ORIGINAL ARTICLE
Mitigation of paracetamol-induced reproductive damage by chrysin in male rats via reducing oxidative stress 2 € € u € r1, E. Eldutar3, S. Ku € cßu € kler3 & S. C E. H. Aksu1, M. Ozkaraca , F. M. Kandemir3, A. D. Om ß omaklı2
€rk University, Erzurum, Turkey; 1 Department of Reproduction and Artificial Insemination, Veterinary Medicine Faculty, Atatu €rk University, Erzurum, Turkey; 2 Department of Pathology, Veterinary Medicine Faculty, Atatu €rk University, Erzurum, Turkey 3 Department of Biochemistry, Veterinary Medicine Faculty, Atatu
Keywords Bax/Bcl-2—caspase-3—male rat—paracetamol—sperm Correspondence Emrah Hicazi Aksu, PhD, Department of Reproduction and Artificial Insemination, €rk Veterinary Medicine Faculty, Atatu University, 25240 Erzurum, Turkey. Tel.: +904422317132; Fax: +904422317244; E-mail: [email protected] Accepted: January 3, 2016 doi: 10.1111/and.12553
Summary Paracetamol (PRC) is a nonsteroidal anti-inflammatory drug used widely as a painkiller for various diseases and as the symptomatic flu cure in several countries worldwide. PRC toxicity may occur under conditions of the overdose usage. Chrysin (CR) is a flavonoid that is naturally present in several plants, honey and propolis. The aim of this study was to investigate the effects of CR (at the doses of 25 mg kg1 and 50 mg kg1) pre-treatment over seven consecutive days against PRC-induced reproductive toxicity in male rats. Our results showed that PRC toxicity decreased the sperm motility, and increased dead sperm rate, abnormal sperm cell rate, apoptosis and MDA levels in testicular tissues. Pre-treatment with CR at the dose of 25 and 50 mg kg1 for 7 days mitigated side effects of acute PRC toxicity in male reproductive system proportionally in a dose-dependent manner. This possible protection mechanism might be dependent on the antioxidant activity of CR. In conclusion, pre-treatment with CR at the dose of 25 and 50 mg kg1 for 7 days can be the beneficial against PRC-induced reproductive toxicity proportionally in a dosedependent manner.
Introduction Paracetamol (N-acetyl-p-aminophenol (PRC)), also known as acetaminophen, is a nonsteroidal anti-inflammatory, analgesic and antipyretic drug used widely as a painkiller for various diseases and as the symptomatic flu cure in several countries worldwide. PRC is generally accepted to be safe if it is provided and used at doses recommended by physicians. Alternatively, this drug may have harmful effects if it is used at the excess dose. Some researchers suggesting that PRC toxicity may occur under conditions of the overdose usage. Regular usage of the drug is also a risk factor for drug toxicity. PRC affects several regions of the body to produce various types of toxicity including hepatotoxicity (Zhao et al., 2011; Scheiermann et al., 2013; El-Shafey et al., 2015), renal toxicity (Zhao et al., 2011; El-Shafey et al., 2015), reproductive toxicity (Ratnasooriya & Jayakody, 2000; Yano & Dolder, 2002) and even cardiomyocyte toxicity (Jin & Park, 2012). The wide use of the drug gives an elevated risk for toxicity. The toxicological effects of PRC are probably related to increased reactive oxygen species (ROS), induced apoptosis and DNA damage (Jin & Park, © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
2012). Ratnasooriya & Jayakody (2000) suggested that treatment with PRC (500 mg kg1 or 1000 mg kg1) for consecutive 30 days caused to decrease in libido, sexual vigour and fertility index in male rats. They also suggested that all these detrimental effects were reversible. Several agents have been well studied that may be able to mitigate or totally protect against some of the side effects by PRC, such as the increase in ROS levels. When PRC is taken by the oral way, it is absorbed quickly in the gastrointestinal system. After 30 min following the drug is taken, it reaches the maximum plasma concentration and disturbed all tissues (Larson, 2007). The half-life of PRC is about 150 min. (Graham & Scott, 2005). To protect or mitigate PRC’s side effects, the use of antioxidant agents is advised by some researchers. For example, Kim et al. (2015) suggested that metformin administration (30 min. before PRC treatment at the dose of 350 mg kg1, p.o.) mitigates PRC-induced hepatotoxicity in mice. El-Shafey et al. (2015) reported that quercetin treatment (at the dose of 15 mg kg1) for twenty-one consecutive days had protective effects against renal toxicity induced by a single injection of PRC (at the dose of 3 g kg1). Furthermore, treatment with interleukin-22 1
Chrysin reduces paracetamol-induced sperm damage
(single dose of 3.5 ug) had a preventive impact against PRC-induced hepatotoxicity according to a study by Scheiermann et al. (2013). Similarly, Kim et al. (2014) suggested that pre-treatment with phospholipase A2, found in bee venom, at the dose of 0.2 mg kg1 for 5 days before a single injection of PRC at 500 mg kg1 had a protective effect against hepatotoxicity in mice. Chrysin (chemically named 5,7-dihydroxyflavone (CR)) is a flavonoid that is naturally present in several plants, honey and propolis. It has some pharmacological and biological properties including antioxidant, anti-inflammatory (Mantawy et al., 2014), anticancer (Khoo et al., 2010), anti-diabetic, anti-allergic (Kasala et al., 2015), anti-apoptotic (Jiang et al., 2014) and antihypertensive (Villar et al., 2002) activities. Especially hydroxyl groups of CR at 5th and 7th carbons give it the antioxidant property (Sathiavelu et al., 2009). The plasma membrane protective and free radical scavenging effects of CR help to keep free radical levels under control (Pushpavalli et al., 2010). Walle et al. (2001) suggested that CR reaches the peak plasma concentration in 1 h. Few studies have examined the effects of acute PRC toxicity on male reproductive system. Furthermore, only a few studies have examined the effects of CR against PRC-induced toxicity. However, the studies have not mentioned any information concerning the male reproductive system. The aim of this study was to investigate the effects of CR pretreatment over seven consecutive days against PRCinduced reproductive toxicity in male rats. Material and methods
tion has toxic effects. Our dosage choice for PRC is similar with these studies (Graham & Scott, 2005; Kim et al., 2014). Ciftci et al. (2012) suggested that treatment with CR at a dose of 50 mg kg1 increased sperm motility, sperm cell density and serum testosterone levels; this dose also decreased abnormal sperm rates. Darwish et al. (2014) suggested that 50 mg kg1 CR is more protective than 25 mg kg1 CR against testicular dysfunction of adjuvant arthritic rats. Rehman et al. (2014) suggested that oral treatment with chrysin (at the dose of 25 and 50 mg kg1 bw1) protected the CP-induced hepatotoxicity by reducing OS. We preferred 50 and 25 mg kg1 CR doses against to acute PRC toxicity. Rats were divided into four groups as; Group I (n = 7); referred as Control (C) group, rats received physiological saline only via oral gavage for consecutive 7 days before oral gavage of physiological saline. Group II (n = 7); named as PRC, the rats received physiological saline only via oral gavage for consecutive 7 days before PRC application (500 mg kg1). Group III (n = 7); referred as PRC+CR-25, rats received 25 mg kg1 CR by oral gavage for consecutive 7 days before oral PRC (500 mg kg1) application. Group IV (n = 7); referred as PRC+CR-50, rats received 50 mg kg1 CR by oral gavage for consecutive 7 days before oral PRC (500 mg kg1) application. After 24 h from the PRC applications, rats were decapitated under inhalation anaesthesia with isoflurane (IsoFloâ; Abbott, Queenborough, UK). The approval of Committee for Institutional Animal Care and Use was provided from Atat€ urk University Local Board of Ethics (the approval number: 2015/61) before the study had been planned. Collection of samples
Chemicals _ TurPRC (Parol 500 mg tablet; Atabay Co., Istanbul, key) was purchased from a pharmacy and CR (97% purity; cat no: C80105 Sigma-Aldrich Co., St. Louis, Missouri, USA) was purchased from a medical market. All other chemicals were analytical purity (Sigma-Aldrich Co.). â
Animals and experimental procedure In this study, twenty-eight male Sprague–Dawley rats (Specific Pathogen Free), 10-week-old and weighing 250– 300 g, were used as the animal material. The rats were purchased from Atat€ urk University Medical Experimental Application and Research Centre. The rats were kept under standard laboratory conditions (40% humidity, 24 °C, a 12 h light: 12 h dark cycle), in the abovementioned centre. Rats were fed with a commercial pellet chow and fresh drinking water was available ad libitum. Some studies suggested that 500 mg kg1 PRC applica2
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Following decapitation procedure, the testes and cauda epididymidis of the rats were removed from the corpse and cleaned from connective tissues such adipose or connective tissues with anatomical scissors and tweezers. Both testes and cauda epididymidis were weighed and recorded as total testes weight (TTW) and total cauda epididymidis weight (TCEW). For the pathological examinations, one of the testes was kept in Bouin’s solution while another one was kept in deep freeze immediately (at 20 °C) for biochemical assays by classifying for their groups. Semen evaluation One of cauda epididymidis was used to obtain semen sample for each animal. For this purpose, randomly selected cauda epididymidis was minced in Petri dish including 5 ml of physiological saline. To provide the migrations of spermatozoa from cauda epididymidis to fluid, 5 min incubation period was obtained on warmed © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
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stage (at 35 °C). Following the incubation period, cauda epididymidis residue was removed by using anatomical tweezers from the Petri dish. The fluid remaining in the Petri dish was used as semen sample. Evaluation of semen was conducted using routine spermatological parameters including motility, density of sperm cells, dead sperm rate and morphological examination of spermatozoa. To evaluate the percentage of sperm motility, light microscope (Primo Star; Carl Zeiss, Oberkochen, Germany) equipped with the heated stage was used. Briefly, a slide was placed on a heated stage warmed up to 35 °C placed on a conventional light microscope. Approximately 20 ll of semen sample was dropped on the slide. The percentage of sperm motility was detected by visual investigation of the sample. To estimate the sperm motility, randomly selected three different fields from each sample were evaluated. The average of three field estimations was calculated as the final motility score of the sample (Turk et al., 2008). To determine of sperm cells concentration, the method described in our previous study (Aksu et al., 2015) was used. Briefly, the semen sample was diluted as the rate of 1/100 with eosin solution (2 g dry eosin dye and 3 g sodium citrate solved in 100 ml of distilled water) in Eppendorf tube. The Eppendorf tubes were vortexed at 2500 rpm for 15 s and sperm suspension was transferred into the counting chambers of Thoma chamber. Then, sperm cells in both chambers were counted under the conventional light microscope (Zeiss Primo Star) at the magnification of 4009. To determine the percentage of morphological abnormality of spermatozoa, the method (with a little modification by using only eosin dye instead of eosin-nigrosin dye) described by Turk et al. (2008) was used. Briefly, the slides were stained with eosin dye. Then, the slides were evaluated under light microscope at 4009 magnification with the help of immersion oil (immersion oil for microscopy type A, no: 1.515; Nikon, Tokyo, Japan). Two hundred and fifty spermatozoa from each slide were observed and the percentages of sperm head, sperm mid, sperm tail and total abnormality of spermatozoa were stated. Biochemical evaluations of testicular tissues For assaying MDA, GSH levels and CAT activity homogenates were centrifuged for 15 min at 1000 g at +4 °C while to assay the GSH-px activity of testicular homogenates were centrifuged for 20 min at 9000 g at +4 °C. Following thecentrifuge process, the obtained supernatant was subjected to enzyme assays as soon as possible. The malondialdehyde (MDA) level of testicular tissues was measured by the thiobarbituric acid reaction method of Placer et al. (1966). The homogenisation of testicular tissues was carried out in Teflon-glass homogenizer with a buffer © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
Chrysin reduces paracetamol-induced sperm damage
containing 1.15% KCl to obtain 1 : 10 (w/v) whole homogenate. The values of MDA were expressed as nmol g1 tissue. The GSH content of testicular homogenates was determined at 412 nm according to the method, described by Sedlak & Lindsay (1968) and GSH levels were expressed as nmol g1 tissue. The CAT activity of testicular tissue was determined according to the method of Aebi (1983). The values of CAT were expressed as catal g1 protein. The GSH-Px activity of testes was determined using the method of Lawrence & Burk (1976). The GSH-px activity of testicular homogenates was expressed as U g1 protein. To assay superoxide dismutase (SOD) activity of testicular tissues, the method of Sun et al. (1988) was used. The SOD activity of testes was measured as the level of decrease in the absorbance at 560 nm and SOD values of testicular homogenates were expressed as U g1 protein. The protein content of the testicular tissues was measured according to the method described by Lowry et al. (1951). Immunohistological examinations The testes were removed instantly, fixed in bouin’s solution, embedded in paraffin blocks, sectioned 5 lm in thickness and stained with immunohistochemistry and then examined under a light microscope at 920 magnification. After deparaffinisation process, the slides were immersed in antigen retrieval solution (pH 6.0) and heated in a microwave for 15 min to unmasked antigens. The sections were then dipped in 3% H2O2 for 10 min to block endogenous peroxidase and then incubated at room temperature with polyclonal rabbit anti-Bax polyclonal antibody (cat. no. bs0127R, dilution 1/200; Bioss, Massachusetts, USA), polyclonal rabbit anti-active caspase 3 (cat. no. NB600-1235, dilution 1/200; Novusbiological, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (cat. no. ab29, dilution 1/100; Abcam, Cambridge, UK) and rabbit anti Bcl-2 polyclonal antibody (cat. no. bs0032R; dilution 1/200; Bioss). Expose mouse and rabbit specific HRP/DAB detection IHC kit (cat. no. ab80436; Abcam) was used as follows and eventually with 3,30 diaminobenzidine + chromogen. The slides were counterstained with haematoxylin. The slides in the sections were graded as 0 (none), 1 (mild), 2 (moderate) and 3 (severe). Similarly, in immunohistological evaluations after the slides prepared they evaluated semi-quantitatively under a light microscope and ranked as 0 (none), 1 (mild), 2 (moderate) and 3 (severe) considering their dye density status. Statistical analyses Spermatological parameters, biochemical values and testicular traits of the groups were presented as mean standard error of means (SEM). The differences were 3
Chrysin reduces paracetamol-induced sperm damage
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regarded significant when P < 0.05. Statistical analyses of the values were done by analysis of variance (One-way ANOVA) and post hoc Duncan test using the IBM SPSS/PC (Version 20.0, IBM Co., North Castle, New York, USA) software program. Also, spermatological parameters were analysed to determine potential correlations exist between them with Pearson─correlation test. Also, the histological evaluations were analysed with the Kruskal─Wallis test in IBM SPSS/PC (Version 20.0) software program. Dual crosschecks between groups displaying significant values were estimated with Mann─Whitney U-test (P < 0.05).
between all groups for TTW and TCEW values. In other words, experimental acute PRC toxicity did not affect TTW and TCEW in male rats. Semen evaluations Spermatological parameters are presented in Table 1. As seen in Table 1, treatment of PRC alone significantly (P < 0.01) decreased the motility rates and increased dead sperm rates, while pre-treatment for seven consecutive days with CR (CR-25 and CR-50) significantly (P < 0.01) mitigated the side effects of PRC on these parameters when compared to the control group. Although there was a decrease in sperm cell density with both the PRC and treatment groups when compared to the control group, this reduction was not statistically significant. With regard to spermatological abnormalities in all groups, PRC toxicity led to an increase (P < 0.01) in the
Results Reproductive organ weights Reproductive organ weights of all groups are presented in Table 1. As shown in Table 1, there was no difference Table 1 Reproductive and biochemical parameters of all groups Parameters/Groups
Control
TTW (g) TCEW (g) Motility (%) Dead sperm rate (%) Density (9106) Abnormal Sperm head (%) Abnormal Sperm mid (%) Abnormal Sperm tail (%) Total of Abnormal (%) MDA (nmol g1 tissue) GSH (nmol g1 tissue) CAT (catal g1 protein) GSH-px (U g1 protein) SOD (U g1 protein)
3.047 0.547 65 14.4 153.214 3.5 0.9 6.9 11.3 26.75 5.34 5.50 11.25 20.16
PRC
0.104 0.017 1.5a 1.4a 12.975 0.2a 0.2a 1.2 1.3ab 0.39a 0.04a 0.17a 1.41a 0.20a
2.924 0.478 40 29.3 135.833 5.9 2.1 6.1 14.0 49.17 3.26 3.27 7.29 13.80
PRC+CR-25
0.123 0.051 1.8c 0.8c 20.572 0.6c 0.5b 1 1.3b 0.83d 0.05d 0.07d 0.11c 0.20c
2.766 0.504 55 22.3 137.083 5.3 1.3 7.3 13.9 38.03 4.23 3.81 8.88 13.75
PRC+CR-50 0.150 0.022 1.8b 1.2b 11.626 0.6bc 0.3ab 0.6 0.5b 0.46c 0.04c 0.06c 0.12bc 0.41c
3.138 0.536 50 18.6 124.583 3.9 1.1 5.1 10.1 29.56 4.90 4.46 10.32 17.46
Sign. 0.880 0.023 2.4b 1.8b 16.563 0.4ab 0.4ab 0.6 0.6a 0.52b 0.07b 0.10b 0.16ab 0.32b
N.S. N.S. P < 0.01 P < 0.01 N.S. P < 0.01 P < 0.05 N.S. P < 0.05 P < 0.001 P < 0.001 P < 0.001 P < 0.01 P < 0.001
(a–c): Different superscript letters in same row indicate statistical differences. Sign., Significant; N.S., Not Significant; PRC, Paracetamol (500 mg kg1); CR-25, Chrysin (25 mg kg1); CR-50, Chrysin (50 mg kg1). Table 2 Correlations between sperm characteristics and biochemical parameters of testes tissues Motility Motility Dead Sperm Head Abn. Mid Abn. Tail Abn. Total Abn MDA GSH CAT GSH-px SOD
** ** *
() () ()
Dead Sperm
Head Abn.
**
**
()
** ** **
() (+)
Mid Abn * **
Tail Abn.
Total Abn.
**
() (+)
** **
(+) (+)
** **
** **
() ** (+) ** (+) ** (+) ** (+)
** ** ** ** **
(+) (+) () () () ()
** ** ** ** * **
(+) (+) () () () ()
** ** * **
(+) (+) () ()
MDA
**
(+) (+) (+) (+)
() (+) ** (+) ** (+) **
GSH ** ** ** *
*
(+) ** * *
(+) () ()
** ** ** **
*
()
**
() () () ()
**
(+) () () () () ()
CAT ** ** ** **
() () ** (+)
(+) (+)
** ** *
(+) () ()
*
**
** **
(+) () () ()
GSH-px
**
(+) (+)
SOD ** ** **
(+) () ()
*
**
() ** (+) ** (+) **
() () ** (+) ** (+) ** (+)
**
(+)
Correlation is significant at the P < 0.05 level (2-tailed). Correlation is significant at the P < 0.01 level (2-tailed). Presence of the minus symbol () indicates negative correlation while plus symbol (+) indicates positive correlation. *
**
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Chrysin reduces paracetamol-induced sperm damage
percentage of sperm-head abnormalities. However, pretreatment with CR-25 had no effect against this side effect, while pre-treatment with CR-50 attenuated the percentage of sperm-head abnormalities. The toxicity led to an increase in mid-sperm abnormalities in the PRC group while it did not affect the percentage of sperm-tail abnormalities (P < 0.05). Although pre-treatment with CR-25 and CR-50 decreased the mid-sperm abnormality rate induced by PRC toxicity, there was no significant difference between the PRC and treatment groups in this parameter. Furthermore, when the total abnormalities of all groups were considered, the PRC+CR-50 group had significantly fewer abnormalities than the PRC alone group. Pre-treatment with CR at a dose of 50 mg kg1 had a protective effect against an increased sperm abnormality percentage induced by PRC toxicity when compared to controls and the lower dose pre-treatment group (CR-25).
CR when compared to the PRC group. There was also a significant difference between treatment groups with both GSH and CAT activity being reduced to a greater extent in the CR-50 group. GSH-px and SOD activity decreased in the PRC group when compared to the control group. Furthermore, while the CR-50 significantly increased (P < 0.01 for GSH-px and P < 0.001 for SOD activities), CR-25 dose was ineffective in altering GSH-px and SOD activities. According to our results, CR has a reductive effect in proportion to the treatment dose against oxidative stress induced by PRC. These results might be attributed to the antioxidant effect of CR.
Biochemical evaluations of testicular tissue As seen in Table 1, MDA levels in the PRC group were significantly higher than in the control and treatment groups. Also, in terms of MDA levels, in treatment groups there was a proportional difference when compared to each other and the lowest level (P < 0.001) of MDA was found in the control group. With regard to GSH and CAT activity, the highest levels were in the control group, while the lowest values were in the PRC group. However, there was a proportionally significant increase in these activities in the treatment groups with
Graph 1 Bax expressions levels of all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
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Fig. 1 (a–d) Bax expression (arrowhead) of Control group (a), PRC group (b), PRC+CR-25 group (c), and PRC+CR-50 group (d).
© 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
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Chrysin reduces paracetamol-induced sperm damage
Correlations between spermatological parameters and biochemical parameters of testicular tissues are presented in Table 2. As shown in Table 2, there was a positive correlation between motility and antioxidant enzyme (including; CAT, GSH-px, and SOD) and GSH levels of the testis, and a negative correlation between motility and dead sperm rates, sperm-head abnormalities, mid-sperm abnormalities and MDA levels of the testes. There was a positive correlation between dead sperm rates and sperm-head abnormalities, mid-sperm abnormalities, total sperm abnormalities and MDA levels of the testes. However, there was a negative correlation between dead sperm rates and motility and antioxidant enzyme levels of testicular tissue. For sperm-head abnormalities, there was a positive correlation between these abnormalities and dead sperm rates, total sperm abnormalities, and MDA levels of the testes, while there was a negative correlation between sperm-head abnormalities and sperm motility, GSH, CAT, GSH-px and SOD activity of the testes. With regard to mid-sperm abnormalities, there was a positive correlation between these and dead sperm rates, total abnormalities and MDA levels of the testes. However, there was a negative correlation between midsperm abnormalities and motility, GSH level and CAT activity of the testes. For the sperm-tail abnormalities, there was a positive correlation between sperm-tail abnormality and total abnormality of spermatozoa. These results suggest that increased lipid peroxidation (MDA level of testicular tissue) caused the decreases in sperm motility and antioxidant enzymes levels in the testes. Alternatively, increased lipid peroxidation led to
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increases in dead sperm rates, sperm-head abnormalities, and mid-sperm abnormalities. The correlation table (Table 2) also indicates that OS did not affect all spermatological parameters equally. Some parameters were affected at more severe levels from OS while other parameters affected at relatively light levels or not affected at all. Immunohistochemical examinations Bax was expressed slightly in the control group (Fig. 1a). In the PRC and PRC+CR-25 groups, Bax expression was
Graph 2 Caspase-3 expression levels of all groups. (a–d) Different letters indicate statistical difference among the groups (P < 0.05).
Fig. 2 (a–d) Caspase 3 expression of Control group (a). Caspase 3 expression of (arrowhead) of PRC group (b), (arrowhead) of PRC+CR-25 group (c) and PRC+CR-50 group (d).
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severe (Fig. 1b,c). However, Bax expression in the PRC+CR-50 group was statistically decreased when compared to both PRC and PRC+CR-25 groups, but it was increased when compared to the control group (Fig. 1d, Graph 1). Caspase-3 activity was expressed slightly in the control group (Fig. 2a). The highest caspase-3 activity was detected in the PRC group, and CR (both CR-25 and CR-50) treatments significantly (P < 0.05) decreased the caspase-3 activity in a dose-dependent manner. There was also a significant difference between the PRC+CR-25 and PRC+CR-50 groups with regard to caspase-3 activity (Fig. 2b,c,d, Graph 2). PCNA was expressed in the control group at very light levels. PCNA in the PRC and PRC+CR-25 groups was expressed as increased with a significant difference (P < 0.05) between the two groups (Fig. 3a,b,c). However, PCNA expression levels in the PRC+CR-50 group significantly decreased when compared to the PRC and PRC+CR-25 groups, but it was not as low as in the control group (Fig. 3d, Graph 3). Bcl-2, known as an anti-apoptotic marker, was expressed at high levels in the control group (Fig. 4a) while Bcl-2 expression decreased significantly (P < 0.05) in both the PRC and PRC+CR-25 groups. However, the Bcl-2 expression level in the PRC+CR-50 group was similar to the control group (Fig. 4b,c,d, Graph 4). Bax, caspase-3 and Bcl-2 were expressed in intertubular fields while PCNA was expressed from both intertubular fields and germinal cells in the seminiferous tubules of all groups.
Discussion PRC is used widely as a painkiller in various diseases and as symptomatic cure for flu in several countries. Although, it is generally accepted to be safe when taken in doses recommended by physicians. The common usage of this drug makes the precautions against its toxicological side effects more important. Toxic effects of the drug appear in certain types of tissues including the liver, kidneys and testes. These side effects may be attributed to ROS increasing the effects of PRC. ROS are produced physiologically and are required for some cellular
Graph 3 PCNA expression levels of all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
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Fig. 3 (a–d) PCNA expression of Control group (a). PCNA expression (arrowhead) of PRC group (b), PRC+CR-25 group (c) and PRC+CR-50 group (d).
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mechanisms, such sperm maturation, sperm-oocyte interactions and capacitation of sperm cells (Ko et al., 2014). It is well known that increased ROS levels cause OS and, generally in this condition, the balance of oxidant/antioxidant status in the body is broken and tissues may be damaged in severe situations if this imbalance is not corrected. Increased ROS levels may stem from lipid peroxidation induced by various physiological factors, they also have negative effects on spermatological parameters, the reproductive system and hence male fertility (Ko et al., 2014). According to results of a study by Ratnasooriya & Jayakody (2000), PRC toxicity for over long periods causes reductions in fertility rates, sexual performance, libido and sexual vigour in male rats. Yano & Dolder (2002) reported that after 5, 10 and 50 days of single PRC injections at doses of 4.4 mmol kg1, testicular alterations in male rats were observed. Although these PRC side effects of the testes are reversible (Ratnasooriya & Jayakody, 2000), treatment with an antioxidant compound can be beneficial in protecting against or mitigating the damage. On the other hand, Ciftci et al. (2012) suggested that treatment with CR at a dose of 50 mg kg1 increased sperm motility, sperm cell density and serum testosterone levels; this dose also decreased abnormal sperm rates. These researchers also found that CR treatment increased antioxidant enzyme levels (CAT, SOD and GSH-px) and GSH levels. Similarly, Dhawan et al. (2002) suggested that administering CR at a dose of 1 mg kg1 for 30 days increased the libido, sperm count and fertilisation potential in 2-year-old male rats.
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Our results showed that PRC treatment caused decreases in sperm motility, live sperm rate and oxidant enzyme levels including, GSH, CAT, GSH-px and SOD activities; however, it also increased sperm abnormality rates, including abnormalities to the sperm head, mid sperm, and total sperm. As a matter of fact, increases in MDA (an indicator of lipid peroxidation) levels were related to an excess production of ROS and probably was responsible for the side effects of PRC. However, some reproductive parameters were not affected by PRC toxicity, including TTW, TCEW and sperm tail abnormalities. Wiger et al. (1995) reported that treatment with PRC at the dose of 400 mg kg1 for 5 days caused the reductions
Graph 4 Bcl-2 expression levels of the all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
Fig. 4 (a–d) Bcl-2 expression (arrowhead) of Control group (a). Bcl-2 expression (arrowhead) of PRC group (b), PRC+CR-25 group (c) and PRC+CR-50 group (d).
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in relative testis weights in mice. Since our study included a single injection of PRC, our results on reproductive organ weights (TTW and TCEW) were not altered. Although there was a numerical decrease in sperm cell density in the PRC and treatment groups when compared to the control group, it was not statistically significant. We detected that pre-treatment with CR-25 had attenuating effects against PRC-induced reproductive damage, including sperm motility, dead sperm rates, MDA and GSH levels and CAT activity. However CR-50 had attenuating effects on sperm motility, dead sperm rates, abnormal sperm heads, total abnormal sperm rates, MDA, GSH and GSH-px levels and CAT and SOD activities. It clearly shows that higher doses of CR (50 mg kg1) were more effective against PRC-induced reproductive toxicity than lower doses (25 mg kg1). Bax and Bcl-2 are two important genes that play a role in the apoptotic process. Bax protein, used as a pro-apoptotic biomarker, stimulates apoptotic cell death, while Bcl-2 protein, an anti-apoptotic marker, inhibits this process (Baell & Huang, 2002; Chan & Yu, 2004; Kirkin et al., 2004) PCNA is a biomarker that appears in the early stages of testicular toxicity and may be used in early estimations of testes toxicity (D’Andrea et al., 2008). Caspase-3 can be defined as the key mediator of apoptosis in mammalian cells because it initiates the apoptotic process by activating other caspase enzymes. However, caspase-3 causes the division of nuclear and cytosolic substrates, chromatin condensation, DNA fragmentation and apoptotic bodies (Salvesen & Dixit, 1997). Alternatively, ROS promote apoptosis by a mitochondrial-dependent pathway (Gach et al., 2015). Various studies have indicated that certain chemicals, such as cisplatin (T€ urk et al., 2011), malathion (Geng et al., 2015) and methotrexate (Vardi et al., 2009) induce the apoptotic process. Although Boulares et al. (2002) reported that PRC induces apoptosis in lymphocytes and hepatoma cells in humans, no study has examined the effect of PRC on apoptotic activity in testicular tissue. Our results showed that there was a significant increase in Bax expression in all the treatment groups (PRC, PRC+CR-25, and PRC+CR-50) when compared to the control group. While Bax expression levels of PRC and PRC+CR-25 groups were similar, the levels with PRC+CR-50 were statistically lower than them. With regard to both caspase-3 activity and PCNA expression, the lowest values were in the control group while the highest values for both belong to the PRC alone group. Although CR treatments against PRC toxicity mitigate caspase-3 activity and PCNA expression when compared to PRC alone, the higher CR dose had significantly better protective effects than CR-25 for the both parameters. © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
Chrysin reduces paracetamol-induced sperm damage
The highest Bcl-2 activities were in the control and PRC+CR-50 groups while the lowest expression levels were in the PRC alone group. Pre-treatment with CR-25 also significantly increased the Bcl-2 expression level compared to PRC alone group. In conclusion, when considering all our results, PRC toxicity decreased the spermatological quality while increasing apoptosis and MDA levels in testicular tissues. As expected, the higher of CR exhibited higher antioxidant activity. This possible protection mechanism might be dependent on the antioxidant activity of CR although its presence increased the absorption rates of PRC. Moreover, pre-treatment with CR against PRC toxicity mitigates some of the harmful side effects of PRC proportionally in a dose-dependent manner by free radical scavenging, anti-apoptotic, plasma membrane protective properties of it. References Aebi H (1983) Catalase. In: Methods in Enzymatic Analysis. Bergmeyer HU (ed). Academic Press, New York, pp 276– 286. € € ur AD, Ucßar O € (2015) Aksu EH, Akman O, Ozkaraca M, Om€ Effect of Maclura Pomifera extract on cisplatin-induced damages in reprodutive system of male rats. Kafkas Univ Vet Fak Derg 21:397–403. Baell JB, Huang DC (2002) Prospects for targeting the bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 64:851–863. Boulares AH, Zoltoski AJ, Stocia BA, Cuvillier O, Smulson ME (2002) Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes. Pharmacol Toxicol 90:38–50. Chan SL, Yu VC (2004) Proteins of bcl-2 family in apoptosis signalling: from mechanistic insights to therapeutic opportunities. Clin Exp Pharmacol Physiol 31:119–128. Ciftci O, Ozdemir I, Aydin M, Beytur A (2012) Beneficial effects of chrysin on the reproductive system of adult male rats. Andrologia 44:181–186. D’Andrea MR, Lawrence D, Nagele RG, Wang CY, Damiano BP (2008) PCNA indexing as preclinical immunohistochemical biomarker for testicular toxicity. Biotech Histochem 83:211–220. Darwish HA, Arab HH, Abdelsalam RM (2014) Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: comparison with celecoxib. Toxicol Appl Pharmacol 279:129–140. Dhawan K, Kumar S, Sharma A (2002) Beneficial effects of chrysin and benzoflavone on virility in 2-year-old male rats. J Med Food 5:43–48. El-Shafey MM, Abd-Allah GM, Mohamadin AM, Harisa GI, Mariee AD (2015) Quercetin protects against acetaminophen-induced hepatorenal toxicity by reducing
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reactive oxygen and nitrogen species. Pathophysiology 22:49– 55. Gach K, Długosz A, Janecka A (2015) The role of oxidative stress in anticancer activity of sesquiterpene lactones. Naunyn Schmiedeberg’s Arch Pharmacol 388:477–486. Geng X, Shao H, Zhang Z, Ng JC, Peng C (2015) Malathioninduced testicular toxicity is associated with spermatogenic apoptosis and alterations in testicular enzymes and hormone levels in male Wistar rats. Environ Toxicol Pharmacol 39:659–667. Graham GG, Scott KF (2005) Mechanism of action of paracetamol. Am J Ther 12:46–55. Jiang Y, Gong FL, Zhao GB, Li J (2014) Chrysin suppressed inflammatory responses and the inducible nitric oxide synthase pathway after spinal cord injury in rats. Int J Mol Sci 15:12270–12279. Jin SM, Park K (2012) Acetaminophen induced cytotoxicity and altered gene expression in cultured cardiomyocytes of h (9)c(2) cells. Environ Health Toxicol 27:e2012011. Kasala ER, Bodduluru LN, Madana RM, V AK, Gogoi R, Barua CC (2015) Chemopreventive and therapeutic potential of chrysin in cancer: mechanistic perspectives. Toxicol Lett 233:214–225. Khoo BY, Chua SL, Balaram P (2010) Apoptotic effects of chrysin in human cancer cell lines. Int J Mol Sci 11:2188–2199. Kim H, Keum DJ, Kwak JW, Chung HS, Bae H (2014) Bee venom phospholipase A2 protects against acetaminopheninduced acute liver injury by modulating regulatory T cells and IL-10 in mice. PLoS One 9:e114726. Kim YH, Hwang JH, Kim KS, Noh JR, Choi DH, Kim DK, Tadi SD, Yim TH, Choi HS, Lee CH (2015) Metformin ameliorates acetaminophen hepatotoxicity via Gadd45b-dependent regulation of JNK signalling in mice. J Hepatol 63:75–82. Kirkin V, Joos S, Z€ ornig M (2004) The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 1644:229–249. Ko EY, Sabanegh ES Jr, Agarwall A (2014) Male infertility testing: reactive oxygen species and antioxidant capacity. Fertil Steril 102:1518–1527. Larson AM (2007) Paracetamol hepatotoxicity. Clin Liver Dis 11:525–548. Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71:952–958. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin reagent. J Biol Chem 193:265. Mantawy EM, El-Bakly WM, Esmat A, Badr AM, ElDemerdash E (2014) Chrysin alleviates acut doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol 728:107–118. Placer ZA, Cushmanni LL, Johnson BC (1966) Estimation of products of lipid peroxidation (as malondialdehyde) in biochemical systems. Anal Biochem 16:359–364. Pushpavalli G, Kalaiarasi P, Veeramani C, Pugalendi KV (2010) Effect of chrysin on hepatoprotective and antioxidant
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status in D-galactosamine-induced hepatitis in rats. Eur J Pharmacol 631:36–41. Ratnasooriya WD, Jayakody JR (2000) Long-term administration of large doses of paracetamol impairs the reproductive competence of male rats. Asian J Androl 2:247– 255. Rehman MU, Ali N, Rashid S, Jain T, Tahir M, Khan AQ, Lateef A, Khan R, Hamiza OO, Kazim S, Qamar W, Sultana S (2014) Allevation of hepatic injury by chrysin in cisplatin administrated rats: probable role of oxidative and inflammatory markers. Pharmacol Rep 66:1050–1059. Salvesen GS, Dixit VM (1997) Caspases: intracellular signalling by proteolysis. Cell 91:443–446. Sathiavelu J, Senapathy GJ, Devaraj R, Namasivayam N (2009) Hepatoprotective effect of chrysin on prooxidantantioxidant status during ethanol-induced toxicity in female albino rats. J Pharm Pharmacol 61:809–817. Scheiermann P, Bachmann M, Goren I, Zwissler B, Pfeilschifter J, M€ uhl H (2013) Application of interleukin-22 mediates protection in experimental acetaminophen-induced acut liver injury. Am J Pathol 182:1107–1113. Sedlak J, Lindsay RHC (1968) Estimation of total protein bound and nonprotein sulfhydryl groups in tissue with Ellmann’s reagent. Anal Biochem 25:192–205. Sun Y, Larry WO, Ying L (1988) A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Turk G, Atessahin A, Sonmez M, Ceribasi AO, Yuce A (2008) Improvement of cisplatin – induced injuries to sperm quality, the oxidant–antioxidant system, and the histologic structure of the rat testis by ellagic acid. Fertil Steril 89:1474–1481. T€ urk G, Ceribasßı AO, Sahna E, Atesßßs ahin A (2011) Lycopene and ellagic acid prevent testicular apoptosis induced by cisplatin. Phytomedicine 8:356–361. Vardi N, Parlakpinar H, Ates B, Cetin A, Otlu A (2009) Antiapoptotic and antioxidant effects of beta-carotene against methotrexate-induced testicular injury. Fertil Steril 92:2028–2033. Villar IC, Jimenez R, Galisteo M, Garcia-Saura MF, Zarzuelo A, Duarte J (2002) Effects of chronic chrysin treatment in spontaneously hypertensive rats. Planta Med 68:847–850. Walle T, Otake Y, Brubaker JA, Walle UK, Halushka PV (2001) Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol 51:143–146. Wiger R, Honglso JK, Evenson DP, De Angelis P, Schwarze PE, Holme JA (1995) Effects of acetaminophen and hydroxyurea on spermatogenesis and sperm chromatin structure in laboratory mice. Reprod Toxicol 9:21–23. Yano CL, Dolder H (2002) Rat testicular structure and ultrastructure after paracetamol treatment. Contraception 66:463–467. Zhao YL, Zhou GD, Yang HB, Wang JB, Shan LM, Li RS, Xiao XH (2011) Rhein protects against acetaminopheninduced hepatic and renal toxicity. Food Chem Toxicol 49:1705–1710.
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Mitigation of paracetamol-induced reproductive damage by chrysin in male rats via reducing oxidative stress 2 € € u € r1, E. Eldutar3, S. Ku € cßu € kler3 & S. C E. H. Aksu1, M. Ozkaraca , F. M. Kandemir3, A. D. Om ß omaklı2
€rk University, Erzurum, Turkey; 1 Department of Reproduction and Artificial Insemination, Veterinary Medicine Faculty, Atatu €rk University, Erzurum, Turkey; 2 Department of Pathology, Veterinary Medicine Faculty, Atatu €rk University, Erzurum, Turkey 3 Department of Biochemistry, Veterinary Medicine Faculty, Atatu
Keywords Bax/Bcl-2—caspase-3—male rat—paracetamol—sperm Correspondence Emrah Hicazi Aksu, PhD, Department of Reproduction and Artificial Insemination, €rk Veterinary Medicine Faculty, Atatu University, 25240 Erzurum, Turkey. Tel.: +904422317132; Fax: +904422317244; E-mail: [email protected] Accepted: January 3, 2016 doi: 10.1111/and.12553
Summary Paracetamol (PRC) is a nonsteroidal anti-inflammatory drug used widely as a painkiller for various diseases and as the symptomatic flu cure in several countries worldwide. PRC toxicity may occur under conditions of the overdose usage. Chrysin (CR) is a flavonoid that is naturally present in several plants, honey and propolis. The aim of this study was to investigate the effects of CR (at the doses of 25 mg kg1 and 50 mg kg1) pre-treatment over seven consecutive days against PRC-induced reproductive toxicity in male rats. Our results showed that PRC toxicity decreased the sperm motility, and increased dead sperm rate, abnormal sperm cell rate, apoptosis and MDA levels in testicular tissues. Pre-treatment with CR at the dose of 25 and 50 mg kg1 for 7 days mitigated side effects of acute PRC toxicity in male reproductive system proportionally in a dose-dependent manner. This possible protection mechanism might be dependent on the antioxidant activity of CR. In conclusion, pre-treatment with CR at the dose of 25 and 50 mg kg1 for 7 days can be the beneficial against PRC-induced reproductive toxicity proportionally in a dosedependent manner.
Introduction Paracetamol (N-acetyl-p-aminophenol (PRC)), also known as acetaminophen, is a nonsteroidal anti-inflammatory, analgesic and antipyretic drug used widely as a painkiller for various diseases and as the symptomatic flu cure in several countries worldwide. PRC is generally accepted to be safe if it is provided and used at doses recommended by physicians. Alternatively, this drug may have harmful effects if it is used at the excess dose. Some researchers suggesting that PRC toxicity may occur under conditions of the overdose usage. Regular usage of the drug is also a risk factor for drug toxicity. PRC affects several regions of the body to produce various types of toxicity including hepatotoxicity (Zhao et al., 2011; Scheiermann et al., 2013; El-Shafey et al., 2015), renal toxicity (Zhao et al., 2011; El-Shafey et al., 2015), reproductive toxicity (Ratnasooriya & Jayakody, 2000; Yano & Dolder, 2002) and even cardiomyocyte toxicity (Jin & Park, 2012). The wide use of the drug gives an elevated risk for toxicity. The toxicological effects of PRC are probably related to increased reactive oxygen species (ROS), induced apoptosis and DNA damage (Jin & Park, © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
2012). Ratnasooriya & Jayakody (2000) suggested that treatment with PRC (500 mg kg1 or 1000 mg kg1) for consecutive 30 days caused to decrease in libido, sexual vigour and fertility index in male rats. They also suggested that all these detrimental effects were reversible. Several agents have been well studied that may be able to mitigate or totally protect against some of the side effects by PRC, such as the increase in ROS levels. When PRC is taken by the oral way, it is absorbed quickly in the gastrointestinal system. After 30 min following the drug is taken, it reaches the maximum plasma concentration and disturbed all tissues (Larson, 2007). The half-life of PRC is about 150 min. (Graham & Scott, 2005). To protect or mitigate PRC’s side effects, the use of antioxidant agents is advised by some researchers. For example, Kim et al. (2015) suggested that metformin administration (30 min. before PRC treatment at the dose of 350 mg kg1, p.o.) mitigates PRC-induced hepatotoxicity in mice. El-Shafey et al. (2015) reported that quercetin treatment (at the dose of 15 mg kg1) for twenty-one consecutive days had protective effects against renal toxicity induced by a single injection of PRC (at the dose of 3 g kg1). Furthermore, treatment with interleukin-22 1
Chrysin reduces paracetamol-induced sperm damage
(single dose of 3.5 ug) had a preventive impact against PRC-induced hepatotoxicity according to a study by Scheiermann et al. (2013). Similarly, Kim et al. (2014) suggested that pre-treatment with phospholipase A2, found in bee venom, at the dose of 0.2 mg kg1 for 5 days before a single injection of PRC at 500 mg kg1 had a protective effect against hepatotoxicity in mice. Chrysin (chemically named 5,7-dihydroxyflavone (CR)) is a flavonoid that is naturally present in several plants, honey and propolis. It has some pharmacological and biological properties including antioxidant, anti-inflammatory (Mantawy et al., 2014), anticancer (Khoo et al., 2010), anti-diabetic, anti-allergic (Kasala et al., 2015), anti-apoptotic (Jiang et al., 2014) and antihypertensive (Villar et al., 2002) activities. Especially hydroxyl groups of CR at 5th and 7th carbons give it the antioxidant property (Sathiavelu et al., 2009). The plasma membrane protective and free radical scavenging effects of CR help to keep free radical levels under control (Pushpavalli et al., 2010). Walle et al. (2001) suggested that CR reaches the peak plasma concentration in 1 h. Few studies have examined the effects of acute PRC toxicity on male reproductive system. Furthermore, only a few studies have examined the effects of CR against PRC-induced toxicity. However, the studies have not mentioned any information concerning the male reproductive system. The aim of this study was to investigate the effects of CR pretreatment over seven consecutive days against PRCinduced reproductive toxicity in male rats. Material and methods
tion has toxic effects. Our dosage choice for PRC is similar with these studies (Graham & Scott, 2005; Kim et al., 2014). Ciftci et al. (2012) suggested that treatment with CR at a dose of 50 mg kg1 increased sperm motility, sperm cell density and serum testosterone levels; this dose also decreased abnormal sperm rates. Darwish et al. (2014) suggested that 50 mg kg1 CR is more protective than 25 mg kg1 CR against testicular dysfunction of adjuvant arthritic rats. Rehman et al. (2014) suggested that oral treatment with chrysin (at the dose of 25 and 50 mg kg1 bw1) protected the CP-induced hepatotoxicity by reducing OS. We preferred 50 and 25 mg kg1 CR doses against to acute PRC toxicity. Rats were divided into four groups as; Group I (n = 7); referred as Control (C) group, rats received physiological saline only via oral gavage for consecutive 7 days before oral gavage of physiological saline. Group II (n = 7); named as PRC, the rats received physiological saline only via oral gavage for consecutive 7 days before PRC application (500 mg kg1). Group III (n = 7); referred as PRC+CR-25, rats received 25 mg kg1 CR by oral gavage for consecutive 7 days before oral PRC (500 mg kg1) application. Group IV (n = 7); referred as PRC+CR-50, rats received 50 mg kg1 CR by oral gavage for consecutive 7 days before oral PRC (500 mg kg1) application. After 24 h from the PRC applications, rats were decapitated under inhalation anaesthesia with isoflurane (IsoFloâ; Abbott, Queenborough, UK). The approval of Committee for Institutional Animal Care and Use was provided from Atat€ urk University Local Board of Ethics (the approval number: 2015/61) before the study had been planned. Collection of samples
Chemicals _ TurPRC (Parol 500 mg tablet; Atabay Co., Istanbul, key) was purchased from a pharmacy and CR (97% purity; cat no: C80105 Sigma-Aldrich Co., St. Louis, Missouri, USA) was purchased from a medical market. All other chemicals were analytical purity (Sigma-Aldrich Co.). â
Animals and experimental procedure In this study, twenty-eight male Sprague–Dawley rats (Specific Pathogen Free), 10-week-old and weighing 250– 300 g, were used as the animal material. The rats were purchased from Atat€ urk University Medical Experimental Application and Research Centre. The rats were kept under standard laboratory conditions (40% humidity, 24 °C, a 12 h light: 12 h dark cycle), in the abovementioned centre. Rats were fed with a commercial pellet chow and fresh drinking water was available ad libitum. Some studies suggested that 500 mg kg1 PRC applica2
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Following decapitation procedure, the testes and cauda epididymidis of the rats were removed from the corpse and cleaned from connective tissues such adipose or connective tissues with anatomical scissors and tweezers. Both testes and cauda epididymidis were weighed and recorded as total testes weight (TTW) and total cauda epididymidis weight (TCEW). For the pathological examinations, one of the testes was kept in Bouin’s solution while another one was kept in deep freeze immediately (at 20 °C) for biochemical assays by classifying for their groups. Semen evaluation One of cauda epididymidis was used to obtain semen sample for each animal. For this purpose, randomly selected cauda epididymidis was minced in Petri dish including 5 ml of physiological saline. To provide the migrations of spermatozoa from cauda epididymidis to fluid, 5 min incubation period was obtained on warmed © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
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stage (at 35 °C). Following the incubation period, cauda epididymidis residue was removed by using anatomical tweezers from the Petri dish. The fluid remaining in the Petri dish was used as semen sample. Evaluation of semen was conducted using routine spermatological parameters including motility, density of sperm cells, dead sperm rate and morphological examination of spermatozoa. To evaluate the percentage of sperm motility, light microscope (Primo Star; Carl Zeiss, Oberkochen, Germany) equipped with the heated stage was used. Briefly, a slide was placed on a heated stage warmed up to 35 °C placed on a conventional light microscope. Approximately 20 ll of semen sample was dropped on the slide. The percentage of sperm motility was detected by visual investigation of the sample. To estimate the sperm motility, randomly selected three different fields from each sample were evaluated. The average of three field estimations was calculated as the final motility score of the sample (Turk et al., 2008). To determine of sperm cells concentration, the method described in our previous study (Aksu et al., 2015) was used. Briefly, the semen sample was diluted as the rate of 1/100 with eosin solution (2 g dry eosin dye and 3 g sodium citrate solved in 100 ml of distilled water) in Eppendorf tube. The Eppendorf tubes were vortexed at 2500 rpm for 15 s and sperm suspension was transferred into the counting chambers of Thoma chamber. Then, sperm cells in both chambers were counted under the conventional light microscope (Zeiss Primo Star) at the magnification of 4009. To determine the percentage of morphological abnormality of spermatozoa, the method (with a little modification by using only eosin dye instead of eosin-nigrosin dye) described by Turk et al. (2008) was used. Briefly, the slides were stained with eosin dye. Then, the slides were evaluated under light microscope at 4009 magnification with the help of immersion oil (immersion oil for microscopy type A, no: 1.515; Nikon, Tokyo, Japan). Two hundred and fifty spermatozoa from each slide were observed and the percentages of sperm head, sperm mid, sperm tail and total abnormality of spermatozoa were stated. Biochemical evaluations of testicular tissues For assaying MDA, GSH levels and CAT activity homogenates were centrifuged for 15 min at 1000 g at +4 °C while to assay the GSH-px activity of testicular homogenates were centrifuged for 20 min at 9000 g at +4 °C. Following thecentrifuge process, the obtained supernatant was subjected to enzyme assays as soon as possible. The malondialdehyde (MDA) level of testicular tissues was measured by the thiobarbituric acid reaction method of Placer et al. (1966). The homogenisation of testicular tissues was carried out in Teflon-glass homogenizer with a buffer © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
Chrysin reduces paracetamol-induced sperm damage
containing 1.15% KCl to obtain 1 : 10 (w/v) whole homogenate. The values of MDA were expressed as nmol g1 tissue. The GSH content of testicular homogenates was determined at 412 nm according to the method, described by Sedlak & Lindsay (1968) and GSH levels were expressed as nmol g1 tissue. The CAT activity of testicular tissue was determined according to the method of Aebi (1983). The values of CAT were expressed as catal g1 protein. The GSH-Px activity of testes was determined using the method of Lawrence & Burk (1976). The GSH-px activity of testicular homogenates was expressed as U g1 protein. To assay superoxide dismutase (SOD) activity of testicular tissues, the method of Sun et al. (1988) was used. The SOD activity of testes was measured as the level of decrease in the absorbance at 560 nm and SOD values of testicular homogenates were expressed as U g1 protein. The protein content of the testicular tissues was measured according to the method described by Lowry et al. (1951). Immunohistological examinations The testes were removed instantly, fixed in bouin’s solution, embedded in paraffin blocks, sectioned 5 lm in thickness and stained with immunohistochemistry and then examined under a light microscope at 920 magnification. After deparaffinisation process, the slides were immersed in antigen retrieval solution (pH 6.0) and heated in a microwave for 15 min to unmasked antigens. The sections were then dipped in 3% H2O2 for 10 min to block endogenous peroxidase and then incubated at room temperature with polyclonal rabbit anti-Bax polyclonal antibody (cat. no. bs0127R, dilution 1/200; Bioss, Massachusetts, USA), polyclonal rabbit anti-active caspase 3 (cat. no. NB600-1235, dilution 1/200; Novusbiological, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (cat. no. ab29, dilution 1/100; Abcam, Cambridge, UK) and rabbit anti Bcl-2 polyclonal antibody (cat. no. bs0032R; dilution 1/200; Bioss). Expose mouse and rabbit specific HRP/DAB detection IHC kit (cat. no. ab80436; Abcam) was used as follows and eventually with 3,30 diaminobenzidine + chromogen. The slides were counterstained with haematoxylin. The slides in the sections were graded as 0 (none), 1 (mild), 2 (moderate) and 3 (severe). Similarly, in immunohistological evaluations after the slides prepared they evaluated semi-quantitatively under a light microscope and ranked as 0 (none), 1 (mild), 2 (moderate) and 3 (severe) considering their dye density status. Statistical analyses Spermatological parameters, biochemical values and testicular traits of the groups were presented as mean standard error of means (SEM). The differences were 3
Chrysin reduces paracetamol-induced sperm damage
E. H. Aksu et al.
regarded significant when P < 0.05. Statistical analyses of the values were done by analysis of variance (One-way ANOVA) and post hoc Duncan test using the IBM SPSS/PC (Version 20.0, IBM Co., North Castle, New York, USA) software program. Also, spermatological parameters were analysed to determine potential correlations exist between them with Pearson─correlation test. Also, the histological evaluations were analysed with the Kruskal─Wallis test in IBM SPSS/PC (Version 20.0) software program. Dual crosschecks between groups displaying significant values were estimated with Mann─Whitney U-test (P < 0.05).
between all groups for TTW and TCEW values. In other words, experimental acute PRC toxicity did not affect TTW and TCEW in male rats. Semen evaluations Spermatological parameters are presented in Table 1. As seen in Table 1, treatment of PRC alone significantly (P < 0.01) decreased the motility rates and increased dead sperm rates, while pre-treatment for seven consecutive days with CR (CR-25 and CR-50) significantly (P < 0.01) mitigated the side effects of PRC on these parameters when compared to the control group. Although there was a decrease in sperm cell density with both the PRC and treatment groups when compared to the control group, this reduction was not statistically significant. With regard to spermatological abnormalities in all groups, PRC toxicity led to an increase (P < 0.01) in the
Results Reproductive organ weights Reproductive organ weights of all groups are presented in Table 1. As shown in Table 1, there was no difference Table 1 Reproductive and biochemical parameters of all groups Parameters/Groups
Control
TTW (g) TCEW (g) Motility (%) Dead sperm rate (%) Density (9106) Abnormal Sperm head (%) Abnormal Sperm mid (%) Abnormal Sperm tail (%) Total of Abnormal (%) MDA (nmol g1 tissue) GSH (nmol g1 tissue) CAT (catal g1 protein) GSH-px (U g1 protein) SOD (U g1 protein)
3.047 0.547 65 14.4 153.214 3.5 0.9 6.9 11.3 26.75 5.34 5.50 11.25 20.16
PRC
0.104 0.017 1.5a 1.4a 12.975 0.2a 0.2a 1.2 1.3ab 0.39a 0.04a 0.17a 1.41a 0.20a
2.924 0.478 40 29.3 135.833 5.9 2.1 6.1 14.0 49.17 3.26 3.27 7.29 13.80
PRC+CR-25
0.123 0.051 1.8c 0.8c 20.572 0.6c 0.5b 1 1.3b 0.83d 0.05d 0.07d 0.11c 0.20c
2.766 0.504 55 22.3 137.083 5.3 1.3 7.3 13.9 38.03 4.23 3.81 8.88 13.75
PRC+CR-50 0.150 0.022 1.8b 1.2b 11.626 0.6bc 0.3ab 0.6 0.5b 0.46c 0.04c 0.06c 0.12bc 0.41c
3.138 0.536 50 18.6 124.583 3.9 1.1 5.1 10.1 29.56 4.90 4.46 10.32 17.46
Sign. 0.880 0.023 2.4b 1.8b 16.563 0.4ab 0.4ab 0.6 0.6a 0.52b 0.07b 0.10b 0.16ab 0.32b
N.S. N.S. P < 0.01 P < 0.01 N.S. P < 0.01 P < 0.05 N.S. P < 0.05 P < 0.001 P < 0.001 P < 0.001 P < 0.01 P < 0.001
(a–c): Different superscript letters in same row indicate statistical differences. Sign., Significant; N.S., Not Significant; PRC, Paracetamol (500 mg kg1); CR-25, Chrysin (25 mg kg1); CR-50, Chrysin (50 mg kg1). Table 2 Correlations between sperm characteristics and biochemical parameters of testes tissues Motility Motility Dead Sperm Head Abn. Mid Abn. Tail Abn. Total Abn MDA GSH CAT GSH-px SOD
** ** *
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Dead Sperm
Head Abn.
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Mid Abn * **
Tail Abn.
Total Abn.
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** ** ** ** **
(+) (+) () () () ()
** ** ** ** * **
(+) (+) () () () ()
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(+) (+) () ()
MDA
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(+) (+) (+) (+)
() (+) ** (+) ** (+) **
GSH ** ** ** *
*
(+) ** * *
(+) () ()
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CAT ** ** ** **
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SOD ** ** **
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Correlation is significant at the P < 0.05 level (2-tailed). Correlation is significant at the P < 0.01 level (2-tailed). Presence of the minus symbol () indicates negative correlation while plus symbol (+) indicates positive correlation. *
**
4
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Chrysin reduces paracetamol-induced sperm damage
percentage of sperm-head abnormalities. However, pretreatment with CR-25 had no effect against this side effect, while pre-treatment with CR-50 attenuated the percentage of sperm-head abnormalities. The toxicity led to an increase in mid-sperm abnormalities in the PRC group while it did not affect the percentage of sperm-tail abnormalities (P < 0.05). Although pre-treatment with CR-25 and CR-50 decreased the mid-sperm abnormality rate induced by PRC toxicity, there was no significant difference between the PRC and treatment groups in this parameter. Furthermore, when the total abnormalities of all groups were considered, the PRC+CR-50 group had significantly fewer abnormalities than the PRC alone group. Pre-treatment with CR at a dose of 50 mg kg1 had a protective effect against an increased sperm abnormality percentage induced by PRC toxicity when compared to controls and the lower dose pre-treatment group (CR-25).
CR when compared to the PRC group. There was also a significant difference between treatment groups with both GSH and CAT activity being reduced to a greater extent in the CR-50 group. GSH-px and SOD activity decreased in the PRC group when compared to the control group. Furthermore, while the CR-50 significantly increased (P < 0.01 for GSH-px and P < 0.001 for SOD activities), CR-25 dose was ineffective in altering GSH-px and SOD activities. According to our results, CR has a reductive effect in proportion to the treatment dose against oxidative stress induced by PRC. These results might be attributed to the antioxidant effect of CR.
Biochemical evaluations of testicular tissue As seen in Table 1, MDA levels in the PRC group were significantly higher than in the control and treatment groups. Also, in terms of MDA levels, in treatment groups there was a proportional difference when compared to each other and the lowest level (P < 0.001) of MDA was found in the control group. With regard to GSH and CAT activity, the highest levels were in the control group, while the lowest values were in the PRC group. However, there was a proportionally significant increase in these activities in the treatment groups with
Graph 1 Bax expressions levels of all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
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Fig. 1 (a–d) Bax expression (arrowhead) of Control group (a), PRC group (b), PRC+CR-25 group (c), and PRC+CR-50 group (d).
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Chrysin reduces paracetamol-induced sperm damage
Correlations between spermatological parameters and biochemical parameters of testicular tissues are presented in Table 2. As shown in Table 2, there was a positive correlation between motility and antioxidant enzyme (including; CAT, GSH-px, and SOD) and GSH levels of the testis, and a negative correlation between motility and dead sperm rates, sperm-head abnormalities, mid-sperm abnormalities and MDA levels of the testes. There was a positive correlation between dead sperm rates and sperm-head abnormalities, mid-sperm abnormalities, total sperm abnormalities and MDA levels of the testes. However, there was a negative correlation between dead sperm rates and motility and antioxidant enzyme levels of testicular tissue. For sperm-head abnormalities, there was a positive correlation between these abnormalities and dead sperm rates, total sperm abnormalities, and MDA levels of the testes, while there was a negative correlation between sperm-head abnormalities and sperm motility, GSH, CAT, GSH-px and SOD activity of the testes. With regard to mid-sperm abnormalities, there was a positive correlation between these and dead sperm rates, total abnormalities and MDA levels of the testes. However, there was a negative correlation between midsperm abnormalities and motility, GSH level and CAT activity of the testes. For the sperm-tail abnormalities, there was a positive correlation between sperm-tail abnormality and total abnormality of spermatozoa. These results suggest that increased lipid peroxidation (MDA level of testicular tissue) caused the decreases in sperm motility and antioxidant enzymes levels in the testes. Alternatively, increased lipid peroxidation led to
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increases in dead sperm rates, sperm-head abnormalities, and mid-sperm abnormalities. The correlation table (Table 2) also indicates that OS did not affect all spermatological parameters equally. Some parameters were affected at more severe levels from OS while other parameters affected at relatively light levels or not affected at all. Immunohistochemical examinations Bax was expressed slightly in the control group (Fig. 1a). In the PRC and PRC+CR-25 groups, Bax expression was
Graph 2 Caspase-3 expression levels of all groups. (a–d) Different letters indicate statistical difference among the groups (P < 0.05).
Fig. 2 (a–d) Caspase 3 expression of Control group (a). Caspase 3 expression of (arrowhead) of PRC group (b), (arrowhead) of PRC+CR-25 group (c) and PRC+CR-50 group (d).
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Chrysin reduces paracetamol-induced sperm damage
severe (Fig. 1b,c). However, Bax expression in the PRC+CR-50 group was statistically decreased when compared to both PRC and PRC+CR-25 groups, but it was increased when compared to the control group (Fig. 1d, Graph 1). Caspase-3 activity was expressed slightly in the control group (Fig. 2a). The highest caspase-3 activity was detected in the PRC group, and CR (both CR-25 and CR-50) treatments significantly (P < 0.05) decreased the caspase-3 activity in a dose-dependent manner. There was also a significant difference between the PRC+CR-25 and PRC+CR-50 groups with regard to caspase-3 activity (Fig. 2b,c,d, Graph 2). PCNA was expressed in the control group at very light levels. PCNA in the PRC and PRC+CR-25 groups was expressed as increased with a significant difference (P < 0.05) between the two groups (Fig. 3a,b,c). However, PCNA expression levels in the PRC+CR-50 group significantly decreased when compared to the PRC and PRC+CR-25 groups, but it was not as low as in the control group (Fig. 3d, Graph 3). Bcl-2, known as an anti-apoptotic marker, was expressed at high levels in the control group (Fig. 4a) while Bcl-2 expression decreased significantly (P < 0.05) in both the PRC and PRC+CR-25 groups. However, the Bcl-2 expression level in the PRC+CR-50 group was similar to the control group (Fig. 4b,c,d, Graph 4). Bax, caspase-3 and Bcl-2 were expressed in intertubular fields while PCNA was expressed from both intertubular fields and germinal cells in the seminiferous tubules of all groups.
Discussion PRC is used widely as a painkiller in various diseases and as symptomatic cure for flu in several countries. Although, it is generally accepted to be safe when taken in doses recommended by physicians. The common usage of this drug makes the precautions against its toxicological side effects more important. Toxic effects of the drug appear in certain types of tissues including the liver, kidneys and testes. These side effects may be attributed to ROS increasing the effects of PRC. ROS are produced physiologically and are required for some cellular
Graph 3 PCNA expression levels of all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
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Fig. 3 (a–d) PCNA expression of Control group (a). PCNA expression (arrowhead) of PRC group (b), PRC+CR-25 group (c) and PRC+CR-50 group (d).
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mechanisms, such sperm maturation, sperm-oocyte interactions and capacitation of sperm cells (Ko et al., 2014). It is well known that increased ROS levels cause OS and, generally in this condition, the balance of oxidant/antioxidant status in the body is broken and tissues may be damaged in severe situations if this imbalance is not corrected. Increased ROS levels may stem from lipid peroxidation induced by various physiological factors, they also have negative effects on spermatological parameters, the reproductive system and hence male fertility (Ko et al., 2014). According to results of a study by Ratnasooriya & Jayakody (2000), PRC toxicity for over long periods causes reductions in fertility rates, sexual performance, libido and sexual vigour in male rats. Yano & Dolder (2002) reported that after 5, 10 and 50 days of single PRC injections at doses of 4.4 mmol kg1, testicular alterations in male rats were observed. Although these PRC side effects of the testes are reversible (Ratnasooriya & Jayakody, 2000), treatment with an antioxidant compound can be beneficial in protecting against or mitigating the damage. On the other hand, Ciftci et al. (2012) suggested that treatment with CR at a dose of 50 mg kg1 increased sperm motility, sperm cell density and serum testosterone levels; this dose also decreased abnormal sperm rates. These researchers also found that CR treatment increased antioxidant enzyme levels (CAT, SOD and GSH-px) and GSH levels. Similarly, Dhawan et al. (2002) suggested that administering CR at a dose of 1 mg kg1 for 30 days increased the libido, sperm count and fertilisation potential in 2-year-old male rats.
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Our results showed that PRC treatment caused decreases in sperm motility, live sperm rate and oxidant enzyme levels including, GSH, CAT, GSH-px and SOD activities; however, it also increased sperm abnormality rates, including abnormalities to the sperm head, mid sperm, and total sperm. As a matter of fact, increases in MDA (an indicator of lipid peroxidation) levels were related to an excess production of ROS and probably was responsible for the side effects of PRC. However, some reproductive parameters were not affected by PRC toxicity, including TTW, TCEW and sperm tail abnormalities. Wiger et al. (1995) reported that treatment with PRC at the dose of 400 mg kg1 for 5 days caused the reductions
Graph 4 Bcl-2 expression levels of the all groups. (a–c) Different letters indicate statistical difference among the groups (P < 0.05).
Fig. 4 (a–d) Bcl-2 expression (arrowhead) of Control group (a). Bcl-2 expression (arrowhead) of PRC group (b), PRC+CR-25 group (c) and PRC+CR-50 group (d).
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in relative testis weights in mice. Since our study included a single injection of PRC, our results on reproductive organ weights (TTW and TCEW) were not altered. Although there was a numerical decrease in sperm cell density in the PRC and treatment groups when compared to the control group, it was not statistically significant. We detected that pre-treatment with CR-25 had attenuating effects against PRC-induced reproductive damage, including sperm motility, dead sperm rates, MDA and GSH levels and CAT activity. However CR-50 had attenuating effects on sperm motility, dead sperm rates, abnormal sperm heads, total abnormal sperm rates, MDA, GSH and GSH-px levels and CAT and SOD activities. It clearly shows that higher doses of CR (50 mg kg1) were more effective against PRC-induced reproductive toxicity than lower doses (25 mg kg1). Bax and Bcl-2 are two important genes that play a role in the apoptotic process. Bax protein, used as a pro-apoptotic biomarker, stimulates apoptotic cell death, while Bcl-2 protein, an anti-apoptotic marker, inhibits this process (Baell & Huang, 2002; Chan & Yu, 2004; Kirkin et al., 2004) PCNA is a biomarker that appears in the early stages of testicular toxicity and may be used in early estimations of testes toxicity (D’Andrea et al., 2008). Caspase-3 can be defined as the key mediator of apoptosis in mammalian cells because it initiates the apoptotic process by activating other caspase enzymes. However, caspase-3 causes the division of nuclear and cytosolic substrates, chromatin condensation, DNA fragmentation and apoptotic bodies (Salvesen & Dixit, 1997). Alternatively, ROS promote apoptosis by a mitochondrial-dependent pathway (Gach et al., 2015). Various studies have indicated that certain chemicals, such as cisplatin (T€ urk et al., 2011), malathion (Geng et al., 2015) and methotrexate (Vardi et al., 2009) induce the apoptotic process. Although Boulares et al. (2002) reported that PRC induces apoptosis in lymphocytes and hepatoma cells in humans, no study has examined the effect of PRC on apoptotic activity in testicular tissue. Our results showed that there was a significant increase in Bax expression in all the treatment groups (PRC, PRC+CR-25, and PRC+CR-50) when compared to the control group. While Bax expression levels of PRC and PRC+CR-25 groups were similar, the levels with PRC+CR-50 were statistically lower than them. With regard to both caspase-3 activity and PCNA expression, the lowest values were in the control group while the highest values for both belong to the PRC alone group. Although CR treatments against PRC toxicity mitigate caspase-3 activity and PCNA expression when compared to PRC alone, the higher CR dose had significantly better protective effects than CR-25 for the both parameters. © 2016 Blackwell Verlag GmbH Andrologia 2016, xx, 1–10
Chrysin reduces paracetamol-induced sperm damage
The highest Bcl-2 activities were in the control and PRC+CR-50 groups while the lowest expression levels were in the PRC alone group. Pre-treatment with CR-25 also significantly increased the Bcl-2 expression level compared to PRC alone group. In conclusion, when considering all our results, PRC toxicity decreased the spermatological quality while increasing apoptosis and MDA levels in testicular tissues. As expected, the higher of CR exhibited higher antioxidant activity. This possible protection mechanism might be dependent on the antioxidant activity of CR although its presence increased the absorption rates of PRC. Moreover, pre-treatment with CR against PRC toxicity mitigates some of the harmful side effects of PRC proportionally in a dose-dependent manner by free radical scavenging, anti-apoptotic, plasma membrane protective properties of it. References Aebi H (1983) Catalase. In: Methods in Enzymatic Analysis. Bergmeyer HU (ed). Academic Press, New York, pp 276– 286. € € ur AD, Ucßar O € (2015) Aksu EH, Akman O, Ozkaraca M, Om€ Effect of Maclura Pomifera extract on cisplatin-induced damages in reprodutive system of male rats. Kafkas Univ Vet Fak Derg 21:397–403. Baell JB, Huang DC (2002) Prospects for targeting the bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 64:851–863. Boulares AH, Zoltoski AJ, Stocia BA, Cuvillier O, Smulson ME (2002) Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes. Pharmacol Toxicol 90:38–50. Chan SL, Yu VC (2004) Proteins of bcl-2 family in apoptosis signalling: from mechanistic insights to therapeutic opportunities. Clin Exp Pharmacol Physiol 31:119–128. Ciftci O, Ozdemir I, Aydin M, Beytur A (2012) Beneficial effects of chrysin on the reproductive system of adult male rats. Andrologia 44:181–186. D’Andrea MR, Lawrence D, Nagele RG, Wang CY, Damiano BP (2008) PCNA indexing as preclinical immunohistochemical biomarker for testicular toxicity. Biotech Histochem 83:211–220. Darwish HA, Arab HH, Abdelsalam RM (2014) Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: comparison with celecoxib. Toxicol Appl Pharmacol 279:129–140. Dhawan K, Kumar S, Sharma A (2002) Beneficial effects of chrysin and benzoflavone on virility in 2-year-old male rats. J Med Food 5:43–48. El-Shafey MM, Abd-Allah GM, Mohamadin AM, Harisa GI, Mariee AD (2015) Quercetin protects against acetaminophen-induced hepatorenal toxicity by reducing
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