Department of Medicine, Memorial-Sloan Kettering Cancer Center,. Cornell University Medical ..... Shaw, S.; Jayakilleke, E.; Ross, W. A.; Gordon, E. R.; Lieber,.
Alcohol,Vol. 12, No. 1, pp. 43-47, 1995 Copyright©1995ElsevierScienceLtd Primed in the USA.All rightsreserved 0741-8329/95 $9.50 + .00
Pergamon 0741-8329(94)00068-9
Acute Ethanol Exposure Alters Hepatic Glutathione Metabolism in Riboflavin Deficiency PURABI DUTTA, l JENNIFER SEIRAFI, DIANE HALPIN, JOHN PINTO AND RICHARD RIVLIN
Department o f Medicine, Memorial-Sloan Kettering Cancer Center, Cornell University Medical College, New York, N Y 10021 Received 11 M a r c h 1994; A c c e p t e d 5 A u g u s t 1994 DUTTA, P., J. SEIRAFI, D. HALPIN, J. PINTO AND R. RIVLIN. Acute ethanol exposure alters hepatic glutathione metabolism in riboflavin deficiency. ALCOHOL 12(1) 43-47, 1995.-Since acute ethanol consumption and riboflavin deficiency each induces oxidative stress within tissues, we examined whether their combined effects compromise the major antioxidative system in liver, namely, reduced glutathione (GSH) metabolism. Four hours before sacrifice, half the riboflavindeficient (RD) and riboflavin-sufficient (RS) rats were treated with ethanol (3 g/kg). Livers were excised and analyzed for GSH and enzymes that control its metabofism. In RD rats, GSH increased while glucose-6-phosphate dehydrogenase (G6PD) activity decreased. El:hanoi had no effect on these measurements in RS rats. In RD rats, ethanol administration decreased GSH along with the activities of GSH peroxidase, glutathione reductase, and G6PD. These data suggest that riboflavin deficiency alone does not compromise hepatic GSH metabolism. By contrast, ethanol consumption together with riboflavin deficiency depletes hepatic GSH, blunts enzyme activities controlling GSH metabolism and may enhance alcohol-induced liver injury. Ethanol
Glutathi~one
Riboflavin deficiency
Alcohol
A L T H O U G H the pathogenesis of alcoholic liver disease is not completely understood, an important factor contributing to hepatic injury may be the result of enhanced peroxidative stress (4,23). Lipid peroxidation has long been implicated in alcohol-induced liver injury (5,13). Acute ethanol ingestion has been reported to produce a marked decrease in hepatic reduced glutathione (GSH) concentration (15,24). Reduced glutathione (GSH) plays an important role in antioxidative defense and detoxification of a variety o f xenobiotic substances. Some studies suggest that depletion of GSH following acute ethanol intoxication is a result of increased lipid peroxidation 02,27). By contrast, other studies have produced conflicting results regarding tile effects of chronic ethanol consumption and the steady-state content of hepatic GSH (8,10). The metabolism of reduced glutathione is markedly influenced by deficiency of riboflavin (vitamin B2). The physiological significance of ribofla,~in resides in the form of the coenzyme, FAD, which is required by glutathione reductase for regenerating reduced GSH! from its oxidized form (GSSG)
Liver
Glucose-6-phosphate dehydrogenase
(21). We have previously shown that during riboflavin deficiency, despite depressed erythrocyte glutathione reductase activity, levels of GSH in erythrocytes and in liver are significantly elevated (6). One possible mechanism for the increased GSH level observed during riboflavin deficiency may be the activation of enzyme(s) responsible for its de novo biosynthesis from its amino acid precursors (6). The purpose of this study is to determine whether riboflavin deficiency exacerbates the effects of acute alcohol administration on GSH metabolism in liver. MATERIALS AND METHODS
Diets and Treatments of Animals Diets in this study were prepared by Dyets, Inc. (Bethlehem, PA). Three-week-old male Holtzman rats (Holtzman Co., Madison, WI) were divided into two experimental groups, designated control and riboflavin-deficient and were housed individually in metabolic cages. Animals in the ribofla-
Requests for reprints should be addressed to: Purabi Dutta, Ph.D., Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 140, New York, NY 10021. 43
44 vin-deficient group were fed a diet that was analyzed in our laboratory and found to contain < 1 ppm of riboflavin. Control animals were pair-fed a diet identical in composition but supplemented with 8.5 mg riboflavin/kg diet. This level represents three times the Recommended Dietary Allowance (RDA) for riboflavin in the rat and approximately 4-5 times that needed to achieve maximum growth of young male rats. After 20-40 weeks of feeding on these diets, half the number of rats from each dietary group received intraperitoneal injections of ethanol (EtOH) (3g/kg body weight) while the other half received an equivalent volume of saline. At 4-h postinjection, rats were anesthetized by exposing them to solid carbon dioxide. Blood samples were drawn by cardiac puncture into tubes containing EDTA as an anticoagulant. Livers were excised and analyzed for levels of GSH and activities of selective enzymes responsible for GSH metabolism.
GSH Concentration GSH was measured by the method of Beutler (2). Briefly, the livers were homogenized at 4°C in 10 vol. of water and centrifuged at 100 000 x g for 15 min. A n aliquot of the supernatant fraction was added to a precipitation solution containing metaphosphoric acid, EDTA and NaCI. After standing for 5 min, the mixture was centrifuged at 5000 x g for 10 min, and an aliquot of the supernatant fraction was added to 0.3 M Na2HPO4. An initial absorbance reading was measured at 412 nm followed by a second reading at the same wavelength following the addition of 5,5' dithiobis-2nitrobenzoic acid. The final concentration of GSH is reported as nanomoles per milligram protein.
Glutathione Reductase Activity and Activity Coefficient (GRAC) The basal activity of glutathione reductase (GSSGR) and the activity coefficient (GRAC) were determined in both erythrocyte lysates and the supernatant fraction of liver homogenates (1). The GRAC is the ratio of the activity in the presence of exogenous FAD in vitro to the basal activity. The GRAC when measured in erythrocytes is a marker for riboflavin nutritional status. We define advanced riboflavin deficiency in our rat model as those animals that have an erythrocyte GRAC >_ 1.7. The basal activity of GSSGR is expressed as milliunits per milligram protein. One milliunit of reductase activity represents the amount of enzyme that oxidizes 1 nmol of N A D P H per minute.
Glutathione Peroxidase Glutathione peroxidase (GSH-Px) activity in erythrocytes and liver was determined by using the method of Paglia and Valentine (16). This method takes advantage of the capability of the flavoenzyme, glutathione reductase, to replenish GSH from GSSG using NADPH. Aliquots from hemolysates or homogenates were added to an assay solution containing N A D P H , sodium azide (catalase inhibitor), GSH and yeast glutathione reductase. The reaction was initiated by addition of H202 to the reaction mixture and the oxidation of N A D P H monitored by recording the decrease in absorbance at 340 nm. One miUiunit of peroxidase activity is defined as the amount of enzyme that oxidizes 1 nmol of N A D P H per minute. The activity of glutathione peroxidase was expressed as milliunits per mg protein.
DUTTA ET AL.
Glucose-6-Phosphate Dehydrogenase Glucose-6-phosphate dehydrogenase (G-6-PD) activity was assayed spectrophotometrically at 340 nm by measuring the rate of formation of N A D P H in the presence of glucose-6phosphate (14).
Protein Assay Protein concentration was determined by incubating diluted protein samples with BCA reagent at 370C for 30 rain. The spectrophotometric quantitation of protein was conducted by determining the absorbance at 562 nm (25).
Statistical Analysis For statistical analysis, we used Student's t-test for paired analysis to compare results in samples from riboflavindeficient and control animals. A p < 0.05 was considered significant. All results were shown as the mean ± SEM. A two-way analysis of variance was also conducted to determine the effect of diets (riboflavin-sufficient or riboflavindeficient), drug (EtOH or saline), and their interaction. RESULTS
Body Weights and Liver Weights Body weights (317 + 43 vs. 362 + 49 g; p < 0.05) of riboflavin-deficient rats were significantly lower than those of pair-fed control rats. There was no difference in the weights of liver between these two groups (10 +_ 1.0 vs. l0 + 1.8 g).
GSH Concentration Rats fed a riboflavin-deficient diet had significantly higher GSH levels in liver as compared to that in riboflavin-sufficient rats (Fig. 1). Rats fed a riboflavin-sufficient diet and treated with EtOH did not show diminished hepatic glutathione content. By contrast, when EtOH was administered to riboflavindeficient rats, the hepatic GSH level was markedly reduced. The concentration of GSH in EtOH-treated riboflavindeficient rats was significantly lower than that in riboflavindeficient rats treated with saline.
GSSGR GSSGR activity in the liver was not significantly changed by either riboflavin deficiency or EtOH treatment alone (Table 1). However, when riboflavin-deficient rats were subjected to EtOH, hepatic GSSGR of these animals was significantly reduced when compared to values obtained from riboflavin sufficient rats not treated with EtOH.
GSH-Px Table 1 shows that riboflavin deficiency alone does not alter the activity of GSH-Px in liver. In riboflavin-sufficient rats, administration of EtOH did not significantly alter GSH-Px activity. However, when EtOH was administered to riboflavin-deficient animals, GSH-Px activity was significantly diminished as compared to values obtained from rats that were not treated with EtOH, regardless o f their riboflavin status.
G-6-PD In riboflavin-deficient rats the activity of G-6-PD was reduced (Table 1). Ethanol treatment did not result in any
ETHANOL, RIBOFLAVIN, AND GSH METABOLISM
45
GLUTATHIONE
150'
I~lmj pFc Sal]ne RDD Saline OH tOH
C a.
O) E W _o 0 E
100'
50'
TREATMENT FIG. 1. Effects of riboflavin deficiency and alcohol treatment upon reduced glutathione concentration in rat liver. Animal treatments and detailed methods are given in the text. Data represent means + SEM of six independent determinations. Means with common superscripts are not significantly different at the < 0.05 level. Means without common superscripts are significantly different from one anot]~er at the < 0.05 level. Abbreviations used for treatments are as follows: PFC = pairfed control diet; RDD = riboflavin deficient diet; PFC EtOH = palrfed control diet + alcohol treatment; RI')D EtOH = riboflavin deficient diet + alcohol treatment.
change in the activity of this hepatic enzyme in riboflavinsufficient rats. However, 'when EtOH was administered to riboflavin-deficient rats, the activity of G-6-PD was further reduced. In fact, in riboflavin-deficient rats treated with EtOH the activity of this enzyme was only 17% of the value obtained from rats which were riboflavin-sufficient and not treated with alcohol. Among two EtOH-treated groups, hepatic G-6-PD activity was four times greater in the riboflavinsufficient groups than that in riboflavin-deficient animals. Analysis of variance indicated that both riboflavin deficiency and EtOH treatment significantly reduced the activity of G6-PD in liver. DISCUSSION
Alcohol increases lipid peroxidation (13) and to protect against this stress, the demand for hepatic GSH may be increased. Since the FAD-dependent enzyme, glutathione reductase, regenerates GSH from its oxidized form (GSSG), we inquired whether riboflavin deficiency exacerbates effects of acute EtOH treatment on GSH metabolism in liver. Results of this study confirmed that unlike the activity in
erythrocytes (6), liver GSSGR is not significantly depressed during riboflavin deficiency. By contrast, the hepatic level of GSH is significantly elevated in this condition. Although it may seem paradoxical that GSH levels are higher in livers obtained from riboflavin-deficient animals as compared to that from riboflavin-sufficient controls, we have previously observed similar findings (6,7). One possible mechanism for the increased GSH level observed in tissues during riboflavin deficiency may be the activation of enzyme(s) involved in de novo biosynthesis of GSH. Under normal conditions a significant portion of GSH is generated through the reduction of oxidized GSH by GSSGR and the remaining GSH is produced by the biosynthetic pathway (17). However, during oxidative stress when the demand for GSH increases, or in riboflavin deficiency when GSSGR activity is limited, the pathway of GSH regeneration through de novo biosynthesis becomes more prominent. This is especially true for liver, which can generate cysteine from methionine for GSH biosynthesis and can readily export GSH for supplying extrahepatic tissues (11). The enzyme, gamma glutamylcysteine synthetase, is recognized as the rate limiting enzyme for GSH synthesis and it is known to be regulated by the
TABLE 1 ACTIVITIES OF H E P A T I C ENZYMES IN A L C O H O L TREATED-CONTROL AND RIBOFLAVIN DEFICIENT RATS
GSSG-R (Units/mg) GSH-Px (mUnit/mg) G6PD(Units/mg)
PFC Saline
RDD Saline
PFC EtOH
RDD EtOH
19.9 + 2.7* 340 + 80*,t 30.1 ± 3.4*
15.0 + 2.2",t 430 + 80* 11.7 ± 3.0
19.1 + 3.7",t 370 ± 40* 21.9 + 3.4*
11.8 + 2.2t 210 + 40t 5.3 + 1.17
Hepatic enzyme activities involved in giutathione metabolism in riboflavin deficiency and after alcohol ~Lreatment. Animal treatments and detailed methods are given in the text. Data represent means :t: SEM of six independent determinations. Means with common superscripts are not significantly different at the < 0.05 level. Means without common superscripts are significantly different from one another at the < 0.05 level. Abbreviations used for treatments are as follows: PFC = palrfed control diet; RDD = riboflavin deficient diet; PFC EtOH = pairfed control diet + alcohol treatment; RDD EtOH = riboflavin deficient diet + alcohol treatment.
DUTTA ET AL. concentration of GSH (20). During riboflavin deficiency the activity of this enzyme may be stimulated to maintain the level of GSH in the normal range (6). However, when an additional oxidative stress is imposed, such as administration of EtOH in combination with riboflavin deficiency, the net effect may overwhelm the compensatory mechanism for meeting the excess demand of GSH. This hypothesis is supported by the finding that although hepatic GSH levels were significantly higher in riboflavin-deficient rats than controls, alcohol treatment to riboflavin-deficient animals drastically reduced their GSH level. By contrast, when animals were riboflavinsufficient, acute EtOH treatment did not significantly change GSH levels in liver. In similar fashion, the activity of GSH-Px, which utilizes GSH as a substrate, also remains unaffected by alcohol treatment in riboflavin-sufficient rats. Riboflavin deficiency by itself does not compromize GSH-Px activity. However, when EtOH was administered to riboflavindeficient animals, GSH-Px activity was significantly diminished as compared to the enzyme activity in either riboflavindeficient rats or control rats. Riboflavin deficiency is one of the major nutrient deficiencies imposed by alcohol (3,22). Our laboratory has previously shown (19) that one mechanism of alcohol-induced riboflavin deficiency is the decreased bioavailability. Alcohol interferes with the digestion and absorption of food sources of riboflavin. Since alcohol induces riboflavin deficiency, the influence of alcohol on riboflavin-deficient subjects could be potentially serious, as riboflavin is required for generating GSH that plays an important role in antioxidative defense especially during alcohol-induced generatation of reactive oxygen species. The effect of riboflavin deficiency on the metabolism of alcohol is not known as we did not measure blood level of alcohol or acetaldehyde in this study. Previous studies by others have shown that a 4-week feeding of a riboflavin-deficient or a riboflavin-supplemented diet to rats on voluntary intake of ethanol did not significantly influence either the amount of
ethanol intake, blood acetaldehyde level, or ethanol elimination rate (18). Maintaining adequate levels of GSH requires NADPH generated by G-6-PD. To convert oxidized GSSG back to its reduced state, GSSGR requires N A D P H as reducing equivalents. G-6-PD activity is reported to be diminished during riboflavin deficiency (26). Our study extends this observation that not only does riboflavin deficiency diminish G-6-PD activity in liver, but EtOH consumption during riboflavin deficiency exerts a synergistic effect on inhibiting G-6-PD activity. The availability of N A D P H in livers of alcohol-treated riboflavin-deficient rats may be diminished by the inactivation of G-6-PD and increased utilization of this coenzyme through induction of cytochrome P450 2El by ethanol (9). Thus, the diminished availability of N A D P H combined with the decreased basal activity of glutathione reductase would result in marked reduction in the level of hepatic GSH in these animals. Compromised levels of GSH eventually would result in diminished GSH-Px activity, as shown in this study. Decreased hepatic concentration of GSH and decreased activity of GSH-Px may have significant deleterious effects on the hepatic defense system against oxidants and xenobiotic substances. Thus, in its entirety, our study shows that EtOH consumption in association with riboflavin deficiency may be especially detrimental to the reducing capacity of liver and eventually may lead to enhanced risk for liver toxicity induced by alcohol. ACKNOWLEDGEMENTS This work was supported by the Clinical Nutrition Research Unit Grant CA 29502 and CA 08748 from National Institutes of Health, and by grants from National Institute of Aging 5 KOI AG00399, Hoffmann-La Roche, Inc., CPC-Best Foods, Inc., and The Stella and Charles Guttman Foundation. The research was performed in the Nutrition Research Laboratory of Sloan-Kettering Institute. Purabi Dutta is a recipient of Research Scientist Development Award (K01 AG00399) from the Institute of Aging.
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