Apricot ameliorates alcohol induced testicular damage in rat model

Food and Chemical Toxicology 47 (2009) 2666–2672

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Apricot ameliorates alcohol induced testicular damage in rat model Meltem Kurus a,*, Murat Ugras b, Burhan Ates c, Ali Otlu a a

Inonu University, Faculty of Medicine, Department of Histology–Embriyology, Turkey Inonu University, School of Medicine, Department of Urology, Turkey c Inonu University, Faculty of Science, Department of Chemistry, Malatya, Turkey b

a r t i c l e

i n f o

Article history: Received 29 April 2009 Accepted 27 July 2009

Keywords: Apricot Alcohol Testes Histopathology Rat

a b s t r a c t In this study, we intended to determine the possible preventive effects of dietary apricot on oxidative stress due to ethanol usage in rat testes. The animals were divided into six groups as follows: Group 1 was control. Group 2 received ethanol. Group 3 were fed with apricot diet for 3 months. Group 4 were fed with apricot diet for 6 months. Group 5 received ethanol and apricot diet for 3 months. Group 6 were fed apricot diet for 3 months, and then ethanol + apricot diet for 3 months. Following sacrification, the testes were treated for morphological (tubular and germ cell histology, Sertoli and Leydig cell counts) and biochemical (superoxide dismutase, glutathion peroxidase, catalase, malondialdehyde) analyses. In Group 2, severe histopathological changes in seminiferous tubules and germ cells were determined as well as tubular degeneration and atrophy. Sertoli and Leydig cell counts in the interstitial tissue were decreased. Biochemical parameters revealed tissue oxidative stress. Similar alterations existed in Group 5, although to a lesser extent. In Groups 1, 3 and 4, no histopathological alterations were noted. Results of Group 6 were similar to the controls. Apricot rich diet may have a preventive role on histopathological changes caused by alcohol in rat testes. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Due to its solubility in both water and lipids, alcohol diffuses to all tissues of the body and affects most vital functions (Lieber, 1997, 2005). Studies have shown that alcohol consumption may result in increased oxidative stress with formation of lipid peroxides and free radicals (Bjorneboe and Bjorneboe, 1993; Nordmann, 1994; El-Sokkary et al., 1999; Gentry-Nielsen et al., 2004). Alcoholinduced oxidative stress is linked to the metabolism of ethanol (Zima et al., 2001). Ethanol-induced oxidative stress is not restricted to the liver where the ethanol is actively oxidized, but can affect various extrahepatic tissues as shown by experimental data obtained in rat models of acute or chronic ethanol intoxication. Most of these data concern the central nervous system, the heart and the testes

Abbreviations: AP1, apricot 1 group; AP2, apricot 2 group; A + AP1, alcohol + apricot 1 group; A + AP2, alcohol + apricot 2 group; CAT, catalase; GSH-Px, glutathione peroxidase; H&E, hematoxylene and eosin; LC, Leydig cell; MDA, malondialdehyde; PAS, Periodic Acid Shiff; ROS, reactive oxygene species; SC, Sertoli cell; STD, seminiferous tubule diamater; SOD, Cu, Zn-superoxide dismutase; Vit A, vitamine A; Vit C, vitamine C; Vit E, vitamine E. * Corresponding author. Address: Inonu Universitesi, Tip Fakultesi, Histoloji ve Embriyoloji Anabilim Dali, 44280 Malatya, Turkey. Tel.: +90 422 3410660/1234; fax: +90 422 3410036. E-mail address: [email protected] (M. Kurus). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.034

(Nordmann et al., 1990; Nordmann, 1994; Lieber, 1997, 2005). Alcohol is toxic to testiscular tissue and its chronic use leads to both endocrine and reproductive failure (Rosenblum et al., 1989; El-Sokkary et al., 1999). Peltola et al. shown that free radical formation and lipid peroxidation are potentially important mediators in testicular physiology and toxicology (Peltola et al., 1996). The human body has various antioxidants as a defense mechanism, some of which are dietary-derived (Catoni et al., 2008; Ruiz et al., 2005a). The human diet contains an array of different compounds that possess antioxidant capacity, including scavenger activity (Diplock et al., 1998). The most prominent representatives of dietary antioxidants are carotenoids, ascorbate, tocopherols and flavinoids (Diplock et al., 1998). Most of the research has focused on carotenoids (vitamin A), neglecting a number of more common, more potent, and thereby possibly more important, antioxidants (Catoni et al., 2008). Despite the wide cultivation, there is very little knowledge about the potential antioxidant benefits of dried fruits, which are within easy reach of public for all seasons. Among them, apricot is mostly suitable for manufacturing in many commercial forms such as fresh fruit, dried fruit and fruit juice. Recent literature reveals that apricot has significant antioxidant potential due to its rich content of vitamins A and C, and some polyphenols such as b-carotene (Parlakpinar et al., 2009; Ruiz et al., 2005a,b). Beta carotene is the precursor of vitamin A that appears to protect against reactive oxygen species (ROS) (Aydilek et al., 2004).

M. Kurus et al. / Food and Chemical Toxicology 47 (2009) 2666–2672

It is imperative to study the responses of the tissue antioxidant defense system in rats chronically exposed to ethanol (Genc et al., 1998). In this study, we intended to determine the possible preventive effects of dietary apricot on oxidative stress caused by chronic ethanol usage in rat testis. 2. Materials and methods 2.1. Animals and diets The experiment was performed in accordance with the guidelines for Animal Research from National Institutes of Health; was approved by the Committee on animal Research at Inonu University (numbered as 2006/007) and was in accordance with ‘Guide for the Care and Use of Laborotory Animals, DHEW Publication No (NIH) 85–23, 1985’. Sixty adult male Sprague–Dawley rats aged 6 months and weighing 300–345 g obtained from the Laboratory of Animal Research Center of Inonu University. The animals were kept in special cages in ventilated rooms with 22 ± 2 °C temperature, 50 ± 10% humidity, 12 h light/12 h dark cycle and fed as described. Each animal was placed in a different cage to ensure the correct calculation of administrations. Following an adaptation period of 7 days, the animals were divided into six groups of 10 rats each. These groups were as follows: Group 1 (control) were fed with a standard diet (Elazig Turkey) and tap water ad libitum for 6 months. Group 2 (alcohol-only) received 6% of ethanol and standard diet ad libitum for 3 months. Group 3 (apricot 1 group, AP1) were fed with tap water and apricot diet ad libitum for 3 months. Group 4 (apricot 2 group, AP2) were fed with tap water and apricot diet ad libitum for 6 months. Group 5 (alcohol + AP1 group, A + AP1) received 6% of ethanol and apricot diet ad libitum for 3 months. Group 6 (Alcohol + AP2 group, A + AP2) were fed initially with tap water for 3 months and then 6% of ethanol for next 3 months ad libitum while on apricot diet. The composition of standard diet and apricot diet is given in Table 1. Metabolic energy for each composition was adjusted to 2650 kcal/kg. A 20% apricot diet was manufactured with apricots obtained from Malatya region, a major apricot producing province of Turkey. The natural and organic sun-dried apricots of Kabaasi variety used in the study were obtained from the region of Akcadag–Malatya. This variety was chosen because of its elevated radical scavenging activity and higher total phenolic content (Akinci and Olmez, 2004). Ethanol administration was done via Lieber–DeCarli liquid diet ad libitum: rats to consume alcohol received 36% of total calories as ethanol (Lieber et al., 1989). Ethanol was introduced into the diet in 7 days dosing initially as 1% and finally as 6% of liquid volume. 2.2. Histological examination Total duration of the experiment was 3 months for Groups 2, 3 and 5 and 6 months for Groups 1, 4 and 6. At the end of the experimental periods, animals were sacrificed by servical dislocation. No anesthesia was used. The testes were harvested, fixed in Bouin’s fixative and then embedded in a paraffin blocks. Cross sections of 4–5 l were obtained and stained either with hematoxylene and eosin (H&E) to observe the structure or with Periodic Acid Shiff (PAS), to count Sertoli and Leydig cells. Sections were examined using a Leica DFC 280 light microscope and Leica

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Q Win Plus Image Analysis System (Leica Micros Imaging Solutions Ltd.; Cambridge, UK); at 10, and 40 magnification for overall histopathological evaluation and at 100 magnification for cell counts. Qualitative histopathological analysis was performed according to the method of Sayim (2007), evaluating 100 tubules per animal, each screened and classified according to the degree of seminiferous tubule degeneration. Tubules that showed disruption of cellular association were clasified as ‘sloughing’. Tubules that has scarce or no germ cells were classified as ‘atrophic’. The tubules with round spermatids exhibiting vacuolated nuclei, round spermatids with a halo appearance and giant cells were classified as ‘with germ cell degeneration’ (Sayim, 2007). Quantitative histopathological evaluation was done according to the methods of Omura et al. (1999) for Sertoli cells and Hussein et al. (2006) and Teerds et al. (1998) for Leydig cells. One hundred seminiferous tubules per animal were evaluated for germinal epithelium height and cell counts. Leydig cells were identified in the interstitial area by their oval-to-round nucleus in combination with the specific blue–purple staining of the cytoplasm.

2.3. Biochemical examination 2.3.1. Tissue antioxidant enzyme activity 2.3.1.1. Homogenization. Testes tissue for enzyme activity were obtained from testicular samples and homogenized (PCV Kinematica Status Homogenizator) in icecold phosphate buffered saline (pH 7.4). The homogenate was sonified with an ultrasonifier (Bronson sonifier 450) by six cycles (20-s sonications and 40-s pause on ice). The homogenate was centrifugated (15,000g, 10 min, 4 °C) and cell-free supernatant was subjected to enzyme assay immediately.

2.3.1.2. Cu, Zn-superoxide dismutase assay (SOD). SOD (Cu, Zn-SOD) activity in the supernatant fraction was measured using the xanthine oxidase/cytochrome c method (McCord and Fridovich, 1969) where one unit of activity is the amount of enzyme needed to cause half-maximal inhibition of cytochrome c reduction. The amount of SOD in the extract was determined as ng of enzyme per mg protein, utilizing a commercial SOD as the standard.

2.3.1.3. Catalase assay (CAT). CAT activity was measured at 37 °C by following the rate of disappearance of hydrogen peroxide (H2O2) at 240 nm (e240 = 40 M 1 cm 1) (Luck, 1963). One unit of catalase activity is defined as the amount of enzyme catalyzing the degradation of 1 lmol of H2O2 per min at 37 °C and specific activity corresponding to transformation of substrate (in lmol) (H2O2) per min per mg protein. 2.3.1.4. Glutathione peroxidase assay (GSH-Px). GSH-Px activity was determined in a coupled assay with glutathione reductase by measuring the rate of NADPH oxidation at 340 nm using H2O2 as the substrate (Lawrance and Burk, 1976). Specific activity is given as the amount of NADPH (lmol) disappeared per min per mg protein. 2.3.2. Lipid peroxidation assay (malondialdehyde = MDA) The analysis of lipid peroxidation was carried out as described (Buege and Aust, 1978) with a minor modification. The reaction mixture was prepared by adding 250 lL homogenate into 2 ml of reactive (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N HCl, 1:1:1, w/v) and heated at 100 °C for 15 min. The mixture was cooled to room temperature, centrifuged (10,000g for 10 min) and the absorbance of the supernatant was recorded at 532 nm. MDA results were expressed as nmol per mg protein in the homogenate. The protein content of samples was determined using the colorimetric method of Lowry et al. (1951) using BSA as the standard. All analyses were performed in duplicates.

Table 1 Composition of pellet diets (1000 kg). Ingredient of the mixture

Standard pellet diet Amount (kg)

20% dried apricot containing diet Amount (kg)

Low–high limit

Corn Wheat Bran Soya-48 Fish flour Molasses Marble Salt V-221 Sen-Met Apricot Metabolic energy (kcal/kg)

300.00 154.81 149.87 260.38 80.00 30.00 10.64 9.95 2.50 1.77 – 2650.00

287.44 – 69.30 380.85 80.00 32.54 10.88 10.00 2.50 2.48 200.00 2650

100.00–300.00 0.00–250.00 0.00–250.00 0.00–350.00 80.00–100.00 30.00–50.00 0.00–40.00 0.00–10.00 2.50–25.0 0.00–5.00 100.00–100.00 2650.00–2800.00

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2.3.3. Determination of the antioxidant capacities of chow extracts 2.3.3.1. Extract preparation. Diet and apricot samples (10 g) were mixed with 90 ml of ethanol (70%), and were crushed by using Ultra-Turrax homogenizer (Ultra-Turrax, Model T25, IKA-Works, Inc., Cincinnati, UK) at the speed of 20,000 rpm. The suspension was kept in refrigerator (4 °C) for 48 h and filtered through Whatman No.: 1 filter paper. Extracts were used in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test and total phenolic content analysis.

2.4. Statistical analysis All parameters were expressed as mean ± standard deviation. Data from all groups were compared by nonparametric Kruskal Wallis test and Mann–Whitney U test. Exact p values were given where available, and p < 0. 05 was accepted as statistically significant.

3. Results 2.3.3.2. DPPH radical scavenging power assay. Radical scavenging power (RSP) of rat food extracts was assessed by the method of Shimada et al. (1992) with slight modifications. Two milliliter of the reaction mixture contained 2.9 mmol DPPH (1.8 ml 1  10 4 DPPH) and 0.2 ml extracts. In control, ethanol (70%) was used in place of the sample. Cuvettes were left in dark at room temperature for 10 min, and the resulting color was measured spectrophotometrically at 520 nm against blanks. A decreasing intensity of purple color was related to higher RSP percentage, which was calculated using the following equation: RSP = [1 (AS:10/AB:10)]  100, where AS:10 is the absorbance of samples and AB:10 is the absorbance of the blank at 10th min of the reaction period.

2.3.3.3. Determination of total phenolic content. Total phenolic contents of the samples were determined by the Folin and Ciocalteu’s reagent method (Durmaz and Alpaslan, 2007). Sample extracts of 0.1 ml were diluted to 1 ml with 70% ethanol and 1 ml of Folin and Ciocalteu’s reagent was added. After a rest of 3 min, 1 ml of 2% sodium carbonate was added. After 5 min incubation at ambient temperature with shaking, the resulting absorbance was measured at 760 nm. The calibration curve was performed with gallic acid, and the results were expressed as l g of gallic acid equivalents (lg GAE/gdw).

2.3.3.4. Ferric-reducing power of extracts. It was determined according to the procedure of Oyaizu (1988). Each 0.2 ml of extract was placed into tubes, and volume was adjusted to 1 ml with ethanol (70%); 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide were added and mixed gently. The mixtures were incubated at 50 °C in water bath for 20 min. Then, 2.5 ml of 10% trichloroacetic acid was added, and centrifugated at 6000 rpm for 10 min. From the top layer of supernatant, 2.5 ml was transferred into tubes containing 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride (FeCl3–6H2O). The color intensity was determined at 700 nm against blanks after shaking and a rest for 5 min. The higher absorbance was related to better reducing power of the sample.

Mean daily ethanol consumption was 4.2 ± 1.1 g per rat. The weights of the rats did not change significantly during the study. At the end of the study period the body weights of the animals were not significantly different between the six groups. No significant side effects like diarrhea of 20% apricot diet was seen. 3.1. Histopathology of testis Histological examination results are given in Tables 2 and 3. No significant histopathological alterations in seminiferous tubular histology, Leydig and Sertoli cell counts were determined in groups 1, 3, and 4 (Figs. 1 and 2). In alcohol-only group, severe histopathological changes such as degeneration and atrophy of seminiferous tubules, sloughing and shortening of seminiferous epithelium, vacuolization and lipid droplets in seminiferous tubules and germ cells were determined. Sertoli and Leydig cell counts were decreased. Decreased mean seminiferous tubule diameter and decreased number of germ cells were prominent along with several germ cells revealing gross morphological abnormality. The seminiferous tubules showed irregular arrangement of spermatogenic cell line and germ cell degeneration. Also, sloughing of germ cells into the tubular lumen was observed (Figs. 3 and 4). All histological alterations were statistically significant when compared to controls. Seminiferous tubular diameter as well as Sertoli and Leydig cell counts were decreased signif-

Table 2 Leydig cell (LC) and Sertoli cell (SC) count and seminiferous tubule diamater (STD) in rat testis.

LC SC STD (lm) a b c d e

Significantly Significantly Significantly Significantly Significantly

Control

Alcohol

AP1

AP2

A + AP1

20.2 ± 1.2

10.4 ± 2.3a,b,c,d,e

20.9 ± 1.1

22.1 ± 0.9

16.2 ± 3.6a,b,c,e

19.5 ± 1.4b,c

77.3 ± 2.5

a,b,c,d,e

77.5 ± 1.4

a,b,c,e

75.3 ± 2.7b,c

a,b,c,e

81.3 ± 6.2b,c

35.6 ± 1.2

89.4 ± 2.1 different different different different different

from from from from from

values values values values values

73.0 ± 1.4

a,b,c,d,e

36.1 ± 1.4 of of of of of

87.2 ± 2.5

c

90.7 ± 2.3

A + AP2

70.4 ± 5.5

74.6 ± 6.5

control (p < 0.05). AP1 (p < 0.05). AP2 (p < 0.05). A + AP1 (p < 0.05). A + AP2 (p < 0.05).

Table 3 Percentage of histopathologic classification of seminiferous tubules in the testis. Control

Alcohol

AP1

AP2

A + AP1

A + AP2

N

97.20 ± 0.9

51.1 ± 6.8a,b,c,d,e

96.20 ± 2.0

97.00 ± 1.2

87.20 ± 4.8a,b,c,e

96.2 ± 1.3

S A

2.8 ± 0.9 –

34.2 ± 7.1a,b,c,d,e 11.5 ± 3.7a,b,c,d,e

3.80 ± 2.0 –

3.00 ± 1.2 –

10.60 ± 3.7 a,b,c,e 1.90 ± 1.5a,b,c,e

3.8 ± 1.3 –

CD

–

3.2 ± 1.5a,b,c,d,e

–

–

0.60 ± 0.7

–

N = normal, S = sloughing, A = atrophy CD = cell degeneration. a Significantly different from values of control (p < 0.05). b Significantly different from values of AP1 (p < 0.05). c Significantly different from values of AP2 (p < 0.05). d Significantly different from values of A + AP1 (p < 0.05). e Significantly different from values of A + AP2 (p < 0.05).

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Fig. 1. Testicular section of control group which show normal spermatogenesis. Normal cell arrangement in the seminiferous tubules. H&E  10.

Fig. 4. Testicular sections of rats administrated with alcohol which show further seminiferous tubule degeneration, loss of all germ cells and enlargement of interstitial spaces due to tubular atrophy. PAS  40.

Fig. 2. Testicular section of apricot 2 group which show normal spermatogenesis. Normal cell arrangement in the seminiferous tubules. H&E  10.

Fig. 5. Testicular section of alcohol + apricot 1 group which show normal spermatogenesis. Normal cell arrangement in the seminiferous tubules, but some of the seminiferous epithelium are shortening. H&E  10.

Fig. 3. Testicular sections of rats administrated with alcohol which show further seminiferous tubule degeneration characterized by severe vacuolization and atrophy of tubules. H&E  10.

Fig. 6. Testicular section of alcohol + apricot 2 group which show normal spermatogenesis. Normal cell arrangement in the seminiferous tubules. H&E  10.

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M. Kurus et al. / Food and Chemical Toxicology 47 (2009) 2666–2672 Table 5 SOD, CAT, GSH-Px and MDA levels in rat testis.

Control Alcohol AP1 AP2 A + AP1 A + AP2 a b c

MDA (nmol/mg prt)

SOD (U/mg prt)

CAT (U/mg prt)

GSH-Px (U/mg prt)

0.12 ± 0.03 0.25 ± 0.03a 0.11 ± 0.03 0.08 ± 0.02 0.14 ± 0.02 0.13 ± 0.02

8.7 ± 0.6 3.1 ± 06a 10.3 ± 0.6 12.0 ± 1.6 6.4 ± 1.1 8.1 ± 1.1 c

6.1 ± 0.4 2.4 ± 0.4a 7.1 ± 1.4 8.5 ± 1.1 5.9 ± 0.8 5.1 ± 0.9

3.9 ± 0.5 1.3 ± 0.4a 4.4 ± 0.9 5.8 ± 0.7 4.8 ± 0.6b 4.5 ± 0.7

Significantly different at p = 0.0001. Significantly higher than control at p = 0.02. Significantly higher than A + AP1 at p = 0.001.

group was similar to controls (p > 0.05). Interestingly, SOD was significantly higher in A + AP2 group when compared to A + AP1 group (p = 0.001). Fig. 7. Testicular section of alcohol + apricot 2 group which show normal spermatogenesis. Normal cell arrangement in the seminiferous tubules. The scale bar on the figure is 20 lm. The arrow indicates a Sertoli cell nucleus. L: Leydig cell nucleus. PAS  40.

icantly in this group when compared to apricot 1, apricot 2, A + AP1 and A + AP2 groups. In A + AP1 group, the morphology was similar to the control group, while sloughing and shortening of the seminiferous epithelium and vacuolization in seminiferous tubules could still be observed. Tubular degeneration and atrophy were determined in some areas (Fig. 5). Occasionally, Sertoli and Leydig cell counts were decreased when compared to controls. Alterations in all parameters but cellular degeneration were statistically significant when compared to control, AP1 and AP2 groups. In A + AP2 group, minimal histopathological changes were determined such as sloughing of seminiferous epithelium in rare areas. No seminiferous tubular vacuolization, tubular degeneration or atrophy was seen (Figs. 6 and 7). No statistically significant alterations were determined when compared to controls while all parameters but cellular degeneration were significantly better when compared to A + AP1 group. 3.2. Biochemical analyses 3.2.1. Antioxidant capacities of food extracts DPPH radical scavenging power, reducing power and total phenolic contents of chow extracts were assessed in vitro to determine their antioxidant capacities. As shown in Table 4, antioxidant capacity of rat chow was significantly increased after supplementation with dried apricot for each ratio. 3.2.2. Oxidative stress parameters Tissue levels of testicular SOD (Unit/mg prt), CAT (Unit/mg prt), GSH-Px (lmol/mg prt), MDA (nmol/mg prt) for each group and statistical comparisons are given in Table 5. All oxidative stress indicators were significantly different in alcohol-only group. In A + AP2 group, none of these parameters was significantly different from controls. Also, MDA of A + AP2

4. Discussion Acute alcohol consumption results in testicular injury (Dahlgren et al., 1989; Kelce et al., 1990), while chronic administration results in testicular atrophy and testesterone imbalance in both humans and animals (Grattagliano et al., 1997; Nordmann et al., 1990; Rosenblum et al., 1989). Also, chronic ethanol consumption causes decreased seminiferous tubular diameter and diminished germ cell numbers (Grattagliano et al., 1997; Weinberg and Vogl, 1988). Both in vivo and in vitro studies indicate that ethanol has direct adverse effects on Leydig cell morphology and function (Weinberg and Vogl, 1988). Ethanol-induced impairment of spermatogenesis could reflect a toxic effect of ethanol on Sertoli cell morphology and/or function (Weinberg and Vogl, 1988). Creasy (2001) reported that one of the most common morphological responses of Sertoli cells to injury is vacuolation and subsequent germ cell degeneration, disorganization or exfoliation. In our study, alcohol-only group had severe histopathological changes such as sloughing and shortening of the seminiferous epithelium, vacuolization and lipid droplets in seminiferous tubules and in germ cells, tubular degeneration and atrophy. Sertoli and Leydig cell counts were also diminished, all in conjunction with the literature. Acute (Lecomte et al., 1994; Schlorff et al., 1999; Oner-Iyidogan et al., 2001) and chronic (Genc et al., 1998; Bekpinar and Tugrul, 1995; Lieber, 2005) exposure to ethanol, either administered orally or intraperitoneally, lead to increased free radical and lipid peroxide formation. Some studies reveal that, free radical generation and lipid peroxidation might be an important mechanism in the toxicity of ethanol in the testes (Oner-Iyidogan et al., 2001; Peltola et al., 1996; Grattagliano et al., 1997; Aitken, 1994; Sanocka et al., 1996; Rosenblum et al. 1989). Decreased GSH-Px, SOD and CAT activity seems to indicate the ethanol-induced oxidative stress (Husain et al., 2001). Chronic alcohol consumption accelerates lipid peroxidation in the testes as measured by MDA production (Schlorff et al., 1999; El-Sokkary et al., 1999; Oner-Iyidogan et al., 2001). Testicular membranes are rich in polyenoic fatty acids that are prone to oxidative decomposition, and it is likely that the resulting

Table 4 DPPH radical scavenging power, ferric-reducing power and total phenolic contents of chow extracts.

Standart chow 20%apricot-added chow a

DPPH scavenging activity (%)

Reducing powder (absorbance)

Total phenolic content (mg/g of GAE)

38.65 ± 0.15 54.57 ± 0.56a

0.23 ± 0.001 0.83 ± 0.020a

0.58 ± 0.012 0.93 ± 0.021a

Significantly different from values of standart chow (p < 0.05).

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lipid peroxidation contributes to the membrane injury and gonadal dysfunction that occurs as a result of alcohol abuse and/or chronic use (Rosenblum et al. 1989; Chainy et al., 1997; El-Sokkary et al., 1999; Aydilek et al., 2004) Biochemical evaluations in our study support the literature. In alcohol-only group, decreased tissue SOD, CAT and GSH-Px and elevated MDA obviously reveal the damage caused by free radicals. Reactive oxygen species generated in the tissues are efficiently scavenged by enzymatic antioxidant system (such as SOD, CAT and GSH-Px) and nonenzymatic antioxidants (such as vitamin A, C, and E) (Shimada et al., 1992; El-Sokkary et al., 1999; Husain et al., 2001) and carotenoids (Genc et al., 1998). The antioxidant capacity of common fruits and vegetables has been analyzed in various studies (Ou et al., 2002; Wu et al., 2004). Some studies demonstrated a wide diversity of antioxidant capacities among the common fruits and vegetables that can largely be attributed to their phenolic compounds such as phenolic acids, flavonoids and carotenoids (Prior et al.,1998; Huang et al., 2007). Apricot is considered as a good source of dietary antioxidants, with its content of flavonoids and carotenoids (Ramos et al., 2005). Carotenoids, especially b-carotene, are found to be the principal pigment in apricot (Ruiz et al., 2005a, 2008). Flavonoids are a large group of polyphenolic antioxidants that exhibit a wide range of biological activities including the inhibition of lipid peroxidation (Ramos et al., 2005). Some studies showed that, apricot is also rich in vitamin C (Mascio et al., 1991; Ruiz et al., 2005b; Catoni et al., 2008). Some authors believe that, vitamin C is an important antioxidant which protect spermatogenesis from free radical damage (Grattagliano et al., 1997). Carotenoids are probably nonfocal antioxidants that indirectly affect the male fertility (Catoni et al., 2008). Mixtures of carotenoids or associations with others antioxidants (e.g. vitamin C and vitamin E) can increase their ability to protect against lipid peroxidation (Sies et al., 1992; Stahl and Sies, 2003). In our study, we realized that SOD, CAT and GSH-Px levels of AP2 group were higher even than the controls while MDA was less than all groups, which can be attributed to the increased antioxidant activity due to chronic ingestion of apricot. The significant elevation of SOD in AP2 group may also be interpreted similarly, indicating less utilization and thus accumulation of SOD. Impaired nutritional status of different vitamins and trace elements have been reported in alcoholics. Research on the role of reactive oxygen species in the pathogenesis of alcohol-related disease, and the role of nutrition in favour of the antioxidant defence mechanisms has been proposed (Bjorneboe and Bjorneboe, 1993). Indeed, studies by Mobarhan et al. (1990) showed that replenishing young men with b-carotene can decrease the level of circulating lipid peroxides. It is also known that vitamin A stabilizes testicular membranes by reducing lipid peroxidation and prevents the alcohol-induced atrophy (Rosenblum et al., 1989; Nordmann et al.,1990), suggesting that the increased peroxidation of testicular lipids with ethanol exposure may be an important factor in the pathogenesis of alcohol-associated gonadal injury (Rosenblum et al., 1989). In our study, the number of degenerated seminiferous tubules was significantly higher as well as the incidence of sloughing in alcohol-only and A + AP1groups. The incidence of intensive tubular damage, classified as sloughing, atrophy and germ cell degeneration were also significantly increased in these groups. Atrophy and germ cell degeneration were not observed in the control, AP1, AP2 and A + AP2-administrated groups. Histopathological alterations in seminiferous tubules were prominent in alcohol-only group when compared to the A + AP1 and the A + AP2 groups. The affected tubules were lined by very few spermatogenic cells or only by cell debris, observed mostly in the alcohol-only group.

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Radical scavenging capacity is an in vitro method to estimate the antioxidant activity. DPPH radical is a stable lipophilic free radical, which has been generally used to estimate the antioxidant activities of food and medicine materials (Chen et al., 2000). We assessed the ferric-reducing power in addition to DPPH scavenging activity to determine the radical scavenging capacity of food extracts in this study. Since the total phenolics have been shown to be closely related to the antioxidant capacity of a sample (Prior et al., 1998; Huang et al., 2007), total phenolic content of extracts was also determined. DPPH scavenging activity, ferric-reducing power and total phenolic contents of rat chow were significantly higher than the regular rat chow after apricot supplementation. In this study, we found that histopathological alterations in alcohol-consuming rats due to the oxidative stress and lipid peroxidation were prominent but reversible in testicular tissue. Apricot addition to diet ameliorated these alterations mainly by radical scavenging activity. Pretreatment with apricot prior to alcohol exposure was mostly effective in face of testicular morphology. Indeed, histological and biochemical results should be emphasized by in vivo mating studies to reveal any potential protective effects on overall reproductive function. Resultantly, antioxidants in apricot may prevent ethanol-induced testicular damage, but further studies including tissue and blood testosterone levels are necessary for humans. Conflict of interest statement All authors declare that they have no conflicts of interest. Acknowledgements This study was supported by Apricot Research Foundation of Malatya Province. The apricot used in this study was obtained from Karapinar Kırlangic Koyleri Tarimsal Kalkinma Kooperatifi Akcadag/Malatya/TURKEY. The apricot was minced for feeding in Toksa Tarim Urunleri Ticaret Sanayi Limited Sirketi Dilek/Malatya/TURKEY. Total caloric composition of the standart diet and diet with dried apricot was assayed by Prof. Dr. Kazim Sahin, who was on duty in Animal Nutrition and Nutritional Diseases department at Firat University in Elazig/Turkey. References Aitken, R.J., 1994. A free radical theory of male infertility. Reprod. Fertil. Dev. 6, 19– 24. Akinci, M.B., Olmez, H.A., 2004. Kayisi. In: Malatya Tarim il Mudurlugu, first ed. Gunes Form, Malatya, pp. 5–7 (in Turkish). Aydilek, N., Aksakal, M., Karakılçık, A.Z., 2004. Effects of testesterone and vitamin E on the antioxidant system in rabbit testis. Andrologia 36, 277–281. Bekpinar, S., Tugrul, Y., 1995. Influence of selenium supplementation in non-toxic doses on testis lipid peroxide and antioxidant levels in chronic alcohol-fed rats. Alcohol Alcohol. 30, 645–650. Bjorneboe, A., Bjorneboe, G.E., 1993. Antioxidant status and alcohol-related diseases. Alcohol Alcohol. 28, 111–116. Buege, A.J., Aust, S.D., 1978. Microsomal lipid peroxidation. Meth. Enzymol. 52, 302– 310. Catoni, C., Peters, A., Schaefer, H.M., 2008. Life history trade-offs are influenced by the diversity, availability and interactions of dietary antioxidants. Anim. Behav. 76, 1107–1119. Chainy, G.B.N., Samantaray, S., Samanta, L., 1997. Testosterone-induced changes in testicular antioxidant system. Andrologia 29, 343–349. Chen, C., Tang, H.R., Sutcliffe, L.H., Belton, P.S., 2000. Gren tea polyphenols react with 1, 1-diphenyl-2-picrylhydrazyl free radicals in the bilayer of liposomes: direct evidence from electron spin resonance studies. J. Agric. Food Chem. 48, 5710– 5714. Creasy, D.M., 2001. Pathogenesis of male reproductive toxicity. Toxicol. Pathol. 29, 64–76. Dahlgren, I.L., Frikson, C.J., Gustafsson, B., Harthon, C., Hard, E., Larsson, K., 1989. Effects of chronic and acute ethanol treatment during prenatal and early postnatal ages on testosterone levels and sexual behaviors in rats. Pharmacol. Biochem. Behav. 33, 867–873.

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