Scholarly article on topic 'Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats – The therapeutic potential of virgin coconut oil'

Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats – The therapeutic potential of virgin coconut oil Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Olufunke O. Dosumu, Oluwole B. Akinola, Edidiong N. Akang

Abstract Objective To evaluate the possible protective effects of virgin coconut oil on alcohol-induced oxidative stress and serum lipid values in rats. Design This is an experimental animal study. Method The animals were gavaged with 30% ethanol (7ml/kg body weight/day) while 6.67ml/kg body weight/day of virgin coconut oil (VCNO) was administered for 4 weeks using a cannulated syringe. Results Animals treated with ethanol alone showed a significant elevation in the level of malondialdehyde (MDA) as measured by thiobarbituric acid reactive substances (TBARS), which lowered the antioxidant defence system such as reduced glutathione (GSH) and catalase activities when compared with the control. Sperm count, sperm motility and serum testosterone levels were also significantly reduced in this group. Levels of total cholesterol/HDL (TC/HDL) ratio increased significantly in the ethanol-only treated group, while HDL level was significantly reduced. Administration of VCNO improved the antioxidant status by decreasing the levels of MDA and altering lipid profile levels to near normal. Sperm count, motility and serum testosterone levels were also significantly increased when compared with the alcohol-only treated group. Conclusion Findings of the present study indicate VCNO has antioxidant activities and does not adversely alter serum lipid levels.

Academic research paper on topic "Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats – The therapeutic potential of virgin coconut oil"

Middle East Fertility Society Journal (2012) 17, 122-128

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats - The therapeutic potential of virgin coconut oil

Olufunke O. Dosumu a, Oluwole B. Akinola , Edidiong N. Akang

a Department of Anatomy, College of Medicine, University of Lagos, Nigeria b Department of Anatomy, College of Health Sciences, University of Ilorin, Nigeria

Received 22 November 2011; accepted 15 December 2011 Available online 12 April 2012

KEYWORDS

Testes;

Oxidative stress; Cholesterol; Coconut oil; Testosterone; Antioxidants

Abstract Objective: To evaluate the possible protective effects of virgin coconut oil on alcohol-induced oxidative stress and serum lipid values in rats.

Design: This is an experimental animal study.

Method: The animals were gavaged with 30% ethanol (7 ml/kg body weight/day) while 6.67 ml/kg body weight/day of virgin coconut oil (VCNO) was administered for 4 weeks using a cannulated syringe.

Results: Animals treated with ethanol alone showed a significant elevation in the level of malondi-aldehyde (MDA) as measured by thiobarbituric acid reactive substances (TBARS), which lowered the antioxidant defence system such as reduced glutathione (GSH) and catalase activities when compared with the control. Sperm count, sperm motility and serum testosterone levels were also significantly

Corresponding author. Address: Department of Anatomy, College of Medicine, University of Lagos, P.M.B. 12003, Lagos, Nigeria. Tel.: + 234 8065299278.

E-mail address: eltyeddy@yahoo.com (E.N. Akang).

1110-5690 © 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

Peer review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2011.12.005

reduced in this group. Levels of total cholesterol/HDL (TC/HDL) ratio increased significantly in the ethanol-only treated group, while HDL level was significantly reduced. Administration of VCNO improved the antioxidant status by decreasing the levels of MDA and altering lipid profile levels to near normal. Sperm count, motility and serum testosterone levels were also significantly increased when compared with the alcohol-only treated group.

Conclusion: Findings of the present study indicate VCNO has antioxidant activities and does not adversely alter serum lipid levels.

© 2012 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Alcohol is widely consumed in alcoholic beverages all over the world and as a result, alcohol-related disorders are becoming increasingly important causes of morbidity and mortality (1). Ethanol is a small molecule soluble in both water and lipids. It can permeate all tissues of the body and affect their vital organs (2).

Reports have demonstrated that excessive ethanol consumption can produce oxidative stress and induce testicular damage (3,4). Moreover, an increased intake of ethanol is known to increase the levels of lipids, leading to hyperlipidemia (5). Although significant progress has been made in understanding the pathogenesis of alcoholic testicular damage, treatment options are limited as well as problematic. Therefore, alternative therapies are needed to supplement the existing synthetic drugs.

Nutritional antioxidants have been reported to play an important role in cellular antioxidative defense mechanisms (6). In fact, various authors have suggested that antioxidants of plant origin could be of value in the prevention of fundamental cellular disturbances resulting from alcohol-induced oxidative stress in the testes and other organs (7-9).

VCNO has been reported to have active polyphenol compounds which have been recognized as a powerful counter measure against lipid peroxidation (10,11). However, it has been suggested that saturated fatty acids and cholesterol each independently elevate blood cholesterol and low density lipo-protein (LDL) concentrations (12). Hence, coconut oil because of its high saturated fatty acid composition (consists of about 90% saturated fat) is believed to elevate blood cholesterol (13) and therefore may be atherogenic (14). In this context, any dietary oil that lowers LDL cholesterol and elevates HDL cholesterol is considered to have health benefits.

In view of this, the present study was conducted to investigate the effects of VCNO on alcohol-induced oxidative stress and cholesterol homeostasis.

2. Materials and methods

2.1. Chemicals

Thirty percent ethanol prepared from absolute ethanol (99.86% v/v) with substance identification number 1170 manufactured by James Burrough (F.A.D. Ltd. UK) was used for the study.

2.2. Plant material

Virgin coconut oil: The solid endosperm of mature coconuts authenticated as Cocos nucifera palmae at the Federal Institute

of Forestry Research Ibadan (Voucher number, 107,825) was used for the study.

Preparation of VCNO: The VCNO was extracted using the wet extraction method as previously described (15). Briefly, the solid endosperm of mature coconuts was crushed and made into a viscous slurry. About 500 ml of water was added to the slurry obtained and squeezed through a fine sieve to obtain coconut milk. The resultant coconut milk was left for about 24 h to facilitate the gravitational separation of the emulsion as previously described (16), after which the oil on top was scooped and heated for about 5 min to remove moisture. The obtained VCNO was then filtered through a fine sieve, stored in plain sample bottles at room temperature for 4 weeks and used for the study.

2.3. Animal experiments

Adult male Sprague-Dawley rats weighing 150-170 g were used for the study. Animals were procured from the Nigerian Institute of Medical Research (NIMR) located in Yaba, Lagos. The animals were housed in the animal holdings of the Anatomy Department in well ventilated plastic cages with 12:12 light-dark cycles at 27 ± 1 0C. Rats were randomized into five groups of five animals each. The mode of administration for all groups was through gastric intubation. Group I control rats received distilled water, group II rats received 30% ethanol for 4 weeks (7 ml/kg body weight), group III rats received 30% ethanol along with VCNO (6.67 ml/kg body weight) for 4 weeks, group IV rats received VCNO for 4 weeks followed by ethanol for 4 weeks, while group V rats received ethanol for 4 weeks followed by VCNO for 4 weeks. At the end of the treatment period, the rats were sacrificed after which blood and tissues (testes) were collected for various assays. All experimental protocols followed the guidelines approved by the Ethics Committee of the College of Medicine, University of Lagos, Nigeria.

2.4. Parameters investigated

Semen analysis: The cauda epididymis of the rats were incised and a drop of epididymal fluid delivered onto a glass slide, covered by a 22 x 22 mm cover slip and examined under the light microscope at a magnification of x100 while evaluating different fields (17). For the purpose of this study, motility was classified as either motile or non-motile (18). After assessing different microscopic fields, the relative percentage of motile sperm was estimated and reported to the nearest 5% using the subjective determination of motility (19).

The sperm count was determined using the Neubauer improved hemocytometer. Epididymal fluid ratio of 1:20 was

Table 1 Effect of alcohol and VCNO on serum lipid levels.

Groups HDL (mmol/L) LDL (mmol/L) TC (mmol/L) TG (mmol/L) TC/HDL

Group I Group II Group III Group IV Group V 1.13 ± 0.31 0.53 ± 0.06* 1.00 ± 0.14 1.08 ± 0.10 0.83 ± 0.26 0.27 ± 0.06 0.25 ± 0.01 0.26 ± 0.08 0.17 ± 0.01* 0.40 ± 0.05 1.42 ± 0.18 1.38 ± 0.13 1.14 ± 0.03 1.13 ± 0.10 1.38 ± 0.08 1.28 ± 0.06 1.31 ± 0.50 1.07 ± 0.04 0.89 ± 0.21 1.01 ± 0.32 1.29 ± 0.23 2.52 ± 0.09* 1.15 ± 0.13 1.14 ± 0.10 1.80 ± 0.52

Key: I - control, II - alcohol 4 weeks, III - alcohol + VCNO 4 weeks, IV - VCNO 4 weeks/alcohol 4 weeks, V - alcohol 4 weeks/VCNO 4 weeks, TC - total cholesterol; TG - total triglycerides; HDL - cholesterol; LDL - cholesterol. Significance at p < 0.05.

i n m iv v

Treatment groups

y: p < 0.05 significance; a: p < 0.001 significance Key

I- Control

II- Alcohol 4 weeks

III- Alcohol + VCNO 4 weeks

IV- VCNO 4 WEEKS / Alcohol 4 week

V- Alcohol 4 weeks / VCNO 4 weeks

Figure 1 The effect of alcohol and VCNO on sperm count and motility.

prepared by adding 0.1 ml of fluid to 1.9 ml of water. The dilution was mixed thoroughly and both sides of the counting chamber were scored and the average taken. Spermatozoa within five of the red blood cell squares including those which lie across the outermost lines at the top and right sides were counted, while those at the bottom and left sides were left out. The number of spermatozoa counted was expressed in millions/ml (19).

2.5. Biochemical estimations

The lipid peroxidation products were estimated by measuring TBARS and were determined by the method of Niehaus and Samuelson (20). Antioxidants such as reduced glutathione and catalase were estimated by the methods of Ellman (21) and Sinha (22), respectively. Total cholesterol, triglycerides

HDL cholesterol and LDL cholesterol were estimated by using Roche/Hitache analyzers.

2.6. Hormone determination

The serum levels of testosterone (TT) were measured using commercially available enzyme-linked immunoassay kit (Diagnostic Automation Inc., CA) according to manufacturer's instructions.

2.7. Statistical analysis

Data were expressed as means ± SD. Statistical significance of data was determined by analysis of variance plus Bonferroni's post hoc test. p < 0.05 was considered significant.

3. Results

3.1. Effect on lipid levels

In the present study, LDL cholesterol was significantly lower in group IV (p < 0.05) while other groups showed no significant difference when compared with the control. HDL cholesterol was, however, significantly reduced (p < 0.05) only in group II when compared with the control. There was no significant reduction in total cholesterol and triglycerides levels in the VCNO-treated rats when compared with the control; however, ratio of TC/HDL was significantly increased in the alcohol-treated group when compared with the control (Table 1).

3.2. Effect on semen quality

Sperm count and motility were not significantly different in groups III (VCNO + Alcohol) and V (Alcohol/VCNO) when compared with the control (Fig. 1), while groups II (Alcohol only) and IV (VCNO/Alcohol) had significantly reduced (p < 0.001, 0.05 respectively) sperm count and motility when compared with the control. However, when compared with the alcohol-only treated group, all VCNO-treated groups had significantly increased (p < 0.05) sperm count and motility. Sperm count in group II (18.00 ± 3.46) was lower than 20 x 106 mP1 which is considered to be normal (19), while all VCNO-treated groups had counts much higher than this value (Fig. 1).

3.3. Effect on testicular malondialdehyde levels

Testicular malondialdehyde level was reduced significantly (p < 0.05) in animals treated with alcohol together with

Table 2 Effect of alcohol and VCNO on testicular malondialdehyde, reduced glutathione and catalase.

Treatment groups tMDA (nmol/min) tCAT (imol/mg protein) tGSH (imol/min)

Group I 8.20 ± 1.11 6.33 ± 0.30 0.37 ± 0.02

Group II 23.57 ± 2.99** 7.29 ± 0.23* 0.15 ± 0.03**

Group III 5.39 ± 2.49* 8.47 ± 0.96* 0.14 ±0.04**

Group IV 13.93 ± 1.60** 4.98 ± 0.60* 0.28 ± 0.03*

Group V 5.08 ± 1.28* 8.94 ± 2.07 0.08 ± 0.02**

Key: I - control, II - alcohol 4 weeks, III - alcohol + VCNO 4 weeks, IV - - VCNO 4 weeks/alcohol 4 weeks, V - - alcohol 4 weeks/VCNO 4

weeks, tMDA - testicular malondialdehyde; tCAT - testicular catalase; tGSH - testicular reduced glutathione.

Significance at p < 0.05.

Significance at p < 0.001.

I II III IV V

Treatment groups

y: p < 0.05 significance Key

I- Control

II- Alcohol 4 weeks

III- Alcohol + VCNO 4 weeks

IV- VCNO 4 WEEKS / Alcohol 4 weeks

V- Alcohol 4 weeks / VCNO 4 weeks

Figure 2 The effect of alcohol and VCNO on serum testosterone level.

VCNO (group III) and the group treated with VCNO following alcohol treatment (group V), while the groups treated with alcohol alone (group II) as well as those treated with alcohol following VCNO treatment showed a significant increase (p < 0.05) in tMDA levels when compared with the control (Table 2). However, when compared with the alcohol-only treated group, all the VCNO-treated groups had significantly reduced (p < 0.05) tMDA values (Table 2).

3.4. Effect on antioxidant enzymes

Glutathione (GSH) level was reduced significantly (p < 0.05) in all groups when compared with the control (Table 2).

Testicular catalase level increased significantly (p < 0.05) in groups II (Alcohol alone) and III (VCNO + Alcohol). The increase in catalase level was not significant in group V (Alcohol/ VCNO) while the reduction in catalase level in group IV (VCNO/Alcohol) was significant (p < 0.05) (Table 2).

3.5. Effect on serum testosterone level

Following the period of administration, only the alcohol-only treated group showed a significant decrease in TT level (p < 0.05) when compared with the control. However, when compared with the alcohol-only treated group, all the VCNO-treated groups showed a significant increase in serum TT level (p < 0.05) (Fig. 2).

4. Discussion

Available scientific reports show that the highly saturated nature of coconut fatty acids increases cholesterol synthesis in the body and this contributes to a higher incidence of heart diseases (23). This contention is however being scientifically refuted as more available reports are showing that VCNO is neither hypercholesterolemic nor atherogenic (24-26).

In the present study, animals treated with VCNO did not show adversely altered lipid levels (cholesterol, lipids) when compared with the control. HDL cholesterol levels in VCNO-treated rats however were found to be higher when compared with the alcohol-only treated group (1.075 ± 0.096; 0.85 ± 0.263; 1.00 ± 0.141), an indication of the beneficial effect of the oil. Saturated fatty acids have been reported to increase the HDL cholesterol concentration, which has been associated with increased lecithin cholesterol acyl transferase (LCAT) (27).

The effects of chronic alcohol exposure on cholesterol homeostasis has not been well studied and underlying mechanisms behind are still very elusive (28). Ethanol has been reported as a powerful indicator of hyperlipidemia in both animals and humans (29). The most common lipid abnormalities during chronic alcohol consumption are known to produce hypercholesterolemia and hypertriglyceridemia (30,31). From the present study, ethanol-only treated groups showed significant increase in the ratio of TC/HDL even though both TC and LDL cholesterol levels were not significantly different from values of control. There is sufficient evidence that ethanol may affect cholesterol synthesis and/or transport (32-34). Specifically, Ashakumari et al. (35) have stated that the increased cholesterol during alcohol ingestion is attributed to the increased a-hydroxyl methyl glutaryl CoA (HMG CoA)

reductase activity, which is the rate limiting step in cholesterol biosynthesis. Going by the report of Kinosian et al. (36) that ''an increase in the ratio of TC/HDL is a better predictor of subsequent coronary heart disease than either TC or LDL cholesterol by themselves'', the results of the present study indicate that alcohol not VCNO may contribute to heart diseases. In addition, reports have indicated that unsaponifiable components of VCNO like vitamin E and polyphenols may play a beneficial role in reducing cholesterol level and lipid peroxida-tion (37).

The present study demonstrates the effects of ethanol on the testes. Ethanol consumption resulted in a significant increase in tMDA level. Different authors have demonstrated that acute and chronic exposure to ethanol either administered orally or intraperitoneally leads to increased free radical and lipid peroxide formation (15,38,39). Some studies have revealed that free radical or ROS generation and lipid peroxida-tion might be an important mechanism in the toxicity of ethanol in the testes (40-42). The significant increase in tMDA levels in the alcohol-only treated groups corroborates these studies. In contrast, however, to the high tMDA levels observed in the alcohol-only treated groups, all the VCNO-trea-ted groups had significantly reduced tMDA levels. Earlier reports indicating that VCNO has active polyphenol compounds, recognized as a powerful counter measure against li-pid peroxidation (13) may account for this.

GSH is an endogenous antioxidant and plays a significant role in the detoxification of xenobiotics and maintenance of the redox status of the cells (43). Low levels of GSH were observed in the present study at all regimen tested. The higher peroxide levels (MDA) and lower antioxidant levels showed an altered oxidant and antioxidant status (44) and depletion of endogenous antioxidants like GSH renders the cell more susceptible to oxidative stress (45). Alcohol has been reported to deplete cellular GSH levels (45). The depletion of GSH in the alcohol-treated groups is consistent with other reports (4,46) and could be explained on the basis of its utilization in scavenging free radicals, acting as a co-factor for glutathi-one-S-transferase (GST) during detoxification of xenobiotics like alcohol, oxidation of glutathione peroxidase in detoxification of H2O2 and/or lipid peroxides and suppression of gluta-thione synthesis by ethanol (45). Reduced glutathione (GSH) is a critical cellular antioxidant and is important in limiting the toxicity of ethanol as well as many other toxic chemicals

(47). A decline in cellular level has been considered to be indicative of oxidative stress (43). Reports have stated that alcohol activates free radical generation and also alters the levels of enzymatic and non-enzymatic endogenous antioxidant systems

(48). This is evident in the low levels of GSH observed in the present study.

The inhibition of catalase activity (evident in the increased MDA levels) in the alcohol-only treated group is suggestive of enhanced synthesis of superoxide radical during alcohol ingestion. Superoxide radicals have been reported as a powerful inhibitor of catalase (49). The observed increase/sustenance in catalase activity in the VCNO-treated groups corroborate the study of Nevin and Rajamohan (50), in which rats fed VCNO showed an increased catalase activity. It is possible that the polyphenolic compounds in VCNO are responsible for this. These compounds have been recognized as a potent scavenger of superoxide anion, hydroxyl radicals and nitric oxide. Hence, VCNO helps to maintain the cellular status of

antioxidant enzymes such as catalase, making it available to detoxify the toxic metabolites produced in the course of etha-nol metabolism and thus reduce oxidative stress, evident in the lower MDA levels of these groups.

The present study also shows that the repeated intake of eth-anol alone or following VCNO treatment resulted in fertility disturbances evident from low sperm count and impaired sperm motility. Aitken and Roman (51) have reported that free radicals attack germ cells within the seminiferous tubules leading to extensive necrosis and disruption of spermatogenesis. This ultimately results in reduced sperm count as observed in the study.

Studies have shown that ROS have detrimental effects on normal sperm function, inhibiting both motility and sperm competence for zona binding (52). Increased MDA levels have been correlated with decreased sperm motility (53,54). From the present study, animals treated with alcohol alone or following VCNO treatment exhibited significantly reduced sperm motil-ity, supporting earlier reports that alcohol-generated ROS decrease sperm motility. ROS affects sperm motility by diffusing across the membrane into cells and inhibiting the activity of some vital enzymes such as glucose-6-phosphate dehydrogenase (6GPD) (55). However, these effects were attenuated by VCNO (in the VCNO-treated groups) which readily scavenged and quenched ROS activities, reduced oxidative stress, thereby increasing sperm count and motility, hence correlating decreased MDA levels to increased sperm motility and count.

Various authors have reported a decrease in testosterone levels upon exposure to ethanol (15,56,57). The present study supports these findings as alcohol decreased serum testosterone levels in the alcohol-only treated groups. Various studies have also pointed out the testes possess the necessary enzymes for the breakdown of alcohol and in the presence of alcohol, these enzymes, rather than facilitate testosterone production are diverted to alcohol breakdown, thus leading to a reduction in testosterone levels over time (58,59). In addition alcohol increases the activity of testosterone reductase, which in turn increases the breakdown activity of testosterone in the liver. Studies have also shown that alcohol consumption resulted in elevated levels of the hormone cortisol. Cortisol acts directly on cells in the testes to inhibit the production and release of testosterone, leading to suppressed testosterone levels (60,61). Calvin et al. (62) have suggested that the metabolic pathway of testosterone synthesis requires protection against peroxidation and polyphenols present in coconut oil have been recognized as a powerful counter measure against lipid peroxidation (10,11,15). This may account for its ability to decrease oxidative stress and protect the metabolic pathway of testosterone against lipid peroxida-tion, thereby preventing the suppression of testosterone (15).

5. Conclusion

From the results, we conclude that VCNO exerts a significant antioxidant activity against ethanol-induced testicular toxicity and demonstrates the potential to maintain cholesterol homeostasis.

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