0145-6008/92/16014001$3.00/0 ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH
Vol. 16, No. 1 January/Febmary 1992
Comparative Effects of Chronic Ethanol Consumption on the Properties of Mitochondria from Rat Brain and Liver William S. Thayer and Hagai Rottenberg
The effects of chronic alcohol consumption on biochemical proper-
Ues of mitochondria isolated from liver and brain were compared in rats. As has been found in previous studies (reviewed in Thayer WS: Ann NY Acad Sci 492193-206, 1987) in liver, ethanol consumption M to a 41% decrease in active phosphorylating (state 3) respiration and a 25% decrease in resting (state 4) respiration. These changes resulted in a 23% decrease in the respiratory control ratio (ratio of respiration rate in state 3 to that in state 4). These effects were associated with a 40% decrease in functional cytochrome oxidase content, determined spectrophotometrically as heme aa3. By contrast, in brain mitochondria isolated from the same rats, ethanol consumption did not result in any significant changes in respiration rates, respiratory control ratio, or cytochrome contents. The findings demonstrate a differential pathobiologic response of brain and liver mitochondria to chronic ethanol consumption. Since the liver is predominant in metabolism of ingested ethanol, the findings of this study suggest that the deleterious effects of chronic alcohol consumption on the structure and function of liver mitochondria may be related to ethanol metabolism. Key Words: Brain, Ethanol, Liver, Mitochondria, Cytochromes.
HRONIC CONSUMPTION of ethanol produces characteristicalterations in the protein and lipid composition and biochemical activities of subcellular organelles of the liver.' Prominent among these effects is a substantial decrease in the rates of respiration and ATP synthesis displayed by mitochondria isolated from livers of ethanol-fed rats as compared with pair-fed controls. The diminution of respiration by ethanol-feeding is largest, typically on the order of 25% to 40%, with NADlinked substrates which donate reducing equivalents at site I of the electron transport chain.2 Studies of the molecular basis for this ethanol effect have identified decreases in the active contents of specific respiratory chain proteins. The active or functional contents of cytochromes aa3 and b,3 A T p a ~ e , ~and , ~ iron-sulfur clusters of NADHdehyrogenase6are decreased by values ranging from 50% to 20%, respectively, per unit total membrane protein after ethanol-feeding. The precise mechanisms by which such alterations in the protein composition of the mitochondrial membrane come about remain to be elucidated.
C
From the departments of Biological Chemistry and Pathology, Hahnemann University, School of Medicine, Philadelphia, Pennsylvania. Received for publication April 8, 1991; accepted August 1, 1991 This work was supported in part by Research Scientist Development Award AA00087 from the National Institute on Alcohol Abuse and Alcoholism to W.S.T. and by Grants AA07238 and GM28173 to H.R. Reprint requests: William S. Thayer, Ph.D., Hahnemann University, MS #411, Broad & Vine Streets, Philadelphia, PA 19102. Copyright 0 1992 by The Research Society on Alcoholism. AIcohoICIin Exp Res, Vol 16, No 1, 1992: pp 1-4
However, the observation that these enzymatic changes occur has been widely reported'-" and can be regarded as an established consequence of chronic ethanol consumption. In this communication, we report the results of comparative studies conducted with both liver and brain mitochondria isolated from ethanol-fed and control rats. Our data indicate that brain mitochondria do not exhibit any decrease in respiratory activity or alteration in cytochrome contents after a period of chronic ethanol consumption that is sufficient to induce the characteristic mitochondria1 changes in the liver. The findings demonstrate organ specificity in the effects of ethanol on subcellular biochemistry and may provide insight concerning factors underlying the pathophysiologic actions of ethanol. MATERIALS AND METHODS Animals Male, Sprague-Dawley littermate rats were pair-fed a totally liquid diet" (Bio-Serv, Inc., Frenchtown, NJ) containing ethanol as 36% of calories for 35 to 40 days as previously described? The nutrient composition of the diet included 16% protein, 35% fat, and the remainder carbohydrate. In the control diet, carbohydrate replaced ethanol isocalorically.
Preparations Rat liver mitochondria were isolated by conventional differential centrifugation of a homogenate prepared in 0.25 M sucrose and 1 mM EDTA, pH 7.0, as previous described." Rat brain mitochondria were isolated by a Ficoll gradient method."
Analytical Procedures Respiration was measured polarographicallywith an oxygen electrode at 25". For liver mitochondria, the medium contained 0.2 M sucrose, 50 mM KCl, 10 mM Tris-HCI, 2 mM Na'P,, and 2 mM MgCI2at pH 7.4. For brain mitochondria, the medium contained 0.2 M sucrose, 100 mM KC1, 5 mM Na+HEPES, 5 mM Na+P,, and 5 mM MgClz at pH 7.5. In both cases, 5 mM glutamate plus 5 mM malate were present as substrates. Mitochondria1protein concentration, determined by the biuret method, was 1.O to 1.5 mg/ml in all assays. Respiratory control ratio (RCR) was determined as the respiration rate in the presence of ADP (state 3) divided by the respiration rate after phosphorylation of ADP to ATP (state 4) according to established procedure^.'^ For determination of cytochrome contents, mitochondria were diluted to 0.5 to 1.5 mg protein/ml in 0.25 M sucrose, 10 mM Na+HEPES, and 5 mM MgClz (pH 7.5) and portioned equally into two cuvettes. A small amount of sodium dithionite was added to one cuvette. Reduced minus oxidized difference spectra were then recorded using an Aminco DW-2A spectrophotometer. Cytochrome contents were determined from the characteristic absorb-
2
THAYER AND ROTTENBERG
ances of the hemes as previously de~cribed.~ In brief, a baseline was drawn between the isosbestic points at 535,511, and 630 nm. Absorbance values at peak wavelengths were then analyzed using extinction coefficient values as follows: cytochrome aag, C605 = 24.0 mM-’cm-’; cytochromes b, 6563 = 23.4 mM-lcrn-’; cytochromes c + cI, €552 = 19.1 rnM-lcm-’. Statistical significance of differences between alcoholic and control preparations was evaluated by Student’s t test for paired samples.
same rats there were only small differences between ethanol-fed and control rats in either state 3 or state 4 respiratory rates. State 3 rates tended to be slightly higher in preparations from ethanol-fed rats while state 4 rates were slightly lower. The combined effect of these alterations was a small apparent increase in the respiratory control ratio in ethanol-fed rats. These differences were not statistically significant, however (Table 1). RESULTS In order to explore the molecular basis for the difference Oxidative phosphorylation is a highly regulated process in response to alcohol consumption between liver and wherein the rate at which substrates are oxidized through brain mitochondria, we measured the cytochrome conthe mitochondrial respiratory chain is determined by the tents of the mitochondrial preparations by difference specavailability of ADP and Pi for synthesis of ATP. This troscopy. For these studies, chemical reduction by dithioregulation is known as respiratory control and is exhibited nite was used to obtain reduced minus oxidized difference by carefully-isolated mitochondria. Respiratory control is spectra. This method measures the total complement of characterized by a transition from a state of slow basal cytochromes in the preparations; these cytochromes are respiration in the presence of substrate, Pi and oxygen but active during both state 3 and state 4 respiration. In liver without ADP, known as “state 4,” to a state of rapid mitochondria, cytochrome aa3 was decreased an average respiration which ensues upon addition of ADP and dur- of 40% in preparations from ethanol-fed rats (Table 2). ing which ATP is formed, termed state 3.14 When all the This change was accompanied by a 12% increase in cytoavailable ADP has been converted to ATP, the rate of chromes c + c1 and no apparent change in cytochromes b respiration again slows spontaneously to the value char- (Table 2). It should be noted that for these particular acteristic of the “resting” state (state 4). The ratio of spectral determinations the respiratory inhibitor antimyrespiratory rates in state 3 and state 4, known as the RCR, cin was not used. As discussed previously, an effect of reflects the degree to which phosphorylation activity (syn- ethanol consumption on cytochrome b can be seen clearly thesis of ATP) is “coupled” to respiration. High RCR when cytochrome b is selectively reduced by addition of values are indicative of “tight” or efficient coupling be- antimycin under aerobic condition^.^ tween oxidation and phosphorylation activities. ExperiBy contrast, brain mitochondria from ethanol-fed rats mentally, high RCR values are found only with intact, showed no significant differences in amounts of dithioniterelatively undamaged, mitochondrial preparations. For a reducible cytochromes aa3, b, or c plus c1 as compared to given mitochondrial preparation, the maximal rate of ATP brain mitochondria from control rats (Table 2). For brain synthesis is attained during state 3 respiration. mitochondria, we also checked spectra recorded aerobiTable I illustrates the effect of chronic ethanol feeding cally with both succinate and antimycin present in order on the respiratory activity of mitochondria from liver and to facilitate selective reduction of cytochromes b. In this brain. In liver mitochondria, the state 3 respiration rate case also, no difference in cytochromes b content between was decreased about 41 % while the state 4 rate was de- ethanol-fed and control rats was seen. creased 25% in preparations from ethanol-fed rats compared with those from pair-fed control rats. The net effect DISCUSSION of these changes was a decrease averaging 23% in the Results of this study demonstrate a differential biochemRCR. The decreases in activities of the liver mitochondrial preparations from alcoholic rats were statistically signifi- ical response between brain and liver mitochondria in rats cant. In addition, the extents of the decreases in respiration fed ethanol chronically. After a period of ethanol conrates and RCR values were comparable to those seen in sumption sufficient to elicit marked decreases in the active complement of cytochromes and associated respiratory earlier studies.12 By contrast, with brain mitochondria isolated from the activities in the liver, there is no effect on mitochondria in the brain. This differential biochemical response sugTable 1. Effect of Ethanol-Feedingon Respiratory Activity of Mitochondria in Liver and Brain
Control Liver mitochondria (n = 7) State 3 rate State 4 rate RCR Brain mitochondria (n = 7) State 3 rate State 4 rate RCR
Ethanol-Fed
OO /
Change
Table 2. Effect of Alcohol Consumption on Mitochondria1 Cytochromes
p
YO
Control 40+5 68C15 12 3 16C4 4.14 0.64 3.19 0.61
+
+ +
76C18 86C24 23+3 25C6 3.11 k 0.62 3.74 f 0.76
-41 -25 -23 +13 -8 +20
Vol. 16, No. 1 January/Febmary 1992
Comparative Effects of Chronic Ethanol Consumption on the Properties of Mitochondria from Rat Brain and Liver William S. Thayer and Hagai Rottenberg
The effects of chronic alcohol consumption on biochemical proper-
Ues of mitochondria isolated from liver and brain were compared in rats. As has been found in previous studies (reviewed in Thayer WS: Ann NY Acad Sci 492193-206, 1987) in liver, ethanol consumption M to a 41% decrease in active phosphorylating (state 3) respiration and a 25% decrease in resting (state 4) respiration. These changes resulted in a 23% decrease in the respiratory control ratio (ratio of respiration rate in state 3 to that in state 4). These effects were associated with a 40% decrease in functional cytochrome oxidase content, determined spectrophotometrically as heme aa3. By contrast, in brain mitochondria isolated from the same rats, ethanol consumption did not result in any significant changes in respiration rates, respiratory control ratio, or cytochrome contents. The findings demonstrate a differential pathobiologic response of brain and liver mitochondria to chronic ethanol consumption. Since the liver is predominant in metabolism of ingested ethanol, the findings of this study suggest that the deleterious effects of chronic alcohol consumption on the structure and function of liver mitochondria may be related to ethanol metabolism. Key Words: Brain, Ethanol, Liver, Mitochondria, Cytochromes.
HRONIC CONSUMPTION of ethanol produces characteristicalterations in the protein and lipid composition and biochemical activities of subcellular organelles of the liver.' Prominent among these effects is a substantial decrease in the rates of respiration and ATP synthesis displayed by mitochondria isolated from livers of ethanol-fed rats as compared with pair-fed controls. The diminution of respiration by ethanol-feeding is largest, typically on the order of 25% to 40%, with NADlinked substrates which donate reducing equivalents at site I of the electron transport chain.2 Studies of the molecular basis for this ethanol effect have identified decreases in the active contents of specific respiratory chain proteins. The active or functional contents of cytochromes aa3 and b,3 A T p a ~ e , ~and , ~ iron-sulfur clusters of NADHdehyrogenase6are decreased by values ranging from 50% to 20%, respectively, per unit total membrane protein after ethanol-feeding. The precise mechanisms by which such alterations in the protein composition of the mitochondrial membrane come about remain to be elucidated.
C
From the departments of Biological Chemistry and Pathology, Hahnemann University, School of Medicine, Philadelphia, Pennsylvania. Received for publication April 8, 1991; accepted August 1, 1991 This work was supported in part by Research Scientist Development Award AA00087 from the National Institute on Alcohol Abuse and Alcoholism to W.S.T. and by Grants AA07238 and GM28173 to H.R. Reprint requests: William S. Thayer, Ph.D., Hahnemann University, MS #411, Broad & Vine Streets, Philadelphia, PA 19102. Copyright 0 1992 by The Research Society on Alcoholism. AIcohoICIin Exp Res, Vol 16, No 1, 1992: pp 1-4
However, the observation that these enzymatic changes occur has been widely reported'-" and can be regarded as an established consequence of chronic ethanol consumption. In this communication, we report the results of comparative studies conducted with both liver and brain mitochondria isolated from ethanol-fed and control rats. Our data indicate that brain mitochondria do not exhibit any decrease in respiratory activity or alteration in cytochrome contents after a period of chronic ethanol consumption that is sufficient to induce the characteristic mitochondria1 changes in the liver. The findings demonstrate organ specificity in the effects of ethanol on subcellular biochemistry and may provide insight concerning factors underlying the pathophysiologic actions of ethanol. MATERIALS AND METHODS Animals Male, Sprague-Dawley littermate rats were pair-fed a totally liquid diet" (Bio-Serv, Inc., Frenchtown, NJ) containing ethanol as 36% of calories for 35 to 40 days as previously described? The nutrient composition of the diet included 16% protein, 35% fat, and the remainder carbohydrate. In the control diet, carbohydrate replaced ethanol isocalorically.
Preparations Rat liver mitochondria were isolated by conventional differential centrifugation of a homogenate prepared in 0.25 M sucrose and 1 mM EDTA, pH 7.0, as previous described." Rat brain mitochondria were isolated by a Ficoll gradient method."
Analytical Procedures Respiration was measured polarographicallywith an oxygen electrode at 25". For liver mitochondria, the medium contained 0.2 M sucrose, 50 mM KCl, 10 mM Tris-HCI, 2 mM Na'P,, and 2 mM MgCI2at pH 7.4. For brain mitochondria, the medium contained 0.2 M sucrose, 100 mM KC1, 5 mM Na+HEPES, 5 mM Na+P,, and 5 mM MgClz at pH 7.5. In both cases, 5 mM glutamate plus 5 mM malate were present as substrates. Mitochondria1protein concentration, determined by the biuret method, was 1.O to 1.5 mg/ml in all assays. Respiratory control ratio (RCR) was determined as the respiration rate in the presence of ADP (state 3) divided by the respiration rate after phosphorylation of ADP to ATP (state 4) according to established procedure^.'^ For determination of cytochrome contents, mitochondria were diluted to 0.5 to 1.5 mg protein/ml in 0.25 M sucrose, 10 mM Na+HEPES, and 5 mM MgClz (pH 7.5) and portioned equally into two cuvettes. A small amount of sodium dithionite was added to one cuvette. Reduced minus oxidized difference spectra were then recorded using an Aminco DW-2A spectrophotometer. Cytochrome contents were determined from the characteristic absorb-
2
THAYER AND ROTTENBERG
ances of the hemes as previously de~cribed.~ In brief, a baseline was drawn between the isosbestic points at 535,511, and 630 nm. Absorbance values at peak wavelengths were then analyzed using extinction coefficient values as follows: cytochrome aag, C605 = 24.0 mM-’cm-’; cytochromes b, 6563 = 23.4 mM-lcrn-’; cytochromes c + cI, €552 = 19.1 rnM-lcm-’. Statistical significance of differences between alcoholic and control preparations was evaluated by Student’s t test for paired samples.
same rats there were only small differences between ethanol-fed and control rats in either state 3 or state 4 respiratory rates. State 3 rates tended to be slightly higher in preparations from ethanol-fed rats while state 4 rates were slightly lower. The combined effect of these alterations was a small apparent increase in the respiratory control ratio in ethanol-fed rats. These differences were not statistically significant, however (Table 1). RESULTS In order to explore the molecular basis for the difference Oxidative phosphorylation is a highly regulated process in response to alcohol consumption between liver and wherein the rate at which substrates are oxidized through brain mitochondria, we measured the cytochrome conthe mitochondrial respiratory chain is determined by the tents of the mitochondrial preparations by difference specavailability of ADP and Pi for synthesis of ATP. This troscopy. For these studies, chemical reduction by dithioregulation is known as respiratory control and is exhibited nite was used to obtain reduced minus oxidized difference by carefully-isolated mitochondria. Respiratory control is spectra. This method measures the total complement of characterized by a transition from a state of slow basal cytochromes in the preparations; these cytochromes are respiration in the presence of substrate, Pi and oxygen but active during both state 3 and state 4 respiration. In liver without ADP, known as “state 4,” to a state of rapid mitochondria, cytochrome aa3 was decreased an average respiration which ensues upon addition of ADP and dur- of 40% in preparations from ethanol-fed rats (Table 2). ing which ATP is formed, termed state 3.14 When all the This change was accompanied by a 12% increase in cytoavailable ADP has been converted to ATP, the rate of chromes c + c1 and no apparent change in cytochromes b respiration again slows spontaneously to the value char- (Table 2). It should be noted that for these particular acteristic of the “resting” state (state 4). The ratio of spectral determinations the respiratory inhibitor antimyrespiratory rates in state 3 and state 4, known as the RCR, cin was not used. As discussed previously, an effect of reflects the degree to which phosphorylation activity (syn- ethanol consumption on cytochrome b can be seen clearly thesis of ATP) is “coupled” to respiration. High RCR when cytochrome b is selectively reduced by addition of values are indicative of “tight” or efficient coupling be- antimycin under aerobic condition^.^ tween oxidation and phosphorylation activities. ExperiBy contrast, brain mitochondria from ethanol-fed rats mentally, high RCR values are found only with intact, showed no significant differences in amounts of dithioniterelatively undamaged, mitochondrial preparations. For a reducible cytochromes aa3, b, or c plus c1 as compared to given mitochondrial preparation, the maximal rate of ATP brain mitochondria from control rats (Table 2). For brain synthesis is attained during state 3 respiration. mitochondria, we also checked spectra recorded aerobiTable I illustrates the effect of chronic ethanol feeding cally with both succinate and antimycin present in order on the respiratory activity of mitochondria from liver and to facilitate selective reduction of cytochromes b. In this brain. In liver mitochondria, the state 3 respiration rate case also, no difference in cytochromes b content between was decreased about 41 % while the state 4 rate was de- ethanol-fed and control rats was seen. creased 25% in preparations from ethanol-fed rats compared with those from pair-fed control rats. The net effect DISCUSSION of these changes was a decrease averaging 23% in the Results of this study demonstrate a differential biochemRCR. The decreases in activities of the liver mitochondrial preparations from alcoholic rats were statistically signifi- ical response between brain and liver mitochondria in rats cant. In addition, the extents of the decreases in respiration fed ethanol chronically. After a period of ethanol conrates and RCR values were comparable to those seen in sumption sufficient to elicit marked decreases in the active complement of cytochromes and associated respiratory earlier studies.12 By contrast, with brain mitochondria isolated from the activities in the liver, there is no effect on mitochondria in the brain. This differential biochemical response sugTable 1. Effect of Ethanol-Feedingon Respiratory Activity of Mitochondria in Liver and Brain
Control Liver mitochondria (n = 7) State 3 rate State 4 rate RCR Brain mitochondria (n = 7) State 3 rate State 4 rate RCR
Ethanol-Fed
OO /
Change
Table 2. Effect of Alcohol Consumption on Mitochondria1 Cytochromes
p
YO
Control 40+5 68C15 12 3 16C4 4.14 0.64 3.19 0.61
+
+ +
76C18 86C24 23+3 25C6 3.11 k 0.62 3.74 f 0.76
-41 -25 -23 +13 -8 +20
