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Toxicity of chronic ethanol ingestion and superoxide radical formation on seminal vesicles of rats
Pergamon
Food and Chemical
Toxicology
34 (1996) 1003-1007
SO278-6915(96)00065-8
BRIEF COMMUNICATIONS
Toxicity of Chronic Ethanol Ingestion and Superoxide Radical Formation on Seminal Vesicles of Rats E. L. B. NOVELLIt, N. L. RODRIGUESt, B. 0. RIBASS and J. L. V. B. NOVELL1 FILHOg TDepartamento de Quimica, Instituto de Biociincias, Universidade Estadual Paulista-UNESP, 18618-000 Botucatu, Slo Paula, Brazil, SInstituto de Salud Carlos III, MinistCrio de Sanidade y Consumo, Madrid, Spain and §Faculdade de Medicina de Botucatu, Universidade Estadual Paulista-UNESP, 18618-000 Botucatu, S%o Paula, Brazil (Accepted 24 June 1996)
Abstract-Tb: toxic effects of chronic ethanol ingestion were evaluated in male adult rats for 300 days. The animals were divided into three groups: the controls received only tap water as liquid diet; the chronic ethanol ingestion group received only ethanol solution (30%) in semivoluntary research; and the withdrawal group received the same treatment as chronic ethanol-treated rats until 240 days, after which they reverted to drinking water. Chronic ethanol ingestion induced increased lipoperoxide levels and acid phosphatase activities in seminal vesicles. Cu-Zn superoxide dismutase (SOD) decreased from its basal level 70.8 f 3.5 to 50.4 k 1.6 U/mg protein at 60 days of chronic ethanol ingestion. As changes in GSH-PX activity were observed in rats after chronic ethanol ingestion, while SOD activities were decreased in these animals, it is assumed that superoxide anion elicits lipoperoxide formation and induces cell damage before being converted to hydrogen peroxide by SOD. Ethanol withdrawal induced increased SOD activity and reduced seminal vesicle damage, indicating that the toxic effects were reversible, since increased SOD activity was adequate to scavenge superoxide radical formation. Superoxide radical is an important intermediate in the toxicity of chronic ethanol ingestion. Copyright 0 1996 Elsevier Science Ltd
Introduction At the present time, much attention is being paid to both consumer products and additive substances in foodstuffs. In many societies, alcoholic beverages are considered as part of the basic food supply. Ethanol is rich in energy, but excessive alcohol drinking is a lifestyle factor associated with physical dependence, severe withdrawal reactions and development of damage (Duffy, 1995). Alcohol consumption increased substantially from the late 1950s to the 1990s. Ethanol is a recognized cause of testicular injury (Rosenblum et al., 1989), hepatocellular damage (Nordmann et al., 1992) and oesophageal tumour development (Aze ef al., 1993). Martinez et al. (1993) *Author for correspondence. ACP = acid phosphatase; ALT = alanine transaminase; EDTA = ethylenediamine tetraacetic acid; GSH-PX = glutathione peroxidase; HIOl = hydrogen peroxide; NBT = nitro blue tetrazolium: SOD = superoxide dismutase; TBA = thiobarbituric acid.
Abbreviations:
showed significantly increased damage to the reproductive systems of rats after chronic ethanol ingestion. The seminal vesicles are implicated in sperm motility and changes in their structure could be related to lowered fertility and increased susceptibility to infection from various micro-organisms (Chyou, 1993). Oxygen-derived radicals are known to be cytotoxic to cells. During oxidative stress, reactive oxygen species, superoxide anion (0; - ), hydroxyl radicals (OH.) and hydrogen peroxide (H,O,) can elicit widespread damage to cell constituents such as membrane lipids. Lipid peroxidation is an important toxic event of 02 - causing final cell death. Indeed, cell and tissue destruction mediated by radicals can often lead to more lipid peroxidation because antioxidants are diluted and transition metal ions that can stimulate the peroxidation process are released from disrupted cells (Novelli et al., 1995b). Ethanol is assumed to be a toxic factor, but there is no clear evidence on the mechanism, phase, initiation or promotion by which ethanol exerts its effects.
0278-6915/96/$15.00 + 0.00 Copyright 0 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII 0278-6915(96)00065-8
1004
E. L.
B. Novelli et a/.
The present study was carried out to determine the toxicity of chronic ethanol ingestion to seminal vesicle lesions, and to clarify the contribution of O? in this effect.
Material and Methods Male, laboratory rats (Rattus noroegicus; Wistar) (150 animals) weighing 18&200 g were divided into three groups. A control group (A) received tap water throughout the experimental period, while the experimental group (B) were allowed to drink only aqueous ethanol (30%). The animals of group B received ethanol solution throughout the experimental period. To induce abstinence, animals of group C received aqueous ethanol for 240 days, following a withdrawal period (days 24&300) when they received only tap water. However, animals do not normally ingest more ethanol than they can metabolize. As a consequence they do not voluntarily become dependent. To establish an analogous animal model of human alcoholism, rats received aqueous ethanol and when adapted to the taste of the solution and metabolic tolerance had developed, the ethanol content of the solutions was gradually raised. Alcohol was introduced as aqueous ethanol to groups B and C: from 5% in the first week, 10% in the subsequent week, and thereafter increased 5%/wk (for example, 30% at wk 6) until the end of the study (Pohorecky, 1981). Animals were then maintained at the highest concentration of ingested ethanol (30%). As Goldman et al. (1980) reported that physical dependence on ethanol can be produced without nutritional impairment in rodents, the same diet was used in the present study for all animals. Food (Purina Ltd, Campinas, SP, Brazil), water and ethanol solutions were provided ad lib. Rats were killed (10 for each group) by decapitation at time intervals of 60, 120, 180, 240 and 300 days after treatment. The seminal vesicles were taken, weighed and homogenized in five volumes of phosphate buffer (0.1 M, pH 7.4) containing 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma, St
Louis, MO, USA), with a motor-driven teflon-glass tissue homogenizer. EDTA powerfully inhibits iron-catalysed lipid peroxidation (Wills, 1966). The homogenate was centrifuged at 1200 g for 15 min. The supernatant was used for total protein (Lowry et al., 1951), alanine transaminase (ALT)(E.C.2.6.1.2) (Wilkinson et a/., 1972), acid phosphatase (ACP)-(E.C.3.1.3.2), (Andersch and Sczypinsky, 1974) and glutathione peroxidase (GSH-PX)(E.C. 1.11.1.9) (Hopkins and Tudhope, 1973) determinations. Lipoperoxide was determined by thiobarbituric acid (TBA)-(Sigma) according to Ohkawa et al. (1979). The reaction mixture, in 20 ml screw-capped vials, consisted of 0.2 ml sample (supernatant), 0.2 ml 8.1% sodium dodecyl sulfate solution, 1.5 ml 20% acetic acid solution (adjusted to pH 3.5 with 1 N NaOH) and 1.5 ml 1% TBA solution. Distilled water was added to make up the volume to 4.0 ml. A tissue blank was prepared for each sample by substituting the TBA solution with distilled water. The solution was mixed and heated in a water-bath at 95°C for 60 min. The vials were allowed to cool to room temperature and 2 ml of the aliquot of tissue blank or sample was transferred to a tube with 2.0 ml 10% trichloroacetic acid. The absorbance was measured spectrophotometrically at 532 nm. Cu-Zn superoxide dismutase (SOD)(E.C. 1.15.1.1.) activity was determined based on the ability of the enzyme to inhibit the reduction of nitro blue tetrazolium (NBT) (Sigma), which was generated by hydroxylamine 37.5 mM (Carlo Erba, Italy) in alkaline solution (Crouch et al., 1982). The assay was performed in 0.5 M sodium carbonate (pH 10.2) to with 0.1 M EDTA. The reduction of NBT by 0: blue formazan was measured spectrophotometrically at 560 nm. The rate of NBT reduction in the absence of tissue was used as the reference rate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition. All data are expressed in units of SOD per mg protein. The mitochondrial Mn superoxide dismutase was inactivated by treatment with mixtures of chloroform and ethanol (Weisiger and Fridovich,
Table I. Total protem. ALT and ACP in the seminal vesiclesof controls (A), rats after chronic ethanol ingestion (B) and rats after ethanol withdrawal (C) Days following treatment 60
120
I80
240
300
Total protein (mg/g tissue) A B C A B C A B C A B C A B C
I .9 I.5 2.4 I .9 3.3 2.6 2.0 2.6 2.8 2.5 3.9 2.3 2.1 2.3 1.6
f f f f f f f f + f f f f + f
0.4 0.6 0.9 0.6 1.8 I.1 0.8 0.8 0.9 I.1 2.2 1.1 0.6 I.2 0.9
ALT (U/mg protein) 1.9 f 1.7 & I .6 f 3.3 * 3.9 * 3.7 f 3.8 + 4.2 + 3.7 * I .4 f I.5 * 1.4 + 2.2 f 2.1 f 2.4 f
‘Values are significantly different from those of the control group by Student’s f-test (P < 0.05).
0.1 0.6 0.9 1.9 2.3 I..( 2.2 2.4 2.0 0.4 0.2 0.2 I.1 I.2 0.9
ACP (U:g tissue) I.5 2.3 2.7 I.6 3.8 2.9 I.5 2.6 2.7 I.1 2.5 2.8 I.5 3.3 I .6
+ + + f i & * k + + * f f f f
0.4 0.1’ 0.3’ 0.4 0.5* 0.18 0.3 0.1’ 0.2’ 0.2 0.1. 0.2’ 0.1 0.2’ 0.2’
Toxic
Table 2. Lipoperoxide, CwZn SOD and GSH-PX
effects
of chronic
ethanol
ingestion
1005
in the seminal vesicles of controls (A), rats after chronic ethanol ingestion (B) and rats
after ethanol withdrawal (C) Days following treatment 60
120
Lipoperoxide (meq/Kg) A B C A B A B C A B C A
2.1 5.7 5.5 2.5 4.6 4.3 2.1 19.5 18.9 2.4 20.9 22.0 2.7
B C
IO.1 + I.21 3.0 f 0.5
C 180
240
300
& f f f f + f f f + f f +
0.7 0.6’ 0.1; 0.3 0.5’ 0.2’ 0.6 1.4’ 1.7* 0.7 2.4’ 2.5’ 0.3
SOD (U/mg protein) 70.8 50.4 59.7 74.8 52.4 51.5 75.6 40.1 39.2 74.9 37.6 39.6 75.3
+ + + f + f + * i & i i k
3.5 1.6; 1.6; 4.9 1.6’ 1.1* 2.0 3.98 2.8* 2.2 1.8; 1.9* 2.9
34.3 & 1.6: 96.3 f 1.5*
GSH-PX
(U/mg 1.6 I.1 0.8 0.9 0.9 0.8 0.9 1.0 0.8 0.9 1.2
f + + * * f + f f f k
protein)
0.3 0.2 0.2 0.2
0.1
0.3 0.2 0.3 0.3 0.2 0.3 I.1 + 0.3
1.3 + 0.4 1.2 i 0.3 I.1 + 0.2
‘Values are significantly different from those of the control group by Student’s t-test (P < 0.05).
1973). This SOD enzyme has all characteristics of human SOD (Hsu et al., 1992). Values are presented as means f SEM. Significance of difference was tested by analysis of variance and Student’s multiple t-test (Zar, 1974). Results Chronic ethanol ingestion induced increased ACP activity and lipoperoxide concentrations throughout the experimental period. On the other hand, ethanol withdrawal (days 240-300) led lipoperoxide and ACP values to approach those of the controls (A) at 300 days following I:reatment. This is supported by the data for ACP activities [ 1.5 + 0.1 in the controls (A), 1.6 & 0.2 in withdrawal rats (C) but 3.3 k 0.2 in rats with chronic eth,anol intake (B)] (Tables 1 and 2). No alterations were observed in total protein concentrations and ALT activities in the seminal vesicles of rats treated with chronic ethanol, and no changes were observed in total protein and ALT with ethanol withdrawal (Table 1). Significantly decreased SOD was observed in rats with chronic ethanol ingestion (Table 2). Ethanol withdrawal induced increased SOD activities in seminal vesicles of rats at 300 days following treatment, (Table 2). No alterations were observed in GSH-PX activity in the seminal vesicles of rats with chronic ethanol withdrawal (C) (Table 2). Discussion Enhanced generation of O* - or reduced levels of SOD have been implicated in a wide range of pathological conditions, such as nickel-induced toxicity (Novelli et dzl., 1995a), cataracta formation and ageing, inflammatory tissue necrosis, tumour proliferation and neurodegenerative disorders (Cayuela, 1995). Free-radical mechanisms also appear to be implicated in the toxicity of ethanol in various extrahepatic tissues (Orsilles et al., 1995). Although clinical studies have not yet demonstrated clearly the role of free radicals in the pathogenesis of
ethanol-induced cellular injury, increased lipoperoxide formation in the seminal vesicles of rats after chronic ethanol ingestion was observed in the present study (Table 2). Oxygen-derived species such as superoxide radical (Ox’-) are produced in mammalian cells during normal aerobic metabolism. Excess generation of these reactive oxygen species ‘in uivo’ results in lipid peroxidation (Novelli et al., 1994). Some studies (Mascio et al., 1996) have shown that sexual dysfunction induced by ethanol is the result of lipoperoxide formation, which is a free-radicalmediated chain reaction since once initiated it is self-perpetuating. The length of the propagation chain depends on chain-breaking antioxidant enzymes, such as Cu-Zn SOD (Mascio et al., 1996). There are further consequences related to membrane lipoperoxidation such as increased activities of AP (Table 1). Miller (1993) showed that ACP is increased in damaged seminal vesicles and is an indispensable marker to diagnose prostate disease. Thus, the fact that chronic ethanol ingestion increased ACP activity from its basal level at 60 days reflects seminal vesicle damage (Table 1). The total daily dose of ethanol varied from day to day, depending on ingestion of diet, but no alterations in total protein concentration and ALT activities were observed throughout the experimental period (Table 1). These observations are consistent with Lum (1995) who showed that protein concentrations and ALT activities were not altered in ethanol related disease. It should be noted (Table 2) that SOD activity was decreased at 60 days after chronic ethanol ingestion, while significantly increased lipoperoxide and ACP activities were observed in this period. As SOD activity was decreased, the unscavenged superoxide could result in lipoperoxide formation. The levels of SOD reported (Table 2) are consistent with these statements. At day 240 the levels of SOD were lower in rats following chronic ethanol ingestion (B) and after ethanol withdrawal (C) than in the controls (A). At day 300, after a withdrawal period
E. L. B. Novelli et al.
1006
(days 24&300), the SOD activities were higher in abstinent rats (C) than in the controls (A). On the other hand, no significant differences in lipoperoxide values were observed between rats subjected to withdrawal (C) and the controls (A), indicating that increased SOD activity scavenged O1 after withdrawal. Lipoperoxide and ACP values were increased throughout the experiment in rats after chronic ethanol ingestion (Tables 1 and 2) indicating that chronic ethanol ingestion induced an initially increased respiratory burst in which oxygen was reduced to O2 - and endogenous SOD activities were overwhelmed to scavenger the vast excess of O2 -. 0~~ and HzO, are known to be cytotoxic to cells causing damage to lipids. GSH-PX catalyses the conversion of H,Oz to water, thus, GSH-PX could reduce tissue injury by removing H202. SOD catalyses the destruction of superoxide radical by dismutation and H,Oz formation. As no changes in GSH-PX activity were observed in rats after chronic ethanol ingestion, while SOD activities were decreased in these animals, it is supposed that O2 _ generated by ethanol is of primary importance in the pathogenesis of seminal vesicle injury. O2 - elicits lipoperoxide formation and induces cell damage before being converted to H,O, by SOD. GSH-PX activity was considered as a means of differentiating between alternative cytotoxic mechanisms. This study allows conclusions to be drawn regarding which reactive oxygen metabolite plays a part in chronic ethanol toxicity. These observations suggest that the superoxide radical has a part in ethanol-related tissue injury. Ethanol withdrawal, which was obtained following a period of 240-300 days, when rats (C) received only reduced Oz - production, decreased tap water, lipoperoxide formation (Table 1) and ACP activities (Table 2) and reduced seminal vesicle damage. These observations are consistent with the hypothesis that Oi- is produced as a mediator of tissue lesions induced by chronic ethanol ingestion. The toxic effects of chronic ethanol ingestion were reversible, since increased SOD was adequate to scavenger superoxide radical formation after a withdrawal period. In conclusion, the toxicity of chronic ethanol ingestion on seminal vesicles involves oxidative reactions, such as ethanol induced lipid peroxidation. Superoxide radical is an important toxic intermediate in the development of chronic ethanol damage. Acknowledgemenfs-This study was supported by FAPESP (Fundacao de Amparo a Pesquisa do Estado de SBo Paulo). The authors thank Mr Guerino S. Bianchi Filho and Mrs Claudete Ezias Grassi for their technical services.
REFERENCES
Andersch M. A. A. and Sczypinsky A. J. (1974) Use of p-nitrophenylphosphate as the substrate in determination
of serum acid phosphatase. American Journal of Clinical Pathology 17, 571-574. Aze Y., Toyota K., Furukawa F., Mitsumori K. and Takahashi M. (1993) Enhancing effect of ethanol on esophageal tumor development in rats by initiation of diethylnitrosamine. Carcinogenesis 14, 47-50. Cayuela M. M. (1995) Oxygen free radicals and human disease. Biochimie 77, 147-161. Chyou P. H. (1993) A prospective study of alcohol and other style factors in relation to obstructive urophaty. Prostate 22, 253-264. Crouch R., Gandy S. E. and Kinsey G. (1982) The inhibition of islet superoxide dismutase by diabetogenic drugs. Diabetes 30, 2355241. Duffy J. C. (1995) Alcohol consumption an all-cause mortality. International Journal qj Epidemiology 24, 10&l 15. Goldman M. E., Miller S. S., Shorey R. L. and Erickson C. K. (1980) Ethanol dependence produced in rats by nutritionally complete diets. Pharmacology Biochemistr! and Behavior 12, 503-507. Hopkins J. and Tudhope G. R. (1973) Glutathione peroxidase in human red cells in health and disease. British Journal of Haemarology 25, 563-575. Hsu J. L., Visner G. A., Burr T. A. and Nick H. S. (1992) Rat copper/zinc superoxide dismutase gene: isolation, characterization and species comparison. Biochemical and Biophysical Research Communications 186, 936943. Lowry 0. H., Rosembrough N. J. and Farr A. L. (1951) Protein measurement with folin-phenol reagent. Journal of Biological Chemisrr_v 193, 265-275. Lum G. (1995) Low activities of aspartate and alanine aminotransferase. Labor Medica 26, 273-276. Martinez F. E., Garcia P. J., Padovani C. R., Cagnon V. H. A. and Martinez M. (1993) Ultrastructural study of the ventral lobe of prostate of rats submitted to experimental chronic alcoholism. Prostate 22, 3 17-324. Mascio P. D., Medeiros M. H. G., Bechara E. J. H. and Catalani J. H. (1996) Singlet molecular oxygen: generation, reactivity, identification and biological effects. Cikia e Cultura 47, 297-3 I I. Miller 0. (1993) Bioquimica do sangue. Enzimas. In Laboratdrio para o Clinico. Atheneu. pp. 63-68. Livraria Atheneu Editora, Sao Paulo. Nordmann R.. Ribiere C. and Rouach H. (1992) Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radical Biology and Medicine 12, 2 19-240. Novelli E. L. B., Rodrigues N. L. and Ribas B. 0. (1995a) Superoxide radical and toxicity of environmental nickel exposure. Human and Esperimenral To\-icolog! 14, 248-25 I. Novelli E. L. B., Silva A. M. M., Novelli Filho J. L. V. B. and Curi P. R. (1995b) Reactive oxygen generation by azomethine H: a new antimalarial drug. Canadian Journal Phjxiologj* and Pharmacology 73, 1189-l 194. Novelli E. L. B., Valente J. P. S. and Rodrigues N. L. (1994) Toxic effects of water eutrophication on pancreatic, hepatic and osteogenic tissue of rats. To.uicon 22, 1270-1274. Ohkawa H., Ohishi N. and Yagi K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analvtical Biorhemistrv $5, 351-358. Orsilles M. A.. Donadio A. C. and Depaoli D. M. (1995) Time course of reactive oxygen intermediates release and histopathological findings during experimental autoimmune prostatitis development. Prostate 27, 5Ck57. Pohorecky A. (198 1) Animal analog of alcohol dependence. Federation Proceedings 40, 2056-2064. Rosenblum E. P., Gavalar J. S. and Van Thiel D. H. (1989)
Toxic effects of chronic Lipid peroxidation, a mechanism for alcohol-induced testicular injury. Free Radical Biology and Medicine 7, 569-577. Weisiger R. A. and Fridovich I. (1973) Superoxide dismutases: organelle specificity. Journal of Biological Chemistry 248, 358.!-3592. Wilkinson J. H., Baron D. N. and Moss D. W. (1972) Standardization of clinical enzymes assays: a reference
ethanol
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method for aspartate and alanine transaminases. Journal of Clinical Pathology 25, 940-949. Wills E. D. (1966) Mechanisms of lipid peroxide formation in animals tissues. Biochemical Journal 99, 667-676. Zar J. H. (1974) Multiple comparisons. In Biosratisrical Analysis. W. 0. McElroy and C. D. Swanson. pp. 12G-138. Prentice Hall, New York.
Food and Chemical
Toxicology
34 (1996) 1003-1007
SO278-6915(96)00065-8
BRIEF COMMUNICATIONS
Toxicity of Chronic Ethanol Ingestion and Superoxide Radical Formation on Seminal Vesicles of Rats E. L. B. NOVELLIt, N. L. RODRIGUESt, B. 0. RIBASS and J. L. V. B. NOVELL1 FILHOg TDepartamento de Quimica, Instituto de Biociincias, Universidade Estadual Paulista-UNESP, 18618-000 Botucatu, Slo Paula, Brazil, SInstituto de Salud Carlos III, MinistCrio de Sanidade y Consumo, Madrid, Spain and §Faculdade de Medicina de Botucatu, Universidade Estadual Paulista-UNESP, 18618-000 Botucatu, S%o Paula, Brazil (Accepted 24 June 1996)
Abstract-Tb: toxic effects of chronic ethanol ingestion were evaluated in male adult rats for 300 days. The animals were divided into three groups: the controls received only tap water as liquid diet; the chronic ethanol ingestion group received only ethanol solution (30%) in semivoluntary research; and the withdrawal group received the same treatment as chronic ethanol-treated rats until 240 days, after which they reverted to drinking water. Chronic ethanol ingestion induced increased lipoperoxide levels and acid phosphatase activities in seminal vesicles. Cu-Zn superoxide dismutase (SOD) decreased from its basal level 70.8 f 3.5 to 50.4 k 1.6 U/mg protein at 60 days of chronic ethanol ingestion. As changes in GSH-PX activity were observed in rats after chronic ethanol ingestion, while SOD activities were decreased in these animals, it is assumed that superoxide anion elicits lipoperoxide formation and induces cell damage before being converted to hydrogen peroxide by SOD. Ethanol withdrawal induced increased SOD activity and reduced seminal vesicle damage, indicating that the toxic effects were reversible, since increased SOD activity was adequate to scavenge superoxide radical formation. Superoxide radical is an important intermediate in the toxicity of chronic ethanol ingestion. Copyright 0 1996 Elsevier Science Ltd
Introduction At the present time, much attention is being paid to both consumer products and additive substances in foodstuffs. In many societies, alcoholic beverages are considered as part of the basic food supply. Ethanol is rich in energy, but excessive alcohol drinking is a lifestyle factor associated with physical dependence, severe withdrawal reactions and development of damage (Duffy, 1995). Alcohol consumption increased substantially from the late 1950s to the 1990s. Ethanol is a recognized cause of testicular injury (Rosenblum et al., 1989), hepatocellular damage (Nordmann et al., 1992) and oesophageal tumour development (Aze ef al., 1993). Martinez et al. (1993) *Author for correspondence. ACP = acid phosphatase; ALT = alanine transaminase; EDTA = ethylenediamine tetraacetic acid; GSH-PX = glutathione peroxidase; HIOl = hydrogen peroxide; NBT = nitro blue tetrazolium: SOD = superoxide dismutase; TBA = thiobarbituric acid.
Abbreviations:
showed significantly increased damage to the reproductive systems of rats after chronic ethanol ingestion. The seminal vesicles are implicated in sperm motility and changes in their structure could be related to lowered fertility and increased susceptibility to infection from various micro-organisms (Chyou, 1993). Oxygen-derived radicals are known to be cytotoxic to cells. During oxidative stress, reactive oxygen species, superoxide anion (0; - ), hydroxyl radicals (OH.) and hydrogen peroxide (H,O,) can elicit widespread damage to cell constituents such as membrane lipids. Lipid peroxidation is an important toxic event of 02 - causing final cell death. Indeed, cell and tissue destruction mediated by radicals can often lead to more lipid peroxidation because antioxidants are diluted and transition metal ions that can stimulate the peroxidation process are released from disrupted cells (Novelli et al., 1995b). Ethanol is assumed to be a toxic factor, but there is no clear evidence on the mechanism, phase, initiation or promotion by which ethanol exerts its effects.
0278-6915/96/$15.00 + 0.00 Copyright 0 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII 0278-6915(96)00065-8
1004
E. L.
B. Novelli et a/.
The present study was carried out to determine the toxicity of chronic ethanol ingestion to seminal vesicle lesions, and to clarify the contribution of O? in this effect.
Material and Methods Male, laboratory rats (Rattus noroegicus; Wistar) (150 animals) weighing 18&200 g were divided into three groups. A control group (A) received tap water throughout the experimental period, while the experimental group (B) were allowed to drink only aqueous ethanol (30%). The animals of group B received ethanol solution throughout the experimental period. To induce abstinence, animals of group C received aqueous ethanol for 240 days, following a withdrawal period (days 24&300) when they received only tap water. However, animals do not normally ingest more ethanol than they can metabolize. As a consequence they do not voluntarily become dependent. To establish an analogous animal model of human alcoholism, rats received aqueous ethanol and when adapted to the taste of the solution and metabolic tolerance had developed, the ethanol content of the solutions was gradually raised. Alcohol was introduced as aqueous ethanol to groups B and C: from 5% in the first week, 10% in the subsequent week, and thereafter increased 5%/wk (for example, 30% at wk 6) until the end of the study (Pohorecky, 1981). Animals were then maintained at the highest concentration of ingested ethanol (30%). As Goldman et al. (1980) reported that physical dependence on ethanol can be produced without nutritional impairment in rodents, the same diet was used in the present study for all animals. Food (Purina Ltd, Campinas, SP, Brazil), water and ethanol solutions were provided ad lib. Rats were killed (10 for each group) by decapitation at time intervals of 60, 120, 180, 240 and 300 days after treatment. The seminal vesicles were taken, weighed and homogenized in five volumes of phosphate buffer (0.1 M, pH 7.4) containing 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma, St
Louis, MO, USA), with a motor-driven teflon-glass tissue homogenizer. EDTA powerfully inhibits iron-catalysed lipid peroxidation (Wills, 1966). The homogenate was centrifuged at 1200 g for 15 min. The supernatant was used for total protein (Lowry et al., 1951), alanine transaminase (ALT)(E.C.2.6.1.2) (Wilkinson et a/., 1972), acid phosphatase (ACP)-(E.C.3.1.3.2), (Andersch and Sczypinsky, 1974) and glutathione peroxidase (GSH-PX)(E.C. 1.11.1.9) (Hopkins and Tudhope, 1973) determinations. Lipoperoxide was determined by thiobarbituric acid (TBA)-(Sigma) according to Ohkawa et al. (1979). The reaction mixture, in 20 ml screw-capped vials, consisted of 0.2 ml sample (supernatant), 0.2 ml 8.1% sodium dodecyl sulfate solution, 1.5 ml 20% acetic acid solution (adjusted to pH 3.5 with 1 N NaOH) and 1.5 ml 1% TBA solution. Distilled water was added to make up the volume to 4.0 ml. A tissue blank was prepared for each sample by substituting the TBA solution with distilled water. The solution was mixed and heated in a water-bath at 95°C for 60 min. The vials were allowed to cool to room temperature and 2 ml of the aliquot of tissue blank or sample was transferred to a tube with 2.0 ml 10% trichloroacetic acid. The absorbance was measured spectrophotometrically at 532 nm. Cu-Zn superoxide dismutase (SOD)(E.C. 1.15.1.1.) activity was determined based on the ability of the enzyme to inhibit the reduction of nitro blue tetrazolium (NBT) (Sigma), which was generated by hydroxylamine 37.5 mM (Carlo Erba, Italy) in alkaline solution (Crouch et al., 1982). The assay was performed in 0.5 M sodium carbonate (pH 10.2) to with 0.1 M EDTA. The reduction of NBT by 0: blue formazan was measured spectrophotometrically at 560 nm. The rate of NBT reduction in the absence of tissue was used as the reference rate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition. All data are expressed in units of SOD per mg protein. The mitochondrial Mn superoxide dismutase was inactivated by treatment with mixtures of chloroform and ethanol (Weisiger and Fridovich,
Table I. Total protem. ALT and ACP in the seminal vesiclesof controls (A), rats after chronic ethanol ingestion (B) and rats after ethanol withdrawal (C) Days following treatment 60
120
I80
240
300
Total protein (mg/g tissue) A B C A B C A B C A B C A B C
I .9 I.5 2.4 I .9 3.3 2.6 2.0 2.6 2.8 2.5 3.9 2.3 2.1 2.3 1.6
f f f f f f f f + f f f f + f
0.4 0.6 0.9 0.6 1.8 I.1 0.8 0.8 0.9 I.1 2.2 1.1 0.6 I.2 0.9
ALT (U/mg protein) 1.9 f 1.7 & I .6 f 3.3 * 3.9 * 3.7 f 3.8 + 4.2 + 3.7 * I .4 f I.5 * 1.4 + 2.2 f 2.1 f 2.4 f
‘Values are significantly different from those of the control group by Student’s f-test (P < 0.05).
0.1 0.6 0.9 1.9 2.3 I..( 2.2 2.4 2.0 0.4 0.2 0.2 I.1 I.2 0.9
ACP (U:g tissue) I.5 2.3 2.7 I.6 3.8 2.9 I.5 2.6 2.7 I.1 2.5 2.8 I.5 3.3 I .6
+ + + f i & * k + + * f f f f
0.4 0.1’ 0.3’ 0.4 0.5* 0.18 0.3 0.1’ 0.2’ 0.2 0.1. 0.2’ 0.1 0.2’ 0.2’
Toxic
Table 2. Lipoperoxide, CwZn SOD and GSH-PX
effects
of chronic
ethanol
ingestion
1005
in the seminal vesicles of controls (A), rats after chronic ethanol ingestion (B) and rats
after ethanol withdrawal (C) Days following treatment 60
120
Lipoperoxide (meq/Kg) A B C A B A B C A B C A
2.1 5.7 5.5 2.5 4.6 4.3 2.1 19.5 18.9 2.4 20.9 22.0 2.7
B C
IO.1 + I.21 3.0 f 0.5
C 180
240
300
& f f f f + f f f + f f +
0.7 0.6’ 0.1; 0.3 0.5’ 0.2’ 0.6 1.4’ 1.7* 0.7 2.4’ 2.5’ 0.3
SOD (U/mg protein) 70.8 50.4 59.7 74.8 52.4 51.5 75.6 40.1 39.2 74.9 37.6 39.6 75.3
+ + + f + f + * i & i i k
3.5 1.6; 1.6; 4.9 1.6’ 1.1* 2.0 3.98 2.8* 2.2 1.8; 1.9* 2.9
34.3 & 1.6: 96.3 f 1.5*
GSH-PX
(U/mg 1.6 I.1 0.8 0.9 0.9 0.8 0.9 1.0 0.8 0.9 1.2
f + + * * f + f f f k
protein)
0.3 0.2 0.2 0.2
0.1
0.3 0.2 0.3 0.3 0.2 0.3 I.1 + 0.3
1.3 + 0.4 1.2 i 0.3 I.1 + 0.2
‘Values are significantly different from those of the control group by Student’s t-test (P < 0.05).
1973). This SOD enzyme has all characteristics of human SOD (Hsu et al., 1992). Values are presented as means f SEM. Significance of difference was tested by analysis of variance and Student’s multiple t-test (Zar, 1974). Results Chronic ethanol ingestion induced increased ACP activity and lipoperoxide concentrations throughout the experimental period. On the other hand, ethanol withdrawal (days 240-300) led lipoperoxide and ACP values to approach those of the controls (A) at 300 days following I:reatment. This is supported by the data for ACP activities [ 1.5 + 0.1 in the controls (A), 1.6 & 0.2 in withdrawal rats (C) but 3.3 k 0.2 in rats with chronic eth,anol intake (B)] (Tables 1 and 2). No alterations were observed in total protein concentrations and ALT activities in the seminal vesicles of rats treated with chronic ethanol, and no changes were observed in total protein and ALT with ethanol withdrawal (Table 1). Significantly decreased SOD was observed in rats with chronic ethanol ingestion (Table 2). Ethanol withdrawal induced increased SOD activities in seminal vesicles of rats at 300 days following treatment, (Table 2). No alterations were observed in GSH-PX activity in the seminal vesicles of rats with chronic ethanol withdrawal (C) (Table 2). Discussion Enhanced generation of O* - or reduced levels of SOD have been implicated in a wide range of pathological conditions, such as nickel-induced toxicity (Novelli et dzl., 1995a), cataracta formation and ageing, inflammatory tissue necrosis, tumour proliferation and neurodegenerative disorders (Cayuela, 1995). Free-radical mechanisms also appear to be implicated in the toxicity of ethanol in various extrahepatic tissues (Orsilles et al., 1995). Although clinical studies have not yet demonstrated clearly the role of free radicals in the pathogenesis of
ethanol-induced cellular injury, increased lipoperoxide formation in the seminal vesicles of rats after chronic ethanol ingestion was observed in the present study (Table 2). Oxygen-derived species such as superoxide radical (Ox’-) are produced in mammalian cells during normal aerobic metabolism. Excess generation of these reactive oxygen species ‘in uivo’ results in lipid peroxidation (Novelli et al., 1994). Some studies (Mascio et al., 1996) have shown that sexual dysfunction induced by ethanol is the result of lipoperoxide formation, which is a free-radicalmediated chain reaction since once initiated it is self-perpetuating. The length of the propagation chain depends on chain-breaking antioxidant enzymes, such as Cu-Zn SOD (Mascio et al., 1996). There are further consequences related to membrane lipoperoxidation such as increased activities of AP (Table 1). Miller (1993) showed that ACP is increased in damaged seminal vesicles and is an indispensable marker to diagnose prostate disease. Thus, the fact that chronic ethanol ingestion increased ACP activity from its basal level at 60 days reflects seminal vesicle damage (Table 1). The total daily dose of ethanol varied from day to day, depending on ingestion of diet, but no alterations in total protein concentration and ALT activities were observed throughout the experimental period (Table 1). These observations are consistent with Lum (1995) who showed that protein concentrations and ALT activities were not altered in ethanol related disease. It should be noted (Table 2) that SOD activity was decreased at 60 days after chronic ethanol ingestion, while significantly increased lipoperoxide and ACP activities were observed in this period. As SOD activity was decreased, the unscavenged superoxide could result in lipoperoxide formation. The levels of SOD reported (Table 2) are consistent with these statements. At day 240 the levels of SOD were lower in rats following chronic ethanol ingestion (B) and after ethanol withdrawal (C) than in the controls (A). At day 300, after a withdrawal period
E. L. B. Novelli et al.
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(days 24&300), the SOD activities were higher in abstinent rats (C) than in the controls (A). On the other hand, no significant differences in lipoperoxide values were observed between rats subjected to withdrawal (C) and the controls (A), indicating that increased SOD activity scavenged O1 after withdrawal. Lipoperoxide and ACP values were increased throughout the experiment in rats after chronic ethanol ingestion (Tables 1 and 2) indicating that chronic ethanol ingestion induced an initially increased respiratory burst in which oxygen was reduced to O2 - and endogenous SOD activities were overwhelmed to scavenger the vast excess of O2 -. 0~~ and HzO, are known to be cytotoxic to cells causing damage to lipids. GSH-PX catalyses the conversion of H,Oz to water, thus, GSH-PX could reduce tissue injury by removing H202. SOD catalyses the destruction of superoxide radical by dismutation and H,Oz formation. As no changes in GSH-PX activity were observed in rats after chronic ethanol ingestion, while SOD activities were decreased in these animals, it is supposed that O2 _ generated by ethanol is of primary importance in the pathogenesis of seminal vesicle injury. O2 - elicits lipoperoxide formation and induces cell damage before being converted to H,O, by SOD. GSH-PX activity was considered as a means of differentiating between alternative cytotoxic mechanisms. This study allows conclusions to be drawn regarding which reactive oxygen metabolite plays a part in chronic ethanol toxicity. These observations suggest that the superoxide radical has a part in ethanol-related tissue injury. Ethanol withdrawal, which was obtained following a period of 240-300 days, when rats (C) received only reduced Oz - production, decreased tap water, lipoperoxide formation (Table 1) and ACP activities (Table 2) and reduced seminal vesicle damage. These observations are consistent with the hypothesis that Oi- is produced as a mediator of tissue lesions induced by chronic ethanol ingestion. The toxic effects of chronic ethanol ingestion were reversible, since increased SOD was adequate to scavenger superoxide radical formation after a withdrawal period. In conclusion, the toxicity of chronic ethanol ingestion on seminal vesicles involves oxidative reactions, such as ethanol induced lipid peroxidation. Superoxide radical is an important toxic intermediate in the development of chronic ethanol damage. Acknowledgemenfs-This study was supported by FAPESP (Fundacao de Amparo a Pesquisa do Estado de SBo Paulo). The authors thank Mr Guerino S. Bianchi Filho and Mrs Claudete Ezias Grassi for their technical services.
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