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Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats
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Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats Gamal Said Abd El-Aziz a , Magdy Omar El-Fark a,b , Raid Mahmoud Hamdy a,∗ a b
Department of Anatomy, Faculty of Medicine, King Abdulaziz University, PO Box 80205, Jeddah 21589, Saudi Arabia Department of Anatomy, Faculty of Medicine, Suez Canal University, Egypt
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 29 March 2016 Accepted 25 May 2016 Available online xxx Keywords: Nicotine Testis Morphometry Histology Rat
a b s t r a c t Nicotine is a pharmacologically active component of the tobacco that adversely affects the male reproductive system and fertility. Nicotine administration in experimental animals was found to affect spermatogenesis, epididymal sperm count, motility and the fertilizing potential of sperms. The goal of this work is to assess the protective or ameliorative effect of Eruca Sativa seed oil against testicular damage induced by oral administration of nicotine in rats. Male adult Sprague-Dawley rats were used and divided into three groups; control, nicotine treated and nicotine and Eruca seed oil treated groups. After three weeks of treatment, the rats were weighed and sacrificed where testes were removed and weighed then calculating relative testis weights. The testes were processed for routine paraffin embedding and staining and the sections were examined for different morphometric and histopathological changes. The results show that nicotine administration had an effect on the body and testis weight and various morphometric parameters of the testis. It also induced varying degrees of structural damage to the seminiferous tubules, with shrinkage and absence of mature spermatids. Disorganized, vacuolization and loss of germinal cells were noticed in the basement membrane. The co-administration of Eruca Sativa seed oil led to improvement in the morphometric and histopathological changes of the seminiferous tubules. In conclusion, Eruca Sativa seed oil treatment in this study had a protective role by reversing, almost completely, all morphometric and histological changes in the testis induced by nicotine administration. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Nicotine, a pharmacologically active alkaloid, is the most important component in tobacco, in addition to other chemicals that exceed 3000 compounds including 28 known carcinogens (Pfeifer et al., 2002). Although the manufactured cigarettes are the most widespread type of tobacco utilization universally; other styles are also common in certain regions of the world in the form of oral nicotine intake (ONI) and smokeless tobacco (ST), which have been employed more than other forms of tobacco consumption (Rogozinski, 1990). Such methods of nicotine intake represent a significant part of the whole world tobacco problem (MPOWER package, 2011). Unlike tobacco smoking, ONI is consumed without burning. Instead, nicotine is placed in contact with mucous membranes of the oral cavity, then absorbed into the blood stream (Walsh and Epstein, 2000).
∗ Corresponding author. E-mail address: [email protected] (R.M. Hamdy).
In some countries, ONI is common in the form of chewing tobacco, while in other countries, tobacco is mostly chewed with other ingredients. Chewing tobacco is coarser than snuff and exists in three forms: loose leaf, plug and twist. Tobacco should be chewed for several hours; so that getting continuous supply of the nicotine; till reaching the satisfying level of nicotine; at such time nicotine is absorbed into the blood through the buccal mucosa (Idris et al., 1998; Walsh and Epstein, 2000). Then concentrations of nicotine in the blood rise gradually and plateau at about 30 min, with levels slowly persisting and declining over 2 h or more. In the bloodstream, at pH 7.4, 69% of nicotine became ionized and 31% became unionized. The drug is distributed extensively with a steady-state volume to body tissues with averaging distribution of 2.6 l/Kg. Nicotine is extensively metabolized by the liver to six well identified primary metabolites, Nicotine N’-oxide, Nicotine, Nicotine glucuronide, Nicotine isomethonium ion, Nornicotine, and 4-Oxo4-(3-pyridyl)-butanoic acid (Benowitz et al., 2009). In animal experiments, rodents are exposed to either, nicotine-containing solutions and water or to a forced-drinking procedure of nicotine solutions, in these experiments nicotine oral self-administration is associated with the amount of nicotine metabolizing enzyme
http://dx.doi.org/10.1016/j.tice.2016.05.006 0040-8166/© 2016 Elsevier Ltd. All rights reserved.
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cytochrome P2A5 as well as the rate at which nicotine is metabolized in vitro (Siu et al., 2006). It is believed that the health hazards associated with ONI are less than those with cigarette smoking. However, several studies have reported some health harmful effects of ONI in the form of increased risk of cheek and gum cancers, reaching about 50-fold among long term users (US Department of Health and Human Services, 1986). Also, ONI can cause oral leukoplakias, gingival recession, and may play a contributing role in the development of hypertension, cardiovascular and peripheral vascular diseases, peptic ulcers, and fetal morbidity and mortality (Critchley and Unal, 2003). Nicotine was reported to adversely affect the male reproductive system and fertility (Oyeyipo et al., 2010), where its administration in experimental animals was found to affect spermatogenesis, epididymal sperm; count, motility, and fertilizing potential; decreased level of testosterone, in addition it resulted in different histopathological changes in the testis (Kavitharaj and Vijayammal, 1999; Aydos et al., 2001; Sarasin et al., 2003; Oyeyipo et al., 2011). In spite of the increased knowledge about the harmful reproductive effects of nicotine, the actual molecular events resulting in male reproductive toxicity remains unclear. Currently, it is clear that the deleterious effects of nicotine are, at least in part, the result of enhanced oxidative stress and increased production of reactive oxygen species (ROS) and lipid peroxidation in the testis which leads to tissue oxidative damage in experimental animals. Therefore, it is supposed that the application of different antioxidants; always used as preventive or counteractive agents; can be used as therapeutic for testicular abnormalities induced by nicotine (Seema et al., 2007; Erat et al., 2007). Sener et al. (2005a,b), reported that taurine supplementation effectively attenuates the oxidative damage caused by chronic nicotine administration on different body organs of rats including; thoracic aorta, heart, urinary bladder and kidney; this possibly due its antioxidant effects. For removing ROS, the biological systems possess a number of mechanisms, of which is the integrated antioxidant defense systems (Seema et al., 2007). The enzymatic scavengers of ROS, such as glutathione peroxidases (GPx), glucose6-phosphate dehydrogenase (G-6-PDH), and glutathione reductase (GR), may have a protective effect on the cellular system from deleterious effects of the free radicals which induced by xenobiotics in the testis (Murugesan et al., 2007). The nonenzymatic antioxidant, GSH, quenches and scavenges free radicals, and inactivates the damage process induced by free radicals. (Murugesan et al., 2007). Many plants have been examined for apply in a wide variety of human diseases. Among them, Eruca sativa (Family: Brassicaceae), commonly known as Rocket plant or “Aljarjeer”, is an herbaceous plant used as a salad vegetable, and it is an annual edible plant originated in the Mediterranean region, but nowadays is found and consumed in many countries. Since last more than 20 centuries, Eruca sativa are most widely used in salads, and it was reported to play an important role among natural antioxidants because it has several antioxidant constituents including, glucosinolates, carotenoids, and flavonoids, (Bennett et al., 2006; Martinez-Sanchez et al., 2008; Varga et al., 2009). In previous studies, pharmacological activities of Eruca sativa rocket plants have been moderately related to their strong antioxidant properties (Barillari et al., 2005). Eruca sativa seed oil administration in rats was reported to stimulate the process of spermatogenesis (Salem and Moustafa, 2001) with a significant rise of sperm activity and increase in testosterone level to reach the normal values (Barillari et al., 2005). Also, Eruca sativa has the ability to stimulate the growth of testes and enhance the proliferation, maturation and differentiation of spermatozoa (Homady et al., 2000).
The aim of the present study was to assess the testicular damage induced by ONI in adult rats, in addition to evaluating the protective/ameliorative effect of Eruca seed oil administration on this testicular damage. 2. Materials and methods 2.1. Chemicals Nicotine bitartrate (Sigma, St. Louis, MO, USA) was purchased from local dealers, Jeddah, Saudi Arabia. Eruca sativa seed oil was purchased from local market. Nicotine solution was prepared freshly (100 ml) by dissolving nicotine powder in distilled water so that every ml contained 5 mg of nicotine. This concentration of nicotine was selected as it produced a similar nicotine serum concentration similar to a heavy cigarette smoker (Hui et al., 1991). All nicotine and Eruca seed oil treatments were given daily at 10 am through oral gavage. 2.2. Animals and experimental design Thirty two young adult male Sprague-Dawley albino rats (8 weeks of age, weighing—250–275 gm at the beginning of the experiment) were used in this study. They were obtained from the Animal House of King Fahd Medical Research Center. At the beginning of the experiment the animals were kept in separate metallic cages under standard temperature (24 ± 2 ◦ C), humidity (55 ± 5%) and lighting (12 h: 12 h Light:Dark) conditions. Food formed of Purina rat chow and drinking water was supplied ad libitum. This study was conducted in King Fahd Center for Medical Research and was approved and registered by the Committee of Animal Investigations in Department of Anatomy, Faculty of Medicine, King Abdulaziz University. The health status of the rats was monitored daily. After a two-week acclimatization period, the animals were randomly divided into four groups (n = 8, each) and receive treatment for four weeks, as followed: • Group I (control group): served as negative control and received distilled water in an amount equivalent to that given to the experimental groups. • Group II (Eruca sativa “seed oil” treated group): the rats of this group were given 0.25 ml/kg/day of Eruca seed oil. This dose was proven to produce activation of spermatogenesis (Salem and Moustafa, 2001). • Group III (Nicotine treated group): the rats of this group received 2.5 mg/kg/day (5% of LD50 ) of nicotine. (MSDS SIGMAALDRICH, 2004) • Group IV (Nicotine and Eruca sativa “seed oil” treated group): the rats of this group received 2.5 mg/kg/day (5% of LD50 ) of nicotine together with 0.25 ml/kg/day of Eruca seed oil. 2.3. Studies 2.3.1. Testis and body weight Body weight assessment was carried out by recording the initial body weight, final body weight, and body weight gain. After four weeks of treatment, the rats of different groups were sacrificed using an over dose of diethyl ether. The testes were immediately removed and each testis was then washed with normal saline to separate the surrounding fat and connective tissue. After drying the surface with filter paper, the dimensions (length and width) of the testis were recorded using vernier caliper. The testis (right and left) weight was measured separately in the different groups. Finally, all the weights were represented as mean ± SD and the relative testis
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Table 1 Effect of nicotine and Eruca sativa on body weight, testis weight and relative body/testis weight. Groups
Initial Body weight (g) n=8
Control Eruca sativa Nicotine Eruca sativa + Nicotine
269.2 268.9 270.0 271.2
± ± ± ±
8.9 8.4 9.5 9.1
Final Body weight (g) n=8 320.9 322.4 308.7 318.6
± ± ± ±
Body weight gain (g) n=8
12.4 12.6 10.1 11.7
51.7 53.5 38.7 47.4
± ± ± ±
Absolute testis weight (g) n = 16
3.5 4.2 4.6a,d,f 3.6c
1.58 1.61 1.25 1.49
± ± ± ±
0.05 0.06 0.03b,e,g 0.04a,d
Relative testis weight (%) n = 16 0.49 0.50 0.40 0.47
± ± ± ±
0.01 0.012 0.007b,e,g 0.009a,d
Results are expressed as Mean ± SD, n = 8. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. a P < 0.001 vs. Control group. b P < 0.0001 vs. Control group. c P < 0.05 vs. Eruca sativa group. d P < 0.001 vs. Eruca sativa group. e P < 0.0001 vs. Eruca sativa group. f P < 0.01 vs. Eruca sativa + Nicotine group. g P < 0.0001 vs. Eruca sativa + Nicotine group. Table 2 Effect of nicotine and Eruca sativa on length and width of the testis, diameter and germinal epithelial height of the seminiferous tubules. Groups
Length of testis (cm) n = 16
Control Eruca sativa Nicotine Eruca sativa+ Nicotine
2.22 2.25 1.94 2.18
± ± ± ±
0.15 0.17 0.12 a,c,e 0.13
Width of testis (cm) n = 16 1.27 1.29 1.02 1.24
± ± ± ±
0.09 0.1 0.06 a,c,e 0.08
Seminiferous tubule diameter (um) 257.44 260.1 205.74 248.21
± ± ± ±
12.68 12.95 11.84 b,d,e 12.75 a,c
Germinal epithelial height (um) 87.72 89.31 67.29 80.76
± ± ± ±
7.1 7.4 8.6 b,d,e 7.9 a,c
Results are expressed as Mean ± SD. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. a P < 0.001 vs. Control group. b P < 0.0001 vs. Control group. c P < 0.001 vs. Eruca sativa group. d P < 0.0001 vs. Eruca sativa group. e P < 0.001 vs. Eruca sativa + Nicotine group.
weights were calculated according to the following formula (testis weight/final body weight X 100). 2.3.2. Tissue processing for light microscopy The testes were cut into small pieces and fixed in Bouin’s fluid for 18 h and processed to prepare paraffin blocks and 5 m-thick sections were cut and stained with hematoxylin and eosin and Masson’s trichrome. Stained sections were examined microscopically for qualitative assessment of the seminiferous tubules and changes in the intertubular tissues. Diameter and germinal epithelium height of seminiferous tubules showed different stages of spermatogenic cycle; stages I–VII which contain spermatogonia, early pachytene spermatocytes, round spermatids, and mature elongated spermatids; and stages IX–XIV which contain spermatogonia, prepachytene spermatocytes, late pachytene spermatocytes, and elongated spermatocytes (Creasy, 1997); (5 seminiferous tubules for each stage) were measured with an ocular micrometer, measurement carried only to those tubules cut at right angle to the axis of seminiferous tubules. One observation in each section was noted. Observations were recorded from two different sections in each slide chosen at random. 2.3.3. Testosterone assay procedure Measurement of testosterone level in serum samples were carried out through using an enzyme-based immunoassay (EIA) system. The kits for EIA system were obtained from immunometrics (London, UK), containing, a testosterone EIA enzyme label, testosterone EIA substrate reagent and EIA quality control sample.To ascertain the acceptability with respect to bias and within batch variation, a quality control was carried out at the beginning and at the end of the assay. The EIA kits used had a sensitivity of approximately 0.3 nmol/M (0.1 g/mL) of testosterone. The intra and inter assay variations were 10.02% and 10.12% respectively.
2.4. Statistical analysis Statistical Package for Social Sciences (SPSS) for Windows version 14.0 software (SPSS, Chicago, IL, USA) was used for the calculations and statistical analyses. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. The data were expressed as mean ± SD, P < 0.05 was considered statistically significant. 3. Results 3.1. Body and testis weights As shown in the Table 1, it was observed that the Eruca sativa treated group showed values slightly higher than that of the control group regarding weight gain, testis weight, and relative testis weight. Nicotine treatment for four weeks caused a highly significant decrease in the body weight gain when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups. Also, there was a highly significant decrease in both absolute and relative testis weights in nicotine treated group when compared to control (P < 0.0001), Eruca sativa (P < 0.0001), and Eruca sativa + Nicotine (P < 0.0001) treated groups. Although the combined administration of Eruca sativa and Nicotine caused an improvement in such parameters towards the control values, a significant decrease still existed in weight gain (P < 0.05) and absolute and relative testis weights (P < 0.001) when compared to both control and Eruca sativa groups. 3.2. Testicular size, seminiferous tubule diameter and germinal epithelial height As shown in the Table 2; it was observed that regarding, testicular size (length and width), seminiferous tubule diameter and
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Fig. 1. Shows the level of serum testosterone in control, Eruca sativa, Nicotine, and Eruca sativa + Nicotine treated groups. Notice that the serum level of testosterone is higher in Eruca sativa group than that of the control group. The Nicotine treated group shows a highly significant decrease in the level of serum testosterone when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups.
germinal epithelial height of testis, the Eruca sativa treated group showed values slightly higher than that of the control group. Nicotine treatment caused a highly significant decrease in length and width of the testis when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.001) treated groups. Also, there was a highly significant decrease in both seminiferous tubule diameter and germinal epithelial height of testis when compared to control (P < 0.0001), Eruca sativa (P < 0.0001), and Eruca sativa + Nicotine (P < 0.001) treated groups. The combined administration of Eruca sativa together with Nicotine caused an improvement in the above mentioned parameters towards the control values, but still with a significant decrease in both seminiferous tubule diameter and germinal epithelial height of testis when compared to control (P < 0.001) and Eruca sativa (P < 0.001) groups.
3.3. Serum testosterone level As shown in Fig. 1, the level of serum testosterone was higher in Eruca sativa group than that of the control group. Nicotine treatment for four weeks caused a highly significant decrease in the level of serum testosterone when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups. The combined administration of Eruca sativa and Nicotine caused an improvement in the above mentioned parameters towards the control values.
3.4. Morphological observations 3.4.1. Control group The light microscopic examination of the testis from rats of this group showed the normal; cytoarchitecture of the seminiferous tubules, the interstitial tissue, spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa, basement membrane, myoepithelial cells, Sertoli cells, Leydig cells, and tunica propria (Figs. 2 and 6).
3.4.2. Eruca sativa treated group The light microscopic examination of the testis from rats of this group showed normal cytoarchitecture of the seminiferous tubules and normal process of spermatogenesis, in the form of the presence of mature spermatids at the lumina, with normal late spermatids both in size and shape. Interstitial cells of Leydig appeared normal and similar to the control, also, tunica propria appeared normal (Figs. 3 and 7). 3.4.3. Nicotine treated group The light microscopic examination of the testis from rats of this group showed marked changes in the testicular tissue, which included vascular, interstitial and tubular alterations. The vascular alterations involved variable degrees of congestion of interstitial blood vessels, while the interstitial tissues showed mild edema, which was evidenced by wide separation of the seminiferous tubules. The interstitial cells of Leydig became scanty. The majority of the seminiferous tubules appeared shrunken with disorganized outline associated with the presence of some degenerated tubules. In some tubules, the spermatogenic cells were reduced to a few layers, so the lumen of such tubules appeared wider than in control (hypo-spermatogenesis). There were multiple focal irregular dilatations of the intercellular spaces (vacuolar spaces) in-between germ cells that were also observed. Further examination of the spermatogenic cells revealed obvious vacuolation of their cytoplasm and nuclear changes as pyknosis. Also, degenerated spermatids were easily observed. In some tubules, the degenerated spermatogenic cells were separated from the germinal epithelium at different levels and sloughed into their lumina forming clumps of cells obliterating the lumina. There was a marked decrease in the tunica propria around somniferous tubules (Figs. 4 and 8). 3.4.4. Eruca sativa + Nicotine treated group The light microscopic examination of the testis from rats of this group showed more or less normal cytoarchitecture of the seminiferous tubules and normal process of spermatogenesis i.e. the presence of mature spermatids at the lumina. Late spermatid
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Fig. 2. Photomicrographs of transverse sections of the testis from control rats (A,B), shows the normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A H&E X 200 “scale bar = 100 m” B H&E X 400 “scale bar = 50 m”).
Fig. 3. Photomicrographs of transverse sections of the testis from Eruca sativa treated group (A,B), shows normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A, H&E X 200 “scale bar = 100 m” B, H&E X 400 “scale bar = 50 m”).
showed no change in size and shape when compared to the control. Interstitial cells of Leydig appeared similar to the control. Also, normal amount of the interstitial tissue, normal basement membrane of the seminiferous tubules and normal blood vessels were observed, also, tunica propria appeared normal (Figs. 5 and 9).
in accordance with some studies in humans and animals, which reported that the observed decrease in body weight gain could be due to inhibitory role on food intake and body weight associated with nicotine administration (Hui et al., 1991; Oyeyipo et al., 2010). Also, the present results showed that nicotine administration caused a decrease in the relative testicular weight, which is an important index of reproductive toxicity in male animals. In accordance, it was reported that chronic nicotine used in mice provoked the reduction in testicular weight and atrophied male accessory sex glands, due to the androgenic depletion (Reddy et al., 1998). However, one recent study showed that nicotine had no adverse effects on body weight, testicular measurements, prostate gland or length of epididymis in rat (Fairuz et al., 2011). Probably, the dosage of injected nicotine was not sufficient to cause metabolic changes which might reduce the food intake and increased in the amount of energy consumption as reported by Liu et al. (2003). In the present study, the histological assessment of the testicular tissues from nicotine treated rats showed a number of structural changes in the form of degeneration in the seminiferous tubules, altered general architecture and reduced number of spermatogenic cells. In accordance, it was previously demonstrated that under experimental conditions in rats the oral administration of nicotine was associated with testicular degeneration, disorganization
4. Discussion Several studies have mentioned that the commencement of ONI takes places in young people. This early starting age means that there is an opportunity for more exposure to the negative health effects of ONI, which has deleterious effects particularly on the reproductive organs (U.S. Department of Health and Human Services, 1994). Some effects of nicotine on reproduction in the form of significant increase in the incidence of infertility rate in both human and laboratory animals were reported by Tuormaa (1995), and Oyeyipo et al. (2011). In the present study, the oral administration of nicotine at a dose of 2.5 mg/kg body weight/day was used for the duration of four weeks to make it equivalent to the constant exposure to nicotine in smokers in humans (Hui et al., 1991). The results of this study showed that there was a significant decrease in weight gain of the rats that received nicotine. This is
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Fig. 5. Photomicrographs of transverse sections of the testis from Eruca sativa + Nicotine treated group (A,B), showing normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A, H&E X 200 “scale bar = 100 m” B, H&E X 400 “scale bar = 50 m”).
Fig. 4. Photomicrographs of transverse sections of the testis from Nicotine treated group (A,B,C), showing degenerated seminiferous tubule (st), reduction of spermatogenic cells to few layers; hypospermatogenesis; (i), multiple focal irregular dilatations of the intercellular spaces; vacuolar spaces; (v), widening of the interstitial spaces between the seminiferous tubules (is) with decreased number of Leydig cells (t), some of which has pyknotic nuclei (Py), also, there is dilated and congested blood vessel (bl). (A, H&E X 200 “scale bar = 100 m” B,C, H&E X 400 “scale bar = 50 m”).
of the cytoarchitecture, and decreased serum testosterone levels (Oyeyipo et al., 2010). Also, Reddy et al. (1998) stated that nicotine caused a reduction in spermatogenic cells (spermatogonia, spermatocytes and spermatids) and a retarded spermatocyte to spermatid conversion process in albino rats directly proportional to the dose
of nicotine administered. Seema et al. (2007) related the previous observations to the effects of nicotine on spermatogenesis. Degeneration of the germ cells by nicotine may be the result of the low intratesticular concentrations of testosterone, which is essential for the normal spermatogenesis as well as for the maintenance of the structural morphology and normal physiology of the seminiferous tubule (Sharpe et al., 1992). Studies on humans reported reduced sperm-fertilizing capacity in smokers, in addition to decreased sperm count and motility, which were explained by the degenerative testicular changes induced by nicotine (Künzle et al., 2003). Another study has reported that the use of chewing tobacco by a group of Indian people was strongly associated with a decrease in sperm quality and to a lesser extent with oligoasthenozoospermia or azoospermia (Said et al., 2005). These reports, however, contradict the results of study on 119 tobacco chewers in which no significant differences were found in sperm parameters between tobacco consumers and nonusers (Dikshit et al., 1987). Our results regarding the hormonal assay of testosterone have shown that it was significantly decreased in the nicotine treated rats when compared to control values. Although there are a lot of controversies on the effect of nicotine on testosterone level, most studies have associated nicotine with decreased serum testos-
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Fig. 6. Photomicrographs of transverse sections of the testis from control rats (A,B) shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule. (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
Fig. 7. Photomicrographs of transverse sections of the testis from Eruca sativa treated group (A,B), shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule. (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
terone (Kavitharaj and Vijayammal, 1999; Sarasin et al., 2003; Oyeyipo et al., 2011). In another respect, some researches have indicated that nicotine decreased the level of testosterone through the inhibition of the multiple steps of testosterone biosynthesis in the rats and the mouse, suggesting that nicotine might have adversely affected Leydig cell number and function leading to decreased serum testosterone level (Yamamoto et al., 1998; Sarasin et al., 2003). Furthermore, it has also been established that nicotine administration decreased the testicular androgenic enzymes along with plasma testosterone and sperm counts in mature male albino rats (Yamamoto et al., 1998). A lot of studies have reported that testosterone is necessary for the development, growth and normal functioning of the testis and the male accessory reproductive gland. Low serum testosterone levels have been reported to adversely affect the structure, weight and functioning of the testis and epididymis. Hence, the reduction in weight of the testis, epididymis and seminal vesicle could be attributed to the decreased serum level of testosterone in the nicotine treated rats (George and Welson, 1986). It has become well known that antioxidant phytochemicals are present in plants, fruits and vegetables (Weiss and Landauer, 2003). Earlier studies on phytochemical analysis of Eruca sativa seeds have shown the presence of a number of compounds including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs)
and their breakdown products, e.g. isothiocyanates to which its antioxidant activity may be ascribed in the form of free radical scavenging activity and protection against oxidative stress (Barillari et al., 2005; Alhowiriny et al., 2013). In the present study, the group of rats treated with Eruca sativa seed oil alone showed features more or less similar to the control group, even it resulted in slightly higher values regarding the testosterone level, testis size and relative testis weight, which may reflect the possible enhancing effect on the testis. In this particularity, Eruca sativa was known to have a strong aphrodisiac property, which was widely used by many males to improve their sexual performance since ancient times (Hussein, 2013). Co-administration of Eruca sativa seed oil and nicotine to rats resulted in a significant improvement of all the testicular parameters towards the control values. In accordance, Eruca sativa seed oil was reported to stimulate the process of spermatogenesis in rats (Salem and Moustafa, 2001). Moreover, our results revealed that Eruca seed oil caused a significant increase in testosterone level to reach the normal values. This result agrees with Barillari et al. (2005) who reported that the presence of many substances that caused a significant increase in sperm activity. Furthermore, histological examination of seminiferous tubules revealed a significant increase in the diameter of these tubules, spermatids and Leydig cells as opposed to the decreased interstitial space. This increase
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Fig. 8. Photomicrographs of transverse sections of the testis from Nicotine treated group (A,B), shows marked decrease in the tunica propria (arrows), irregularity of the basement membrane of the seminiferous tubules (B), widening of the interstitial spaces between the seminiferous tubules (is), decreased number of Leydig cells (t),and degenerated seminiferous tubule (st). (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
Fig. 9. Photomicrographs of transverse sections of the testis from Eruca sativa + Nicotine treated group (H,I), shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
might be due to the ability of Eruca sativa to stimulate the growth of testes and enhance the proliferation, maturation and differentiation of spermatozoa (Homady et al., 2000). In conclusion, the present study confirmed that nicotine taken orally harmfully affected the testicular morphometric parameters and structure. In addition, Eruca seed oil treatment in the present study was successful in reversing (almost completely) all morphometric and histological changes of nicotine testicular damage.
They ensure that there are no conflicts of interest (including specific financial interest and relationships and affiliation).
Ethical statement This study was approved and registered by the Committee of Animal Investigations in the Department of Anatomy, Faculty of Medicine, King Abdulaziz University. During all the steps of the study the animals were cared for in King Fahd Center for Medical Research in accordance with the regulations of the Research Ethical Committee, Unit of Biomedical Ethics, Faculty of Medicine, King Abdulaziz University, Saudi Arabia. Acknowledgments The authors are grateful to the referees for suggestions. All authors had important contribution during conduction of the research work and in consequent elaboration of the manuscript.
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Protective effect of Eruca sativa seed oil against oral nicotine induced testicular damage in rats Gamal Said Abd El-Aziz a , Magdy Omar El-Fark a,b , Raid Mahmoud Hamdy a,∗ a b
Department of Anatomy, Faculty of Medicine, King Abdulaziz University, PO Box 80205, Jeddah 21589, Saudi Arabia Department of Anatomy, Faculty of Medicine, Suez Canal University, Egypt
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 29 March 2016 Accepted 25 May 2016 Available online xxx Keywords: Nicotine Testis Morphometry Histology Rat
a b s t r a c t Nicotine is a pharmacologically active component of the tobacco that adversely affects the male reproductive system and fertility. Nicotine administration in experimental animals was found to affect spermatogenesis, epididymal sperm count, motility and the fertilizing potential of sperms. The goal of this work is to assess the protective or ameliorative effect of Eruca Sativa seed oil against testicular damage induced by oral administration of nicotine in rats. Male adult Sprague-Dawley rats were used and divided into three groups; control, nicotine treated and nicotine and Eruca seed oil treated groups. After three weeks of treatment, the rats were weighed and sacrificed where testes were removed and weighed then calculating relative testis weights. The testes were processed for routine paraffin embedding and staining and the sections were examined for different morphometric and histopathological changes. The results show that nicotine administration had an effect on the body and testis weight and various morphometric parameters of the testis. It also induced varying degrees of structural damage to the seminiferous tubules, with shrinkage and absence of mature spermatids. Disorganized, vacuolization and loss of germinal cells were noticed in the basement membrane. The co-administration of Eruca Sativa seed oil led to improvement in the morphometric and histopathological changes of the seminiferous tubules. In conclusion, Eruca Sativa seed oil treatment in this study had a protective role by reversing, almost completely, all morphometric and histological changes in the testis induced by nicotine administration. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Nicotine, a pharmacologically active alkaloid, is the most important component in tobacco, in addition to other chemicals that exceed 3000 compounds including 28 known carcinogens (Pfeifer et al., 2002). Although the manufactured cigarettes are the most widespread type of tobacco utilization universally; other styles are also common in certain regions of the world in the form of oral nicotine intake (ONI) and smokeless tobacco (ST), which have been employed more than other forms of tobacco consumption (Rogozinski, 1990). Such methods of nicotine intake represent a significant part of the whole world tobacco problem (MPOWER package, 2011). Unlike tobacco smoking, ONI is consumed without burning. Instead, nicotine is placed in contact with mucous membranes of the oral cavity, then absorbed into the blood stream (Walsh and Epstein, 2000).
∗ Corresponding author. E-mail address: [email protected] (R.M. Hamdy).
In some countries, ONI is common in the form of chewing tobacco, while in other countries, tobacco is mostly chewed with other ingredients. Chewing tobacco is coarser than snuff and exists in three forms: loose leaf, plug and twist. Tobacco should be chewed for several hours; so that getting continuous supply of the nicotine; till reaching the satisfying level of nicotine; at such time nicotine is absorbed into the blood through the buccal mucosa (Idris et al., 1998; Walsh and Epstein, 2000). Then concentrations of nicotine in the blood rise gradually and plateau at about 30 min, with levels slowly persisting and declining over 2 h or more. In the bloodstream, at pH 7.4, 69% of nicotine became ionized and 31% became unionized. The drug is distributed extensively with a steady-state volume to body tissues with averaging distribution of 2.6 l/Kg. Nicotine is extensively metabolized by the liver to six well identified primary metabolites, Nicotine N’-oxide, Nicotine, Nicotine glucuronide, Nicotine isomethonium ion, Nornicotine, and 4-Oxo4-(3-pyridyl)-butanoic acid (Benowitz et al., 2009). In animal experiments, rodents are exposed to either, nicotine-containing solutions and water or to a forced-drinking procedure of nicotine solutions, in these experiments nicotine oral self-administration is associated with the amount of nicotine metabolizing enzyme
http://dx.doi.org/10.1016/j.tice.2016.05.006 0040-8166/© 2016 Elsevier Ltd. All rights reserved.
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cytochrome P2A5 as well as the rate at which nicotine is metabolized in vitro (Siu et al., 2006). It is believed that the health hazards associated with ONI are less than those with cigarette smoking. However, several studies have reported some health harmful effects of ONI in the form of increased risk of cheek and gum cancers, reaching about 50-fold among long term users (US Department of Health and Human Services, 1986). Also, ONI can cause oral leukoplakias, gingival recession, and may play a contributing role in the development of hypertension, cardiovascular and peripheral vascular diseases, peptic ulcers, and fetal morbidity and mortality (Critchley and Unal, 2003). Nicotine was reported to adversely affect the male reproductive system and fertility (Oyeyipo et al., 2010), where its administration in experimental animals was found to affect spermatogenesis, epididymal sperm; count, motility, and fertilizing potential; decreased level of testosterone, in addition it resulted in different histopathological changes in the testis (Kavitharaj and Vijayammal, 1999; Aydos et al., 2001; Sarasin et al., 2003; Oyeyipo et al., 2011). In spite of the increased knowledge about the harmful reproductive effects of nicotine, the actual molecular events resulting in male reproductive toxicity remains unclear. Currently, it is clear that the deleterious effects of nicotine are, at least in part, the result of enhanced oxidative stress and increased production of reactive oxygen species (ROS) and lipid peroxidation in the testis which leads to tissue oxidative damage in experimental animals. Therefore, it is supposed that the application of different antioxidants; always used as preventive or counteractive agents; can be used as therapeutic for testicular abnormalities induced by nicotine (Seema et al., 2007; Erat et al., 2007). Sener et al. (2005a,b), reported that taurine supplementation effectively attenuates the oxidative damage caused by chronic nicotine administration on different body organs of rats including; thoracic aorta, heart, urinary bladder and kidney; this possibly due its antioxidant effects. For removing ROS, the biological systems possess a number of mechanisms, of which is the integrated antioxidant defense systems (Seema et al., 2007). The enzymatic scavengers of ROS, such as glutathione peroxidases (GPx), glucose6-phosphate dehydrogenase (G-6-PDH), and glutathione reductase (GR), may have a protective effect on the cellular system from deleterious effects of the free radicals which induced by xenobiotics in the testis (Murugesan et al., 2007). The nonenzymatic antioxidant, GSH, quenches and scavenges free radicals, and inactivates the damage process induced by free radicals. (Murugesan et al., 2007). Many plants have been examined for apply in a wide variety of human diseases. Among them, Eruca sativa (Family: Brassicaceae), commonly known as Rocket plant or “Aljarjeer”, is an herbaceous plant used as a salad vegetable, and it is an annual edible plant originated in the Mediterranean region, but nowadays is found and consumed in many countries. Since last more than 20 centuries, Eruca sativa are most widely used in salads, and it was reported to play an important role among natural antioxidants because it has several antioxidant constituents including, glucosinolates, carotenoids, and flavonoids, (Bennett et al., 2006; Martinez-Sanchez et al., 2008; Varga et al., 2009). In previous studies, pharmacological activities of Eruca sativa rocket plants have been moderately related to their strong antioxidant properties (Barillari et al., 2005). Eruca sativa seed oil administration in rats was reported to stimulate the process of spermatogenesis (Salem and Moustafa, 2001) with a significant rise of sperm activity and increase in testosterone level to reach the normal values (Barillari et al., 2005). Also, Eruca sativa has the ability to stimulate the growth of testes and enhance the proliferation, maturation and differentiation of spermatozoa (Homady et al., 2000).
The aim of the present study was to assess the testicular damage induced by ONI in adult rats, in addition to evaluating the protective/ameliorative effect of Eruca seed oil administration on this testicular damage. 2. Materials and methods 2.1. Chemicals Nicotine bitartrate (Sigma, St. Louis, MO, USA) was purchased from local dealers, Jeddah, Saudi Arabia. Eruca sativa seed oil was purchased from local market. Nicotine solution was prepared freshly (100 ml) by dissolving nicotine powder in distilled water so that every ml contained 5 mg of nicotine. This concentration of nicotine was selected as it produced a similar nicotine serum concentration similar to a heavy cigarette smoker (Hui et al., 1991). All nicotine and Eruca seed oil treatments were given daily at 10 am through oral gavage. 2.2. Animals and experimental design Thirty two young adult male Sprague-Dawley albino rats (8 weeks of age, weighing—250–275 gm at the beginning of the experiment) were used in this study. They were obtained from the Animal House of King Fahd Medical Research Center. At the beginning of the experiment the animals were kept in separate metallic cages under standard temperature (24 ± 2 ◦ C), humidity (55 ± 5%) and lighting (12 h: 12 h Light:Dark) conditions. Food formed of Purina rat chow and drinking water was supplied ad libitum. This study was conducted in King Fahd Center for Medical Research and was approved and registered by the Committee of Animal Investigations in Department of Anatomy, Faculty of Medicine, King Abdulaziz University. The health status of the rats was monitored daily. After a two-week acclimatization period, the animals were randomly divided into four groups (n = 8, each) and receive treatment for four weeks, as followed: • Group I (control group): served as negative control and received distilled water in an amount equivalent to that given to the experimental groups. • Group II (Eruca sativa “seed oil” treated group): the rats of this group were given 0.25 ml/kg/day of Eruca seed oil. This dose was proven to produce activation of spermatogenesis (Salem and Moustafa, 2001). • Group III (Nicotine treated group): the rats of this group received 2.5 mg/kg/day (5% of LD50 ) of nicotine. (MSDS SIGMAALDRICH, 2004) • Group IV (Nicotine and Eruca sativa “seed oil” treated group): the rats of this group received 2.5 mg/kg/day (5% of LD50 ) of nicotine together with 0.25 ml/kg/day of Eruca seed oil. 2.3. Studies 2.3.1. Testis and body weight Body weight assessment was carried out by recording the initial body weight, final body weight, and body weight gain. After four weeks of treatment, the rats of different groups were sacrificed using an over dose of diethyl ether. The testes were immediately removed and each testis was then washed with normal saline to separate the surrounding fat and connective tissue. After drying the surface with filter paper, the dimensions (length and width) of the testis were recorded using vernier caliper. The testis (right and left) weight was measured separately in the different groups. Finally, all the weights were represented as mean ± SD and the relative testis
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Table 1 Effect of nicotine and Eruca sativa on body weight, testis weight and relative body/testis weight. Groups
Initial Body weight (g) n=8
Control Eruca sativa Nicotine Eruca sativa + Nicotine
269.2 268.9 270.0 271.2
± ± ± ±
8.9 8.4 9.5 9.1
Final Body weight (g) n=8 320.9 322.4 308.7 318.6
± ± ± ±
Body weight gain (g) n=8
12.4 12.6 10.1 11.7
51.7 53.5 38.7 47.4
± ± ± ±
Absolute testis weight (g) n = 16
3.5 4.2 4.6a,d,f 3.6c
1.58 1.61 1.25 1.49
± ± ± ±
0.05 0.06 0.03b,e,g 0.04a,d
Relative testis weight (%) n = 16 0.49 0.50 0.40 0.47
± ± ± ±
0.01 0.012 0.007b,e,g 0.009a,d
Results are expressed as Mean ± SD, n = 8. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. a P < 0.001 vs. Control group. b P < 0.0001 vs. Control group. c P < 0.05 vs. Eruca sativa group. d P < 0.001 vs. Eruca sativa group. e P < 0.0001 vs. Eruca sativa group. f P < 0.01 vs. Eruca sativa + Nicotine group. g P < 0.0001 vs. Eruca sativa + Nicotine group. Table 2 Effect of nicotine and Eruca sativa on length and width of the testis, diameter and germinal epithelial height of the seminiferous tubules. Groups
Length of testis (cm) n = 16
Control Eruca sativa Nicotine Eruca sativa+ Nicotine
2.22 2.25 1.94 2.18
± ± ± ±
0.15 0.17 0.12 a,c,e 0.13
Width of testis (cm) n = 16 1.27 1.29 1.02 1.24
± ± ± ±
0.09 0.1 0.06 a,c,e 0.08
Seminiferous tubule diameter (um) 257.44 260.1 205.74 248.21
± ± ± ±
12.68 12.95 11.84 b,d,e 12.75 a,c
Germinal epithelial height (um) 87.72 89.31 67.29 80.76
± ± ± ±
7.1 7.4 8.6 b,d,e 7.9 a,c
Results are expressed as Mean ± SD. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. a P < 0.001 vs. Control group. b P < 0.0001 vs. Control group. c P < 0.001 vs. Eruca sativa group. d P < 0.0001 vs. Eruca sativa group. e P < 0.001 vs. Eruca sativa + Nicotine group.
weights were calculated according to the following formula (testis weight/final body weight X 100). 2.3.2. Tissue processing for light microscopy The testes were cut into small pieces and fixed in Bouin’s fluid for 18 h and processed to prepare paraffin blocks and 5 m-thick sections were cut and stained with hematoxylin and eosin and Masson’s trichrome. Stained sections were examined microscopically for qualitative assessment of the seminiferous tubules and changes in the intertubular tissues. Diameter and germinal epithelium height of seminiferous tubules showed different stages of spermatogenic cycle; stages I–VII which contain spermatogonia, early pachytene spermatocytes, round spermatids, and mature elongated spermatids; and stages IX–XIV which contain spermatogonia, prepachytene spermatocytes, late pachytene spermatocytes, and elongated spermatocytes (Creasy, 1997); (5 seminiferous tubules for each stage) were measured with an ocular micrometer, measurement carried only to those tubules cut at right angle to the axis of seminiferous tubules. One observation in each section was noted. Observations were recorded from two different sections in each slide chosen at random. 2.3.3. Testosterone assay procedure Measurement of testosterone level in serum samples were carried out through using an enzyme-based immunoassay (EIA) system. The kits for EIA system were obtained from immunometrics (London, UK), containing, a testosterone EIA enzyme label, testosterone EIA substrate reagent and EIA quality control sample.To ascertain the acceptability with respect to bias and within batch variation, a quality control was carried out at the beginning and at the end of the assay. The EIA kits used had a sensitivity of approximately 0.3 nmol/M (0.1 g/mL) of testosterone. The intra and inter assay variations were 10.02% and 10.12% respectively.
2.4. Statistical analysis Statistical Package for Social Sciences (SPSS) for Windows version 14.0 software (SPSS, Chicago, IL, USA) was used for the calculations and statistical analyses. Significance of differences among groups was evaluated by one-way ANOVA followed by Bonferroni Post Hoc Test. The data were expressed as mean ± SD, P < 0.05 was considered statistically significant. 3. Results 3.1. Body and testis weights As shown in the Table 1, it was observed that the Eruca sativa treated group showed values slightly higher than that of the control group regarding weight gain, testis weight, and relative testis weight. Nicotine treatment for four weeks caused a highly significant decrease in the body weight gain when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups. Also, there was a highly significant decrease in both absolute and relative testis weights in nicotine treated group when compared to control (P < 0.0001), Eruca sativa (P < 0.0001), and Eruca sativa + Nicotine (P < 0.0001) treated groups. Although the combined administration of Eruca sativa and Nicotine caused an improvement in such parameters towards the control values, a significant decrease still existed in weight gain (P < 0.05) and absolute and relative testis weights (P < 0.001) when compared to both control and Eruca sativa groups. 3.2. Testicular size, seminiferous tubule diameter and germinal epithelial height As shown in the Table 2; it was observed that regarding, testicular size (length and width), seminiferous tubule diameter and
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Fig. 1. Shows the level of serum testosterone in control, Eruca sativa, Nicotine, and Eruca sativa + Nicotine treated groups. Notice that the serum level of testosterone is higher in Eruca sativa group than that of the control group. The Nicotine treated group shows a highly significant decrease in the level of serum testosterone when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups.
germinal epithelial height of testis, the Eruca sativa treated group showed values slightly higher than that of the control group. Nicotine treatment caused a highly significant decrease in length and width of the testis when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.001) treated groups. Also, there was a highly significant decrease in both seminiferous tubule diameter and germinal epithelial height of testis when compared to control (P < 0.0001), Eruca sativa (P < 0.0001), and Eruca sativa + Nicotine (P < 0.001) treated groups. The combined administration of Eruca sativa together with Nicotine caused an improvement in the above mentioned parameters towards the control values, but still with a significant decrease in both seminiferous tubule diameter and germinal epithelial height of testis when compared to control (P < 0.001) and Eruca sativa (P < 0.001) groups.
3.3. Serum testosterone level As shown in Fig. 1, the level of serum testosterone was higher in Eruca sativa group than that of the control group. Nicotine treatment for four weeks caused a highly significant decrease in the level of serum testosterone when compared to control (P < 0.001), Eruca sativa (P < 0.001), and Eruca sativa + Nicotine (P < 0.01) treated groups. The combined administration of Eruca sativa and Nicotine caused an improvement in the above mentioned parameters towards the control values.
3.4. Morphological observations 3.4.1. Control group The light microscopic examination of the testis from rats of this group showed the normal; cytoarchitecture of the seminiferous tubules, the interstitial tissue, spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa, basement membrane, myoepithelial cells, Sertoli cells, Leydig cells, and tunica propria (Figs. 2 and 6).
3.4.2. Eruca sativa treated group The light microscopic examination of the testis from rats of this group showed normal cytoarchitecture of the seminiferous tubules and normal process of spermatogenesis, in the form of the presence of mature spermatids at the lumina, with normal late spermatids both in size and shape. Interstitial cells of Leydig appeared normal and similar to the control, also, tunica propria appeared normal (Figs. 3 and 7). 3.4.3. Nicotine treated group The light microscopic examination of the testis from rats of this group showed marked changes in the testicular tissue, which included vascular, interstitial and tubular alterations. The vascular alterations involved variable degrees of congestion of interstitial blood vessels, while the interstitial tissues showed mild edema, which was evidenced by wide separation of the seminiferous tubules. The interstitial cells of Leydig became scanty. The majority of the seminiferous tubules appeared shrunken with disorganized outline associated with the presence of some degenerated tubules. In some tubules, the spermatogenic cells were reduced to a few layers, so the lumen of such tubules appeared wider than in control (hypo-spermatogenesis). There were multiple focal irregular dilatations of the intercellular spaces (vacuolar spaces) in-between germ cells that were also observed. Further examination of the spermatogenic cells revealed obvious vacuolation of their cytoplasm and nuclear changes as pyknosis. Also, degenerated spermatids were easily observed. In some tubules, the degenerated spermatogenic cells were separated from the germinal epithelium at different levels and sloughed into their lumina forming clumps of cells obliterating the lumina. There was a marked decrease in the tunica propria around somniferous tubules (Figs. 4 and 8). 3.4.4. Eruca sativa + Nicotine treated group The light microscopic examination of the testis from rats of this group showed more or less normal cytoarchitecture of the seminiferous tubules and normal process of spermatogenesis i.e. the presence of mature spermatids at the lumina. Late spermatid
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Fig. 2. Photomicrographs of transverse sections of the testis from control rats (A,B), shows the normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A H&E X 200 “scale bar = 100 m” B H&E X 400 “scale bar = 50 m”).
Fig. 3. Photomicrographs of transverse sections of the testis from Eruca sativa treated group (A,B), shows normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A, H&E X 200 “scale bar = 100 m” B, H&E X 400 “scale bar = 50 m”).
showed no change in size and shape when compared to the control. Interstitial cells of Leydig appeared similar to the control. Also, normal amount of the interstitial tissue, normal basement membrane of the seminiferous tubules and normal blood vessels were observed, also, tunica propria appeared normal (Figs. 5 and 9).
in accordance with some studies in humans and animals, which reported that the observed decrease in body weight gain could be due to inhibitory role on food intake and body weight associated with nicotine administration (Hui et al., 1991; Oyeyipo et al., 2010). Also, the present results showed that nicotine administration caused a decrease in the relative testicular weight, which is an important index of reproductive toxicity in male animals. In accordance, it was reported that chronic nicotine used in mice provoked the reduction in testicular weight and atrophied male accessory sex glands, due to the androgenic depletion (Reddy et al., 1998). However, one recent study showed that nicotine had no adverse effects on body weight, testicular measurements, prostate gland or length of epididymis in rat (Fairuz et al., 2011). Probably, the dosage of injected nicotine was not sufficient to cause metabolic changes which might reduce the food intake and increased in the amount of energy consumption as reported by Liu et al. (2003). In the present study, the histological assessment of the testicular tissues from nicotine treated rats showed a number of structural changes in the form of degeneration in the seminiferous tubules, altered general architecture and reduced number of spermatogenic cells. In accordance, it was previously demonstrated that under experimental conditions in rats the oral administration of nicotine was associated with testicular degeneration, disorganization
4. Discussion Several studies have mentioned that the commencement of ONI takes places in young people. This early starting age means that there is an opportunity for more exposure to the negative health effects of ONI, which has deleterious effects particularly on the reproductive organs (U.S. Department of Health and Human Services, 1994). Some effects of nicotine on reproduction in the form of significant increase in the incidence of infertility rate in both human and laboratory animals were reported by Tuormaa (1995), and Oyeyipo et al. (2011). In the present study, the oral administration of nicotine at a dose of 2.5 mg/kg body weight/day was used for the duration of four weeks to make it equivalent to the constant exposure to nicotine in smokers in humans (Hui et al., 1991). The results of this study showed that there was a significant decrease in weight gain of the rats that received nicotine. This is
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Fig. 5. Photomicrographs of transverse sections of the testis from Eruca sativa + Nicotine treated group (A,B), showing normal cytoarchitecture of the seminiferous tubules (ST) and the interstitial tissue (IS), normal, spermatogonia (g), primary spermatocytes (p), spermatids (c), mature spermatozoa (z), lumen of seminiferous tubules (d), basement membrane (b), myoepithelial cells (m), and Sertoli cells (s), also, normal interstitial tissue between the seminiferous tubule containing Leydig cells (L). (A, H&E X 200 “scale bar = 100 m” B, H&E X 400 “scale bar = 50 m”).
Fig. 4. Photomicrographs of transverse sections of the testis from Nicotine treated group (A,B,C), showing degenerated seminiferous tubule (st), reduction of spermatogenic cells to few layers; hypospermatogenesis; (i), multiple focal irregular dilatations of the intercellular spaces; vacuolar spaces; (v), widening of the interstitial spaces between the seminiferous tubules (is) with decreased number of Leydig cells (t), some of which has pyknotic nuclei (Py), also, there is dilated and congested blood vessel (bl). (A, H&E X 200 “scale bar = 100 m” B,C, H&E X 400 “scale bar = 50 m”).
of the cytoarchitecture, and decreased serum testosterone levels (Oyeyipo et al., 2010). Also, Reddy et al. (1998) stated that nicotine caused a reduction in spermatogenic cells (spermatogonia, spermatocytes and spermatids) and a retarded spermatocyte to spermatid conversion process in albino rats directly proportional to the dose
of nicotine administered. Seema et al. (2007) related the previous observations to the effects of nicotine on spermatogenesis. Degeneration of the germ cells by nicotine may be the result of the low intratesticular concentrations of testosterone, which is essential for the normal spermatogenesis as well as for the maintenance of the structural morphology and normal physiology of the seminiferous tubule (Sharpe et al., 1992). Studies on humans reported reduced sperm-fertilizing capacity in smokers, in addition to decreased sperm count and motility, which were explained by the degenerative testicular changes induced by nicotine (Künzle et al., 2003). Another study has reported that the use of chewing tobacco by a group of Indian people was strongly associated with a decrease in sperm quality and to a lesser extent with oligoasthenozoospermia or azoospermia (Said et al., 2005). These reports, however, contradict the results of study on 119 tobacco chewers in which no significant differences were found in sperm parameters between tobacco consumers and nonusers (Dikshit et al., 1987). Our results regarding the hormonal assay of testosterone have shown that it was significantly decreased in the nicotine treated rats when compared to control values. Although there are a lot of controversies on the effect of nicotine on testosterone level, most studies have associated nicotine with decreased serum testos-
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Fig. 6. Photomicrographs of transverse sections of the testis from control rats (A,B) shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule. (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
Fig. 7. Photomicrographs of transverse sections of the testis from Eruca sativa treated group (A,B), shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule. (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
terone (Kavitharaj and Vijayammal, 1999; Sarasin et al., 2003; Oyeyipo et al., 2011). In another respect, some researches have indicated that nicotine decreased the level of testosterone through the inhibition of the multiple steps of testosterone biosynthesis in the rats and the mouse, suggesting that nicotine might have adversely affected Leydig cell number and function leading to decreased serum testosterone level (Yamamoto et al., 1998; Sarasin et al., 2003). Furthermore, it has also been established that nicotine administration decreased the testicular androgenic enzymes along with plasma testosterone and sperm counts in mature male albino rats (Yamamoto et al., 1998). A lot of studies have reported that testosterone is necessary for the development, growth and normal functioning of the testis and the male accessory reproductive gland. Low serum testosterone levels have been reported to adversely affect the structure, weight and functioning of the testis and epididymis. Hence, the reduction in weight of the testis, epididymis and seminal vesicle could be attributed to the decreased serum level of testosterone in the nicotine treated rats (George and Welson, 1986). It has become well known that antioxidant phytochemicals are present in plants, fruits and vegetables (Weiss and Landauer, 2003). Earlier studies on phytochemical analysis of Eruca sativa seeds have shown the presence of a number of compounds including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs)
and their breakdown products, e.g. isothiocyanates to which its antioxidant activity may be ascribed in the form of free radical scavenging activity and protection against oxidative stress (Barillari et al., 2005; Alhowiriny et al., 2013). In the present study, the group of rats treated with Eruca sativa seed oil alone showed features more or less similar to the control group, even it resulted in slightly higher values regarding the testosterone level, testis size and relative testis weight, which may reflect the possible enhancing effect on the testis. In this particularity, Eruca sativa was known to have a strong aphrodisiac property, which was widely used by many males to improve their sexual performance since ancient times (Hussein, 2013). Co-administration of Eruca sativa seed oil and nicotine to rats resulted in a significant improvement of all the testicular parameters towards the control values. In accordance, Eruca sativa seed oil was reported to stimulate the process of spermatogenesis in rats (Salem and Moustafa, 2001). Moreover, our results revealed that Eruca seed oil caused a significant increase in testosterone level to reach the normal values. This result agrees with Barillari et al. (2005) who reported that the presence of many substances that caused a significant increase in sperm activity. Furthermore, histological examination of seminiferous tubules revealed a significant increase in the diameter of these tubules, spermatids and Leydig cells as opposed to the decreased interstitial space. This increase
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Fig. 8. Photomicrographs of transverse sections of the testis from Nicotine treated group (A,B), shows marked decrease in the tunica propria (arrows), irregularity of the basement membrane of the seminiferous tubules (B), widening of the interstitial spaces between the seminiferous tubules (is), decreased number of Leydig cells (t),and degenerated seminiferous tubule (st). (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
Fig. 9. Photomicrographs of transverse sections of the testis from Eruca sativa + Nicotine treated group (H,I), shows normal cytoarchitecture of the seminiferous tubules (ST), tunica propria (arrows) greenish in color, basement membrane of the seminiferous tubules (b), and the interstitial tissue (IS) in between the seminiferous tubule (A, MT X 200 “scale bar = 100 m” B, MT X 400 “scale bar = 50 m”).
might be due to the ability of Eruca sativa to stimulate the growth of testes and enhance the proliferation, maturation and differentiation of spermatozoa (Homady et al., 2000). In conclusion, the present study confirmed that nicotine taken orally harmfully affected the testicular morphometric parameters and structure. In addition, Eruca seed oil treatment in the present study was successful in reversing (almost completely) all morphometric and histological changes of nicotine testicular damage.
They ensure that there are no conflicts of interest (including specific financial interest and relationships and affiliation).
Ethical statement This study was approved and registered by the Committee of Animal Investigations in the Department of Anatomy, Faculty of Medicine, King Abdulaziz University. During all the steps of the study the animals were cared for in King Fahd Center for Medical Research in accordance with the regulations of the Research Ethical Committee, Unit of Biomedical Ethics, Faculty of Medicine, King Abdulaziz University, Saudi Arabia. Acknowledgments The authors are grateful to the referees for suggestions. All authors had important contribution during conduction of the research work and in consequent elaboration of the manuscript.
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