Alcohol, Vol. 13, No. 1, pp. 41-45, 1996 Published 1996by ElsevierScienceInc. Printed in the USA. All rights reserved 0741-8329/96 $15.00 + .00 ELSEVIER O741-8329(95)O2OO7-Q
Effect of Red Wine Consumption on Rat Liver Peroxidation P . S I M O N E T T I , .1 G . C E R V A T O , t A. BRUSAMOLINO,* N. PELLEGRINI* A N D B. C E S T A R O t
*Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, and tDipartimento di Chimica e Biochimica Medica, Universitft degli Studi di Milano, Italy R e c e i v e d 14 J u l y 1994; A c c e p t e d 9 J u n e 1995 SIMONETTI, P., G. CERVATO, A. BRUSAMOLINO, P. GATTI, N. PELLEGRINI AND B. CESTARO. Effect ofred wine consumption on rat liverperoxidation. ALCOHOL 13(1) 41-45, 1996. - T o evaluate the role of wine polyphenols and that of alcohol on lipid peroxidation indexes and membrane composition in the liver, 40 Sprague-Dawley rats were fed for 28 days with a commercial AIN-76 diet to which was added one of four different beverages: red wine, alcohol solution, dealcoholated wine, or water. The beverage provided 26°7o of the caloric intake. Peroxidation indexes and antioxidative enzymes wel e determined: no significant differences were detected in catalase and glutathione peroxidase whereas superoxide dismutase was significantly lower in the wine-treated animals (220.3 + 15.4 vs. 342.2 + 43.0 U/mg protein of controls). The following significant differences in hepatic variables were observed: increased ct-tocopherol concentration in the alcohol group (0.17 + 0.02 vs. 0.11 + 0.01 #g/rag protein of controls); increased concentration of cytochrome P450 in the rats given wine (0.75 + 0.06 vs. 0.51 + 0.08 nmol/mg protein of the alcohol group); increased concentration of cytochrome b5 in wine and dealcoholated wine treatment groups (0.30 _ 0.01 vs. 0.23 _+ 0.02 nmol/mg protein of controls). The liver membrane fatty acid composition of the wine and dealcoholated wine groups was similar and showed an increase in the saturated fatty acid percentage and a decrease in the polyunsaturated one. The data presented indicate that the main action of polyphenols seems to be an induction of cytochrome activity and that the modality of red wine administration adopted combined with an adequate diet does not provoke any apparent physiological effect on the animals. Rats Liver Polyphenols
Cytochromes P450 and b5
IN R E C E N T years interest has increased in the antioxidant properties of polyphenolic compounds, a class of substances including flavonoids, anthocyanins, and tannins, which are distributed throughout food sources and ingested in significant amounts in everyday diets (14,19). Some authors have reported that various polyphenols are able to inhibit lipoperoxidation induced in cell cultures or food (7,31). Wine is a typical Mediterranean product that contains a fair quantity of these substances (30) and may have beneficial properties if consumed moderately, as shown in epidemiologic studies (23). The consequences o f chronic heavy alcohol ingestion have been widely investigated in both experimental animals and man, and it is accepted that alcohol provokes tissue injury; one hypothesis implicates free radicals (32). Many experimental studies have been conducted to ascertain the effects of chronic alcohol administration on the main liver antioxidant
substrates and enzymes, but discrepancies exist in the reported hepatic levels of antioxidants like glutathione (GSH) (5,12, 26), c~-tocopherol (28), and enzymes like catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSHPx) (33,34), probably depending on the type o f administration that influences the rate and extent to which alcohol is metabolized. The metabolism of substances like ethanol and polyphenols can lead to modifications, through the generation of reactive oxygen species, in the detoxification systems of the multienzymatic hepatic complex cytochrome P450. Therefore, when evaluating cellular activity it is important to verify whether or not there is any variation in cytochrome P450 concentrations due to substances generated during wine metabolism. Another important index in assessing membrane function is its lipid composition. Ethanol consumption produces changes in the
1 Requests for reprints should be addressed to Dr. Paolo Simonetti, Dpt Food Science & Technology, Via Celoria, 2, 20133 Milano, Italy. 41
SIMONETTI ET AL.
essential fatty acid composition of the phospholipid structure (15), with a subsequent variation in membrane permeability, but it is not clear if polyphenols also have any effect. The present study determined the liver antioxidant status and the variations in the concentration of the detoxifying hepatic multienzymatic system of cytochrome P450 in rats given wine, dealcoholated wine, and 1207o alcohol solution, to evaluate the effect of alcohol and the phenolic compounds of wine. METHOD Forty male Sprague-Dawley rats weighing approximately 160 g were randomly divided into four groups, maintained five in a cage in an air-conditioned room (24 _+ 20C, lights on from 0800 to 2000 h), and fed ad lib for 28 days with a commercial AIN-76 diet (American Institute of Nutrition, 1977) purchased from Laboratori Piccioni (Gessate, Italy); the carbohydrate/lipid/protein ratio was 65/5/20. Diet provided 74% of the daily caloric intake and the beverages provided the remaining 26%. The four groups of rats were watered with a 12070 alcohol solution, red wine (Oltrep6 Barbera, 12070 v/v), dealcoholated red wine with added sucrose, or distilled water with added sucrose. The added sucrose was isocaloric with the alcohol percentage of the red wine and alcohol solution. The animals were weighed twice weekly to monitor weight gain, and after 4 weeks of treatment they were lightly anesthetized with diethyl ether and killed by bleeding from the abdominal aorta. The liver was immediately perfused in situ with a cold 1.15070 KC1 solution and homogenized using a motor-driven Teflon pestle-glass homogenizer system (IKA-Werk model RWl8NR). Peroxidation was monitored as TBARS, both endogenous and after stimulation (2 h with FeSO4/ascorbic acid 10/50 /~M), in microsomal fractions, using Buege and Aust's method (3) with slight modifications (6). SOD, CAT, and GSH-Px activities were determined in the supernatant obtained after centrifugation for 10 rain at 7000 x g of homogenates treated with Triton X-100 (final concentration 0.08070) for 1 h at 4°C (17). SOD activity was measured as inhibition of nitroblue tetrazolium reduction by the xanthine/xanthine oxidase superoxide generating system (37), CAT by monitoring hydrogen peroxide ammonium molybdate complex formation (l 1), and GSH-Px activity by determining N A D P H consumption using hydrogen peroxide as substrate in the presence of reduced GSH and GSH reductase (39). For the cytochrome P450 and b5 determinations each liver homogenate was centrifuged at 10,000 x g at 0-4°C for 25 min (Sorvall model RC-B5 centrifuge). The floating fat layer was carefully aspirated, and the underlying supernatant fraction decanted and centrifuged at 105,000 x g at 0-4°C for 1 h (Beckman model L5-50 ultracentrifuge). The resulting microsomal pellet was resuspended in fresh 1.15070 KC1 solution and centrifuged at 105,000 x g for 1 h at 0-4°C. The washed microsomes were resuspended in KCI using the above homogenizer, and the protein content was determined (22). Microsomal preparations were diluted to a protein concentration of 2.0 mg/ml with 0.1 M phosphate buffer, pH 7.0, and the cytochrome P450 concentration was determined by the Omura and Sato method (29) partially modified (8). Oxidized GSH (GSSX; X = any thiol) and total GSH (GSH + GSSX) were determined (27). Total GSH was measured immediately in liver homogenates by reducing all disulfides with dithiothreitol in a prederivatization step and precipitating the proteins with 5-sulfosalicylic acid. GSSX was measured by eliminating GSH with N-ethylmaleimide, reduc-
TABLE 1 BASAL AND STIMULATEDTHIOBARBITURIC ACID REACTIVE SUBSTANCES(TBARS) BasalTBARS StimulatedTBARS (nmol/mg protein) (nmol/mgprotein)
Treatment Deaicoholated wine Alcohol solution Wine Control
0.05 0.16 0.15 0.13
± ± ± ±
0.02 0.07 0.06 0.05
6.14 7.57 8.93 9.76
+ ± ± +
1.00 2.69 2.86 3.52
Values are mean ± SE of 10 rats. ing the disulfides with dithiothreitol, and precipitating the proteins with 5-sulfosalicylic acid. GSH was then reacted with orthophthalaldehyde to form a stable adduct that was separated and quantitated by high performance liquid chromatography (HPLC). Protein content was estimated by the method of Lowry et al. (22) using bovine serum albumin (BSA) as reference standard. Fat-soluble vitamins were extracted from liver homogenares (4) and measured by HPLC (37). Fatty acid composition of liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined in liver homogenates after chloroform/methanol (2/1, v/v) extraction (9). The lipid extract was completely dried under nitrogen and then redissolved in 50/~1 chloroform for thin-layer chromatography separation of PE and PC (10). The phospholipid bands were visualized by exposure to iodine vapor and the PE and PC bands were scraped. Fatty acid methylation was performed (21) and the methyl acids were separated by the gas-liquid chromatography (Varian 3300) procedure of the Association of Official Analytical Chemists (1).
Statistical Analysis The significance of differences among the groups was evaluated using analysis of variance (ANOVA), t-test, and MannWhitney test as appropriate. RESULTS
Growth Performance No significant differences in food intake were observed among the four groups (mean intake was 18.0 + 0.4 g/day). When the consumption of beverages was considered, that of the alcoholic solution was significantly lower than the others (34.2 + 0.8 ml/day vs. 38.1 + 0.8 ml/day); consequently,
TABLE 2 SUPEROXIDE DISMUTASE(SOD), CATALASE(CAT), AND GLUTATHIONE PEROXIDASE (GSH-Px) ACTIVITIES Treatment
SOD (U/mg protein)
Dealcoholated wine Alcohol solution Wine Control
298.2 + 322.9 + 220.3 + 342.2 +
31.1 38.7 15.4" 43.0
CAT (U/mgprotein) 1072.1 + 1242.8 + 1052.3 + 1227.2 +
108.0 21.7 + 2.8 146.4 19.0 + 2.3 75.7 20.6 + 2.03 138.6 25.1 + 3.0
Values are mean + SE of 10 rats. *p < 0.05 vs. other groups (Mann-Whitney test).
RED WINE EFFECTS ON LIVER PEROXIDATION
compared with the wine-treated animals and controls. Hepatic t~-tocopherol was significantly influenced by alcohol consumption; it was higher in the wine and alcohol groups than the other two, with a significant difference between the rats given wine and the controls (p < 0.05).
TABLE 3 HEPATIC MICROSOMALCONCENTRATION OF CYTOCHROMESP450 AND b5 CytochromeP450 Cytochromeb5 (nmol/mgprotein) (nmol/mgprotein)
Treatment Dealcoholated wine Alcohol solution Wine Control
0.68 0.51 0.75 0.63
± ± ± ±
0.04 0.08 0.06* 0.08
0.30 0.29 0.31 0.23
± + + ±
Fatty Acid Composition of Liver PE and PC
0.0H" 0.04 0.01t" 0.02
Values are mean ± SE of 10 rats. *p < 0.05 vs. alcohol solution-treated group. "~p < 0.05 vs. control.
the weight gain of the alcohol group was lower (148 _+ 3 g/30 days) compared with the other groups (168 +_ 8 g/30 days), although the difference was not significant.
Lipid Peroxidation and Antioxidant Enzymes Peroxidation evaluated as basal and stimulated TBARS did not show any significant differences among the groups, although both these indexes were markedly decreased in the dealcoholated wine group (Table 1). This could be due to the high intragroup variability. Antioxidant enzymatic activities are reported in Table 2. No significant differences were observed in CAT and GSH-Px activities among the four groups; with regard to SOD activity the t-test showed no significant differences whereas the M a n n Whitney test revealed a significant decrease in the wine-treated animals vs. all the other groups.
Cytochrome P450 and b5 Activities in Liver Microsomes The values observed in the different groups are reported in Table 3. Cytochrome P450 concentration was significantly higher in the wine than in the alcohol group (p < 0.05) whereas that of cytochrome b5 was higher in all three treated groups compared with controls, significantly so in the animals given wine and dealcoholated wine (p < 0.05).
GSH and Antioxidant Vitamin Status No significant differences were detected in GSH and GSSX among the four groups of animals (Table 4), although, interestingly, the GSSX values were higher in the treated groups than the controls. Hepatic retinol was lower in the dealcoholated wine group
~-Saturated fatty acid (~-SFA), r--monounsaturated fatty acid (~-MUFA), and ~-polyunsaturated fatty acid (~-PUFA) of liver PE and PC are reported in Table 5. Statistical analysis of the percent fatty acid composition of PE revealed that r.-MUFA concentration was significantly higher (p < 0.05) in the animals treated with alcohol or wine with respect to the controls. There was a counterbalancing significant decrease of r~-PUFA in the wine-trcated rats and of v.-SFA in those given alcohol. Administration of wine, dealcoholated or not, had a significant and opposite effect to that of alcohol on the percentages of ]:-SFA and v~-PUFA in PC; compared with the controls, the wine and dealcoholated wine groups showed an increase of ~-SFA and decrease of r.-PUFA whereas a decrease of the former and increase of the latter was observed in animals given alcohol. DISCUSSION Diet acceptability, assessed by measuring daily consumption, was not affected by the inclusion of red wine, dealcoholated wine, or alcohol solution; however, beverage intake was lowest in the group given alcohol, in which growth performance was, consequently, the worst. The various treatments had no significant effect on the peroxidative status of hepatic microsomes. Interestingly, stimulated and particularly basal TBARS were decreased, although not significantly in the dealcoholated wine group. This is probably due to the strong antioxidative power of some organoleptic components of wine, which is particularly evident in the absence of alcohol. Of the antioxidative enzymes, CAT and GSH-Px activities were not significantly different in the various groups whereas SOD activity was significantly lower in the wine-treated rats than in the others. In our experimental conditions, we observed an increased P450 concentration in the wine-treated group. Wine contains "workload" alcohol and flavonoids, which are detoxified by P450. This could cause an increase for SOD and result in wastage of the enzyme. On the other hand, antioxidant enzymes are reported to be inducible by free radicals (16) and in this case free radical formation could be insufficient for SOD induction or the mechanism of H202 inhibition of SOD (36) could be implicated.
TABLE 4 HEPATIC CONCENTRATIONOF a-TOCOPHEROL, RETINOL, GLUTATHIONE(GSH), AND OXIDIZEDGLUTATHIONE(GSSX) Treatment Dealcoholatedwine Alcoholsolution Wine Control
Retinol (ttg/mgprotein) 0.23 0.26 0.29 0.27
+ 0.01 + 0.03 +_ 0.02 ± 0.02
~-Tocopherol GSH GSSX 0zg/mgprotein) (nmol/mgprotein) (nmol/mgprotein) 0.12 0.17 0.15 0.11
+ ± ± ±
0.01 0.02* 0.02 0.01
16.78 + 1.93 15.22 + 1.02 13.56 _+ 1.16 16.09 ± 1.09
Values are mean + SE of 10 rats. *p < 0.05 vs. control and dealcoholated wine-treated groups.
1.70 =l=0.22 1.90 ± 0.50 1.89 ± 0.36 1.04 ± 0.16
S I M O N E T T I E T AL. TABLE 5 PERCENT FATTY ACID COMPOSITION OF HEPATIC PHOSPHATIDYLEHANOLAMINE (PE) AND PHOSPHATIDYLCHOLINE (PC) Variables
Dealcoholated Alcohol Solution
PE E-SFA E-MUFA ~-PUFA ~-w9 ~-o:7 E-w6 r,-o:3
45.3 7.6 47.4 4.4 3.2 36.1 6.6
_+ 1.9" ± 0.4 ± 2.0 ± 0.3~ ± 0.2 ± 1.3 ± 0.6
41.4 8.2 50.4 4.3 4.0 37.0 7.5
_+ 0.7 ± OAt ± 0.8 ± 0.1~ ± 0.4 _+ 0.7 _+ 0.2
45.0 9.0 46.0 4.7 4.3 34.7 6.6
± 1.5" ± 0.7~: ± 1.8*t ± 0.4~ ± 0.4t +_ 1.3t ± 0.4
43.2 6.3 50.5 3.3 2.9 38.3 7.1
± 0.5 ± 0.6 _+ 0.6 ± 0.1 ± 0.5 _+ 0.4 ± 0.1
PC Z-SFA 2-MUFA Z-PUFA r~-o~9 ~-w7 Z-o~6 ~-o:3
52.2 10.2 37.6 5.7 4.5 33.5 2.3
± ± ± + ± ± +
44.2 10,8 45,2 5.1 5,7 39,4 2,9
± 0.4§ ± 0.8 +_ 1.0 ± 0.4 ± 0.5~§ ± 1.1 ± 0.1
47.4 11.5 41.1 5.6 5.9 36.0 2.7
± 1.9 ± 0.4~ ± 2.1 ± 0.3 ± 0.4~§ _+ 1.8 ± 0.2
47.3 9.0 43,7 5.2 3.8 36,6 2.5
± ± + ± ± ± ±
3.4 0.5 3.5* 0.3 0.4 2.9* 0.4
1.5 0.8 1.8 0.4 0.4 1.4 0.2
Values are mean _+ SE of l0 rats. *p < 0.05 vs. alcohol solution. tP < 0.05 vs. control. ~:p < 0.01 vs. control. §p < 0.01 vs. dealcoholated wine.
The lack of evident peroxidative damage attributable to the different treatments is confirmed by the hepatic G S H content, which was similar in the four groups. This result accords with those of other authors (25) who have proposed a mechanism by which chronic ethanol administration increases liver G S H turnover, probably due to increased cellular G S H requirements; the subsequently enhanced activities o f the G S H synthesizing enzyme are then followed by metabolic adaptation. In our experiment the alcoholic beverage was administered with a solid diet to shorten the time required for metabolic adaptation. Some reports have described an interaction between ethanol and retinol (20,35); in general, chronic ethanol intake modifies the hepatic concentration of retinol and, subsequently, changes in the plasma levels. In our study we did not detect any significant variations in liver retinol levels attributable to the presence of alcohol whereas hepatic a-tocopherol levels were significantly increased in both the wine and the alcohol group. Data in the literature are conflicting: some authors observed no ethanol effect on liver vitamin E content in rats receiving adequate dietary vitamin E (18), whereas others found it increased 03). Thus, it appears that the mechanism of chronic ethanol effects on the a-tocopherol level in the liver is still uncertain, although it is known to depend largely on the dose of ethanol administered and the vitamin E content o f the diet (2,28). We did not observe any influence, positive or negative, o f wine phenolic compounds on vitamin status, and the main effect seemed due to ethanol content. Analysis o f the hepatic status of membrane phospholipids and their acid composition revealed some significant differences attributable to the different beverages. In our opinion, the most important finding was the rise o f the monounsaturated fraction in the wine-treated group; the main fatty acids involved were 16 : 1 and 18 : I of the w7 series o f both PE and PC. The relation between these fatty acids and alcohol
consumption is strengthened by some literature data demonstrating a close, positive association between alcohol consumption and the concentrations of some monounsaturated fatty acids (24). In our study alcohol solution and dealcoholated wine induced a similar trend, although statistical significance was not reached. The increase o f the monounsaturated fatty acids fraction was counterbalanced, in the alcoholtreated group, by a nonsignificant decrease of the saturated fraction, whereas there was a decrease of the polyunsaturated fraction of both PE and P C in the animals treated with wine and an even greater one in those given dealcoholated wine. Detailed analysis of the individual series of fatty acids allowed us to distinguish further between the wine and dealcoholated wine treatments. The latter resulted in a reduction of the polyunsaturated fraction due to a decrease o f some long chain fatty acids (20 : 4o~6, 22 : 40;6, and 22 : 6o~3). The wine treatment caused a similar decrease in the ~06 fraction (including a significant reduction of 20 : 4oJ6 o f PE), which was not accompanied by any differences in the ~3 series. Finally, it is important that the various treatments did not have an apparent physiological effect on the animals. We believe that the method of administering alcohol and the time required to metabolize it are fundamental. If administration is constant over the whole study period, as in our experiment, a continuous metabolic washout may be hypothesized, which prevents harmful alcohol concentrations being reached in the organism. The effect of concomitant administration o f a balanced diet supplying an adequate quantity of the ant±oxidative factors considered must not be underestimated. ACKNOWLEDGEMENT This work was supported by C.N.R. grant (Target project "Prevention and Control Disease Factors" subproject "Nutrition" contr. No. 93.00696.PF41).
RED WINE EFFECTS ON LIVER PEROXIDATION
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