapiro-Wilk, the variance of homogeneity was measured by Leveneins test. The data was statistically analyzed by one-way ANOVA after checking the normal distribution of the data by Kruskall-Wallis. Thereafter, t-test or Mann-Whitney test was used for statistical analysis of the data. Duncan’s test or Mann-Whitney U test with Bonferroni correction were used to determine the differences between the groups. Results were presented as mean ± S.D and differences were considered to be significant at P < 0.05 [21].
2.2.3. Superoxide dismutase activity SOD activity was determined by adapting the method of Sun et al. [15]. 2.2.4. Catalase activity CAT activity was measured using the method of Bergmeer et al. [16].
3. Results
2.2.5. Glutathione activity GSH activity values were obtained by using the method of Tietz [17].
The final body weights of mice in BA treated subgroups were not 2
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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significant. GSH level decreased in the 115 mg/kg, 250 mg/kg and 450 mg/kg BA groups compared to the control group (P < 0.001).
Table 1 Changes in the body weight of control and Boric acid (BA) treated mice. Time period
4-week
6-week
Groups (n = 10)
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
Weight (g)
P
Before
Finally
40.20 ± 0.84 38.37 ± 0.83 38.97 ± 1,60 41.65 ± 1.45 NS 36.90 ± 2.38 42.83 ± 1.48 42.06 ± 1.56 41.34 ± 1.10 NS
41.20 ± 0.84 39.59 ± 1.15 39.99 ± 1.17 40.86 ± 1.15 NS 39.40 ± 2.25 44.32 ± 1.41 42.97 ± 1.28 40.30 ± 1.00 NS
4. Discussion Boron is a trace element that is taken for the continuos normal metabolic functions of animals and humans [22]. High doses or prolonged exposure to boron-containing compounds causes toxic effects. BA causes testicular atrophy and sterility in male rats [7]. Although, the mechanism of the toxic effects has not been fully explained yet, it is beleived that the damage is directly proportional to the level of accumulation in the testis tissue [5,12]. Studies have shown that rat body weight reduces by 8 % with the dose level of 9000 ppm BA administered for 28 days [8]. Similary, 275–550 mg applied for 32 weeks caused weight reduction of 10–17 % in mice [23]. In this study, body weights did not change significantly with the application of BA between the 4 and 6-week groups. We can not make one to one comparison with the results of the previous studies, because either our treatment doses were lower or the duration of the exposure was shorter. Chemical compounds causing reproductive toxicity are reported to adversely affect male reproductive health, and a leading cause of reduced fertility or sterility [24]. Application of Copper chloride (CuCl2) twice a week for 30 days at doses of 1.25, 2.5 and 5 mg/kg causes higher sperm anomalies in mice [25]. Similarly, exposure to high-doses (1170 ppm) of BA causes sterility, but low doses (117 and 350 ppm) produce no side effects [7]. Moreover, BA at the dose level of 9000 ppm reduces sperm production as early as at 7th day [8]. In the present study, sperm motility and membrane integrity ratio were significantly lower in both 4 and 6-groups (Table 2). In both periods, the highest reduction was observed in the membrane integrity (P < 0.001). In the 6-week group, live sperm rate was also reduced. Reproductive toxicities of boron-containing compounds are closely related to their application duration. Long-term exposure of BA at high dose causes histopathological changes and atrophy in testicular tissue [8,10,12]. However, low-dose, long-term exposure may not produce changes in testis, prostate and v seminalis weights [26]. Ku at al [12]. reported that 6000–9000 ppm BA administration caused irreversible atrophy in rats. In the present study, no difference was found in testicular weight, though v. seminalis weight decreased only in the 4 week groups (P < 0.05). The data showed that adverse effects in the reproductive system and damage were moderate. BA has been reported to affect the oxidative metabolism [27]. This effect is related to the dose and duration of administration. Increased antioxidant capacity was demonstrated after 5−20 ppm of BA application in erythrocyte suspension [28]. BA induced antioxidant activity in blood and liver tissue when administered orally in the chronic phase (28 days) at the dose of 100 mg/kg [29]. With the same dose, BA induced positive effects on the healing of experimental nerve injury
NS NS NS NS NS NS NS NS
NS: Not Significant.
statistically different compared to the corresponding controls in both 4 and 6 weeks groups (Table 1). Testes weights (right and left together) at the end of the study decreased, but no difference was not statistically significant (Table 2). However, v seminalis weight decreased in all the groups significantly (P < 0.05). The weight of seminal glands were not equal througout the groups corresponding to the proportional variability of the seminal fluid presence or absence. Results showed that the decrease in sperm motility in the 4 and 6week group was statistically significant in the 450 mg/kg group (P < 0.05) and in the 6-week group 250 and 450 mg/kg BA group were (P < 0.001) statistically significant. The decrease in live sperm rate was mild at 4 week period, but after 6-weeks treatment there was a sudden decrease compared to the control (P < 0.001). The membrane integrity of spermatozoa reduced significantly (P < 0.001) in both 4-week and 6week groups (Table 2). Result of comet assay showed that there was no DNA damage in mice spermatozoa in 4-week BA-treated group. The DNA damage was detected in the 6-week BA-treated groups in a dose dependent manner (P < 0.001). DNA damage levels in spermatozoa were seen during the 6 week long term treatment of BA. In the group receiving 450 mg/kg, moderate and advanced DNA damages (3rd and 4th degree) were observed, while in groups 115 and 250 mg/kg, mild and moderate (2nd and 3rd degree) damages were detected (P < 0.001), (Table 4, Fig. 1). At 4 weeks of BA-treatment, MDA levels increased in 250 and 450 mg/kg groups and it was found to be significant compared to the control (P < 0.001). SOD and CAT levels were not statistically significant (Table 3). GSH level significantly decreased in 450 mg/kg BA group (P < 0.05). In 6-week groups, MDA levels in 450 mg/kg BA group were statistically higher than the other groups (P < 0.05). SOD levels were decreased in 250 mg/kg and 450 mg/kg BA groups, compared to 115 mg/ kg and control groups (P < 0.01). CAT levels were not statistically
Table 2 Semen parameters of control and Boric acid (BA) treated mice at the 4th and 6th week of the study. Groups (n = 10)
Parameters Testis weight (mg)
4-week
6-week
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
192.60 ± 5.98 171.80 ± 4.43 174.10 ± 5.98 178.40 ± 9.73 NS 194.30 ± 6.85 207.90 ± 3.88 180.80 ± 0.07 177.20 ± 1.54 NS
V. seminalis weight (mg) a
Sperm motility (%) 77.50 ± 2.38 79.00 ± 1.94 73.00 ± 1.85 69.00 ± 2.66 * 78.00 ± 1.52 72.50 ± 4.36 68.50 ± 2.24 54.00 ± 4.33 ***
356.90 ± 7,44 270.50 ± 8,43 b 311.00 ± 17.57a,b 268.90 ± 20.63 b * 280.30 ± 25.65 b,c 342.10 ± 17.83 a 320.70 ± 15.76 a,b 264.60 ± 12.52 c *
a,b a b,c c
a a,b b c
Live sperm rate (%) 73.50 ± 2.00 78.80 ± 1.44 74.30 ± 1.39 70.70 ± 1.57 ** 74,00 ± 0,93 68,00 ± 2,33 68,20 ± 1,93 57,00 ± 3,09 ***
b,c a b,c c
a b b c
Membrane integrity (%) 75.60 ± 1.78 67.90 ± 2.20 62.00 ± 2.16 64.30 ± 1.85 *** 74,70 ± 1,32 58,30 ± 2,25 60,00 ± 1,54 55,10 ± 3,26 ***
a, b, c: Different letters in the same column indicate statistically significant difference *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant. 3
a b b b
a b b b
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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Fig. 1. Sample fluorescence microscope images of DNA damage in spermatozoa (magnification ×50).
model at the acute (3 days) phase of the injury [30]. The current study again showed statistically insignificant increases in the values of (SOD, CAT, GSH, motility, live sperm rate), somehow implying antioxidant activity, with the dose of 115 mg closest to 100 mg at the 4-week group. But, this was not true in the 6-week group as the changes in these measurements were negatively significant. These findigns suggest that prolonged application time may lead to increased oxidant activity. In male infertility, Reactive oxygen species (ROS) compounds damage the spermatozoal membrane and cause subseqent infertility [24,31,32]. Although, testicular tissue has defensive system against oxidative stress, but it is not as efficient as other somatic cells [33]. Xanthine and Xanthine oxidase caused oxidative stress in sperm, SOD and CAT enzymes showed protective effect against this [34]. Swiss Albino male mice were given dimethoate in a 30-day study and MDA levels were shown to be significantly increased [35]. Kumar et al. [36] applied 15–60 nanomol (100 g live weight) T-butyl and c-butyl peroxides for 1, 2, 3 and 5 weeks. Organic hydrogen peroxide induced oxidative stress to testicular tissue and dose depent increases in the level of MDA along with the abnormal sperm rate and spermatozoon DNA damage have been reported. In the present study, increased levels of MDA at the 4 and 6-week of BA application indicated the production of oxidative stress (Table 3). Likewise, higher doses of BA applied for an extensive period, inflicted negative effects on the sperm quality. Spermatozoons may be subjected to cellular damage during the
Table 4 Boric acid (BA) administered mice at 4th and 6th weeks of sperm damage degree and DNA damage percentage. Time period
4-week
6-week
Groups (n = 10)
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
DNA damage degree (0–4)
DNA damaged cell (%)
00.00 00.00 00.00 00.00 NS 0.00 d 0.12 ± 0.04 0.32 ± 0.07 0.66 ± 0.14 ***
00.00 00.00 00.00 00.00 NS 0.00 d 3.30 ± 1.43 c 6.20 ± 1.84 b,c 14.40 ± 2.89 a ***
c b a
a, b, c: Different letters in the same column indicate statistically significant differences *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant.
spermatogenesis phase or post-testicular sperm maturation [37]. Similary, ingestion of boron-containing compounds may cause damage during cell cycle of mitosis and meiosis (spermiogenesis) [10]. Boron mine workers had no significant differences in semen quality when compared with low, medium and high levels of boron exposure
Table 3 MDA, SOD, CAT and GSH levels in testicular tissue at the 4th and 6th weeks in Boric acid (BA) treated mice. Time period
Groups (n = 10)
Parameters MDA (nmol/mg protein)
4-week
6-week
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
a
143.97 ± 5.16 128.84 ± 12.23 a 183.39 ± 6.24 b 192.07 ± 11.32 b *** 149.29 ± 13.90 b 157.53 ± 8.70 b 167.00 ± 16.50 a,b 216.91 ± 35.34 a *
SOD (U/mg protein)
CAT (k/mg protein)
GSH (mg/g protein)
8.21 ± 1.52 13.80 ± 5.39 10.34 ± 2.29 6.79 ± 0.62 NS 7.04 ± 0.31a 7.03 ± 0.52 a 5.62 ± 0.39 b 5.47 ± 1.22 b **
0.91 ± 0.20 1.11 ± 0.47 0.98 ± 0.22 0.61 ± 0.13 NS 0.79 ± 0.17 0.53 ± 0.07 0.54 ± 0.10 0.73 ± 0.10 NS
15.40 ± 2.25 a 20.90 ± 8.09 a 9.77 ± 1.54 a,b 7.31 ± 2.02 b * 16.78 ± 1.40 a 11.85 ± 0.74 b 8.20 ± 0.98 c 7.62 ± 1.19 c ***
a, b, c: Different letters in the same column indicate statistically significant differences *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant. 4
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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(according to blood boron levels) or those who were not exposed [38]. Male rats were administered three different doses (125, 250 and 500 mg/kg) BA for 60 days. While no significant change was observed in the 125 mg administered group, there was a decrease in testicular tissue DNA levels in the 250 and 500 mg/kg BA treated samples [39]. In the present study, DNA damage observed in the 6-week group, but not in the 4-week group. Increased oxidative stress and elevated MDA levels in testicular tissue as well as the depletion of antioxidant defence molecules (SOD, CAT, GSH) in semen are thought to be detrimental to DNA synthesis. It is seen that the DNA damage is closely related to the duation of BA administration.
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5. Conclusions Although exposure is uncommon, but when subjected to a long time (> six weeks) and at high doses (> 115 mg/kg), BA causes oxidative stress in testicular tissue. Morever, the exposure effects the sperm parameters negatively and damages the spermatozoon DNA. Future research may focus on testing chemical agents with potential to protect against BA and to treat its toxicity. Declaration of Competing Interest None. Acknowledgement This investigation was supported by Adnan Menderes University Scientific Research Projects Unit (ADU-BAP, Grant number VTF13013). References [1] K. Warington, The effect of boric acid and borax on the broad bean and certain other plants, Ann. Bot. 37 (148) (1923) 629–672. [2] C.J. Rainey, L.A. Nyquist, R.E. Christensen, P.L. Strong, B.D. Culver, J.R. Coughlin, Daily boron intake from the American diet, J. Am. Diet. Assoc. 99 (3) (1999) 335–340. [3] W.H. Organization, Trace Elements in Human Nutrition in Health. Report of the WHO Expert Committee on Trace Elemnts in Human Nutrition, Boron in, Geneva, Switzerland, (1996), pp. 175–179. [4] F.H. Nielsen, Nutritional requirements for boron, silicon, vanadium, nickel, and arsenic: current knowledge and speculation, FASEB J. 5 (12) (1991) 2661–2667. [5] F.J. Murray, A comparative review of the pharmacokinetics of boric acid in rodents and humans, Biol. Trace Elem. Res. 66 (1-3) (1998) 331–341. [6] S.A. Hubbard, Comparative toxicology of borates, Biol. Trace Elem. Res. 66 (1–3) (1998) 343–357. [7] R.J. Weir Jr., R.S. Fisher, Toxicologic studies on borax and boric acid, Toxicol. Appl. Pharmacol. 23 (3) (1972) 351–364. [8] K.A. Treinen, R.E. Chapin, Development of testicular lesions in F344 rats after treatment with boric acid, Toxicol. Appl. Pharmacol. 107 (2) (1991) 325–335. [9] R.E. Linder, L.F. Strader, G.L. Rehnberg, Effect of acute exposure to boric acid on the male reproductive system of the rat, J. Toxicol. Environ. Health Part A: Curr. Issues 31 (2) (1990) 133–146. [10] W.W. Ku, R.E. Chapin, Mechanism of the testicular toxicity of boric acid in rats: in vivo and in vitro studies, Environ. Health Perspect. 102 (Suppl. 7) (1994) 99–105. [11] P.A. Fail, J.D. George, J.C. Seely, T.B. Grizzle, J.J. Heindel, Reproductive toxicity of boric acid in Swiss (CD-1) mice: assessment using the continuous breeding protocol, Fundam. Appl. Toxicol. 17 (2) (1991) 225–239. [12] W.W. Ku, R.E. Chapin, R.N. Wine, B.C. Gladen, Testicular toxicity of boric acid (BA): relationship of dose to lesion development and recovery in the F344 rat, Reprod. Toxicol. 7 (4) (1993) 305–319. [13] T. Yoshioka, K. Kawada, T. Shimada, M. Mori, Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the
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Effects of boric acid feeding on the oxidative stress parameters in testes, sperm parameters and DNA damage in mice
T
Serdar Aktasa,*, Cavit Kuma, Melih Aksoyb a b
Adnan Menderes University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Aydın, Turkey Adnan Menderes University, Faculty of Veterinary Medicine Department of Reproduction and AI, Aydin, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Boric acid Oxidative stress Sperm parameters DNA damage
This study was aimed to determine the effects of boric acid on oxidative stress, testicular tissue and spermatozoon DNA. Experiments were performed with Swiss Albino mice divided equally into two groups based on the tratment period: one for 4 and the other for 6-week duration. These groups were further divided into subgroups as Control and those administered daily at oral doses of 115 mg/kg, 250 mg/kg and 450 mg/kg of boric acid. Then, testicular tissue were examined postmortem and analyzed using ex-vivo biochemical tools for oxidative stress, spermatozoon membrane integrity, sperm motility and live cell rate (%). In both 4 and 6-week groups, v. seminalis weight, membrane integrity, motility, live cells and GSH levels exhibited a decreasing trent compared to the controls. In addition, 6-week group had a decrease in SOD level. MDA level was higher in controls in both 4 and 6-week groups. Spermatozoon DNA was intact in the 4-week group, but damaged in the 6-week group, and the degree of the damage dependent on the administered dose. Boric acid induces oxidative stress in testicular tissue, and its long-term application (only 6 weeks) caused damage in spermatozoon DNA.
1. Introduction Boron is an essential trace element for plants [1], but, both humans and animals also need certain amounts per day to maintain normal vital functions [2]. Average daily intake of boron is reported to be between total 0.5–1 mg [3]. Although the role of boron in metabolic activities is not fully understood, it is effective in membrane function and stability, hormone receptors and transmembrane signaling in the cell [4]. Boron is not present as a single element in nature, and is always found together with either hydrogen, oxygen, sodium or calcium. The most common boron minerals in nature are borax and kernite. Boric acid (BA; H3BO3) is the most popular boron compound used in medicine, cosmetics and other industries. BA is an inorganic compound and contains about 17.48 % elemental boron in its structure. It is weakly acidic and easily soluble in water. Albeit it is not absorbed by skin, but can easly enter the body from damaged skin or via dust particles. BA taken orally is absorbed 90 % through the digestive system. The half plasma rate (t½) is about 21 h and is excreted unchanged from the kidneys without metabolism in the body with a detection time up to 96 h in urine [5,6]. Existing data on the toxic level of BA acid in humans is not clear. Information about dosage and duration in humans is limited only to accidental high ingestion and suicide cases. In rats, Weir and Fisher [7] ⁎
reported that 1170 ppm dose was harmful to the reproductive system. In another study, BA administered to F344 rats at a dose of 9000 ppm for 28 days resulted in testicular tissue lesions, decreased spermiation, and extended period of BA application caused testicular atrophy [8]. Linder et al. [9] reported acute, subchronic and chronic effects of BA in rats, and sperm samples were collected by applying 2000 mg/kg/day BA for 2, 14, 28, 57 days. In the sperm samples taken on day 28; spermatozoon maturation disorders and morphological abnormalities were comparatively higher in BA treated rats compared to the control group. There is limited information about toxicity and cytodestructive nature of BA in the body except reproductive system. Neverthless boron contents alone can be toxic. Major toxicity of BA appears during reproductive and developmental stages [8–10] and it also reported to potentiate the mutagenicity of thermal neutron irradiation in Chinese hamster ovary cells [40]. The studies dealing with the effects of BA feeding on sperm cell DNA integrity in mice and rats are scarce. Furthermore, in mice, wide range of dose applications of BA and its effects on the oxidative stress parameters in testicular tissue are not avialable. Therefore; this study was aimed to evaluate the effects of different dose levels of BA fed by oral gavage on the oxidative stress markers and DNA damage in testicular tissue, and the sperm quality parameters in mice.
Corresponding author. E-mail address: [email protected] (S. Aktas).
https://doi.org/10.1016/j.jtemb.2019.126447 Received 12 September 2019; Received in revised form 6 December 2019; Accepted 16 December 2019 0946-672X/ © 2019 Published by Elsevier GmbH.
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2. Materials and methods
2.3. Assessment of sperm quality parameters
2.1. Animals and treatments
2.3.1. Sperm cell motilty and viability Sperm samples were taken from cauda epididymis and were diluted with PBS (10 μl). A 3 μl sperm suspension was covered with a coverslip. The ratio of motile spermatozoa was determined by evaluating at least five different microscopic fields observed under phase contrast microscope attached to a heating table, pre-set to 37 °C. The motility and velocity parameters of neat sperm samples were quantified by using computer assisted semen analyzer (CASA, Sperm Class Analyser, Microptic, Barcelona, Spain), and the values were given as percentage (%) for each sample. The rate of the dead-alive spermatozoon was charterized by supravital staining technique. Briefly, 10 μl of semen suspension was carefully mixed with 10 μl of eosin-nigrosin dye (3 % sodium citrate, 3 % nigrosin and 1 % eosin) and smear was prepared using coverslip and observed under light microscope at 40X magnification. The total number of unstained spermatozoa were counted out of 200 cells and the percentage of the live spermatozoon was calculated for each mouse.
In this present study, 80 sexually mature Swiss Albino male mice (12 weeks of age, and weighed between, 35–40 g) were used as sperm donors. Animals were purchased from Veterinary Control and Research Institute in Bornova/Izmir, Turkey. The experiments were performed at Adnan Menderes University, Faculty of Medicine, Laboratory of Experimental The study was approved by local animal ethics committee of Aydın Adnan Menderes University (Approval number: B.30.2.ADU.0.00.00.00./50.04/2012/084). The animals were fed regular diet and provided water ad libitum. Throughout the study duration, 12/12 h light dark cycles with temperature 22 ± 2 °C were applied. Mice divided equally into two groups (n = 40) based on the duration of the experiment: either 4 or 6-week. Each group was further divided into equal subgroups as Control (n = 10) and those administered with BA dailly at doses of 115 mg/kg (n = 10), 250 mg/kg (n = 10) and 450 mg/kg (n = 10). Daily doses were determined based on the previous publications [10–12,39]. Animal weights were recorded at the beginning of the study and weekly. The amounts of boric acid to be given daily to each mouse were dosed. Boric Acid (Sigma-B0252, USA) dissolved in 0.5 ml of water was given orally by gavage (22 G, Harvard Apparatus). At the end of either 4 or 6 weeks, the mice were fasted overnight and euthanized for postmortem analysis.
2.3.2. Sperm membrane integrity To evaluate the membrane integrity of spermatozoa, hypoosmotic swelling test (HE-test) was performed in combination with eosin nigrosin staining technique [18]. For this purpose, 20 μl of sperm sample was incubated in 180 μl fructose solution with an osmotic pressure of 80 mOsm/L for 15 min at 37 °C. After incubation, sperm cells were well mixed and eosin nigrosin staining was performed. A minimum of 200 spermatozoon were counted under the light microscope at 40× magnification, and the percentage of the spermatozoon with curved tails and white heads (maintaining membrane integrity) was determined.
2.2. Measurement of antioxidant enzyme activity 2.2.1. Sampling Mice were euthanized by cervical dislocation under anaesthesia with Ketamine (Ketasol %10 Ricterpharma/Austria) and Xylazine (Xylazınbio %2 Bioveta/Czech Republic). Epididymal sperm samples, v seminalis and testicular tissues were isolated. The testicular tissues were homogenized in PBS (150 mM, pH 7.4) in an ice cube for 2000 cycles for 1 min. Later, the homogenates were centrifuged at 7000 rpm in a cooled centrifuge for 10 min at 4 °C and the supernatants were stored at −80 °C (NU 9668E, Nuaire, Japan) until the further analysis. Tissue homogenized (80) testes samples were prepared and examined for measuring total protein (TP), malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT) values. To determine the amount of TP, a commercial test kit was utilized (A2300, Archem Health Ind. Co., Turkey) and the results were expressed per mg of soluable protein.
2.3.3. Sperm cell DNA integrity Alkaline comet assay method was used to determine the percentage of DNA damaged spermatozoa. In this method, intact (negative control) and DNA damaged (positive control) sperm samples were observed and compared for morphology of the sperm DNA mass after electrophoresis. In the positive control, DNA damage was induced by 3 h incubation at 44 °C in water bath. Afterwards, comet and round shaped DNA with different tail length in the positive control and negative control groups were observed respectively. A number of replicates were performed with to find the consistent results for validation of the method before primary experiments. After the pretreatment, preparations were dried, stained with 70 μl DAPI (40,6-diamidino-2-phenyindole-dilactate), and covered with coverslip [19]. Randomly drawn images containing at least 100 sperm cells were recorded for evaluation using a QuickcamPro camera of fluorescence microscope. The rates of DNA damaged spermatozoa were determined by the assessment of the images using, “Image J software” (Version 1.47v; NIH. USA) program [20]. The degree of DNA damage was ranged between rating scale of 0–4.
2.2.2. Malondialdehyde activity To determine the level of lipid peroxidation in testicular tissue MDA (end product of lipid peroxidation) content was measured using thiobarbituric acid method as described by Yoshioka et al. [13], Draper and Hadley [14]. MDA forms a pink complex by incubating with TBA at 90 °C in an aerobic environment. The complex was read by spectrophotometer to measure the MDA concentration by the absorbance at a wavelength of 532 nm.
2.4. Statisical analysis The data was evaluated using SPSS (version 11.5), and after determining the normal distribution of the parameters by Shapiro-Wilk, the variance of homogeneity was measured by Leveneins test. The data was statistically analyzed by one-way ANOVA after checking the normal distribution of the data by Kruskall-Wallis. Thereafter, t-test or Mann-Whitney test was used for statistical analysis of the data. Duncan’s test or Mann-Whitney U test with Bonferroni correction were used to determine the differences between the groups. Results were presented as mean ± S.D and differences were considered to be significant at P < 0.05 [21].
2.2.3. Superoxide dismutase activity SOD activity was determined by adapting the method of Sun et al. [15]. 2.2.4. Catalase activity CAT activity was measured using the method of Bergmeer et al. [16].
3. Results
2.2.5. Glutathione activity GSH activity values were obtained by using the method of Tietz [17].
The final body weights of mice in BA treated subgroups were not 2
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significant. GSH level decreased in the 115 mg/kg, 250 mg/kg and 450 mg/kg BA groups compared to the control group (P < 0.001).
Table 1 Changes in the body weight of control and Boric acid (BA) treated mice. Time period
4-week
6-week
Groups (n = 10)
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
Weight (g)
P
Before
Finally
40.20 ± 0.84 38.37 ± 0.83 38.97 ± 1,60 41.65 ± 1.45 NS 36.90 ± 2.38 42.83 ± 1.48 42.06 ± 1.56 41.34 ± 1.10 NS
41.20 ± 0.84 39.59 ± 1.15 39.99 ± 1.17 40.86 ± 1.15 NS 39.40 ± 2.25 44.32 ± 1.41 42.97 ± 1.28 40.30 ± 1.00 NS
4. Discussion Boron is a trace element that is taken for the continuos normal metabolic functions of animals and humans [22]. High doses or prolonged exposure to boron-containing compounds causes toxic effects. BA causes testicular atrophy and sterility in male rats [7]. Although, the mechanism of the toxic effects has not been fully explained yet, it is beleived that the damage is directly proportional to the level of accumulation in the testis tissue [5,12]. Studies have shown that rat body weight reduces by 8 % with the dose level of 9000 ppm BA administered for 28 days [8]. Similary, 275–550 mg applied for 32 weeks caused weight reduction of 10–17 % in mice [23]. In this study, body weights did not change significantly with the application of BA between the 4 and 6-week groups. We can not make one to one comparison with the results of the previous studies, because either our treatment doses were lower or the duration of the exposure was shorter. Chemical compounds causing reproductive toxicity are reported to adversely affect male reproductive health, and a leading cause of reduced fertility or sterility [24]. Application of Copper chloride (CuCl2) twice a week for 30 days at doses of 1.25, 2.5 and 5 mg/kg causes higher sperm anomalies in mice [25]. Similarly, exposure to high-doses (1170 ppm) of BA causes sterility, but low doses (117 and 350 ppm) produce no side effects [7]. Moreover, BA at the dose level of 9000 ppm reduces sperm production as early as at 7th day [8]. In the present study, sperm motility and membrane integrity ratio were significantly lower in both 4 and 6-groups (Table 2). In both periods, the highest reduction was observed in the membrane integrity (P < 0.001). In the 6-week group, live sperm rate was also reduced. Reproductive toxicities of boron-containing compounds are closely related to their application duration. Long-term exposure of BA at high dose causes histopathological changes and atrophy in testicular tissue [8,10,12]. However, low-dose, long-term exposure may not produce changes in testis, prostate and v seminalis weights [26]. Ku at al [12]. reported that 6000–9000 ppm BA administration caused irreversible atrophy in rats. In the present study, no difference was found in testicular weight, though v. seminalis weight decreased only in the 4 week groups (P < 0.05). The data showed that adverse effects in the reproductive system and damage were moderate. BA has been reported to affect the oxidative metabolism [27]. This effect is related to the dose and duration of administration. Increased antioxidant capacity was demonstrated after 5−20 ppm of BA application in erythrocyte suspension [28]. BA induced antioxidant activity in blood and liver tissue when administered orally in the chronic phase (28 days) at the dose of 100 mg/kg [29]. With the same dose, BA induced positive effects on the healing of experimental nerve injury
NS NS NS NS NS NS NS NS
NS: Not Significant.
statistically different compared to the corresponding controls in both 4 and 6 weeks groups (Table 1). Testes weights (right and left together) at the end of the study decreased, but no difference was not statistically significant (Table 2). However, v seminalis weight decreased in all the groups significantly (P < 0.05). The weight of seminal glands were not equal througout the groups corresponding to the proportional variability of the seminal fluid presence or absence. Results showed that the decrease in sperm motility in the 4 and 6week group was statistically significant in the 450 mg/kg group (P < 0.05) and in the 6-week group 250 and 450 mg/kg BA group were (P < 0.001) statistically significant. The decrease in live sperm rate was mild at 4 week period, but after 6-weeks treatment there was a sudden decrease compared to the control (P < 0.001). The membrane integrity of spermatozoa reduced significantly (P < 0.001) in both 4-week and 6week groups (Table 2). Result of comet assay showed that there was no DNA damage in mice spermatozoa in 4-week BA-treated group. The DNA damage was detected in the 6-week BA-treated groups in a dose dependent manner (P < 0.001). DNA damage levels in spermatozoa were seen during the 6 week long term treatment of BA. In the group receiving 450 mg/kg, moderate and advanced DNA damages (3rd and 4th degree) were observed, while in groups 115 and 250 mg/kg, mild and moderate (2nd and 3rd degree) damages were detected (P < 0.001), (Table 4, Fig. 1). At 4 weeks of BA-treatment, MDA levels increased in 250 and 450 mg/kg groups and it was found to be significant compared to the control (P < 0.001). SOD and CAT levels were not statistically significant (Table 3). GSH level significantly decreased in 450 mg/kg BA group (P < 0.05). In 6-week groups, MDA levels in 450 mg/kg BA group were statistically higher than the other groups (P < 0.05). SOD levels were decreased in 250 mg/kg and 450 mg/kg BA groups, compared to 115 mg/ kg and control groups (P < 0.01). CAT levels were not statistically
Table 2 Semen parameters of control and Boric acid (BA) treated mice at the 4th and 6th week of the study. Groups (n = 10)
Parameters Testis weight (mg)
4-week
6-week
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
192.60 ± 5.98 171.80 ± 4.43 174.10 ± 5.98 178.40 ± 9.73 NS 194.30 ± 6.85 207.90 ± 3.88 180.80 ± 0.07 177.20 ± 1.54 NS
V. seminalis weight (mg) a
Sperm motility (%) 77.50 ± 2.38 79.00 ± 1.94 73.00 ± 1.85 69.00 ± 2.66 * 78.00 ± 1.52 72.50 ± 4.36 68.50 ± 2.24 54.00 ± 4.33 ***
356.90 ± 7,44 270.50 ± 8,43 b 311.00 ± 17.57a,b 268.90 ± 20.63 b * 280.30 ± 25.65 b,c 342.10 ± 17.83 a 320.70 ± 15.76 a,b 264.60 ± 12.52 c *
a,b a b,c c
a a,b b c
Live sperm rate (%) 73.50 ± 2.00 78.80 ± 1.44 74.30 ± 1.39 70.70 ± 1.57 ** 74,00 ± 0,93 68,00 ± 2,33 68,20 ± 1,93 57,00 ± 3,09 ***
b,c a b,c c
a b b c
Membrane integrity (%) 75.60 ± 1.78 67.90 ± 2.20 62.00 ± 2.16 64.30 ± 1.85 *** 74,70 ± 1,32 58,30 ± 2,25 60,00 ± 1,54 55,10 ± 3,26 ***
a, b, c: Different letters in the same column indicate statistically significant difference *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant. 3
a b b b
a b b b
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
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Fig. 1. Sample fluorescence microscope images of DNA damage in spermatozoa (magnification ×50).
model at the acute (3 days) phase of the injury [30]. The current study again showed statistically insignificant increases in the values of (SOD, CAT, GSH, motility, live sperm rate), somehow implying antioxidant activity, with the dose of 115 mg closest to 100 mg at the 4-week group. But, this was not true in the 6-week group as the changes in these measurements were negatively significant. These findigns suggest that prolonged application time may lead to increased oxidant activity. In male infertility, Reactive oxygen species (ROS) compounds damage the spermatozoal membrane and cause subseqent infertility [24,31,32]. Although, testicular tissue has defensive system against oxidative stress, but it is not as efficient as other somatic cells [33]. Xanthine and Xanthine oxidase caused oxidative stress in sperm, SOD and CAT enzymes showed protective effect against this [34]. Swiss Albino male mice were given dimethoate in a 30-day study and MDA levels were shown to be significantly increased [35]. Kumar et al. [36] applied 15–60 nanomol (100 g live weight) T-butyl and c-butyl peroxides for 1, 2, 3 and 5 weeks. Organic hydrogen peroxide induced oxidative stress to testicular tissue and dose depent increases in the level of MDA along with the abnormal sperm rate and spermatozoon DNA damage have been reported. In the present study, increased levels of MDA at the 4 and 6-week of BA application indicated the production of oxidative stress (Table 3). Likewise, higher doses of BA applied for an extensive period, inflicted negative effects on the sperm quality. Spermatozoons may be subjected to cellular damage during the
Table 4 Boric acid (BA) administered mice at 4th and 6th weeks of sperm damage degree and DNA damage percentage. Time period
4-week
6-week
Groups (n = 10)
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
DNA damage degree (0–4)
DNA damaged cell (%)
00.00 00.00 00.00 00.00 NS 0.00 d 0.12 ± 0.04 0.32 ± 0.07 0.66 ± 0.14 ***
00.00 00.00 00.00 00.00 NS 0.00 d 3.30 ± 1.43 c 6.20 ± 1.84 b,c 14.40 ± 2.89 a ***
c b a
a, b, c: Different letters in the same column indicate statistically significant differences *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant.
spermatogenesis phase or post-testicular sperm maturation [37]. Similary, ingestion of boron-containing compounds may cause damage during cell cycle of mitosis and meiosis (spermiogenesis) [10]. Boron mine workers had no significant differences in semen quality when compared with low, medium and high levels of boron exposure
Table 3 MDA, SOD, CAT and GSH levels in testicular tissue at the 4th and 6th weeks in Boric acid (BA) treated mice. Time period
Groups (n = 10)
Parameters MDA (nmol/mg protein)
4-week
6-week
Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P Control 115 mg/kg/day 250 mg/kg/day 450 mg/kg/day P
a
143.97 ± 5.16 128.84 ± 12.23 a 183.39 ± 6.24 b 192.07 ± 11.32 b *** 149.29 ± 13.90 b 157.53 ± 8.70 b 167.00 ± 16.50 a,b 216.91 ± 35.34 a *
SOD (U/mg protein)
CAT (k/mg protein)
GSH (mg/g protein)
8.21 ± 1.52 13.80 ± 5.39 10.34 ± 2.29 6.79 ± 0.62 NS 7.04 ± 0.31a 7.03 ± 0.52 a 5.62 ± 0.39 b 5.47 ± 1.22 b **
0.91 ± 0.20 1.11 ± 0.47 0.98 ± 0.22 0.61 ± 0.13 NS 0.79 ± 0.17 0.53 ± 0.07 0.54 ± 0.10 0.73 ± 0.10 NS
15.40 ± 2.25 a 20.90 ± 8.09 a 9.77 ± 1.54 a,b 7.31 ± 2.02 b * 16.78 ± 1.40 a 11.85 ± 0.74 b 8.20 ± 0.98 c 7.62 ± 1.19 c ***
a, b, c: Different letters in the same column indicate statistically significant differences *: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: Not Significant. 4
Journal of Trace Elements in Medicine and Biology 58 (2020) 126447
S. Aktas, et al.
(according to blood boron levels) or those who were not exposed [38]. Male rats were administered three different doses (125, 250 and 500 mg/kg) BA for 60 days. While no significant change was observed in the 125 mg administered group, there was a decrease in testicular tissue DNA levels in the 250 and 500 mg/kg BA treated samples [39]. In the present study, DNA damage observed in the 6-week group, but not in the 4-week group. Increased oxidative stress and elevated MDA levels in testicular tissue as well as the depletion of antioxidant defence molecules (SOD, CAT, GSH) in semen are thought to be detrimental to DNA synthesis. It is seen that the DNA damage is closely related to the duation of BA administration.
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