Alcohol,Vol. 7, pp. 69-74. ~ Pergamon Press plc, 1990. Printed in the U.S.A.
0741-8329/90 $3.00 + .00
Developmental Profile of Hepatic Alcohol and Aldehyde Dehydrogenase Activities in Long-Sleep and Short-Sleep Mice T O N I N E S S S M O L E N , * A N D R E W S M O L E N ? A N D J E N N I F E R L. VAN I~E K A M P *
*Institute for Behavioral Genetics and ~University of Colorado Drug Abuse Research Center Campus Box 447, University of Colorado, Boulder, CO 80309-0447 R e c e i v e d 24 April 1989; A c c e p t e d 29 July 1989
SMOLEN, T. N., A. SMOLEN AND J. L. VAn DE KAMP. Developmentalprofile of hepatic alcohol and aldehyde dehydrogenase activities in Long-Sleep and Short-Sleep mice. ALCOHOL 7(1) 69-74, 1990.--Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low K m (AHD-5), cytosolic high Km (AHD-7), cytosolic low K m (AHD-2) and microsomal high K,~ (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high K m mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice. Alcohol dehydrogenase
Aldehyde dehydrogenase
Long-Sleep mice
ETHANOL is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (alcohol: NAD ÷ oxidoreductase, E.C. 1.1.1.1, ADH) (16). The product of that oxidation, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (aldehyde: NAD ÷ oxidoreductase, E.C. 1.2.1.3, AHD) which are found in cytosolic, mitochondrial and microsomal fractions of liver homogenates (20, 25, 27, 29, 30). The subcellular distribution of AHD in liver varies among species. In rat liver 50-80% of the total aldehyde oxidizing capacity can be accounted for in the mitochondrial fraction (6, 9, 21, 26, 33). The remainder of the AHD activity is found in cytosolic and microsomal fractions, which contribute some 10-20% each to the total activity of the liver homogenates. In mouse liver 70% of total AHD activity is contained in the cytosol, 25% is mitochondrial, and only 5-10% can be found in the microsomal fraction (14, 29, 30), although other distribution patterns have been reported by at least one other group (12). In humans, most of the AHD activity is found in the cytosol, as it is in mice (8,18). Aldehyde dehydrogenase is found in multiple forms, and several systems of terminology have been proposed. We shall employ the system used by Holmes and co-workers (4, 5, 24, 32) since it best fits the system of gene terminology proposed for the mouse genome. For species other than mouse, the alternate terminologies used by other investigators will be used as necessary. Mouse liver contains five major constitutive AHD isoen-
Short-Sleep mice
Enzyme development
zymes: mitochondrial high Kr, (AHD-1), mitochondrial low K m (AHD-5), cytosolic high K m (AHD-7), cytosolic low K m (AHD2), and microsomal high K m (AHD-3), where high and low K m refer to the K m of the enzymes for acetaldehyde (approximately 1 mM and 0.01 raM, respectively). In rat liver most of the acetaldehyde produced from ethanol metabolism is oxidized by the low Km mitochondrial AHD, generally referred to as ALDH-I (11, 13, 21) (equivalent to mouse AHD-5). The subcellular site of acetaldehyde oxidation is not as clearly worked out for the mouse as for the rat, although the literature suggests that acetaldehyde is metabolized by cytosolic enzymes, especially AHD-2, to a greater degree in the mouse (22), although this may depend on genotype (28). We have been investigating the role of genetics on age-related behavioral and physiological responses to ethanol in Long-Sleep (LS) and Short-Sleep (SS) mice from birth until young adulthood. The LS and SS mice were bred for maximal differences in acute response to ethanol as measured by duration of loss of righting response (sleep time) (17). These mice differ in their sleep time response as early as 10 days of age (2), and this difference increases with increasing age. Although we believe these differences are due to an alteration in CNS sensitivity to ethanol, ageand genotype-related differences in metabolism could also account for the pattern of responses seen in these mice. Because of the importance of metabolism in understanding the 69
SMOLEN, SMOLEN AND VAN DE KAMP
70
effects of ethanol in vivo, and because there is little information concerning activities of alcohol metabolizing enzymes during postnatal development in the mouse, we measured the activity of hepatic ADH and AHD in male and female LS and SS mice from 3 days of age to adulthood. We found that both ADH and AHD reach adult levels of activity at about 14 days of age, and that there were no significant sex or line differences in developmental profiles. A preliminary report of these studies has been presented (31 ).
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METHOD
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Animals The animals used in this study were male and female LS and SS mice 3, 6, 12, 20, 24, 28, 32, and 55--65 days of age. Mice were bred at the Institute for Behavioral Genetics, kept on a 12-hour light cycle, and were given free access to food (Wayne Lab Blox) and water. Preweanling mice (
0741-8329/90 $3.00 + .00
Developmental Profile of Hepatic Alcohol and Aldehyde Dehydrogenase Activities in Long-Sleep and Short-Sleep Mice T O N I N E S S S M O L E N , * A N D R E W S M O L E N ? A N D J E N N I F E R L. VAN I~E K A M P *
*Institute for Behavioral Genetics and ~University of Colorado Drug Abuse Research Center Campus Box 447, University of Colorado, Boulder, CO 80309-0447 R e c e i v e d 24 April 1989; A c c e p t e d 29 July 1989
SMOLEN, T. N., A. SMOLEN AND J. L. VAn DE KAMP. Developmentalprofile of hepatic alcohol and aldehyde dehydrogenase activities in Long-Sleep and Short-Sleep mice. ALCOHOL 7(1) 69-74, 1990.--Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low K m (AHD-5), cytosolic high Km (AHD-7), cytosolic low K m (AHD-2) and microsomal high K,~ (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high K m mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice. Alcohol dehydrogenase
Aldehyde dehydrogenase
Long-Sleep mice
ETHANOL is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (alcohol: NAD ÷ oxidoreductase, E.C. 1.1.1.1, ADH) (16). The product of that oxidation, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (aldehyde: NAD ÷ oxidoreductase, E.C. 1.2.1.3, AHD) which are found in cytosolic, mitochondrial and microsomal fractions of liver homogenates (20, 25, 27, 29, 30). The subcellular distribution of AHD in liver varies among species. In rat liver 50-80% of the total aldehyde oxidizing capacity can be accounted for in the mitochondrial fraction (6, 9, 21, 26, 33). The remainder of the AHD activity is found in cytosolic and microsomal fractions, which contribute some 10-20% each to the total activity of the liver homogenates. In mouse liver 70% of total AHD activity is contained in the cytosol, 25% is mitochondrial, and only 5-10% can be found in the microsomal fraction (14, 29, 30), although other distribution patterns have been reported by at least one other group (12). In humans, most of the AHD activity is found in the cytosol, as it is in mice (8,18). Aldehyde dehydrogenase is found in multiple forms, and several systems of terminology have been proposed. We shall employ the system used by Holmes and co-workers (4, 5, 24, 32) since it best fits the system of gene terminology proposed for the mouse genome. For species other than mouse, the alternate terminologies used by other investigators will be used as necessary. Mouse liver contains five major constitutive AHD isoen-
Short-Sleep mice
Enzyme development
zymes: mitochondrial high Kr, (AHD-1), mitochondrial low K m (AHD-5), cytosolic high K m (AHD-7), cytosolic low K m (AHD2), and microsomal high K m (AHD-3), where high and low K m refer to the K m of the enzymes for acetaldehyde (approximately 1 mM and 0.01 raM, respectively). In rat liver most of the acetaldehyde produced from ethanol metabolism is oxidized by the low Km mitochondrial AHD, generally referred to as ALDH-I (11, 13, 21) (equivalent to mouse AHD-5). The subcellular site of acetaldehyde oxidation is not as clearly worked out for the mouse as for the rat, although the literature suggests that acetaldehyde is metabolized by cytosolic enzymes, especially AHD-2, to a greater degree in the mouse (22), although this may depend on genotype (28). We have been investigating the role of genetics on age-related behavioral and physiological responses to ethanol in Long-Sleep (LS) and Short-Sleep (SS) mice from birth until young adulthood. The LS and SS mice were bred for maximal differences in acute response to ethanol as measured by duration of loss of righting response (sleep time) (17). These mice differ in their sleep time response as early as 10 days of age (2), and this difference increases with increasing age. Although we believe these differences are due to an alteration in CNS sensitivity to ethanol, ageand genotype-related differences in metabolism could also account for the pattern of responses seen in these mice. Because of the importance of metabolism in understanding the 69
SMOLEN, SMOLEN AND VAN DE KAMP
70
effects of ethanol in vivo, and because there is little information concerning activities of alcohol metabolizing enzymes during postnatal development in the mouse, we measured the activity of hepatic ADH and AHD in male and female LS and SS mice from 3 days of age to adulthood. We found that both ADH and AHD reach adult levels of activity at about 14 days of age, and that there were no significant sex or line differences in developmental profiles. A preliminary report of these studies has been presented (31 ).
'
I
I
I
I
I
I
I
I
1.6 m y
1.4
~.2 ~
1.()
~
0.8
~
0.6 0.4 0.2
METHOD
0
Animals The animals used in this study were male and female LS and SS mice 3, 6, 12, 20, 24, 28, 32, and 55--65 days of age. Mice were bred at the Institute for Behavioral Genetics, kept on a 12-hour light cycle, and were given free access to food (Wayne Lab Blox) and water. Preweanling mice (
