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Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract
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Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract Rachid Mosbah a,b,∗ , Mokhtar Ibrahim Yousef c , Alberto Mantovani d a
Laboratoy of Animal Physiology, Department of Biology, University of Boumerdes, Boumerdes 35000, Algeria Laboratory of Animal Eco-biology, High School of Teachers, ENS-Kouba, Algiers, Algeria c Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, 163 Horreya Avenue, Chatby, PO Box 832, Alexandria 21526, Egypt d Istituto Superiore di Sanità, 00161 Rome, Italy b
a r t i c l e
i n f o
Article history: Received 18 July 2014 Received in revised form 29 October 2014 Accepted 2 January 2015 Keywords: Nicotine Green tea Haematotoxicity Oxidative stress Reproductive toxicity Rats
a b s t r a c t Nicotine is an active substance present in tobacco that causes oxidative stress and tissues damages leading to several diseases. Natural antioxidants that prevent or slow the progression and severity of nicotine toxicity may have a significant health impact. We have analyzed the effects of green tea extract (GTE) on nicotine (NT)-induced reproductive toxicity, oxidative damage and haematotoxicity in adult Wistar male rats. Thirty-two rats were randomly divided into four groups: control, nicotine (NT, 1 mg/kg i.p.), green tea extract (GTE, 2% w/v as the sole beverage) and (NT + GTE) group. After 2 months of treatment, blood samples were collected for measuring the haematological and oxidative stress parameters and testosterone level, while the reproductive organs were weighed and used for the semen analysis and histopathology. NT induced oxidative damage as indicated by a significant reduction in the activities of antioxidant enzymes and an elevation in TBARS levels. NT also caused reproductive toxicity as shown by a decline in testosterone levels, the weights of reproductive organs and sperm characteristics; the histological examination of testes revealed atrophy, degenerative alterations and perturbation of spermatogenesis in several seminiferous tubules, together with increased interstitial spaces and reduced number of Leydig cells. Both NT and GTE altered white blood cell count and red blood cells parameters, albeit with somewhat different effect, no protective action being seen upon NT + GTE treatment. On the contrary, GTE played a protective role against NT-induced oxidative stress as well as the reproductive effects by improving the oxidative status, semen quality and the testicular histological damage. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Currently, 1.3 billion people worldwide smoke or use other tobacco products. While smoking prevalence has declined in many
Abbreviations: NT, nicotine; GTE, green tea extract; TBARS, thiobarbituric acidreactive substances; SC, sperm count; SN, spermatid number; DSP, sperm daily production; STR, sperm transit rate; ROS, reactive oxygen species; WBC, white blood cells; RBC, red blood cells; Hb, haemoglobin; Ht, haematocrit; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume. ∗ Corresponding author at: Laboratory of Animal Physiology, Department of Biology, Faculty of Sciences, University of Boumerdes, Boumerdes 35000, Algeria. Tel.: +213 71 73 14 32. E-mail addresses: mosbah [email protected], [email protected] (R. Mosbah).
developed countries, it remains high in others and is increasing among women and in developing countries (WHO, 2010). There is growing evidence that cigarette smoking and tobacco chewing have a profound negative impact on general health associated with higher morbidity and mortality. Nicotine is the most abundant volatile alkaloid extracted from the dried leaves and stems of the Nicotiana tabacum and Nicotiana rustica and it features prominently among the over 4000 chemicals found in tobacco products (Rustemeier et al., 2002). Nicotine is well absorbed through the skin, the mucosal lining of the respiratory tract and the lungs and it can rapidly reach peak levels in the bloodstream and brain. It is an active compound on the nervous system, including the neuroendocrine axis, as it binds stereo-selectively to nicotinic cholinergic receptors in the autonomic ganglia, the chromaffin cells of the adrenal medulla, the neuromuscular junctions and the brain (Tundulawessa et al., 2010). Besides its pharmacodynamics
http://dx.doi.org/10.1016/j.etp.2015.01.001 0940-2993/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Mosbah R, et al. Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract. Exp Toxicol Pathol (2015), http://dx.doi.org/10.1016/j.etp.2015.01.001
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activities, nicotine is the main toxic component of cigarette smoking, with genotoxic, immunotoxic as well as reproductive effects in both sexes; it plays a detrimental role in the development of cardiovascular diseases and of respiratory and digestive tract cancers (Polyzos et al., 2009). Green tea (Camellia sinensis) has a long history of use worldwide. The beverage is made from boiling the naturally dried leaves and is considered as the major popular consumed drink other than water in many countries, e.g., Algeria and Egypt. Green tea is a very rich source of a specific kind of antioxidant called polyphenolic flavonoids, the catechins which include catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate (Chengelis et al., 2008). Over the last decade, green tea and polyphenols have attracted a great deal of attention because of its potential health benefits, which include anti-cancer, anti-inflammatory, antioxidant and antimicrobial effects; tea consumption has been linked to a possible risk reduction in some cardiovascular, dermatological, haematological, metabolic and neurological disorders, as well as obesity (Naganuma et al., 2009; Upaganlawar et al., 2009). However, there is currently little evidence about whether green tea may reduce the adverse effects of toxicants. Nicotine is known to generate reactive oxygen species (ROS), while green tea is known by its anti-oxidant activities and ability to scavenge ROS and trap hydroxyl, peroxyl and superoxide anion radicals due to the presence of catechins. Our study was carried out to investigate the possible protective role of Green tea extract against the oxidative stress, haematotoxicity and reproductive toxicity, induced by nicotine in adult Wistar male rats. 2. Materials and methods 2.1. Nicotine and green tea extract Nicotine, S-3-(1-methyl-2-pyrrolidinyl) pyridine, (C10 H14 N2 , CAS registry number 54-11-5, purity 98%) was purchased from Sigma Chemical Co. (St. Louis. France). The dose and the administration route were selected according to previous studies (Valenc¸a et al., 2004; Jana et al., 2010; Rehan et al., 2012). The green tea used in this work is a locally grown cultivar usually consumed in Algeria and purchased at Boumerdes market. Green tea extract was prepared from the dried leaves according to Kang et al. (2000) method. Briefly, twenty grams of green tea leaves were infused for 5 min in 1000 ml of boiling water (90 ◦ C), allowed to cool to room temperature and then filtered and stored in a brown bottle. The resulting aqueous extract (2% w/v) from the consumer product is similar to tea brews for human consumption and used as the sole drinking source according to Mehana et al. (2012). 2.2. Animals and experimental design The local Algerian ethical committee approved the design of the experiment and its compliance with general international principles of laboratory animal welfare. Thirty two adult male Wistar rats aged between 8 and 12 weeks were obtained from Pasteur Institute of Kouba-Algeria (internally bred strain) and acclimatized for 1 week before the experiment. The animals were housed in plastic cages with sawdust bedding and maintained in an air-conditioned animal house at a controlled temperature (22 ± 2 ◦ C) and relative humidity (60 ± 10%) with a photoperiod of 12 h light/12 h dark with free access to pellet feed (purchased from the National Office of Food Livestock, Algeria) and fresh tap water, except groups III and IV (see below). The animals were randomly assigned into four groups of eight animals as follows:
Group I: untreated, served as control (C). Group II: injected intraperitoneally (i.p.) with nicotine (NT in aqueous solution; 1 mg/kg body weight/day). Group III: received aqueous green tea extract (GTE) ad libitum as the sole drinking fluid at a concentration of 2% (w/v). Group IV: animals were simultaneously injected NT (1 mg/kg body weight/day i.p.) and given GTE ad libitum as the sole drinking fluid at a concentration of 2% (w/v) (NT + GTE). The animals were treated for 2 months. 2.3. Body and reproductive organs weights Body weights were recorded weekly during the experimental period. At the end of the experiment, the animals were sacrificed by decapitation under ether anaesthesia. Body weights were recorded before sacrifice; male reproductive organs (testes, epididymes and seminal vesicles) were quickly removed, cleared from adipose and connective tissues and weighed, then relative organ weights were calculated. 2.4. Haematological analysis At the end of the treatment, blood samples were collected from the orbital sinus and placed immediately on ice. EDTA was used as an anticoagulant agent to determine the selected haematological parameters. Red blood cells (RBC) and white blood cells (WBC) count, haematocrit value (Ht), haemoglobin level (Hb) were measured manually by routine methods, namely haemocytometry and spectrophotometry, respectively. Then, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were calculated. 2.5. Determination of oxidative stress indicators The content of reduced glutathione (GSH) was determined by the enzymatic method of Jollow et al. (1974). Thiobarbituric acid reactive substances (TBARS) were determined according to the method of Tappel and Zalkin (1959). The antioxidant enzymes, superoxide dismutase (SOD, EC. 1.15.1.1) and Catalase (CAT, EC. 1.11.1.6) activities were measured according to Flohe and Otting (1984) and Aebi (1984), respectively. 2.6. Semen characteristics 2.6.1. Sperm and spermatid number The left testis and epididymis from each rat were excised. After removal of tunica albuginea, the testis was minced with scissor and homogenized in 10 ml 0.9% NaCl containing 0.5% Triton X-100; the homogenate was gently mixed by using vortex mixer. The number of homogenization-resistant spermatids (spermatids number, SN) was counted by a haemocytometer (Mallassez) chamber; daily sperm production (DSP) was calculated by dividing the number of homogenization-resistant spermatids by 6.1 (Blazak et al., 1993). The cauda epididymis was cut into small pieces by a disposable blade in 10 ml of 0.9% NaCl containing 0.5% Triton X-100, homogenized and spermatozoa were counted (sperm count, SC) as described above; the epididymal sperm transit rate (STR) was estimated by dividing the epididymal sperm number by the daily sperm production (Amman et al., 1976; Blazak et al., 1993). 2.6.2. Sperm motility and morphology analysis The right cauda epididymis of each animal was excised and placed in a warmed petri dish containing 2 ml of Hanks’s solution at 37 ◦ C. The tissue was cut with scalpel to release sperm and placed in a 37 ◦ C incubator for 15 min prior to determining
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sperm motility. The suspension was stirred and 20 l were placed between a warmed microscope slide and coverslip. Motile and nonmotile sperms were manually counted with a microscope at 400× magnification in at least 10 separate and randomly selected fields. The coverslip was removed and the spermatozoa suspension was allowed to dry in air. The sample was stained with 1% eosinY/5% nigrosin and examined microscopically at 400× for morphological abnormalities. Three hundred spermatozoa from different fields were examined for each sample as described previously (Liobet et al., 1995).
Table 3 summarizes the effects of NT, GTE and NT + GTE on plasma TBARS and GSH levels and the activities of CAT and SOD. The results showed a significant elevation in the plasma TBARS levels (p < 0.01) and a reduction in the levels of GSH and in the CAT and SOD activities in the NT group; in the GTE group, CAT and SOD activities were significantly increased. On the other hand, the mean values of oxidative stress indicators in the NT–GTE group were not significantly different from control.
2.7. Testosterone measurement
3.4. Sperm analysis and testosterone levels
Plasma testosterone concentration was measured using the enzyme linked immuno-sorbant assay (ELISA) kit purchased from DRG diagnostics, GmbH, Germany.
The effects of NT, GTE and NT + GTE on semen characteristics are given in Table 4. NT markedly affected sperm quality, as evidenced by a significant reduction (p < 0.01 and p < 0.001) in spermatids number, sperm count, sperm motility, daily sperm production as well as in testosterone levels; meanwhile, sperm abnormality was increased significantly (p < 0.01). On the other hand, no alterations in sperm parameters and testosterone levels were observed in either the GTE or NT + GTE groups as compared to controls.
2.8. Histopathological examination A specimen from the right testis of each animal was removed and dropped in aqueous Bouin’s fluid. After fixation for 48 h, tissues were dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin. Sections of 5 m thick were obtained from paraffin blocks using a rotatory microtome. Then, they were mounted on a microscope slide, stained with haematoxylin–eosin (HE) and examined under light microscopy. 2.9. Statistical analysis All values were expressed as mean ± SD and analysed according to Steel and Torrie (1981). Statistical significance of the difference in values of control and treated animals was calculated by (F) test at 5% significance level. Multiple comparisons of means were done by means of Duncan’s multiple range test with the least significant difference (LSD) (SAS, 1986). 3. Results 3.1. Body and reproductive organs weights The effects of NT, GTE and NT + GTE on body weights as well as the absolute and relative weights of reproductive organs are summarized in Table 1 and Fig. 1. NT alone caused a significant decrease (p < 0.01) in mean body weight gains (68.15 ± 16.35) as compared to control (88.53 ± 10.80). Meanwhile, in the GTE and NT + GTE groups the body weight gains were not different from control (Table 1). As shown in Fig. 1A and B, the NT group showed a significant decrease in absolute and relative weight of testes (p < 0.05), epididymes and seminal vesicles (p < 0.01) compared to the control group. Conversely, the absolute and relative weights of all reproductive organs in both the GTE and NT + GTE groups showed no significant differences as compared to control group (Fig. 1B). 3.2. Haematological parameters The findings on haematological parameters are shown in Table 2. Treatment with either GTE and/or NT did alter haematological parameters, albeit with different effects. As compared to the control group, the WBC was significantly (p < 0.05) decreased in rats treated with GTE alone, while in the NT group WBC was markedly augmented (p < 0.001). Yet, in the NT + GTE group the WBC value was not different from controls. All three treatments, NT, GTE and NT + GTE, reduced significantly the mean RBC, Ht and Hb values; MCV was also increased significantly in all three treatment groups, with the highest value in the NT + GTE group.
3.3. Oxidative stress indicators
3.5. Histopathology of testis The histopathological examination of testes of control and GTE groups (Fig. 2A and C), showed a normal tissue structure and architecture of the spermatogenic cycle, while NT-treated rats (Fig. 2B) exhibited evident signs of testicular injury such as atrophy and degeneration of the seminiferous epithelium with absence of spermatozoa, presence of cell debris and loss of the Sertoli cells in some seminiferous tubules, as well as widening of the interstitial spaces with reduced presence of Leydig cells. No such changes or any other appreciable abnormal findings were present in the NT + GTE group (Fig. 2D). 4. Discussion Overall, the findings of this study show how GTE can reduce or reverse the effects of a toxic substance affecting several body functions like NT. GTE prevented the NT-induced reduction in body weight gain. Perkins et al. (1991) reported that NT administration to rats decreased body weight gain by both affecting metabolism (decreased fat stores) and reducing appetite. Indeed, NT can increase the metabolic rate and energy expenditure by sympathoadrenal activation (Nunn, 1993). NT exhibited a marked testicular toxicity as evidenced by the most tested parameters such as the reduced weight of testes, epididymes and seminal vesicles, overall impaired semen quality and testicular histopathology. Cigarette smoking and nicotine administration are recognized to reduce the male sexual function in animals and humans: a number of epidemiological and experimental studies show effects similar to those observed in NT-treated rats by our study (Patterson et al., 1990; Polyzos et al., 2009; Jana et al., 2010; Gaur et al., 2007; Oyeyipo et al., 2011; Wong et al., 2000; Sankako et al., 2013). We observed decreased testosterone levels as well as reduced presence of Leydig and Sertoli cells in the testes of NT-treated rats, which are findings suggestive of an impaired androgen production. Kavitharaj and Vijayammal (1999) also observed that NT-induced reductions of sperm production, maturation and fertilizing potential in rats were accompanied by a decrease in testosterone and oestradiol levels. Londonkar et al. (2000) observed a reduction in the weight of testis, epididymis, seminal vesicle, prostate gland and vas deferens in rats treated with 3 and 4 mg NT/kg body weight either orally or intraperitoneally for 30 days; the authors attributed the changes to endocrine-related
Please cite this article in press as: Mosbah R, et al. Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract. Exp Toxicol Pathol (2015), http://dx.doi.org/10.1016/j.etp.2015.01.001
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Table 1 Effects of nicotine (NT), green tea extract (GTE) and their combination on body weight gain after 2 months of treatment. Parameter
Groups Control
Initial body weights (g) Final body weights (g) Body weight gains (g) Weight change (%)
217.49 306.01 88.53 28.87
NT ± ± ± ±
10.04a 15.33a 10.80a 2.68a
GTE
219.74 287.89 68.15 23.60
± ± ± ±
12.87a 8.84a 16.35b 5.20b
NT + GTE
216.50 300.68 84.18 27.91
± ± ± ±
10.48a 9.44a 15.46a 4.52a
219.93 298.28 78.35 26.25
± ± ± ±
15.39a 9.28a 15.11a 4.96a
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b) were significantly different, p < 0.05.
Fig. 1. Effects of nicotine (NT), green tea extract (GTE) and their combination on absolute (A) and relative weights (B) of reproductive organs after 2 months of treatment. All data were expressed as means ± SD, * significantly different from control group at (p < 0.05), ** significantly different from control group at (p < 0.01).
Table 2 Effects of nicotine (NT), green tea extract (GTE) and their combination on some haematological parameters after 2 months of treatment. Parameter
Groups Control
3
−3
WBC (10 mm ) RBC (106 mm−3 ) Ht (%) Hb (g/dl) MCH (pg) MCV (fl) MCHC (%)
7.33 5.43 43.75 13.98 25.75 80.55 31.99
NT
± ± ± ± ± ± ±
b
0.87 0.13a 1.71a 0.44a 1.22b 1.26c 1.83a
GTE
9.08 4.31 40 11.43 28.88 95.3 30.35
± ± ± ± ± ± ±
a
0.45 0.21c 1.04b 0.70b 2.01a 4.79a 2.36a
NT + GTE
5.84 4.8 40.25 11.44 25.99 86.05 30.15
± ± ± ± ± ± ±
c
0.22 0.20b 0.96b 0.97b 2.90b 4.18b 2.20a
7.49 4.26 39.75 10.69 28.15 95.81 29.37
± ± ± ± ± ± ±
0.27b 0.11c 1.24b 0.76b 2.29a 3.86a 1.95b
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c) were significantly different, p < 0.05.
Table 3 Effects of nicotine (NT), green tea extract (GTE) and their combination on plasma thiobarbituric acid-reactive substances (TBARS, nmol/ml), superoxide dismutase (SOD, U/ml), catalase (CAT, U/ml) and glutathione (GSH, U/ml). Parameters
Groups Control
TBARS SOD CAT GSH
2.23 2.79 47.69 4.11
± ± ± ±
NT 0.045b 0.061b 2.91b 0.13a
5.31 0.78 24.53 2.28
GTE ± ± ± ±
0.23a 0.097d 1.78d 0.20c
NT + GTE
2.44 3.42 68.26 4.37
± ± ± ±
0.032b 0.057a 1.65a 0.23a
2.89 1.60 41.34 3.10
± ± ± ±
0.052b 0.081c 1.52c 0.15b
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c, d) were significantly different, p < 0.05.
Table 4 Effects of nicotine (NT), green tea extract (GTE) and their combination on spermatids number (SN), sperm count (SC), motility, abnormal sperms, daily sperm production (DSP), sperm transit rate (STR) and testosterone level in rats treated for 2 months. Parameters
Groups Control
6
−1
SN (10 g testis) SC (106 g−1 cauda epididymis) Motility (%) Abnormal sperms (%) DSP (106 g−1 testis) STR (106 day−1 ) Testosterone (ng/ml)
177 314.28 75.53 3.00 29.01 10.84 3.79
NT ± ± ± ± ± ± ±
a
14.45 29.08a 4.80b 0.82c 2.37a 0.73a 0.46a
145.86 239.11 54.15 9.50 23.91 10.03 2.63
GTE ± ± ± ± ± ± ±
c
9.18 15.16c 5.25c 2.51a 1.50d 0.88a 0.27b
165.75 307.94 79.41 2.00 27.17 11.36 3.75
NT + GTE ± ± ± ± ± ± ±
b
6.65 26.24b 3. 64a 0.82d 1.09a 1.22a 0.28a
163.13 285.09 73. 53 4.50 26.74 10.71 3.45
± ± ± ± ± ± ±
9.32b 23.70b 4.21b 1.51b 1.53b 1.30a 0.31a
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c) were significantly different, p < 0.05.
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Fig. 2. Light microscopic sections of testes from various examined groups, control (A), nicotine treated animals (B), green tea extract (C), and combination NT + GTE (D) for 2 months. Sections (5 m thickness) were stained with haematoxylin–eosin (100×). ST: seminiferous tubule; IT: interstitial tissue; L: lumen; SE: seminal epithelium; SG: spermatogonia; SZ: spermatozoa; BV: blood vessel; arrows: disorganization of germinal cells and atrophy of tubules; Star: widened interstitial spaces with few number of Leydig cells.
mechanisms, such as impaired androgen synthesis and reduced output of pituitary FSH and LH. In non-smoking humans, NT intravenous treatment increased serum prolactin levels (Newhouse et al., 1990), further supporting that NT might primarily act on neuroendocrine and pituitary regulation. Studies on men and male rats indicate that cigarette smoking and nicotine administration increase prolactin levels and inhibit the release of GnRH, thus impairing the gonadotropins FSH and LH: these hormones control the function of Sertoli and Leydig cells that are critical for initiating and completing spermatogenesis and steroidgenesis in the testis (Mendelson et al., 2003; Oyeyipo et al., 2013). The concurrent exposure to GTE fully prevented the reproductive toxicity of NT. Overall, the findings on weight gain and reproductive effects may suggest that substances, like catechins, contained in green tea might exert a protective action also towards oxidative stress and neuroendocrine disruption (El-Shahat et al., 2009; Abbas and Wink, 2014; Heikal et al., 2014). Our data clearly show that NT-treatment increases the indicators of oxidative stress and that this finding is prevented by the concurrent intake of GTE, consistent with the known antioxidant role of GTE polyphenols. Indeed, oxidative stress may be a concurrent mechanism of NT testicular toxicity. Nicotine increases ROS by breaking the mitochondrial respiratory chain (Guan et al., 2003) and inhibits testosterone biosynthesis in mouse Leydig cells (Patterson et al., 1990); the NT-enhanced ROS production impairs steroidogenesis at the first step of cholesterol transfer to the mitochondria by suppressing the steroidogenic acute regulatory (StAR) protein expression (Jana et al., 2010; Seema et al., 2007). Spermatozoa, being rich in polyunsaturated fatty acids, are liable to
lipid peroxidation by ROS that will result in changes of membrane structural integrity and fluidity. Seminal plasma and testicular cells are well equipped with antioxidant defences; however the NT-induced overproduction of ROS in the testis may over-ride the antioxidant defence mechanisms (Hammadeh et al., 2009; ElSweedy et al., 2007). Catechins do improve lipid peroxidation and quality parameters in boar semen (Boonsorn et al., 2010). The limited available data also indicate that GTE or specific GT polyphenols may ameliorate the liver or testicular toxicity of substances like chlorpyrifos and doxorubicyn, presumably through an antioxidant action (Heikal et al., 2013; Sato et al., 2010; Kalender et al., 2012). In addition, tea contains zinc, selenium and manganese which act as co-factors in antioxidant enzymes, while polyphenols can chelate metal ions such as iron and copper which leading to the formation of hydroxyl radical, inhibit the redox-sensitive transcription factors and the pro-oxidant enzymes, such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), lipoxygenase 2 (LOX-2) and xanthine oxidase over than stimulate the antioxidant enzymes, such as glutathione S-transferases and superoxide dismutases (Frei and Higdon, 2003). To our best knowledge, our study is the first one showing a preventive effect towards a substance with complex, ROS—as well as endocrine-mediated mechanisms like NT. At the moment, we cannot determine with certainty whether the protective effect of GTE is solely a result of its antioxidant effect, or whether it may mitigate also the adverse effects of NT on neuroendocrine and endocrine regulation. The effect on blood parameters were less straightforward as a decrease in red blood cells, haemoglobin and haematocrit which
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was observed in all the three treated groups, NT, GTE as well as NT + GTE. Consistent with our results, Karafakiogly et al. (2009) observed that after the harvest season of tobacco, a significant decrease of red blood cells, haemoglobin and haematocrit was detected in women workers. Erythrocytes are particularly susceptible to oxidative damage as a result of high polyunsaturated fatty acid content in cell membranes and high concentration of oxygen and haemoglobin, the latter being a potential strong promoter of oxidative processes; thus, the NT-reduction of red blood cells may be due to peroxidative membrane damages via ROS production (Tedesco et al., 2000) and/or by ROS reacting with haemoglobin, altering its structure and releasing free iron ions that, in turn increase the generation of ROS (Cimen, 2008). According to our study, GTE do not afford protection against the NT haematotoxicity contrary to testicular toxicity. This might be at least partly attributable to the erythrocytes being much less equipped by antioxidant enzymes to cope with ROS as compared to seminiferous epithelium and the sperm: thus, erythrocytes could be unable to reverse the NT damage when supported by GTE supplementation contrary to seminiferous cells. Finally, NT and GTE appear to have antagonistic effects on white blood cells (reducing and increasing the WBC number, respectively) that are compensated upon combined treatment: in fact, in the NT + GTE group, the value was comparable to controls. We recognize that the NT-treated group has been exposed to a daily manipulation (the i.p. treatment) that, in principle, might have introduced an additional stress to the animals. However, the findings obtained in the NT group in our study were fully consistent with those reported in the literature as regards NT-effects on analogous parameters: thus, the manipulation bias had at the most a slight effect, if any. 5. Conclusion Green tea extract beverage may protect against nicotineinduced oxidative stress, reduced body growth and reproductive toxicity, including endocrine-mediated effects; a protective effect against nicotine haematotoxicity was not observed in our experimental conditions. The intake of green tea, and possibly of individual green tea catechins, may be considered in order to ameliorate several adverse health effects in subjects that have been exposed to nicotine through active or passive smoking or other scenarios, such as the workplace. References Abbas S, Wink M. Green tea extract induces the resistance of Caenorhabditis elegans against oxidative stress. Antioxidants 2014;3:129–43. Aebi HE. Catalase in vitro. Methods Enzymol 1984;105:121–6. Amman RP, Johnson L, Thompson DL, Pickett BW. Daily spermatozoal production, epididymal spermatozoal reserves and transit time of spermatozoa through the epididymides of the rhesus monkey. Biol Reprod 1976;15:586–92. Blazak WF, Treinen KA, Juniewicz PE. Male reproductive toxicology, Part A. In: Chapin RE, Heindel JJ, editors. Methods in toxicology. San Diego, CA: Calif. Academic Press; 1993. p. 86–94. Boonsorn T, Kongbuntad W, Narklong N, Aengwanich W. Effects of catechin addition to extender on sperm quality and lipid peroxidation in boar semen AmericanEurasian. J Agric Environ Sci 2010;7(3):283–8. Chengelis CP, Kirkpatrick JB, Regan KS, Radovsky AE, Beck MJ, Morita O. 28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats. Food Chem Toxicol 2008;46:978–89. Cimen MY. Free radical metabolism in human erythrocytes. Clin Chim Acta 2008;390:1–11. El-Shahat AR, Gabr A, Meki AR, Mehana E. Altered testicular morphology and oxidative stress induced by cadmium in experimental rats and protective effect of simultaneous green tea extract. Int J Morphol 2009;27(3):757–64. El-Sweedy M, Abdel-Hamid N, El-Moselhy M. The role of a mixture of green tea, turmeric and chitosan in the treatment of obesity-related testicular disorders. J Appl Biomed 2007;5:131–8. Flohe L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:101–4. Frei B, Higdon JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J Nutrition 2003;133:3275S–84S.
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Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract Rachid Mosbah a,b,∗ , Mokhtar Ibrahim Yousef c , Alberto Mantovani d a
Laboratoy of Animal Physiology, Department of Biology, University of Boumerdes, Boumerdes 35000, Algeria Laboratory of Animal Eco-biology, High School of Teachers, ENS-Kouba, Algiers, Algeria c Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, 163 Horreya Avenue, Chatby, PO Box 832, Alexandria 21526, Egypt d Istituto Superiore di Sanità, 00161 Rome, Italy b
a r t i c l e
i n f o
Article history: Received 18 July 2014 Received in revised form 29 October 2014 Accepted 2 January 2015 Keywords: Nicotine Green tea Haematotoxicity Oxidative stress Reproductive toxicity Rats
a b s t r a c t Nicotine is an active substance present in tobacco that causes oxidative stress and tissues damages leading to several diseases. Natural antioxidants that prevent or slow the progression and severity of nicotine toxicity may have a significant health impact. We have analyzed the effects of green tea extract (GTE) on nicotine (NT)-induced reproductive toxicity, oxidative damage and haematotoxicity in adult Wistar male rats. Thirty-two rats were randomly divided into four groups: control, nicotine (NT, 1 mg/kg i.p.), green tea extract (GTE, 2% w/v as the sole beverage) and (NT + GTE) group. After 2 months of treatment, blood samples were collected for measuring the haematological and oxidative stress parameters and testosterone level, while the reproductive organs were weighed and used for the semen analysis and histopathology. NT induced oxidative damage as indicated by a significant reduction in the activities of antioxidant enzymes and an elevation in TBARS levels. NT also caused reproductive toxicity as shown by a decline in testosterone levels, the weights of reproductive organs and sperm characteristics; the histological examination of testes revealed atrophy, degenerative alterations and perturbation of spermatogenesis in several seminiferous tubules, together with increased interstitial spaces and reduced number of Leydig cells. Both NT and GTE altered white blood cell count and red blood cells parameters, albeit with somewhat different effect, no protective action being seen upon NT + GTE treatment. On the contrary, GTE played a protective role against NT-induced oxidative stress as well as the reproductive effects by improving the oxidative status, semen quality and the testicular histological damage. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Currently, 1.3 billion people worldwide smoke or use other tobacco products. While smoking prevalence has declined in many
Abbreviations: NT, nicotine; GTE, green tea extract; TBARS, thiobarbituric acidreactive substances; SC, sperm count; SN, spermatid number; DSP, sperm daily production; STR, sperm transit rate; ROS, reactive oxygen species; WBC, white blood cells; RBC, red blood cells; Hb, haemoglobin; Ht, haematocrit; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume. ∗ Corresponding author at: Laboratory of Animal Physiology, Department of Biology, Faculty of Sciences, University of Boumerdes, Boumerdes 35000, Algeria. Tel.: +213 71 73 14 32. E-mail addresses: mosbah [email protected], [email protected] (R. Mosbah).
developed countries, it remains high in others and is increasing among women and in developing countries (WHO, 2010). There is growing evidence that cigarette smoking and tobacco chewing have a profound negative impact on general health associated with higher morbidity and mortality. Nicotine is the most abundant volatile alkaloid extracted from the dried leaves and stems of the Nicotiana tabacum and Nicotiana rustica and it features prominently among the over 4000 chemicals found in tobacco products (Rustemeier et al., 2002). Nicotine is well absorbed through the skin, the mucosal lining of the respiratory tract and the lungs and it can rapidly reach peak levels in the bloodstream and brain. It is an active compound on the nervous system, including the neuroendocrine axis, as it binds stereo-selectively to nicotinic cholinergic receptors in the autonomic ganglia, the chromaffin cells of the adrenal medulla, the neuromuscular junctions and the brain (Tundulawessa et al., 2010). Besides its pharmacodynamics
http://dx.doi.org/10.1016/j.etp.2015.01.001 0940-2993/© 2015 Elsevier GmbH. All rights reserved.
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activities, nicotine is the main toxic component of cigarette smoking, with genotoxic, immunotoxic as well as reproductive effects in both sexes; it plays a detrimental role in the development of cardiovascular diseases and of respiratory and digestive tract cancers (Polyzos et al., 2009). Green tea (Camellia sinensis) has a long history of use worldwide. The beverage is made from boiling the naturally dried leaves and is considered as the major popular consumed drink other than water in many countries, e.g., Algeria and Egypt. Green tea is a very rich source of a specific kind of antioxidant called polyphenolic flavonoids, the catechins which include catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate (Chengelis et al., 2008). Over the last decade, green tea and polyphenols have attracted a great deal of attention because of its potential health benefits, which include anti-cancer, anti-inflammatory, antioxidant and antimicrobial effects; tea consumption has been linked to a possible risk reduction in some cardiovascular, dermatological, haematological, metabolic and neurological disorders, as well as obesity (Naganuma et al., 2009; Upaganlawar et al., 2009). However, there is currently little evidence about whether green tea may reduce the adverse effects of toxicants. Nicotine is known to generate reactive oxygen species (ROS), while green tea is known by its anti-oxidant activities and ability to scavenge ROS and trap hydroxyl, peroxyl and superoxide anion radicals due to the presence of catechins. Our study was carried out to investigate the possible protective role of Green tea extract against the oxidative stress, haematotoxicity and reproductive toxicity, induced by nicotine in adult Wistar male rats. 2. Materials and methods 2.1. Nicotine and green tea extract Nicotine, S-3-(1-methyl-2-pyrrolidinyl) pyridine, (C10 H14 N2 , CAS registry number 54-11-5, purity 98%) was purchased from Sigma Chemical Co. (St. Louis. France). The dose and the administration route were selected according to previous studies (Valenc¸a et al., 2004; Jana et al., 2010; Rehan et al., 2012). The green tea used in this work is a locally grown cultivar usually consumed in Algeria and purchased at Boumerdes market. Green tea extract was prepared from the dried leaves according to Kang et al. (2000) method. Briefly, twenty grams of green tea leaves were infused for 5 min in 1000 ml of boiling water (90 ◦ C), allowed to cool to room temperature and then filtered and stored in a brown bottle. The resulting aqueous extract (2% w/v) from the consumer product is similar to tea brews for human consumption and used as the sole drinking source according to Mehana et al. (2012). 2.2. Animals and experimental design The local Algerian ethical committee approved the design of the experiment and its compliance with general international principles of laboratory animal welfare. Thirty two adult male Wistar rats aged between 8 and 12 weeks were obtained from Pasteur Institute of Kouba-Algeria (internally bred strain) and acclimatized for 1 week before the experiment. The animals were housed in plastic cages with sawdust bedding and maintained in an air-conditioned animal house at a controlled temperature (22 ± 2 ◦ C) and relative humidity (60 ± 10%) with a photoperiod of 12 h light/12 h dark with free access to pellet feed (purchased from the National Office of Food Livestock, Algeria) and fresh tap water, except groups III and IV (see below). The animals were randomly assigned into four groups of eight animals as follows:
Group I: untreated, served as control (C). Group II: injected intraperitoneally (i.p.) with nicotine (NT in aqueous solution; 1 mg/kg body weight/day). Group III: received aqueous green tea extract (GTE) ad libitum as the sole drinking fluid at a concentration of 2% (w/v). Group IV: animals were simultaneously injected NT (1 mg/kg body weight/day i.p.) and given GTE ad libitum as the sole drinking fluid at a concentration of 2% (w/v) (NT + GTE). The animals were treated for 2 months. 2.3. Body and reproductive organs weights Body weights were recorded weekly during the experimental period. At the end of the experiment, the animals were sacrificed by decapitation under ether anaesthesia. Body weights were recorded before sacrifice; male reproductive organs (testes, epididymes and seminal vesicles) were quickly removed, cleared from adipose and connective tissues and weighed, then relative organ weights were calculated. 2.4. Haematological analysis At the end of the treatment, blood samples were collected from the orbital sinus and placed immediately on ice. EDTA was used as an anticoagulant agent to determine the selected haematological parameters. Red blood cells (RBC) and white blood cells (WBC) count, haematocrit value (Ht), haemoglobin level (Hb) were measured manually by routine methods, namely haemocytometry and spectrophotometry, respectively. Then, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were calculated. 2.5. Determination of oxidative stress indicators The content of reduced glutathione (GSH) was determined by the enzymatic method of Jollow et al. (1974). Thiobarbituric acid reactive substances (TBARS) were determined according to the method of Tappel and Zalkin (1959). The antioxidant enzymes, superoxide dismutase (SOD, EC. 1.15.1.1) and Catalase (CAT, EC. 1.11.1.6) activities were measured according to Flohe and Otting (1984) and Aebi (1984), respectively. 2.6. Semen characteristics 2.6.1. Sperm and spermatid number The left testis and epididymis from each rat were excised. After removal of tunica albuginea, the testis was minced with scissor and homogenized in 10 ml 0.9% NaCl containing 0.5% Triton X-100; the homogenate was gently mixed by using vortex mixer. The number of homogenization-resistant spermatids (spermatids number, SN) was counted by a haemocytometer (Mallassez) chamber; daily sperm production (DSP) was calculated by dividing the number of homogenization-resistant spermatids by 6.1 (Blazak et al., 1993). The cauda epididymis was cut into small pieces by a disposable blade in 10 ml of 0.9% NaCl containing 0.5% Triton X-100, homogenized and spermatozoa were counted (sperm count, SC) as described above; the epididymal sperm transit rate (STR) was estimated by dividing the epididymal sperm number by the daily sperm production (Amman et al., 1976; Blazak et al., 1993). 2.6.2. Sperm motility and morphology analysis The right cauda epididymis of each animal was excised and placed in a warmed petri dish containing 2 ml of Hanks’s solution at 37 ◦ C. The tissue was cut with scalpel to release sperm and placed in a 37 ◦ C incubator for 15 min prior to determining
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sperm motility. The suspension was stirred and 20 l were placed between a warmed microscope slide and coverslip. Motile and nonmotile sperms were manually counted with a microscope at 400× magnification in at least 10 separate and randomly selected fields. The coverslip was removed and the spermatozoa suspension was allowed to dry in air. The sample was stained with 1% eosinY/5% nigrosin and examined microscopically at 400× for morphological abnormalities. Three hundred spermatozoa from different fields were examined for each sample as described previously (Liobet et al., 1995).
Table 3 summarizes the effects of NT, GTE and NT + GTE on plasma TBARS and GSH levels and the activities of CAT and SOD. The results showed a significant elevation in the plasma TBARS levels (p < 0.01) and a reduction in the levels of GSH and in the CAT and SOD activities in the NT group; in the GTE group, CAT and SOD activities were significantly increased. On the other hand, the mean values of oxidative stress indicators in the NT–GTE group were not significantly different from control.
2.7. Testosterone measurement
3.4. Sperm analysis and testosterone levels
Plasma testosterone concentration was measured using the enzyme linked immuno-sorbant assay (ELISA) kit purchased from DRG diagnostics, GmbH, Germany.
The effects of NT, GTE and NT + GTE on semen characteristics are given in Table 4. NT markedly affected sperm quality, as evidenced by a significant reduction (p < 0.01 and p < 0.001) in spermatids number, sperm count, sperm motility, daily sperm production as well as in testosterone levels; meanwhile, sperm abnormality was increased significantly (p < 0.01). On the other hand, no alterations in sperm parameters and testosterone levels were observed in either the GTE or NT + GTE groups as compared to controls.
2.8. Histopathological examination A specimen from the right testis of each animal was removed and dropped in aqueous Bouin’s fluid. After fixation for 48 h, tissues were dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin. Sections of 5 m thick were obtained from paraffin blocks using a rotatory microtome. Then, they were mounted on a microscope slide, stained with haematoxylin–eosin (HE) and examined under light microscopy. 2.9. Statistical analysis All values were expressed as mean ± SD and analysed according to Steel and Torrie (1981). Statistical significance of the difference in values of control and treated animals was calculated by (F) test at 5% significance level. Multiple comparisons of means were done by means of Duncan’s multiple range test with the least significant difference (LSD) (SAS, 1986). 3. Results 3.1. Body and reproductive organs weights The effects of NT, GTE and NT + GTE on body weights as well as the absolute and relative weights of reproductive organs are summarized in Table 1 and Fig. 1. NT alone caused a significant decrease (p < 0.01) in mean body weight gains (68.15 ± 16.35) as compared to control (88.53 ± 10.80). Meanwhile, in the GTE and NT + GTE groups the body weight gains were not different from control (Table 1). As shown in Fig. 1A and B, the NT group showed a significant decrease in absolute and relative weight of testes (p < 0.05), epididymes and seminal vesicles (p < 0.01) compared to the control group. Conversely, the absolute and relative weights of all reproductive organs in both the GTE and NT + GTE groups showed no significant differences as compared to control group (Fig. 1B). 3.2. Haematological parameters The findings on haematological parameters are shown in Table 2. Treatment with either GTE and/or NT did alter haematological parameters, albeit with different effects. As compared to the control group, the WBC was significantly (p < 0.05) decreased in rats treated with GTE alone, while in the NT group WBC was markedly augmented (p < 0.001). Yet, in the NT + GTE group the WBC value was not different from controls. All three treatments, NT, GTE and NT + GTE, reduced significantly the mean RBC, Ht and Hb values; MCV was also increased significantly in all three treatment groups, with the highest value in the NT + GTE group.
3.3. Oxidative stress indicators
3.5. Histopathology of testis The histopathological examination of testes of control and GTE groups (Fig. 2A and C), showed a normal tissue structure and architecture of the spermatogenic cycle, while NT-treated rats (Fig. 2B) exhibited evident signs of testicular injury such as atrophy and degeneration of the seminiferous epithelium with absence of spermatozoa, presence of cell debris and loss of the Sertoli cells in some seminiferous tubules, as well as widening of the interstitial spaces with reduced presence of Leydig cells. No such changes or any other appreciable abnormal findings were present in the NT + GTE group (Fig. 2D). 4. Discussion Overall, the findings of this study show how GTE can reduce or reverse the effects of a toxic substance affecting several body functions like NT. GTE prevented the NT-induced reduction in body weight gain. Perkins et al. (1991) reported that NT administration to rats decreased body weight gain by both affecting metabolism (decreased fat stores) and reducing appetite. Indeed, NT can increase the metabolic rate and energy expenditure by sympathoadrenal activation (Nunn, 1993). NT exhibited a marked testicular toxicity as evidenced by the most tested parameters such as the reduced weight of testes, epididymes and seminal vesicles, overall impaired semen quality and testicular histopathology. Cigarette smoking and nicotine administration are recognized to reduce the male sexual function in animals and humans: a number of epidemiological and experimental studies show effects similar to those observed in NT-treated rats by our study (Patterson et al., 1990; Polyzos et al., 2009; Jana et al., 2010; Gaur et al., 2007; Oyeyipo et al., 2011; Wong et al., 2000; Sankako et al., 2013). We observed decreased testosterone levels as well as reduced presence of Leydig and Sertoli cells in the testes of NT-treated rats, which are findings suggestive of an impaired androgen production. Kavitharaj and Vijayammal (1999) also observed that NT-induced reductions of sperm production, maturation and fertilizing potential in rats were accompanied by a decrease in testosterone and oestradiol levels. Londonkar et al. (2000) observed a reduction in the weight of testis, epididymis, seminal vesicle, prostate gland and vas deferens in rats treated with 3 and 4 mg NT/kg body weight either orally or intraperitoneally for 30 days; the authors attributed the changes to endocrine-related
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Table 1 Effects of nicotine (NT), green tea extract (GTE) and their combination on body weight gain after 2 months of treatment. Parameter
Groups Control
Initial body weights (g) Final body weights (g) Body weight gains (g) Weight change (%)
217.49 306.01 88.53 28.87
NT ± ± ± ±
10.04a 15.33a 10.80a 2.68a
GTE
219.74 287.89 68.15 23.60
± ± ± ±
12.87a 8.84a 16.35b 5.20b
NT + GTE
216.50 300.68 84.18 27.91
± ± ± ±
10.48a 9.44a 15.46a 4.52a
219.93 298.28 78.35 26.25
± ± ± ±
15.39a 9.28a 15.11a 4.96a
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b) were significantly different, p < 0.05.
Fig. 1. Effects of nicotine (NT), green tea extract (GTE) and their combination on absolute (A) and relative weights (B) of reproductive organs after 2 months of treatment. All data were expressed as means ± SD, * significantly different from control group at (p < 0.05), ** significantly different from control group at (p < 0.01).
Table 2 Effects of nicotine (NT), green tea extract (GTE) and their combination on some haematological parameters after 2 months of treatment. Parameter
Groups Control
3
−3
WBC (10 mm ) RBC (106 mm−3 ) Ht (%) Hb (g/dl) MCH (pg) MCV (fl) MCHC (%)
7.33 5.43 43.75 13.98 25.75 80.55 31.99
NT
± ± ± ± ± ± ±
b
0.87 0.13a 1.71a 0.44a 1.22b 1.26c 1.83a
GTE
9.08 4.31 40 11.43 28.88 95.3 30.35
± ± ± ± ± ± ±
a
0.45 0.21c 1.04b 0.70b 2.01a 4.79a 2.36a
NT + GTE
5.84 4.8 40.25 11.44 25.99 86.05 30.15
± ± ± ± ± ± ±
c
0.22 0.20b 0.96b 0.97b 2.90b 4.18b 2.20a
7.49 4.26 39.75 10.69 28.15 95.81 29.37
± ± ± ± ± ± ±
0.27b 0.11c 1.24b 0.76b 2.29a 3.86a 1.95b
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c) were significantly different, p < 0.05.
Table 3 Effects of nicotine (NT), green tea extract (GTE) and their combination on plasma thiobarbituric acid-reactive substances (TBARS, nmol/ml), superoxide dismutase (SOD, U/ml), catalase (CAT, U/ml) and glutathione (GSH, U/ml). Parameters
Groups Control
TBARS SOD CAT GSH
2.23 2.79 47.69 4.11
± ± ± ±
NT 0.045b 0.061b 2.91b 0.13a
5.31 0.78 24.53 2.28
GTE ± ± ± ±
0.23a 0.097d 1.78d 0.20c
NT + GTE
2.44 3.42 68.26 4.37
± ± ± ±
0.032b 0.057a 1.65a 0.23a
2.89 1.60 41.34 3.10
± ± ± ±
0.052b 0.081c 1.52c 0.15b
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c, d) were significantly different, p < 0.05.
Table 4 Effects of nicotine (NT), green tea extract (GTE) and their combination on spermatids number (SN), sperm count (SC), motility, abnormal sperms, daily sperm production (DSP), sperm transit rate (STR) and testosterone level in rats treated for 2 months. Parameters
Groups Control
6
−1
SN (10 g testis) SC (106 g−1 cauda epididymis) Motility (%) Abnormal sperms (%) DSP (106 g−1 testis) STR (106 day−1 ) Testosterone (ng/ml)
177 314.28 75.53 3.00 29.01 10.84 3.79
NT ± ± ± ± ± ± ±
a
14.45 29.08a 4.80b 0.82c 2.37a 0.73a 0.46a
145.86 239.11 54.15 9.50 23.91 10.03 2.63
GTE ± ± ± ± ± ± ±
c
9.18 15.16c 5.25c 2.51a 1.50d 0.88a 0.27b
165.75 307.94 79.41 2.00 27.17 11.36 3.75
NT + GTE ± ± ± ± ± ± ±
b
6.65 26.24b 3. 64a 0.82d 1.09a 1.22a 0.28a
163.13 285.09 73. 53 4.50 26.74 10.71 3.45
± ± ± ± ± ± ±
9.32b 23.70b 4.21b 1.51b 1.53b 1.30a 0.31a
Values are expressed as mean ± SD. Mean values within a row not sharing a common superscript letters (a, b, c) were significantly different, p < 0.05.
Please cite this article in press as: Mosbah R, et al. Nicotine-induced reproductive toxicity, oxidative damage, histological changes and haematotoxicity in male rats: The protective effects of green tea extract. Exp Toxicol Pathol (2015), http://dx.doi.org/10.1016/j.etp.2015.01.001
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Fig. 2. Light microscopic sections of testes from various examined groups, control (A), nicotine treated animals (B), green tea extract (C), and combination NT + GTE (D) for 2 months. Sections (5 m thickness) were stained with haematoxylin–eosin (100×). ST: seminiferous tubule; IT: interstitial tissue; L: lumen; SE: seminal epithelium; SG: spermatogonia; SZ: spermatozoa; BV: blood vessel; arrows: disorganization of germinal cells and atrophy of tubules; Star: widened interstitial spaces with few number of Leydig cells.
mechanisms, such as impaired androgen synthesis and reduced output of pituitary FSH and LH. In non-smoking humans, NT intravenous treatment increased serum prolactin levels (Newhouse et al., 1990), further supporting that NT might primarily act on neuroendocrine and pituitary regulation. Studies on men and male rats indicate that cigarette smoking and nicotine administration increase prolactin levels and inhibit the release of GnRH, thus impairing the gonadotropins FSH and LH: these hormones control the function of Sertoli and Leydig cells that are critical for initiating and completing spermatogenesis and steroidgenesis in the testis (Mendelson et al., 2003; Oyeyipo et al., 2013). The concurrent exposure to GTE fully prevented the reproductive toxicity of NT. Overall, the findings on weight gain and reproductive effects may suggest that substances, like catechins, contained in green tea might exert a protective action also towards oxidative stress and neuroendocrine disruption (El-Shahat et al., 2009; Abbas and Wink, 2014; Heikal et al., 2014). Our data clearly show that NT-treatment increases the indicators of oxidative stress and that this finding is prevented by the concurrent intake of GTE, consistent with the known antioxidant role of GTE polyphenols. Indeed, oxidative stress may be a concurrent mechanism of NT testicular toxicity. Nicotine increases ROS by breaking the mitochondrial respiratory chain (Guan et al., 2003) and inhibits testosterone biosynthesis in mouse Leydig cells (Patterson et al., 1990); the NT-enhanced ROS production impairs steroidogenesis at the first step of cholesterol transfer to the mitochondria by suppressing the steroidogenic acute regulatory (StAR) protein expression (Jana et al., 2010; Seema et al., 2007). Spermatozoa, being rich in polyunsaturated fatty acids, are liable to
lipid peroxidation by ROS that will result in changes of membrane structural integrity and fluidity. Seminal plasma and testicular cells are well equipped with antioxidant defences; however the NT-induced overproduction of ROS in the testis may over-ride the antioxidant defence mechanisms (Hammadeh et al., 2009; ElSweedy et al., 2007). Catechins do improve lipid peroxidation and quality parameters in boar semen (Boonsorn et al., 2010). The limited available data also indicate that GTE or specific GT polyphenols may ameliorate the liver or testicular toxicity of substances like chlorpyrifos and doxorubicyn, presumably through an antioxidant action (Heikal et al., 2013; Sato et al., 2010; Kalender et al., 2012). In addition, tea contains zinc, selenium and manganese which act as co-factors in antioxidant enzymes, while polyphenols can chelate metal ions such as iron and copper which leading to the formation of hydroxyl radical, inhibit the redox-sensitive transcription factors and the pro-oxidant enzymes, such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), lipoxygenase 2 (LOX-2) and xanthine oxidase over than stimulate the antioxidant enzymes, such as glutathione S-transferases and superoxide dismutases (Frei and Higdon, 2003). To our best knowledge, our study is the first one showing a preventive effect towards a substance with complex, ROS—as well as endocrine-mediated mechanisms like NT. At the moment, we cannot determine with certainty whether the protective effect of GTE is solely a result of its antioxidant effect, or whether it may mitigate also the adverse effects of NT on neuroendocrine and endocrine regulation. The effect on blood parameters were less straightforward as a decrease in red blood cells, haemoglobin and haematocrit which
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was observed in all the three treated groups, NT, GTE as well as NT + GTE. Consistent with our results, Karafakiogly et al. (2009) observed that after the harvest season of tobacco, a significant decrease of red blood cells, haemoglobin and haematocrit was detected in women workers. Erythrocytes are particularly susceptible to oxidative damage as a result of high polyunsaturated fatty acid content in cell membranes and high concentration of oxygen and haemoglobin, the latter being a potential strong promoter of oxidative processes; thus, the NT-reduction of red blood cells may be due to peroxidative membrane damages via ROS production (Tedesco et al., 2000) and/or by ROS reacting with haemoglobin, altering its structure and releasing free iron ions that, in turn increase the generation of ROS (Cimen, 2008). According to our study, GTE do not afford protection against the NT haematotoxicity contrary to testicular toxicity. This might be at least partly attributable to the erythrocytes being much less equipped by antioxidant enzymes to cope with ROS as compared to seminiferous epithelium and the sperm: thus, erythrocytes could be unable to reverse the NT damage when supported by GTE supplementation contrary to seminiferous cells. Finally, NT and GTE appear to have antagonistic effects on white blood cells (reducing and increasing the WBC number, respectively) that are compensated upon combined treatment: in fact, in the NT + GTE group, the value was comparable to controls. We recognize that the NT-treated group has been exposed to a daily manipulation (the i.p. treatment) that, in principle, might have introduced an additional stress to the animals. However, the findings obtained in the NT group in our study were fully consistent with those reported in the literature as regards NT-effects on analogous parameters: thus, the manipulation bias had at the most a slight effect, if any. 5. Conclusion Green tea extract beverage may protect against nicotineinduced oxidative stress, reduced body growth and reproductive toxicity, including endocrine-mediated effects; a protective effect against nicotine haematotoxicity was not observed in our experimental conditions. The intake of green tea, and possibly of individual green tea catechins, may be considered in order to ameliorate several adverse health effects in subjects that have been exposed to nicotine through active or passive smoking or other scenarios, such as the workplace. References Abbas S, Wink M. Green tea extract induces the resistance of Caenorhabditis elegans against oxidative stress. Antioxidants 2014;3:129–43. Aebi HE. Catalase in vitro. Methods Enzymol 1984;105:121–6. Amman RP, Johnson L, Thompson DL, Pickett BW. Daily spermatozoal production, epididymal spermatozoal reserves and transit time of spermatozoa through the epididymides of the rhesus monkey. Biol Reprod 1976;15:586–92. Blazak WF, Treinen KA, Juniewicz PE. Male reproductive toxicology, Part A. In: Chapin RE, Heindel JJ, editors. Methods in toxicology. San Diego, CA: Calif. Academic Press; 1993. p. 86–94. Boonsorn T, Kongbuntad W, Narklong N, Aengwanich W. Effects of catechin addition to extender on sperm quality and lipid peroxidation in boar semen AmericanEurasian. J Agric Environ Sci 2010;7(3):283–8. Chengelis CP, Kirkpatrick JB, Regan KS, Radovsky AE, Beck MJ, Morita O. 28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats. Food Chem Toxicol 2008;46:978–89. Cimen MY. Free radical metabolism in human erythrocytes. Clin Chim Acta 2008;390:1–11. El-Shahat AR, Gabr A, Meki AR, Mehana E. Altered testicular morphology and oxidative stress induced by cadmium in experimental rats and protective effect of simultaneous green tea extract. Int J Morphol 2009;27(3):757–64. El-Sweedy M, Abdel-Hamid N, El-Moselhy M. The role of a mixture of green tea, turmeric and chitosan in the treatment of obesity-related testicular disorders. J Appl Biomed 2007;5:131–8. Flohe L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:101–4. Frei B, Higdon JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J Nutrition 2003;133:3275S–84S.
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