Scholarly article on topic 'Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague–Dawley rats'

Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague–Dawley rats Academic research paper on "Clinical medicine"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{"Sperm count" / "Sperm motility" / Testosterone / "Oxidative stress" / "Luteinizing hormone" / Alcohol / Infertility}

Abstract of research paper on Clinical medicine, author of scientific article — A.A. Oremosu, E.N. Akang

Abstract Objective To investigate the impact of alcohol on the reproductive hormones, oxidative stress and semen parameters. Design This is an experimental animal study. Materials and methods Adult male Sprague–Dawley rats weighing between 170 and 200g received 30% v/v ethanol at a concentration of 2g/kg body weight for a period of 4weeks, 8weeks and 16weeks. Parameters tested include: testosterone, estrogen, luteinizing hormone (LH), follicle stimulating hormone (FSH), gonadotropin hormone releasing hormone (GnRH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), sperm count and sperm motility. Results After the 4week study, there was a significant decrease (p <0.05) in estrogen levels, sperm count and sperm motility. Testosterone levels also decreased while MDA levels increased significantly. After the 8week study, testosterone levels decreased significantly, LH and FSH also decreased but GnRH levels increased significantly. MDA and SOD levels increased significantly but sperm count and sperm motility decreased significantly compared to controls. After the 16week study, testosterone and GnRH levels decreased significantly compared to controls. MDA levels increased significantly while sperm count and motility decreased significantly. Conclusion Acute and chronic administration of alcohol depletes testosterone levels, increases oxidative stress and decreases semen parameters. This impact of alcohol on testosterone levels is mediated by direct testicular toxicity and by altering the hormone feedback system in the pituitary gland and the hypothalamus.

Academic research paper on topic "Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague–Dawley rats"

Middle East Fertility Society Journal (2014) xxx, xxx-xxx

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague-Dawley rats

A.A. Oremosu a'*, E.N. Akang b

a Department of Anatomy, College of Medicine, University of Lagos, Lagos State, Nigeria b Department of Anatomy, College of Health Sciences, Benue State University, Makurdi, Benue State, Nigeria

Received 11 March 2014; revised 28 May 2014; accepted 1 July 2014

Middle East Fertility Society Journal

KEYWORDS

Sperm count; Sperm motility; Testosterone; Oxidative stress; Luteinizing hormone; Alcohol; Infertility

Abstract Objective: To investigate the impact of alcohol on the reproductive hormones, oxidative stress and semen parameters.

Design: This is an experimental animal study.

Materials and methods: Adult male Sprague-Dawley rats weighing between 170 and 200 g received 30% v/v ethanol at a concentration of 2 g/kg body weight for a period of 4 weeks, 8 weeks and 16 weeks. Parameters tested include: testosterone, estrogen, luteinizing hormone (LH), follicle stimulating hormone (FSH), gonadotropin hormone releasing hormone (GnRH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), sperm count and sperm motility.

Results: After the 4 week study, there was a significant decrease (p < 0.05) in estrogen levels, sperm count and sperm motility. Testosterone levels also decreased while MDA levels increased significantly. After the 8 week study, testosterone levels decreased significantly, LH and FSH also decreased but GnRH levels increased significantly. MDA and SOD levels increased significantly but sperm count and sperm motility decreased significantly compared to controls. After the 16 week study, testosterone and GnRH levels decreased significantly compared to controls. MDA levels increased significantly while sperm count and motility decreased significantly.

Conclusion: Acute and chronic administration of alcohol depletes testosterone levels, increases oxidative stress and decreases semen parameters. This impact of alcohol on testosterone levels is mediated by direct testicular toxicity and by altering the hormone feedback system in the pituitary gland and the hypothalamus.

© 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

* Corresponding author. Tel.: +234 8059222269.

E-mail address: aaoremosu@cmul.edu.ng (A.A. Oremosu).

Peer review under responsibility of Middle East Fertility Society.

1. Introduction

Infertility is the inability of a couple to conceive after a year of regular unprotected sexual intercourse (1). Treatment and management of infertility has become of global concern as

http://dx.doi.org/10.1016/j.mefs.2014.07.001

1110-5690 © 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

the need to have children is of great priority in families. It is reported that about 15% of couples of reproductive age are infertile and about 50% of these cases are male related (2,3).

The problem of infertility is that it produces stress, a couple failing to achieve the expected goal of reproduction, experiences the feelings of frustration and disappointment. These psychological stresses will lead to generalized increased oxidative stress levels (3). A strong body of evidence suggests that small amounts of reactive oxygen species (ROS) are necessary for spermatozoa to acquire fertilizing capabilities however, it has been reported that stress has a negative effect on sperm concentration, motility and morphology (4,5). It has also been reported that about 25-80% of males with infertility record high levels of ROS (6-8).

Chia et al. (9), reports that about 42% of men with infertility cases consume alcohol. This presents alcohol as a potent precursor of male factor infertility. Moreover, researchers have reported an association between alcohol consumption and decreased testosterone levels (10,11). Testosterone is a hormone produced by the Leydig cells in the interstitium of the testes (12). Its synthesis is triggered by a negative feedback to the anterior pituitary gland which releases luteinizing hormone which in turn is regulated by gonadotropin releasing hormone (GnRH) of the hypothalamus (13). There must be a balanced interplay between these hormones from the hypothalamus, pituitary gland and the testis for a successful germ cell development (14). Decrease in sperm density, eventually leading to azoospermia, has been found to be associated with raised follicle stimulating hormone (FSH), LH and normal or low testosterone level (15).

It is still uncertain if alcohol reduces semen parameters by acting directly on testicular tissue or via the hypothalamic-pituitary-gonadal (HPG) axis. This study is targeted at distinguishing the path of action by which alcohol reduces semen parameters.

2. Materials and methods

2.1. Chemicals

Thirty percent (30% v/v) of ethanol prepared from absolute ethanol (99.86%) with substance identification number 1170 manufactured by James Burrough (F.A.D. Ltd. UK) was used at a concentration of 2 g/kg body weight.

2.2. Animal experiments

Thirty-six adult male Sprague-Dawley rats weighing between 170 and 200 g were procured from the Nigerian Institute of Medical Research (NIMR) located in Yaba, Lagos. The animals were housed in the animal holdings of the Laboratory Animal Center, College of Medicine, University of Lagos, in well ventilated plastic cages with 12:12 light-dark cycles at 27 ± 1 0C. All procedures guiding the use of the animals were in accordance with the standard international guidelines on the use of animals for research. Approval for the study was obtained from the Departmental Ethics Committee and also granted by the Experimental Ethics Committee on Animals Use of College of Medicine, University of Lagos, Nigeria. The animals for the experiments were randomly divided into 3 groups to represent the 3 phase duration. The first phase

A.A. Oremosu, E.N. Akang

lasted 4 weeks, the second, lasted 8 weeks while the third lasted 16 weeks. In each phase the animals were divided into 2 subgroups: A and B. The mode of administration for all groups was through gastric gavage. Subgroup A represented control that received distilled water while subgroup B represented animals that received 30% v/v of alcohol. At the end of each of the phases, the rats were sacrificed after which blood was collected for biochemical analysis and the testes harvested for his-tological studies.

2.3. Parameters investigated

2.3.1. Reproductive hormones

The blood specimens from the animals were collected via ocular puncture of orbital vein into plain sample bottles, cen-trifuged at 3000g and assayed for testosterone, LH, FSH, Estrogen and GnRH levels using the enzyme-linked immuno-sorbent assay kits.

2.3.2. Biochemical parameters estimation

2.3.2.1. Testicular malondialdehyde. Malondialdehyde (MDA) levels in the testicular tissue were measured by the method developed by Ohkawa et al. (16). This is based on the measurement of thiobarbituric acid malondialdehyde absorbance. The tissue MDA levels were expressed as nmol/ml tissue.

2.3.2.2. Superoxide Dismutase (SOD) activity. Superoxide Dis-mutase activity was determined by modifications of methods described by Beauchamp and Fridovich (17) and Sun and Zig-ma (18) using its ability to inhibit the auto-oxidation of epi-nephrine determined by the increase in absorbance at 480 nm. The reaction mixture (3 ml) containing 2.95 ml 0.05 M sodium carbonate buffer pH 10.2, 0.02 ml of the homogenate and 0.03 ml of epinephrine in 0.005 N HCl was used to initiate the reaction. The reference cuvette contained 2.95 ml buffer, 0.03 ml of substrate and 0.02 ml of water. Enzyme activity was calculated by measuring the change in absorbance at 480 nm for 5 min.

2.3.3. Semen parameters

2.3.3.1. Sperm count. The sperm count was determined using the Neubauer improved hemocytometer. Epididymal fluid ratio of 1:20 was prepared by adding 0.1ml 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).

2.3.3.2. Sperm motility. 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 (20). For the purpose of this study, motility was classified as either motile or non-motile (21). 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).

Impact of alcohol on the reproductive hormones, oxidative stress and semen parameters 3

2.4. Statistics

The data obtained from all the groups were compiled and statistically analyzed and expressed as mean ± Standard deviation. Differences between groups were compared using one-way ANOVA with p < 0.05 considered significant and a Bonferroni's post hoc test on SPSS.

3. Results

3.1. Effect of alcohol on reproductive hormones

After the first phase of study, the testosterone levels of subgroup B (alcohol) decreased but not significantly (p > 0.05) compared to subgroup A (control). Estrogen levels in the subgroup B decreased significantly (p < 0.05) compared to subgroup A. There were no significant differences in LH, FSH and GnRH alcohol group compared to control (Table 1). After the second phase study, testosterone levels in subgroup B decreased significantly compared to subgroup A. There was no significant difference in the estrogen, LH and FSH levels of subgroup B compared to subgroup A. GnRH increased significantly in subgroup B compared to subgroup A (Table 2). After the third phase of the study, there was a significant decrease in testosterone levels of subgroup B compared to subgroup A. There was no significant difference in estrogen, LH and FSH of subgroup B compared to subgroup A. GnRH of subgroup B decreased significantly compared to subgroup A (Table 3).

3.2. Effect of alcohol on MDA and SOD levels

Table 2 Reproductive hormones, MDA, SOD and semen

parameters after 8 weeks.

Parameters measured A (Control) B (Alcohol)

Testosterone (ng/ml) 4.72 ± 0.75 1.42 ± 0.33*

Estrogen (pg/ml) 17.57 ± 0.76 16.69 ± 4.52

LH (mIU/ml) 0.86 ± 0.36 0.71 ± 0.20

FSH (mIU/ml) 0.45 ± 0.04 0.24 ± 0.03

GnRH (pg/ml) 98.72 ± 2.72 142.05 ± 5.95*

MDA (nmol/ml) 9.42 ± 0.95 16.75 ± 2.90*

SOD (min/mg/protein) 32.27 ± 0.91 54.07 ± 15.3*

Sperm count (106/ml) 48.95 ± 1.06 31.2 ± 1.27*

Sperm motility (%) 67.5 ± 3.54 32.5 ± 3.54*

Values are expressed as mean ± SD.

p < 0.05 compared with A.

Table 3 Reproductive hormones, parameters after 16 weeks. MDA, SOD and semen

Parameters measured A (Control) B (Alcohol)

Testosterone (ng/ml) 3.07 ± 0.23 0.37 ± 0.04*

Estrogen (pg/ml) 17.23 ± 2.03 15.94 ± 1.92

LH (mIU/ml) 0.69 ± 0.10 1.3 ± 0.28

FSH (mIU/ml) 0.58 ± 0.65 0.40 ± 0.43

GnRH (pg/ml) 93.34 ± 4.30 39.75 ± 1.03*

MDA (nmol/ml) 26.25 ± 2.86 48.64 ± 2.28*

SOD (min/mg/protein) 51.65 ± 4.23 49.49 ± 1.84

Sperm count (106/ml) 84.75 ± 6.71 39.45 ± 3.99*

Sperm motility (%) 77.5 ± 3.54 45 ± 3.07*

Values are expressed as mean ± SD. p < 0.05 compared with A.

After the first phase of the study, there was a significant increase in MDA levels of subgroup B compared to subgroup A. There was no significant difference between the SOD levels of subgroups A and B (Table 1). After the second phase, there was a significant increase in MDA and SOD levels of subgroup B compared to subgroup A (Table 2). At the end of the third phase, MDA levels of subgroup B increased significantly compared to subgroup A (Table 3).

3.3. Effect of alcohol on semen parameters

At the end of the first, second and third phases, there was a significant decrease in sperm count and sperm motility of subgroup B compared to subgroup A (Tables 1-3).

4. Discussion

In this study, testosterone which is essential for spermatogen-esis is decreased by chronic consumption of alcohol. This is in concert with reports from Muthusami and Chinnaswamy (22) that alcohol has a direct toxic effect on the testis which leads to decreased seminiferous tubular function. Contrary to some authors who report that the negative feedback of testosterone on the hypothalamo-pituitary-gonadal (HPG) axis promotes an increase in LH (22-24), our present study shows that the animals that were treated with alcohol after 8 weeks had low testosterone and low LH levels. Alcohol's effects on the anterior pituitary gland produced a decrease in the production of LH and FSH. This finding correlates with the reports of Ren et al. (25) that alcohol does not only affect LH and FSH synthesis but impedes their secretion. After the sixteen week study, alcohol decreased GnRH levels significantly. This implies that chronic alcohol consumption affected the hypothalamic cells resulting in decreased secretion of GnRH. This is in concert with reports from our earlier study (26). Although some authors opine that alcohol acts directly on testicular tissue (22,27) others report that it acts via the HPG axis (25,28,29), our study reveals that alcohol alters Leydig cell function in the test is directly after 4 weeks of administration but it decreased the pituitary gonadotropic hormone secretions after 8 weeks and diminished the hypothalamic cell secretion of releasing hormones after 16 weeks. These findings indicate that

Table 1 Reproductive hormones, MDA, SOD and semen

parameters after 4 weeks.

Parameters measured A (Control) B (Alcohol)

Testosterone (ng/ml) 5.6 ± 1.4 2.9 ± 1.2

Estrogen (pg/ml) 17.02 ± 2.3 13.82 ± 5.0*

LH (mIU/ml) 0.69 ± 0.1 0.85 ± 0.4

FSH (mIU/ml) 0.11 ± 0.1 1.92 ± 2.6

GnRH (pg/ml) 25.57 ± 2.8 42.08 ± 4.9

MDA (nmol/ml) 6.87 ± 0.19 12.10 ± 0.52*

SOD (min/mg/protein) 107.71 ± 5.20 120.66 ± 3.92

Sperm count (106/ml) 88.3 ± 9.90 24.7 ± 1.27*

Sperm motility (%) 98.5 ± 0.71 42.0 ± 1.41*

Values are expressed as mean ± SD.

* p < 0.05 compared with A.

the effects of alcohol action are both directly on the testis and via the HPG axis.

This study shows that acute and chronic administration of alcohol increased lipid peroxidation. Our findings are in consonant with reports from Emanuele and Emanuele (13) and Dosumu et al. (11). Studies adduce increased free radicals or reactive oxygen species and lipid peroxidation for the mechanism by which alcohol causes testicular toxicity (3032). The significant increase in MDA levels of the alcohol treated group in this study supports these earlier findings. It is demonstrated in this study that testicular superoxide dis-mutase increased in response to increased lipid peroxidation. In spite of this increased antioxidant activity, the spermato-genic cells were not protected from the deleterious effects of alcohol.

The significant decrease in sperm count and sperm motility of alcohol treated animals in this study is a proof that alcohol is inimical to male fertility. This corroborates the report by Chia et al. (9) that, about 42% of men with infertility consume alcohol. Moreover, it has been reported that both acute and chronic consumption of alcohol increases oxidative stress (8). Increased and prolonged oxidative stress causes testicular damage which impedes spermatogenesis resulting in decreased sperm count (11,33).

Furthermore, spermatozoa are particularly susceptible to oxidative stress-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFAs) and their cytoplasm contains low concentrations of scavenging enzymes (34-36). In addition, ethanol affects mito-chondrial function. Mitochondria produce ATP required for the movement of the flagella of sperm cells. Hence, a reduced or impaired mitochondrial function will impede sperm motility as observed in the alcohol treated groups of this study. It has been reported that mitochondria are targets for oxidative stress and also contribute to the mechanism by which oxidative stress-related signals control cell fate.(37) Since alcohol increases oxidative stress which adversely affects mitochon-drial function, it is implied that the mechanism by which alcohol decreases sperm motility is related to its effect on mitochondrial function.

5. Conclusion

This study has demonstrated that alcohol induced male infertility by acting directly on the testis and also via disruption of the feedback mechanism of the HPG axis. These findings suggest a cautious use of alcohol among males undergoing various treatments for infertility.

References

(1) WHO. Manual for the standardized investigation and diagnosis for the infertile couple. Cambridge University Press; 2000.

(2) McLachlan R, de Krester D. Male infertility: the case for continued research. MJA 2001;174:116-7.

(3) Shukla KK, Mahdi AA, Ahmad MK, Jaiswar MP, Shankwar SN, Tiwari SC. Mucuna pruriens reduces stress and improves the quality of semen in infertile men. eCAM 2010;7:137-44.

(4) Aitken RJ. The amoroso lecture. The human spermatozoon-a cell in crisis? J Reprod Fertil 1999;115:1-7.

(5) McGrady AV. Effects of psychological stress on male reproduction: a review. Arch Androl 1984;13:1-7.

A.A. Oremosu, E.N. Akang

(6) de Lamirande E, Leduc BE, Iwasaki A, Hassouna M, Gagnon C. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steril 1995;64:637-42.

(7) Padron OF, Brackett NL, Sharma RK, Kohn S, Lynne CM, Thomas Jr AJ. Seminal reactive oxygen species, sperm motility and morphology in men with spinal cord injury. Fertil Steril 1997;67:1115-20.

(8) Kefer JC, Agarwal A, Sabanegh E. Role of antioxidants in the treatment of male infertility. Int J Urol 2009;16:449-57.

(9) Chia SE, Lim ST, Tay SK, Lim ST. Factors associated with male infertility: a case control study of 218 infertile and 240 fertile men. BJOG 2000;107:55-61.

(10) Maneesh M, Dutta S, Chakrabarti A, Vasudevan DM. Alcohol abuse-duration dependent decrease in plasma testosterone and antioxidants in males. Indian J Physiol Pharmacol 2006;50:291-6.

(11) Dosumu OO, Akinola OB, Akang EN. Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats - the therapeutic potential of virgin coconut oil. Middle East Fertil Society J 2012;17:122-8.

(12) Martinez M, Macera S, de Assis GF, Pinheiro PFF, Almeida CCD, Tirapelli LF, et al. Structural evaluation of the effects of chronic ethanol ingestion on the testis of Calomys callosus. Tissue Cell 2009;41:199-205.

(13) Emanuele M, Emanuele N. Alcohol and male reproductive system. Natl Inst Alcohol Abuse Alcohol 2001;25:282-7.

(14) Khan MS, Ali I, Khattak AM, Tahir F, Subhan F, Kazi BM, et al. Role of estimating serum luteinizing hormone and testosterone in infertile males. Gom J Med Sci 2005:61-5.

(15) Martinin FH. Hormones of reproductive system. 5th Ed. Prentice Hall: Upper Saddle River, New Jersey; 2001. p. 1957.

(16) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.

(17) Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276-87.

(18) Sun M, Zigma S. An improved spectrophotometric assay of superoxide dismutase based on epinephrine antioxidation. Anal Biochem 1978;90:81-9.

(19) Keel BA, Webster BW, editors. CRC handbook of the laboratory diagnosis and treatment of infertility. Boca Raton: CRC Press Incorporation; 1990, p. 37.

(20) WHO. Laboratory manual for the examination of human semen and sperm cervical mucus interaction. Cambridge University Press; 1999.

(21) Osinubi AA, Daramola AO, Noronha CC, Okanlawon AO, Ashiru OA. The effect of quinine and ascorbic acid on rat testes. West Afr J Med 2007;26:217-21.

(22) Muthusami KR, Chinnaswamy P. Effect of chronic alcoholism on male fertility hormones and semen quality. Fertil Steril 2005;84:919-24.

(23) Sengupta SN, Ray R, Shetty KT, Desai NG. Pituitary gonadal functioning in male alcoholics in an Indian psychiatric hospital. Alcohol Alcohol 1991;26:47-51.

(24) Heinz A, Rommelspacher H, Graf KJ, Kurten I, Otto M, Baumgartner A. Hypothalamic-pituitary-gonadal axis, prolactin, and cortisol in alcoholics during withdrawal and after three weeks ofabstinence: comparison with health control subjects. Psychiatry Res 1995;56:81-95.

(25) Ren J, Banan A, Keshavarzian A, Zhu Q, LaPaglia N, McNulty J, et al. Exposure to ethanol induces oxidative damage in the pituitary gland. Alcohol 2005;35:91-101.

(26) Akang EN, Oremosu AA, Dosumu OO, Ejiwunmi AB. The role of Telfairia occidentals in protecting the testis against alcohol induced damage. Maced J Med Sci 2011;4:380-7.

(27) Van Thiel DH, Galaver PK, Rosenblum E, Eagon YB. Effects of ethanol on endocrine cells: testicular effects. Ann N Y Acad Sci 1987;492:287-302.

Impact of alcohol on the reproductive hormones, oxidative stress and semen parameters

(28) Hadley ME. Endocrinology. 2nd Ed. Englewoods Cliff, NJ: Prentice Hall; 1988.

(29) Frias J, Torres JM, Miranda MT, Ruiz E, Ortega E. Effects of acute alcohol intoxication on pituitary-gonadal axis hormones, pituitary-adrenal axis hormones, B-Endorphin and prolactin in human adults of both sexes. Alcohol Alcohol 2002;37:169-73.

(30) Peltola V, Huhtaniemi I, Ketala TM, Ahotupa M. Induction of lipid peroxidation during steroidogenesis in the rat testis. Endocrinology 1996;137:105-12.

(31) Oner-Iyidogan Y, Gurdol F, Oner P. The effects of acute melatonin and ethanol treatment on antioxidant enzyme activities in rat testes. Pharmacol Res 2001;44:44-9.

(32) Saleh RA, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl 2002;23:737-52.

(33) Eskenazi B, Wyrobek AJ, Sloter E, Kidd SA, Moore L, Young S, Moore D. The association of age and semen quality in healthy men. Hum Reprod 2003;18:447-54.

(34) Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod Dev 1995;42:334-46.

(35) De Lamirande E, Gagnon C. Capacitation-associated production of superoxide anion by human spermatozoa. Free Rad Biol Med 1995;18:487-95.

(36) Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology 1996;48:835-50 [Review].

(37) Hoek JB, Cahill A, Pastorino JG. Alcohol and mitochondria: a dysfunctional relationship. Gastroenterology 2002;122:2049-63.