Drug and Chemical Toxicology

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Ameliorative effect of quercetin against arsenicinduced sperm DNA damage and daily sperm production in adult male rats Sarwat Jahan, Saima Rehman, Hizb Ullah, Asma Munawar, Qurat Ul Ain & Tariq Iqbal To cite this article: Sarwat Jahan, Saima Rehman, Hizb Ullah, Asma Munawar, Qurat Ul Ain & Tariq Iqbal (2015): Ameliorative effect of quercetin against arsenic-induced sperm DNA damage and daily sperm production in adult male rats, Drug and Chemical Toxicology, DOI: 10.3109/01480545.2015.1101772 To link to this article: http://dx.doi.org/10.3109/01480545.2015.1101772

Published online: 02 Nov 2015.

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Date: 10 November 2015, At: 07:41

http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–7 ! 2015 Taylor & Francis. DOI: 10.3109/01480545.2015.1101772

RESEARCH ARTICLE

Ameliorative effect of quercetin against arsenic-induced sperm DNA damage and daily sperm production in adult male rats Sarwat Jahan, Saima Rehman, Hizb Ullah, Asma Munawar, Qurat Ul Ain, and Tariq Iqbal

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Reproductive Physiology Lab, Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan

Abstract

Keywords

In this study, the protective effect of quercetin was evaluated against arsenic induced reproductive ailments in male rats. For this purpose, male rats (n ¼ 5/group) weighing 180–250 g were used. First group served as control, second group received arsenic (50 ppm) in drinking water. Third group was treated with quercetin (50 mg/kg) alone, while fourth group received arsenic + quercetin. All treatments were carried out for 49 days. After treatment, animals were killed by decapitation; testis and epididymis were dissected out. Right epididymis was minced immediately for comet assay, while left epididymis was processed for histology. Similarly, right testis was homogenized for estimation of daily sperm production (DSP) and detection of metal concentration. The results of our research revealed that arsenic treatment did not cause any significant change in body weight and testicular volume. Quercetin treatment significantly prevented tissue deposition of arsenic within the testis. Arsenic treatment caused a significant reduction in DSP, however, in the arsenic + quercetin-treated group and quercetin alone-treated group, DSP was significantly high as compared to the arsenic-treated group. Histological study of epididymis showed empty lumen in arsenic-treated group while in arsenic + quercetin-treated group and quercetin alone-treated group, lumen were filled with sperm and were comparable to control. Sperm DNA damage, induced by arsenic, was significantly reversed toward control levels by supplementation of quercetin. These results suggest that quercetin not only prevents deposition of arsenic in tissues, but can also protect the sperm DNA damage.

Arsenic, DNA damage, epididymis, quercetin, sperm production

Introduction Arsenic is a toxic element in the environment to which humans are exposed (Hughes & Kitchin, 2006). Elevated levels of arsenic consumption in water have been observed for a couple of years in numerous regions of the world, including China, India and some Central and South American countries (Focazio et al., 1999; Lewis et al., 1999; Nordstrom, 2002; Steinmaus et al., 2003). Arsenic is a major toxicant causing skin cancer (Rossman et al., 2004), damage to kidney, liver, bladder and lungs (Abernathy et al., 1999; Kitchin, 2001; Tchounwou et al., 1999). Arsenic disrupts male reproduction by affecting spermatogenesis, decrease testosterone levels and testicular enzymes (Sarkar et al., 2003). Arsenic either affects male gonads directly or modulates pituitary activity causing changes in the gonadotropins levels (Ghosh et al., 2001), which in turn causes decline in testosterone levels (Jana et al., 1999) within the testis that disrupt spermatogenesis and integrity of seminiferous tubule (Sharpe et al., 1992). It has been reported Address for correspondence: Dr. Sarwat Jahan, Reproductive Physiology Laboratory, Department of Animal Sciences, Quaid-i-Azam University, Islamabad 44000, Pakistan. Tel: +92 5190643070. Fax: +92 512601176. E-mail: [email protected]

History Received 9 February 2015 Revised 21 August 2015 Accepted 27 September 2015 Published online 30 October 2015

that arsenic produces reactive oxygen species (ROS) and oxidative stress (Chang et al., 2007). High levels of ROS cause cell death and apoptosis in the testis (Das et al., 2009), impair spermatogenesis, low sperm count, motility and compromised sperm quality (Pant et al., 2001; Sarkar et al., 2003). Arsenic accumulates in male reproductive tissues, including prostate, seminal vesicles, epididymis and testes. Accumulation of arsenic within the epididymis results in low viability of sperm (Danielsson et al., 1984) by causing sperm DNA damage and spermicidal activity (Recio et al., 2001; Sakkas et al., 1998). Tissue damage caused by arsenic can be minimized by reducing arsenic deposition to the low levels. Enhancing immunity of the individual by boosting antioxidant levels of the body also provides protection against arsenic toxicity. Naturally occurring substances like flavonoids have been reported and used for their protective effects against toxic heavy metals. Flavonoids have several natural and chemical functions, including antioxidant (Clegg & Morton, 1968; Pratt & Watts, 1964), metal chelating properties and anticancer properties (Salup et al., 1992). Quercetin, a flavonoid, found in several types of vegetables and fruits (Denis et al., 2013; Walker & Reddish, 1964), has been reported as a strong antioxidant, which is capable of reducing oxidative stress and cell death by causing chelation

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of metal ions, depletion of oxygen free radicals and provides defense against lipid peroxidation (Bu et al., 2011; Ciftci et al., 2012). Sperm DNA integrity is important for fertilization and development. Arsenic has been reported as an inducer of DNA damage through oxidative stress (Balakumar et al., 2010; Nava-Hernandez et al., 2009). Similarly, sperm DNA integrity is considered as a marker of male fertility (Zhu et al., 2004). In a previous experiment, quercetin treatment was found effective against arsenic induced tissue damage and oxidative stress (Jahan et al., 2015). However, no study has examined the effect of quercetin on arsenic mediated toxicity in epididymis sperm of adult rats. The present study was designed to investigate the effect of quercetin on epididymal sperm DNA integrity and daily sperm production (DSP) in arsenic-treated adult rats.

Materials and methods Animals Twenty male Sprague Dawley rats having age 70–90 days and weight 180–250 g were obtained from Quaid-i-Azam University’s Animal house. During the time of experiment, all the rats were kept under 12/12 h dark/light cycle, at room temperature of 22 ± 3  C. All the animals were kept in stainless steel cages and fed with standard laboratory food and water was available ad libitum. The experimental protocol was approved by the Department Ethical committee of Quaidi-Azam University, Islamabad, Pakistan. Animals were divided into four groups (n ¼ 5). Group I served as control group and remained untreated, while Group II received arsenic in drinking water (50 ppm). Group III was treated with quercetin (50 mg/kg body weight) orally and Group IV received arsenic (50 ppm) in drinking water and quercetin (50 mg/kg body weight) orally for 49 days. On the 50th day of experiment, animals were weighed and sacrificed. Quercetin (Catalog No. Q4951) was purchased from Sigma Aldrich, Munich, Germany and sodium arsenite (Case No.: 7784-46-5) was purchased from Fisher Scientific, Leicestershire, UK. Selection of dose of quercetin (50 mg/ kg body weight) was in accordance with the previous study of Prabu et al. (2013) in which quercetin was found effective against cadmium induced oxidative stress and toxicity in the cardiac tissue of rats (Prabu et al., 2013). While 50 ppm dose of arsenic was selected according to Akram et al. (2009), arsenic (50 ppm) caused DNA damage in ovarian tissues of rats (Akram et al., 2009). Testicular tissues and epididymis were dissected out and placed in ice-cold saline. Right testis was weighed and was stored at 80  C. Tissue was cut into two pieces; half was homogenized for estimation of antioxidant enzymes (Jahan et al., 2015) and estimation of DSP. Remaining half was used for estimation of metal concentration. Right epididymis was washed and minced immediately for further processing of Comet assay, while left epididymis was processed for histology. Daily sperm production Testicular tissue was thawed at room temperature for a few minutes prior to the homogenization process. Spermatids, which were resistant to homogenization (19th stage of

Drug Chem Toxicol, Early Online: 1–7

spermatogenesis) in the homogenate, were counted by the method followed by Robb et al. (1978) and Cooke et al. (1991). Tunica albuginea was removed and then parenchyma was weighed. It was homogenized in 5 ml of 0.9% NaCl for 30 s. 0.5% Triton X-100 was added and the homogenate was diluted fivefold. Twenty microliters of sample was put on Neubauer chambers, and counting of late spermatids was done under microscope at 40 magnification. Each sample was run in triplicate and average number of spermatids in each sample was counted. This value was used to get the number of all spermatids per testis and it was then divided by the testis weight to determine the number of spermatids per gram testis, which is the efficiency of sperm production. For the calculation of DSP the number of spermatids which were resistant to homogenization (per testis and per gram of testis) was divided by 6.3, which shows the number of days these spermatids remained in the seminiferous epithelium. Formula for DSP Y¼

X  100  5  20  1000, 10

Where Y is the number of spermatids present in homogenate, X is the number of spermatids that are counted in Neubauer chambers, 10 is the number of observed squares in one reading, 100 is the number of total squares in chamber, 5 is dilutions made with physiological saline, 20 ml is homogenate for loading the chamber, 1000 is to convert ml into ml, DSP, is Y/6.3 and efficiency of sperm production is DSP/weight of decapsulated testis. Atomic absorption spectrophotometry Flame atomic absorption spectrophotometry is a technique, which is commonly used for analyzing metals. Testes for arsenic analysis were dried in the oven at 60  C. Tissue was put in 65% nitric acid (Merck, Darmstadt, Germany) for 24 h. Ten milliliters HNO3 was added and samples were digested by boiling them with HNO3 on hot plate until half of the solution was left and then were filtered through 135 mm filter papers. The filtrate was diluted up to 15 ml with the help of dH2O. Samples were processed through atomic absorption spectrophotometry through atomic absorption spectrophotometer (Varian, AA 240 FS, Palo Alto, CA). Microelements of arsenic were determined. Results were expressed in ppm and were converted to mg/g of tissue using following formula. Metal concentration ðmg=gÞ ¼

Final volume of solution  Metal concentration ðppmÞ : Sample weight

Assessment of DNA damage DNA damage of individual spermatozoa was assessed by using a modified neutral single cell electrophoresis (SCGE/ comet assay) according to the method of Boe-Hansen et al. (2005). Single strand breaks (SSBs) are not more important because they are quickly repaired and are not regarded as a significant mutagenic lesion. Some of genotoxic substances do not create breaks but produce AP sites. These sites break

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DOI: 10.3109/01480545.2015.1101772

Quercetin protects against arsenic-induced damage to DNA

when the DNA is in alkaline electrophoresis solution (Collins et al., 1997). Neutral comet assay was used to measure double strand break in DNA strands. Right epididymis was minced with forceps in phosphate buffer saline (PBS) in order to collect sperm and kept at 37  C for comet assay. The sperm was diluted in PBS to get 10 000 sperm in 10 ml in the suspension. Slides were gently heated on slide warmer, covered with 100 ml of 1% regular melting point agarose prepared in distilled water at 40  C and immediately covered with a large (22 mm  50 mm) coverslip. The slides were placed in a chilled tray and left at 4  C for at least 30 min to allow the agarose to solidify. The coverslips were then removed and second layer of 85 ml low melting point agarose (LMPA) was spread on top of the first layer containing 20 ml of sperm suspension and 65 ml of 1% LMPA at 37  C coverslip and allowed to solidify. For cell lysing, the coverslip was removed and slides were placed in histology jar containing freshly prepared cold lysis buffer [pH 10.3, 2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% (w/v) Triton X-100]. The slides were incubated for 24 h at room temperature. After the incubation the slides were washed with distilled water three times (20 min each) to remove traces of salt and detergent. For electrophoresis, slides were uniformly placed in columns in the electrophoresis tray facing toward anode containing neutral electrophoresis buffer (54 g/l Tris base, 27.5 g/l boric acid, 0.5 M EDTA, pH 8.0). Electrophoresis was performed for 20 min at 25 V (0.714 V/cm). Slides were removed and were covered with aluminum foil and air-dried overnight at 5  C. The slides were rehydrated with distilled water, stained with acridine orange (300–400 ml of 20 mg/ml of distilled water) and observed under epifluorescent microscopy (400, Nikon AFX-1 Optiphot, Tokyo, Japan) and digital images were captured for subsequent analyses/scoring with comet assay TriTek Comet Score software (V. 1.5). For analysis, 200 cells were counted from four fields of each slide counting the intact DNAs and the number of comets. Pictures obtained were presented in front of student (five students) to identify comet and intact DNA. Following sperm DNA comet parameters were recorded. Comet length (CL, mm), % DNA in head (%H), tail length (TL, mm), tail DNA (TDNA, %), tail moment (TM, mm) and olive moment (OL, mm) were included in this study.

Statistical analysis

Epididymis histology After dissection, left epididymis was washed in cold physiological saline; caput and cauda were cut and placed in sera. Following dehydration in the descending grades of alcohol, tissues were clarified in cedar wood oil and then embedded in paraffin. Five-micrometer thick sections were cut out of paraffin block using Reichert Microtome. Sections were then affixed to pre-cleaned albumenized glass slides and stretched at 60  C on Fisher slide warmer. Slides were transferred to a paraffin oven for the next 12 h for the complete deparafinization. Slides were stained with hematoxylin and eosin and were examined under a Nikon optishot research microscope equipped with an automatic micro photographic system. Every 25th section was studied and morphological changes were noted.

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Data are represented as mean ± SEM. Comparison of groups was done using one-way analysis of variance followed by Tukey’s test with the help of Graph pad prism software, San Diego, CA. Level of significance was set at p50.05.

Results Body weight Animals were weighed before and after 49 days of treatment. Final body weight was not significantly different in all treated groups as compared to the control group (Table 1). Measurement of arsenic concentration in testes Arsenic exposure elevated arsenic levels in the testicular tissues of rats. A highly significant increase (p50.001) in arsenic concentration was found in the testicular tissues of rats treated with arsenic as compared to control group. In the quercetin-treated group, the levels of arsenic were decreased in the testes. Significant increase (p50.05) was found in arsenic levels in the testes of the arsenic-treated group when compared to the arsenic + quercetin-treated group. In the quercetin-treated group, there was a significant decrease (p50.001) in arsenic level in testes when compared to arsenic + quercetin group as described in Table 2. DSP and efficiency of sperm production (DSP/weight of decapsulated testis) Mean ± SEM DSP in arsenic-treated animals was significantly (p50.001) reduced compared to control animals. Quercetin co-treated animals had significantly (p50.001) high DSP as compared to the arsenic-treated group, however, DSP in quercetin co-treated group was significantly less as compared to the control group. In the quercetin alone-treated Table 1. Mean ± SEM body weights (g) of control, arsenic-treated, quercetin treated and arsenic + quercetin treated adult male rats before and after 49 days of treatment. Groups (n ¼ 5/group) Controls Arsenic Quercetin Arsenic + quercetin

Before treatment

After treatment

202.50 ± 28.68 214.00 ± 16.31 180.40 ± 4.08 251.00 ± 17.77

315.75 ± 28.54 300.00 ± 17.25 323.40 ± 11.23 311.40 ± 21.90

Values are expressed as mean ± SEM. Table 2. Mean ± SEM of arsenic concentration in the testes of control, arsenic treated, quercetin treated and arsenic + quercetin treated adult rats after 49 days of treatment. Groups (n 5/group)

Arsenic conc. in testis (mg/g)

Control Arsenic Quercetin Arsenic + quercetin

127.22 ± 0.38 406.92 ± 1.21*** 84.00 ± 0.25+++ 259.14 ± 1.30+

Values are expressed as mean ± SEM. ***Indicates significance from the control group at p50.001 probability level. +,+++Indicate significance from the Arsenic group at p50.05 and p50.001 probability levels, respectively.

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group, DSP was not significantly different as compared to the control group (Table 3). Efficiency of sperm production in arsenic-treated animals were significantly reduced (p50.001) as compared to the control group. However, in the quercetin co-treated animals, efficiency of sperm production was significantly high (p50.001) as compared to the arsenic alone-treated animals. Efficiency of sperm production in arsenic + quercetin-treated animals was significantly less (p50.001) than control animals (Table 3).

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Comet assay Damage in spermatozoa DNA was determined by comet assay. The number of comets/200 cells in different treatment groups is presented in Table 4 and photo micrographs of different groups are presented in Figure 1. The number of comets in the arsenic-treated group was significantly high (p50.01) as compared to the control group. The number of comets was significantly reduced (p50.01) in the animals receiving quercetin supplementation as oral dose. DNA damage, estimated by considering different parameters, showed statistically significant increase (p50.001) in comet length and tail length in arsenic-treated animals compared to control animals, quercetin-treated animals and arsenic + quercetin-treated animals. Comet length and tail length of quercetin-treated animals were comparable to control animals (Table 4). Tail moment was not significantly different in all groups. However, percent DNA in head (p50.05), percent DNA in tail (p50.05) and olive moment values were significantly different in arsenic-treated animals as compared to the control animals. Quercetin Table 3. Mean ± SEM sperm production in control, arsenic treated, quercetin treated and arsenic uercetin treated adult rats after 49 days of treatment.

Groups Control Arsenic Quercetin Arsenic + quercetin

DSP  106/testis

Efficiency (DSP/g of testis)

20.81 ± 0.64 5.69 ± 0.31*** 18.25 ± 0.23+++ 11.97 ± 0.39***, +++

13.92 ± 0.04 3.72 ± 0.25*** 13.00 ± 0.61*** 7.88 ± 0.40***, +++

Values are expressed as mean ± SEM. ***Indicate significance from the control group at p50.001 probability level. +++Indicates significance from the arsenic group at p50.001 probability level.

supplementation sheltered DNA damage induced by arsenic in the arsenic + quercetin-treated group. Values of percent DNA in tail, percent DNA in head and olive moment were significantly different (p50.05) in quercetin alone-treated animals as compared to arsenic-treated animals and was comparable to control (Table 4). Histological results The sections of epididymis from control group revealed normal morphology with compactly arranged tubules. The epithelium of caput and cauda in control group was thick and pseudo stratified, lined with stereo cilia and lumen was filled with spermatozoa. The tubules were surrounded by stroma. In arsenic-treated animals, there was a significant reduction (p50.001) in ductal diameter in caput and cauda compared to the control group. Tubular lumen was empty and sperm concentration was reduced in the arsenic-treated group. In the quercetin co-treated animals, tubular diameter was significantly high (p50.001) as compared to the arsenic alonetreated animals. In the quercetin alone-treated group, these changes were comparable to control group (Figure 2, Table 5).

Discussion Exposure to arsenic is a major contributing factor that leads to toxicity of male reproductive system (Sarkar et al., 2003). In the present study, treatment with arsenic for 49 days caused reduction in DSP along with alteration in epididymis histology. Arsenic concentration within the tissue was higher in the arsenic-treated group, however, quercetin supplementation was found effective against arsenic induced reduction in sperm production and exhibited metal chelating properties by reducing the tissue content of arsenic. DNA damage in the sperm was significantly reduced in the quercetin co-treated group when compared to the arsenic alone-treated animals. Testis morphological studies have shown that seminiferous tubule is composed of different types of proliferating cells, including spermatogonia, primary and secondary spermatocytes and spermatids. These cells are in close contact with the Sertoli cells that provides nourishment to the germinating cells (Holstein et al., 2003). In the first part of our experiment, lesion within the seminiferous tubule epithelium, especially in the sertoli cells were observed along with the reduction in the number of secondary spermatocytes in the arsenic-treated

Table 4. Mean ± SEM DNA damage in control, arsenic treated, quercetin treated and arsenic + uercetin treated adult rats after 49 days of treatment. Parameters Number of comets Comet length Tail length % DNA in head % DNA in tail Tail moment Olive moment

Control

Arsenic

Arsenic + quercetin

Quercetin

42 ± 5.07 148.65 ± 6.02 13.26 ± 0.82 97.45 ± 0.22 2.47 ± 0.22 1.39 ± 0.44 2.47 ± 0.42

72 ± 3.86** 191.13 ± 20.19*** 15.78 ± 0.35*** 95.99 ± 0.40* 4.01 ± 0.40* 1.63 ± 0.25 2.82 ± 0.32*

45 ± 5.13++ 146.42 ± 5.47+++ 13.80 ± 1.07+++ 98.27 ± 0.15+ 1.63 ± 0.15+ 1.37 ± 0.50 2.43 ± 0.40++

39 ± 7.35+++ 153.11 ± 12.45+++ 13.69 ± 0.75+++ 96.95 ± 0.47 3.05 ± 0.47 1.45 ± 0.50 2.51 ± 0.50+

Values are expressed as mean ± SEM. *, **, ***Indicate significance from the control group at p50.05, p50.01 and p50.001 probability levels, respectively. +, ++, +++Indicate significance from the arsenic group at p50.05, p50.01 and p50.001 probability levels, respectively.

Quercetin protects against arsenic-induced damage to DNA

DOI: 10.3109/01480545.2015.1101772

Figure 1. Fluorescent photomicrograph (40) of rat sperm DNA after 49 days of treatment using comet assay, stained with acridine orange, showing (a) control with more intact DNA, (b) comets with short tails in arsenictreated, (c) intact DNA with arsenic + quercetin-treated rats and (d) Intact DNA in quercetin alone-treated animals.

(A)

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(B)

Intact Tail

(C)

Head

(D)

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Intact

Intact

Figure 2. Photomicrograph of cauda epididymis in (A) control, (B) arsenic-treated, showing tubules with empty lumen (arrow), (C) As + quercetin; seminiferous tubule with sperms filled lumen (arrow) and (D) quercetin; showing filled lumen (arrow) 40.

(A)

(B)

(C)

(D)

group (Jahan et al., 2015), suggesting that arsenic exposure effect spermatogenesis. The present part of the study confirmed that these findings and reduced DSP was observed in the arsenic-treated group as compared to the control group. However, quercetin co-treatment protected this reduction in DSP as compared to the arsenic alone-treated group, suggesting that quercetin treatment improves DSP either by protecting the tissue damage or by reducing the metal

deposition within the tissue. Similarly, arsenic treatment affected ductal diameter of epididymis and the lumen was empty as compared to the other treated groups. These findings are in accordance with the previous observation in which tubular lesion was protected by quercetin co-treatment along with the improvement in the secondary spermatocytes within the seminiferous tubule (Ciftci et al., 2010; Jahn et al., 2014).

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Table 5. Mean ± SEM ductal diameter (mm) of epididymis in control and treated groups. Groups Control Arsenic Arsenic + quercetin Quercetin

Caput

Cauda

192.45 ± 1.28 156.80 ± 1.32*** 188.78 ± 1.28+++ 189.49 ± 1.27+++

286.03 ± 1.04 245.49 ± 0.84*** 274.55 ± 1.04***,+++ 269.48 ± 1.46+++

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Values are expressed as mean ± SEM. ***Indicates significance from the control group at p50.001 probability level. +++Indicates significance from the arsenic group at p50.001 probability level.

Mammalian sperm contains thiol protamines, which are found in their chromatin and sulfhydryl group is present in their flagellum, which confers overall maintenance of sperm. However, arsenic causes thiol inhibition and interacts with the process of maturation of sperm (Khan et al., 2013; Working et al., 1985). This supports the reduction in the number of spermatids in the present study. However, quercetin significantly averted decline in DSP and the number of spermatid/testis. Same results were reported in the previous study (Farombi et al., 2012), using 10 mg/kg dose. Oxidative stress plays central role in male infertility (Agarwal et al., 2008). Reduction in DSP might be due to the adverse changes in antioxidant system and ultimate accumulation of oxygen reactive species that cause damage to the integrity of seminiferous epithelium and germinal layer constituents, resulting in low values of daily sperm count and number of spermatids/testis (Jahan et al., 2015). Oxidative stress triggers damage to the sperm plasma membrane flexibility as well as adversely affects nuclear DNA (Farombi et al., 2012). Quercetin treatment not only improved antioxidant enzyme level in the tissue (Jahan et al., 2015), but also increased DSP. Significant decrease was found in the plasma testosterone in rats treated with arsenic, which are in accordance with the earlier study in rats (Sarkar et al., 2003), mice (Pant et al., 2001) and fish (Shukla & Pandey, 1984). It was reported that arsenic elevates adrenocortioid action and causes high levels of corticosterone (Biswas et al., 1994), which reduces level of gonadotropins and testosterone in serum (Philips et al., 1989). However, quercetin co-treatment resulted in increase in testosterone level in arsenic-treated groups (Jahan et al., 2015), hence increased DSP because high levels of testosterone are required for spermatogenesis. Arsenic levels were highly significantly high in the testicular tissues of arsenic-treated rats, however, arsenic deposition was significantly prevented by quercetin cotreatment within the tissue suggesting the metal chelating property of quercetin. In previous studies, elevated levels of arsenic were found in the arsenic-treated animals (Sarkar et al., 2003). Significant increase in arsenic concentration has been reported in the in the lungs, liver and kidney of rats after 28 days of treatment with sodium arsenite (10 mg/kg) (Islam et al., 2009). Arsenic levels within the testis were significantly reduced in quercetin co-treated groups, which are in accordance with the previous findings in which quercetin was reported as a powerful chelating agent (Leopoldini et al., 2006).

Evidence regarding DNA damage is that monomethylarsenic acid and dimethylarsenic acid cause detrimental effect to DNA through a transitional product related to the formation of reactive species of oxygen (Nesnow et al., 2002). Different parameters of comet assay showed a significant increase in the mean values of DNA damage in arsenic-treated rats. Tail length and percent DNA in the tail showed significant increase, while percent DNA was significantly reduced. These results are in agreement with the earlier study and possess injurious effects to genetic material (Patlolla et al., 2012). Quercetin treatment reduced tail length, tail moment, percent DNA in tail and olive moment. In the previous study, 50 and 100 mg/kg doses of quercetin caused decrease in broken strands of DNA. These healing effects were pronounced when treated with higher doses (Bakheet, 2011). These effects may be either due to the metal chelating potential or because of antioxidant potential of quercetin. Further studies are needed to confirm the exact mechanism underlying these findings. However, in quercetin alonetreated animals, percent DNA in the tail was increased but this increase was not significantly high as compared to control. These findings are in accordance with the in vitro models of quercetin induced DNA damage (Caria et al., 1995; Rueff et al., 1992) but contrasting results were found in in vivo model (Barcelos et al., 2011). This partial increase in DNA damage in the quercetin-treated group needs to be investigated again with different doses and in vivo experimentation.

Conclusion From this study, it is concluded that sodium arsenite has the ability to cause male reproductive organs toxicity and adverse effects on DSP and DNA damage. Treatment with quercetin rectified injurious effects caused by arsenic. The present findings suggest that quercetin can be used as a potent drug against arsenic induced reproductive toxicity.

Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This piece of work was funded by the Department of Animal Sciences Quaid-i-Azam University Islamabad, Pakistan.

References Abernathy CO, Liu YP, Longfellow D, et al. (1999). Arsenic: health effects, mechanisms of actions, and research issues. Environ Health Perspect 107:593–597. Agarwal A, Makker K, Sharma R. (2008). Clinical relevance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol 59: 2–11. Akram Z, Jalali S, Shami S, et al. (2009). Genotoxicity of sodium arsenite and DNA fragmentation in ovarian cells of rats. Toxicol Lett 1:81–85. Bakheet SA. (2011). Assessment of anti-cytogenotoxic effects of quercetin in animals treated with topotecan. Oxid Med Cell Longev 2011;2011:824597. Balakumar B, Ramanathan K, Kumaresan S, et al. (2010). DNA damage by sodium arsenite in experimental rats: ameliorative effects of antioxidant vitamins C and E. Ind J Sci Tech 3:322–327. Barcelos GRM, Grotto G, Serpeloni JM, et al. (2011). Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats. Arch Toxicol 85:1151–1157.

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Quercetin protects against arsenic-induced damage to DNA

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