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Biochemical and genetic studies on mouse aldehyde dehydrogenases
Alcohol, Vol. 2, pp. 67-71, 1985.©AnkhoInternational Inc. Printed in the U.S.A.
0741-8329/85$3.00 + ,00
Biochemical and Genetic Studies on Mouse Aldehyde Dehydrogenases R O G E R S. H O L M E S , E L I Z A B E T H M. A L G A R A N D P E T E R B. M A T H E R
School o f Science, Griffith University, Nathan, 4111, Queensland, Australia
HOLMES, R. S., E. M. ALGAR AND P. B. MATHER. Biochemical and genetic studies on mouse aldehyde dehydrogenases. ALCOHOL 2(I) 67-71, 1985.mAldehyde dehydrogenase (AHD) exists as isozymes which are differentially distributed among tissues and subcellular fractions of mouse tissues. Genetic variants for liver mitochondrial (AHD-!) and cytoplasmic (AHD-2) isozymes have been used to map the responsible loci (Ahd-I and Ahd-2) on chromosomes 4 and 19 respectively. Evidence for a regulatory locus (Ahd-3r) controlling the inducibility of the mouse liver microsomal isozyme (AHD-3) has also been obtained. More recent studies have described genetic and biochemical evidence for three additional AHD isozymes: a stomach isozyme (AHD-4);another liver mitochondrial enzyme (AHD-5);and a testis isozyrne (AHD-6). Genetic analyses have indicated that AHD-4 and AHD-6 are encoded by distinct but closely linked loci on the mouse genome (Ahd-4 andAhd-6), which segregate independently ofAhd-I andAhd-2. Liver mitochondrial isozymes, AHD-I and AHD-5, have been purified to homogeneity using aff'mitychromatography. The very high affinity of AHD-5 for acetaldehyde suggests that this enzyme is predominantly responsible for acetaldehyde oxidation in mouse liver mitochondria. Mouse Aldehydedehydrogenase Liver S t o m a c h Testis
lsozymes
Alcohol
THE house mouse (Mus musculus) has served as a useful laboratory subject for alcohol research following early behavioural studies, which demonstrated differences in the amount of voluntary alcohol consumption by inbred strains of mice [23, 26, 39]. The organism has many attributes which make it most suitable for genetic research. Its small size, rapid reproductive rate and the availability of a large number of inbred strains including recombinant and congenic strains, render it as an ideal organism for genetic studies on behavioural, biochemical, pathological and physiological responses to alcohol. Moreover, the availability of biochemical and genetic markers for the 20 pairs of chromosomes found in the mouse karyotype provide an excellent base for gene mapping studies [7]. Many of these features have been exploited in recent studies examining the genetic basis for differential behavioural responses towards alcohol among inbred strains [3, 4, 9, 28]. Investigations have also been undertaken on the biochemical genetics of alcohol metabolism in the mouse. Metabolic studies on rodents have demonstrated that the liver provides the major site of ethanol oxidation, which proceeds via a three stage process: ethanol to acetaldehyde; acetaldehyde to acetate; and acetate to carbon dioxide and water, with the third stage being predominantly an extrahepatic process [ 18, 19, 30]. Liver contains at least three major enzyme systems capable of oxidizing alcohol in vitro: alcohol dehydrogenase (ADH); the microsomai ethanol oxidizing system and catalase [19,30]. It is generally recognized, however, that at ethanol concentrations less than 0.2 percent, at least 80 percent of alcohol oxidation proceeds via ADH [11,16]. Aldehyde dehydrogenase (AHD; E.C.1.2.1.3) catalyses the second step of ethanol metabolism and functions in the oxidation of acetaldehyde to acetate [19]. In contrast to
Acetaldehyde
Mitochondria
Cytoplasm
ADH which is localized exclusively in the cytoplasm, aldehyde dehydrogenase is differentially distributed between liver subcellular fractions [5, 12, 14, 17, 20, 24, 31, 35, 36-37, 40]. Mitochondrial and cytosolic rat liver aldehyde dehydrogenases have been partially purified and characterized kinetically, with both "low" and "high" Michaelis constant forms being described for both sources [14, 29, 33, 38]. Microsomal aldehyde dehydrogenase has also been purified from rat liver and shown to be biochemically distinct form of this enzyme [26]. Aldehyde dehydrogenase isozymes from other mammalian livers have been purified and characterized, including horse [6,8], human [10], and sheep [21] cytoplasmic and mitochondrial enzymes. The role of aldehyde dehydrogenases in the metabolism of ethanol has been demonstrated by the increase in alcohol sensitivity found in oriental populations which show a widespread lack of one of the isozymes [1]. The mitochondria have been considered to be the major site of acetaldehyde oxidation in man, however, more recent studies using human liver biopsy samples suggest that the liver cytosol may play a more significant role [15]. This communication reviews biochemical and genetic evidence concerning the isozymic status of aldehyde dehydrogenases in mouse tissues. Present findings suggest that at least six genetic loci encode aldehyde dehydrogenases in this organism, with the resultant isozymes being differentially distributed among liver subcellular fractions and in mouse stomach and testis extracts. TISSUEDISTRIBUTIONOF MOUSE ALDEHYDEDEHYDROGENASES The electrophoretic patterns for aldehyde dehydrogenase
67
68
HOLMES, ALGAR AND MATHER
4"
--In F
m
•
m 14J.LUJ
m
St
Lu K L
St
Lu K
AHD 3 AHD 5 AHD4 AHD 1 AHD 2
L
FIG. 1. Cellulose acetate zymogram and diagrammatic illustration of aldehyde dehydrogenase activity from tissues of 101/H inbred mice. Aldehyde dehydrogenase (AHD) activity is represented by closed box; non-specific oxidase activity by diagonally striped box; aldehyde oxidase activity by crossed box; and xanthine oxidase activity by vertically striped box. The tissue extracts examined included liver (L); kidney (K); lung (Lu); and stomach (St). AHD isozymes are designated at the side of the diagram. Buffer conditions were 25 mM Tris-glyeine (pH 8.5); electrophoresis was for 200 V for 20 minutes. Staining conditions are described in reference [12].
TABLE 1 BIOCHEMICAL VARIANTS FOR ALDEHYDE DEHYDROGENASES AMONG MOUSE INBRED STRAINS AHD-I (Ahd-l)*
Isozyme
(locus) Variant Strain Distribution
A
17 strains e.g., A/J C57BL/6J 101/H
B
38 strains e.g., BALB/c CBA/H DBA/J 129/MA
AHD-4 (Ahd-4)~.
AHD-2 (Ahd-2)t C
A
castaneus 52 strains
e.g., A/J BALB/c C57BL/6J DBA/J
B
C
3 strains castaneus NZB/BI NZC/BI NZW/B1
A
B
53 strains 3 strains e.g., A/J 'Danish' BALB/c LIII C57BI.,/6J S L / N i A DBA/J
AHD-6 (Ahd-6) § A
B
37 strains 5 strains e.g., ddN e.g., A/J GRS/A BALB/c C57BL/6J DBA/J
*Eiectrophoretic variants described in references [12,25]. ?Electrophorctic variants described in references [25,28]. :l:Electrophoreticvariants described in reference [25]. §Activity variants described in reference [25].
(AHD) isozymes extracted from various tissues of 101/H inbred mice are shown in Fig. 1. Four AHD activity zones were resolved for liver extracts, as well as several oxidase zones, that exhibited activity in the absence of coenzyme. Subcellular fractionation studies have shown that these enzymes are differentially distributed among mitochondrial (AHD-I and AHD-5 isozymes); cytoplasmic (AHD-2); and microsomai (AHD-3) liver preparations [2, 17, 36, 37]. Kidney extracts exhibited two forms of activity which corresponded in terms of electrophoretic migration to the AHD-I and AHD-3 liver isozymes. In contrast, mouse lung extracts showed only a single form of activity, coincident with AHD-5 on the cellulose acetate zymogram. Mouse stomach extracts exhibited an electrophoretically distinct form of aldehyde dehydrogenase activity (designated AHD-4).
GENETIC VARIANTS FOR ALDEHYDE DEHYDROGENASES
Liver Mitochondrial Aldehyde Dehydrogenase (AHD-I ) Three electrophoretic variants for mouse liver mitochondrial aldehyde dehydrogenase (AHD-1 isozyme) have been described [12,25]. The AHD-1A and 1B all¢lic isozymes are widely distributed among common inbred strains, and are found, for example, in C57BL/6J and BALB/c mice respectively (Table l). In contrast, AHD-IC was observed only in castaneus mice, an inbred strain derived from a South-East Asian subspecies of this organism. Electrophoretic phenotypes for F1 mice, heterozygous for the proposed locus encoding these ailelic forms of AHD-1 (designated Ahd-l), showed three-banded patterns of activity, which is consistent with the dimeric subunit structure
MOUSE A L D E H Y D E D E H Y D R O G E N A S E S
TESTIS
69
(+)
STOMACH
AHD-5 AHD-6 AHD-2
="
123
'
- AHD 4
123
FIG. 2. Cellulose acetate zymograms of testis and stomach aldehyde dehydrogenase activity from inbred mice, LIII (i) and ddN (3), and their F~(ddNxLIII) hybrid (2). AHD isozyme designation is based on previous studies [25] and is shown at the side of the zymograms. The fastest anodally migrating zone of activity for the testis zymogram was non-specific oxidase activity. The cathodally migrating minor zone of activity for LIII (1) stomach extracts was recognized as stomach alcohol dehydrogenase C2. Note the activity variation for AHD-6 in testis extracts and the electrophoretic variants for AHD-4 in stomach extracts. Buffer conditions for stomach AHD electrophoresis were 25 mM Tris-glycine (pH 8.5), 300 V for 20 minutes; for testis AHD electrophoresis, buffer conditions were 75 mM Tris-citrate (pH 7.0), 150 V for 90 minutes. Staining conditions are described in reference [12].
reported for this enzyme [2]. Yamazaki and coworkers [40] have recently confirmed these findings for two inbred strains of mice (C57BL/6J and DBA/2), which exhibited electrophoretically distinct forms for one of the mouse liver mitochondrial aldehyde dehydrogenases (AHD-I).
Liver Cytosolic Aldehyde Dehydrogenase (AHD-2) Three electrophoretic variants have also been reported for mouse liver cytosolic aldehyde dehydrogenase (AHD-2) • [25,37]. The AHD-2A allelic form represented the predominant phenotype among the 56 inbred strains examined thus far, and was observed in all strains with the exception of the NZ and castaneus strains (Table I). New Zealand mice (NZB, NZC and NZW strains) exhibited a high activity, electrophoretically distinct form of AHD-2 (AHD-2B), which migrated on the anodal side of the common AHD-2A allelic isozyme [36], whereas castaneus mice showed a low activity AHD-2 phenotype, migrating more rapidly towards the cathode, as compared with AHD-2A [24].
AHD-4A) was coincident with the ddN pattern of activity, whereas the variant phenotype (AHD-2B) was found in the LIII and SL/NiA strains (Table 1).
Testis Aldehyde Dehydrogenase (AHD-6) Evidence has been recently described for a testis-specific form of aldehyde dehydrogenase, designated AHD-6 [25]. Figure 2 illustrates the activity variation observed for the AHD-6 isozyme between LIII and ddN mouse strains, ddN male mice exhibited a much higher activity for AHD-6, as compared with LIII males, whereas Fl(ddNxLIII) males showed an intermediate activity for this isozyme. In contrast, no detectable activity variation was observed for the other testis aldehyde dehydrogenases, AHD-2 and AHD-5. The predominant phenotype observed among 42 inbred strains was similar to that of the LIII mice (designated AHD-6A), with only 5 strains resembling that of the ddN high activity phenotypes (AHD-6B) (Table 1). GENETIC ANALYSES AND GENE MAPPING STUDIES
Stomach Aldehyde Dehydrogenase (AHD-4) A cellulose acetate zymogram illustrating the dectrophoretic variation of stomach aldehyde dehydrogenase (AHD-4) is shown in Fig. 2. LIII mice exhibited a faster migrating form as compared with ddN inbred mice, with Fl(ddNxLIII) animals showing a three-banded pattern. The distribution of these electrophoretic variants for stomach AHD have been examined [25]. The predominant phenotype observed in 52 of the 56 inbred strains examined (designated
Genetic analyses and gene mapping studies have been undertaken using the previously described variants for mouse liver mitochondrial (AHD-I) and cytosolic (AHD-2) aldehyde dehydrogenases [12,37]. These results have shown that the electrophoretic variants, in each case, behaved as though encoded by codominant alleles at the respective loci, Ahd-I and Ahd-2, which are separately localized on the mouse genome on chromosomes 4 and 19 respectively. More recent studies have examined the genetics of the stomach
70 (AHD-4) and testis (AHD-6) specific forms, using the variant phenotypes previously described, as biochemical markers for the proposed respective loci involved (Ahd-# and Ahd-6) [24]. Segregation analyses showed that the variant phenotypes inherited as though encoded by codominant alleles at these loci. Moreover, the proposed Ahd-4 and Ahd-6 loci segregated independently of Ahd-I and Ahd-2, but showed a 7 percent recombination frequency, which is consistent with Ahd-4 and Ahd-6 being separately, but closely, localized on the mouse genome. Variation in the inducibility of the liver microsomal isozyme (AHD-3) by phenobarbital administration has been reported among mouse inbred strains. The phenotypes were inherited in a normal Mendelian fashion, with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r) [36]. BIOCHEMISTRYOF MOUSEALDEHYDEDEHYDROGENASES The biochemical properties of mouse liver mitochondrial aldehyde isozymes, designated AHD-I and AHD-5 on the basis of previous biochemical genetic analyses [ 12,25] have been recently described [2]. These isozymes have been isolated in a highly purified state from extracts of mouse liver mitochondria and characterized by both structural and kinetic analyses. These studies have fh'mly established the presence of two biochemically and kinetically distinct aldehyde dehydrogenase isozymes in mouse liver mitochondria. The subunit sizes of the enzymes were determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis to be 63,000 for AHD-I and 49,000 for AHD-5. Moreover, gel exclusion chromatography indicated that both isozymes are dimers, although there was some evidence for a monomeric form of AHD-I as well. The mouse liver aldehyde dehydrngenase isozymes also exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-I, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate (1.4 mM and 0.78 mM respectively), whereas AHD-5 exhibited a low Km value, 0.2 ~M with acetaldehyde. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-I, pH range 6.5-7.5, AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50 percent inhibition occurring at 13/zM and 0.1/~M for the AHD-I and AHD-5 isozymes respectively. These studies have strongly supported the proposition that AHD-5 is predominantly responsible for mitochondrial acetaldehyde oxidation, in vivo. Sheppard and coworkers [32] have previously described the partial purification and kinetic characterization of aldehyde dehydrogenases from livers of C57BL/6J and DBA/2J inbred mice and their F1 offspring. These enzymes were prepared from whole liver acetone powder extracts and it was not possible to conclude which of the aldehyde dehydrogenase isozymes were being investigated in this study. Little and Peterson [20] reported at this meeting on the partial purification and kinetic properties of mouse liver mitochondrial and cytoplasmic aldehyde dehydrogenases from eight inbred strains of mice. Their results for the mitochondrial isozymes confirmed the existence of separate low and high Km terms corresponding to AHD-5 and AHD-I isozymes respectively. In addition, their study suggested that distinct 'low-Kin' and 'high-Km' forms of aldehyde dehydrogenase also occur within the liver cytoplasm. A proposal was presented correlating genotype and ethanol elimination rates on the basis of variations in the kinetic charac-
HOLMES, ALGAR AND MATHER teristics of the 'low-Km' cytosolic isozyme occurring between inbred strains. DISCUSSION Phenotypic differences for most of the aldehyde dehydrogenase isozymes have been observed among inbred strains of mice. Electrophoretic variants have been reported for a liver mitochondrial isozyme (AHD-I); a liver cytosolic isozyme (AHD-2) (for which one of the variant forms (AHD-2B) exists as a high activity phenotype as well); and the stomach-specific aldehyde dehydrogenase (AHD-4) [12, 25, 37]. Genetic analyses, using these variants as biochemical markers for the corresponding loci, have provided evidence that the genes encoding these enzymes (designated Ahd-l ; Ahd-2; and Ahd-4) are unlinked. Variation in the inducibility of the liver microsomal isozyme (AHD-3) by phenobarbital administration has been also described and a regulatory locus (Ahd-3r) proposed [36]. Moreover, activity variants for the testis specific form of aldehyde dehydrogenase (AHD-6) have been found among mouse inbred strains [25]. Genetic analyses using this activity variant as a biochemical marker for the proposed gene controlling this differential phenotype (designated Ahd-6) have indicated that this locus is inherited in a normal Mendelian fashion and is distinct from the other Ahd loci, although evidence for close linkage withAhd-4 was reported [25]. Thus, in contrast to the presently mapped loci encoding alcohol dehydrogenase isozymes in the mouse, which exist as a gene cluster [13], aldehyde dehydrogenase loci are separately localized on the mouse genome. Biochemical studies on purified preparations of mouse liver mitochondrial aldehyde dehydrogenases have demonstrated that AHD-5 is the most likely catalyst of acetaldehyde oxidation within mouse liver mitochondria [2]. It should be noted, however, that in contrast to rat liver, mouse liver aldehyde dehydrogenase activity is predominantly localized in the cytosol [35]. Studies by Smolen and coworkers [34] have suggested that the mouse cytosolic enzyme is important in acetaldehyde metabolism. In addition, a report at this meeting of the International Society for Biomedical Research on Alcoholism by Little and Peterson [21] supports this view and has indicated that there exists in both liver mitochondria and cytosol, "low-Km" aldehyde dehydrogenases, capable of oxidizing physiological concentrations of acetaldehyde. The relative metabolic capacities of these aldehyde dehydrogenases, under in vivo conditions remain to be determined.
ACKNOWLEDGEMENTS
This research was supported by grants from the Australian Associated Brewers, the National Health and Medical Research Council of Australia and the Australian Research Grants Scheme. We are grateful to the following for supplying mice used in these studies: Dr. J. Hilgers and Dr. J. Hilkens, Netherlands Cancer Institute, Amsterdam; Dr. S. Imal, Nara Medical University, Japan; Dr. V. Chapman, Roswell Memorial Institute, Buffalo; Dr. M. Lyon, M.R.C. Radiobiology Unit, Harwell, England; Dr. E. Eicher. The Jackson Laboratory, Maine, USA.
MOUSE ALDEHYDE DEHYDROGENASES
71 REFERENCES
1. Agarwal, D. P., S. Harada and H. P. Goedde. Racial differences in biological sensitivity to ethanol. Alcoholism Clin Exp Res 5: 12-16, 1981. 2. Algar, E. M. and R. S. Holmes. Mouse mitochondrial aldehyde dehydrogenase isozymes: purification and molecular properties. lnt J Biochem, in press, 1984. 3. Anderson, S. M., G. E. McClearn and V. G. Erwin. Ethanol ~onsumption and hepatic enzyme activity. Pharmacol Biochem Behav 11: 83-88, 1979. 4. Crabbe, J. C., A. Kosobud, E. R. Young and J. S. Janowsky. Polygenic and single gene determination of responses to ethanol in BXD/Ty recombinant inbred mouse strains. Neurobehav Toxicol Teratol 5: 181-187, 1983. 5. Eckfeldt, J. H. and T. Yonetani. SubceHular localization of the F~ and F2 isozymes of horse liver aldehyde dehydrogenase. Arch Biochem Biophys 175: 717-722, 1976. 6. Eckfeidt, J., L. Mope, K. Takio and T. Yonetani. Horse liver aldehyde dehydrogenase: purification and characterization of two isozymes. J Biol Chem 251: 236--240, 1976. 7. Eicher, E. M. Foundation for the future. Formal genetics of the mouse. In: Mammalian Genetics and Cancer: The Jackson Laboratory Fiftieth Anniversary Symposium. edited by E. S. Russell. New York: Alan R. Liss Inc., 1981, pp. 7-49. 8. Feldman, R. I. and H. Weiner. Horse liver aldehyde dehydrogenase. I. Purification and characterization. J Biol Chem 247: 260-266, 1972. 9. Fuller, J. L. and R. L. Collins. Ethanol consumption and preference in mice: A genetic analysis. Ann N Y Acad Sci 197: 42-48, 1972. 10. Greenfield, N. J. and R. Pietruszko. Two aldehyde dehydrogenases from human liver: isolation and characterization of the isozymes. Biol Chim Biophys Acta 483: 35-45, 1977. 11. Havre, P., M. A. Abrams, R. J. M. Corrall, C. Y. Ling, P. A. Szczepanik, H. E. Feldman, P. Klein, M. S. Kong, J. M. Margolis and B. R. Landau. Quantitiation of pathways of ethanol metabolism. Arch Biochem Biophys 182: 14-24, 1977. 12. Holmes, R. S. Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: localization of Ahd-I encoding the mitochondrial isozyme on chromosome 4. Biochem Genet 16: 1207-1218, 1978. 13. Holmes, R. S., J. A. Duley and J. N. Burneil. The alcohol dehydrogenase gene complex on chromosome 3 of the mouse. In: ISOZYMES: Current Topics in Biological and Medical Research, vol 8, edited by M. C. Rattazzi, J. G. Scandalios and G. S. Whitt. New York: Alan R. Liss Inc., 1983, pp. 155--174. 14. Horton, A. A. and M. C. Barrett. The subcellular localization of aldehyde dehydrogenase in rat liver. Arch Biochem Biophys 167: 426-436, 1975. 15. Jenkins, W. J. and T. J. Peters. Subcellular localization of acetaldehyde dehydrogenase in human liver. Cell Biochem Function 1: 37-40, 1983. 16. Khanna, J. M. and Y. Israel. Ethanol metabolism. In: Liver and Bilary Tract Physiology 1, edited by N. B. Jovitt. Baltimore: University Park Press, 1980, pp. 275-315. 17. Koivula, T. and M. Koivusalo. Different forms of rat liver aldehyde dehydrogenase and the subcellular distribution. Biochim Biophys Acta 297: 9-23, 1975. 18. Larsen, J. A. Extrahepatic metabolism in ethanol in man. Nature 184: 1236-1238, 1959. 19. Lieber, C. S. The metabolism of alcohol. In: Metabolic Aspects of Alcoholism, edited by C. S. Lieber. Baltimore: University of Park Press, 1977, pp. 1-29. 20. Lindahl, R. Subcellular distribution and properties of rabbit liver aldehyde dehydrogenases. Biochem Pharmacol 30: 441446, 1981. 21. Little, R. C. and D. R. Peterson. Variation of hepatic aldehyde dehydrogenase among inbred strains of mice. Second Congress of the International Society for Biomedical Research on Alcoholism. Abstract 151, 1984.
22. MacGibbon, A. K. H., L. R. Morton, K. E. Crow, P. D. Buckley and L. F. Blackwell. Purification and properties of sheep liver aldehyde dehydrogenases. Eur J Biochem 96: 585-595, 1979. 23. McLearn, G. E. and D. A. Rodgers. Differences in alcohol preference among inbred strains of mice. Q J Stud Alcohol 20: 691695, 1959. 24. Marjenen, L. Intracellular localization of aldehyde dehydrogenase in rat liver. J Biochem (Tokyo) 127: 633-639, 1972. 25. Mather, P. B. and R. S. Holmes. Biochemical genetics of aldehyde dehydrogenase isozymes in the mouse: evidence for stomach and testis specific isozymes. Biochem Genet 22: 981995, 1984. 26. Mirone, L. The effect of ethyl alcohol on growth, fecundity and voluntary consumption of alcohol in mice. Q J Stud Alcohol 13: 365-369, 1952. 27. Nakayasu, H., K. Mihara and R. Sato. Purification and properties of a membrane-bound aldehyde dehydrogenase from rat liver microsomes. Biochem Biophys Res Commun 83: 697-703, 1978. 28. Oliverio, A. and B. E. Elefiheriou. Motor activity and alcohol: Genetic analysis in the mouse. PhysioIBehav 16: 577-581, 1976. 29. Pietruszko, R. Aldehyde dehydrogenase isozymes. In: Isozymes: Current Topics in Biological and Medical Research, vol 8, edited by M. C. Rattazzi, J. G. Scandalios and G. S. Whitt. New York: Alan R. Liss Inc., 1983, pp. 195-217. 30. Rognstad, R. and N. Grunnet. Enzymatic pathways of ethanol metabolism. In: Biochemistry and Pharmacology of Ethanol, edited by N. Majchrowicz. New York: Plenum Press, 1979, pp. 65-85. 3 I. Schum, G. T. and A. H. Blair. Aldehyde dehydrogenases in rat liver. Can J Bioehem 50: 741-748, 1972. 32. Sheppard, J. R., P. Albershein and G. McClearn. Aldehyde dehydrogenase and ethanol preference in mice. J Biol Chem 245: 2876-2882, 1970. 33. Slew, C., R. A. Deitrich and V. G. Erwin. Localization and characterization of rat liver mitochondrial aldehyde dehydrogenases. Arch Biochem Biophys 176: 638-648, 1976. 34. Smolen, A., N. Atkinson and D. R. Peterson. The role of liver cytosolic aldehyde dehydrogenase in ethanol metabolism in DBA and C57BL mice. In: Alcohol and Aldehyde Metabolizing Systems, vol 4, edited by R. G. Thurman. New York: Plenum Press, 1980, pp. 627-634. 35. Smolen, A., T.-N. Smolen and A. C. Collins. Subcellular distribution of hepatic aldehyde aldehyde dehydrogenase activity in four mouse strains. Comp Biochem Physiol (A) 73" 815-822, 1981. 36. Timms, G. P. and R. S. Holmes. Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: evidence for a locus controlling the inducibility of the liver microsomal isozyme. Biochem Genet 19: 1223-1236, 1981. 37. Timms, G. P. and R. S. Holmes. Genetics of aldehyde dehydrogenase isozymes in the mouse: evidence for multiple loci and localization of Ahd-2 on chromosome 19. Genetics 97: 327-336, 1981. 38. Tottmar, S. O. C., H. Pettersson and K.-H. Kiessling. The subcellular distribution and properties of aldehyde dehydrogenases in rat liver.Biochem J 135: 515-523, 1973. 39. Williams, R. J., L. J. Berry and F. Beerstecher. Biochemical individuality III. Genototrophic factors in the etiology of alcoholism. Arch Biochem 23: 275-290, 1949. 40. Yamazaki, H., K. Nishiguchi, R. Miyamoto, Z. Ogita and S. Nakanishi. Activity and electropboretic profiles of liver aldehyde dehydrogenases from mice of inbred strains with different alcohol preference. Int J Biochem 15- 179-184, 1983.
0741-8329/85$3.00 + ,00
Biochemical and Genetic Studies on Mouse Aldehyde Dehydrogenases R O G E R S. H O L M E S , E L I Z A B E T H M. A L G A R A N D P E T E R B. M A T H E R
School o f Science, Griffith University, Nathan, 4111, Queensland, Australia
HOLMES, R. S., E. M. ALGAR AND P. B. MATHER. Biochemical and genetic studies on mouse aldehyde dehydrogenases. ALCOHOL 2(I) 67-71, 1985.mAldehyde dehydrogenase (AHD) exists as isozymes which are differentially distributed among tissues and subcellular fractions of mouse tissues. Genetic variants for liver mitochondrial (AHD-!) and cytoplasmic (AHD-2) isozymes have been used to map the responsible loci (Ahd-I and Ahd-2) on chromosomes 4 and 19 respectively. Evidence for a regulatory locus (Ahd-3r) controlling the inducibility of the mouse liver microsomal isozyme (AHD-3) has also been obtained. More recent studies have described genetic and biochemical evidence for three additional AHD isozymes: a stomach isozyme (AHD-4);another liver mitochondrial enzyme (AHD-5);and a testis isozyrne (AHD-6). Genetic analyses have indicated that AHD-4 and AHD-6 are encoded by distinct but closely linked loci on the mouse genome (Ahd-4 andAhd-6), which segregate independently ofAhd-I andAhd-2. Liver mitochondrial isozymes, AHD-I and AHD-5, have been purified to homogeneity using aff'mitychromatography. The very high affinity of AHD-5 for acetaldehyde suggests that this enzyme is predominantly responsible for acetaldehyde oxidation in mouse liver mitochondria. Mouse Aldehydedehydrogenase Liver S t o m a c h Testis
lsozymes
Alcohol
THE house mouse (Mus musculus) has served as a useful laboratory subject for alcohol research following early behavioural studies, which demonstrated differences in the amount of voluntary alcohol consumption by inbred strains of mice [23, 26, 39]. The organism has many attributes which make it most suitable for genetic research. Its small size, rapid reproductive rate and the availability of a large number of inbred strains including recombinant and congenic strains, render it as an ideal organism for genetic studies on behavioural, biochemical, pathological and physiological responses to alcohol. Moreover, the availability of biochemical and genetic markers for the 20 pairs of chromosomes found in the mouse karyotype provide an excellent base for gene mapping studies [7]. Many of these features have been exploited in recent studies examining the genetic basis for differential behavioural responses towards alcohol among inbred strains [3, 4, 9, 28]. Investigations have also been undertaken on the biochemical genetics of alcohol metabolism in the mouse. Metabolic studies on rodents have demonstrated that the liver provides the major site of ethanol oxidation, which proceeds via a three stage process: ethanol to acetaldehyde; acetaldehyde to acetate; and acetate to carbon dioxide and water, with the third stage being predominantly an extrahepatic process [ 18, 19, 30]. Liver contains at least three major enzyme systems capable of oxidizing alcohol in vitro: alcohol dehydrogenase (ADH); the microsomai ethanol oxidizing system and catalase [19,30]. It is generally recognized, however, that at ethanol concentrations less than 0.2 percent, at least 80 percent of alcohol oxidation proceeds via ADH [11,16]. Aldehyde dehydrogenase (AHD; E.C.1.2.1.3) catalyses the second step of ethanol metabolism and functions in the oxidation of acetaldehyde to acetate [19]. In contrast to
Acetaldehyde
Mitochondria
Cytoplasm
ADH which is localized exclusively in the cytoplasm, aldehyde dehydrogenase is differentially distributed between liver subcellular fractions [5, 12, 14, 17, 20, 24, 31, 35, 36-37, 40]. Mitochondrial and cytosolic rat liver aldehyde dehydrogenases have been partially purified and characterized kinetically, with both "low" and "high" Michaelis constant forms being described for both sources [14, 29, 33, 38]. Microsomal aldehyde dehydrogenase has also been purified from rat liver and shown to be biochemically distinct form of this enzyme [26]. Aldehyde dehydrogenase isozymes from other mammalian livers have been purified and characterized, including horse [6,8], human [10], and sheep [21] cytoplasmic and mitochondrial enzymes. The role of aldehyde dehydrogenases in the metabolism of ethanol has been demonstrated by the increase in alcohol sensitivity found in oriental populations which show a widespread lack of one of the isozymes [1]. The mitochondria have been considered to be the major site of acetaldehyde oxidation in man, however, more recent studies using human liver biopsy samples suggest that the liver cytosol may play a more significant role [15]. This communication reviews biochemical and genetic evidence concerning the isozymic status of aldehyde dehydrogenases in mouse tissues. Present findings suggest that at least six genetic loci encode aldehyde dehydrogenases in this organism, with the resultant isozymes being differentially distributed among liver subcellular fractions and in mouse stomach and testis extracts. TISSUEDISTRIBUTIONOF MOUSE ALDEHYDEDEHYDROGENASES The electrophoretic patterns for aldehyde dehydrogenase
67
68
HOLMES, ALGAR AND MATHER
4"
--In F
m
•
m 14J.LUJ
m
St
Lu K L
St
Lu K
AHD 3 AHD 5 AHD4 AHD 1 AHD 2
L
FIG. 1. Cellulose acetate zymogram and diagrammatic illustration of aldehyde dehydrogenase activity from tissues of 101/H inbred mice. Aldehyde dehydrogenase (AHD) activity is represented by closed box; non-specific oxidase activity by diagonally striped box; aldehyde oxidase activity by crossed box; and xanthine oxidase activity by vertically striped box. The tissue extracts examined included liver (L); kidney (K); lung (Lu); and stomach (St). AHD isozymes are designated at the side of the diagram. Buffer conditions were 25 mM Tris-glyeine (pH 8.5); electrophoresis was for 200 V for 20 minutes. Staining conditions are described in reference [12].
TABLE 1 BIOCHEMICAL VARIANTS FOR ALDEHYDE DEHYDROGENASES AMONG MOUSE INBRED STRAINS AHD-I (Ahd-l)*
Isozyme
(locus) Variant Strain Distribution
A
17 strains e.g., A/J C57BL/6J 101/H
B
38 strains e.g., BALB/c CBA/H DBA/J 129/MA
AHD-4 (Ahd-4)~.
AHD-2 (Ahd-2)t C
A
castaneus 52 strains
e.g., A/J BALB/c C57BL/6J DBA/J
B
C
3 strains castaneus NZB/BI NZC/BI NZW/B1
A
B
53 strains 3 strains e.g., A/J 'Danish' BALB/c LIII C57BI.,/6J S L / N i A DBA/J
AHD-6 (Ahd-6) § A
B
37 strains 5 strains e.g., ddN e.g., A/J GRS/A BALB/c C57BL/6J DBA/J
*Eiectrophoretic variants described in references [12,25]. ?Electrophorctic variants described in references [25,28]. :l:Electrophoreticvariants described in reference [25]. §Activity variants described in reference [25].
(AHD) isozymes extracted from various tissues of 101/H inbred mice are shown in Fig. 1. Four AHD activity zones were resolved for liver extracts, as well as several oxidase zones, that exhibited activity in the absence of coenzyme. Subcellular fractionation studies have shown that these enzymes are differentially distributed among mitochondrial (AHD-I and AHD-5 isozymes); cytoplasmic (AHD-2); and microsomai (AHD-3) liver preparations [2, 17, 36, 37]. Kidney extracts exhibited two forms of activity which corresponded in terms of electrophoretic migration to the AHD-I and AHD-3 liver isozymes. In contrast, mouse lung extracts showed only a single form of activity, coincident with AHD-5 on the cellulose acetate zymogram. Mouse stomach extracts exhibited an electrophoretically distinct form of aldehyde dehydrogenase activity (designated AHD-4).
GENETIC VARIANTS FOR ALDEHYDE DEHYDROGENASES
Liver Mitochondrial Aldehyde Dehydrogenase (AHD-I ) Three electrophoretic variants for mouse liver mitochondrial aldehyde dehydrogenase (AHD-1 isozyme) have been described [12,25]. The AHD-1A and 1B all¢lic isozymes are widely distributed among common inbred strains, and are found, for example, in C57BL/6J and BALB/c mice respectively (Table l). In contrast, AHD-IC was observed only in castaneus mice, an inbred strain derived from a South-East Asian subspecies of this organism. Electrophoretic phenotypes for F1 mice, heterozygous for the proposed locus encoding these ailelic forms of AHD-1 (designated Ahd-l), showed three-banded patterns of activity, which is consistent with the dimeric subunit structure
MOUSE A L D E H Y D E D E H Y D R O G E N A S E S
TESTIS
69
(+)
STOMACH
AHD-5 AHD-6 AHD-2
="
123
'
- AHD 4
123
FIG. 2. Cellulose acetate zymograms of testis and stomach aldehyde dehydrogenase activity from inbred mice, LIII (i) and ddN (3), and their F~(ddNxLIII) hybrid (2). AHD isozyme designation is based on previous studies [25] and is shown at the side of the zymograms. The fastest anodally migrating zone of activity for the testis zymogram was non-specific oxidase activity. The cathodally migrating minor zone of activity for LIII (1) stomach extracts was recognized as stomach alcohol dehydrogenase C2. Note the activity variation for AHD-6 in testis extracts and the electrophoretic variants for AHD-4 in stomach extracts. Buffer conditions for stomach AHD electrophoresis were 25 mM Tris-glycine (pH 8.5), 300 V for 20 minutes; for testis AHD electrophoresis, buffer conditions were 75 mM Tris-citrate (pH 7.0), 150 V for 90 minutes. Staining conditions are described in reference [12].
reported for this enzyme [2]. Yamazaki and coworkers [40] have recently confirmed these findings for two inbred strains of mice (C57BL/6J and DBA/2), which exhibited electrophoretically distinct forms for one of the mouse liver mitochondrial aldehyde dehydrogenases (AHD-I).
Liver Cytosolic Aldehyde Dehydrogenase (AHD-2) Three electrophoretic variants have also been reported for mouse liver cytosolic aldehyde dehydrogenase (AHD-2) • [25,37]. The AHD-2A allelic form represented the predominant phenotype among the 56 inbred strains examined thus far, and was observed in all strains with the exception of the NZ and castaneus strains (Table I). New Zealand mice (NZB, NZC and NZW strains) exhibited a high activity, electrophoretically distinct form of AHD-2 (AHD-2B), which migrated on the anodal side of the common AHD-2A allelic isozyme [36], whereas castaneus mice showed a low activity AHD-2 phenotype, migrating more rapidly towards the cathode, as compared with AHD-2A [24].
AHD-4A) was coincident with the ddN pattern of activity, whereas the variant phenotype (AHD-2B) was found in the LIII and SL/NiA strains (Table 1).
Testis Aldehyde Dehydrogenase (AHD-6) Evidence has been recently described for a testis-specific form of aldehyde dehydrogenase, designated AHD-6 [25]. Figure 2 illustrates the activity variation observed for the AHD-6 isozyme between LIII and ddN mouse strains, ddN male mice exhibited a much higher activity for AHD-6, as compared with LIII males, whereas Fl(ddNxLIII) males showed an intermediate activity for this isozyme. In contrast, no detectable activity variation was observed for the other testis aldehyde dehydrogenases, AHD-2 and AHD-5. The predominant phenotype observed among 42 inbred strains was similar to that of the LIII mice (designated AHD-6A), with only 5 strains resembling that of the ddN high activity phenotypes (AHD-6B) (Table 1). GENETIC ANALYSES AND GENE MAPPING STUDIES
Stomach Aldehyde Dehydrogenase (AHD-4) A cellulose acetate zymogram illustrating the dectrophoretic variation of stomach aldehyde dehydrogenase (AHD-4) is shown in Fig. 2. LIII mice exhibited a faster migrating form as compared with ddN inbred mice, with Fl(ddNxLIII) animals showing a three-banded pattern. The distribution of these electrophoretic variants for stomach AHD have been examined [25]. The predominant phenotype observed in 52 of the 56 inbred strains examined (designated
Genetic analyses and gene mapping studies have been undertaken using the previously described variants for mouse liver mitochondrial (AHD-I) and cytosolic (AHD-2) aldehyde dehydrogenases [12,37]. These results have shown that the electrophoretic variants, in each case, behaved as though encoded by codominant alleles at the respective loci, Ahd-I and Ahd-2, which are separately localized on the mouse genome on chromosomes 4 and 19 respectively. More recent studies have examined the genetics of the stomach
70 (AHD-4) and testis (AHD-6) specific forms, using the variant phenotypes previously described, as biochemical markers for the proposed respective loci involved (Ahd-# and Ahd-6) [24]. Segregation analyses showed that the variant phenotypes inherited as though encoded by codominant alleles at these loci. Moreover, the proposed Ahd-4 and Ahd-6 loci segregated independently of Ahd-I and Ahd-2, but showed a 7 percent recombination frequency, which is consistent with Ahd-4 and Ahd-6 being separately, but closely, localized on the mouse genome. Variation in the inducibility of the liver microsomal isozyme (AHD-3) by phenobarbital administration has been reported among mouse inbred strains. The phenotypes were inherited in a normal Mendelian fashion, with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r) [36]. BIOCHEMISTRYOF MOUSEALDEHYDEDEHYDROGENASES The biochemical properties of mouse liver mitochondrial aldehyde isozymes, designated AHD-I and AHD-5 on the basis of previous biochemical genetic analyses [ 12,25] have been recently described [2]. These isozymes have been isolated in a highly purified state from extracts of mouse liver mitochondria and characterized by both structural and kinetic analyses. These studies have fh'mly established the presence of two biochemically and kinetically distinct aldehyde dehydrogenase isozymes in mouse liver mitochondria. The subunit sizes of the enzymes were determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis to be 63,000 for AHD-I and 49,000 for AHD-5. Moreover, gel exclusion chromatography indicated that both isozymes are dimers, although there was some evidence for a monomeric form of AHD-I as well. The mouse liver aldehyde dehydrngenase isozymes also exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-I, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate (1.4 mM and 0.78 mM respectively), whereas AHD-5 exhibited a low Km value, 0.2 ~M with acetaldehyde. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-I, pH range 6.5-7.5, AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50 percent inhibition occurring at 13/zM and 0.1/~M for the AHD-I and AHD-5 isozymes respectively. These studies have strongly supported the proposition that AHD-5 is predominantly responsible for mitochondrial acetaldehyde oxidation, in vivo. Sheppard and coworkers [32] have previously described the partial purification and kinetic characterization of aldehyde dehydrogenases from livers of C57BL/6J and DBA/2J inbred mice and their F1 offspring. These enzymes were prepared from whole liver acetone powder extracts and it was not possible to conclude which of the aldehyde dehydrogenase isozymes were being investigated in this study. Little and Peterson [20] reported at this meeting on the partial purification and kinetic properties of mouse liver mitochondrial and cytoplasmic aldehyde dehydrogenases from eight inbred strains of mice. Their results for the mitochondrial isozymes confirmed the existence of separate low and high Km terms corresponding to AHD-5 and AHD-I isozymes respectively. In addition, their study suggested that distinct 'low-Kin' and 'high-Km' forms of aldehyde dehydrogenase also occur within the liver cytoplasm. A proposal was presented correlating genotype and ethanol elimination rates on the basis of variations in the kinetic charac-
HOLMES, ALGAR AND MATHER teristics of the 'low-Km' cytosolic isozyme occurring between inbred strains. DISCUSSION Phenotypic differences for most of the aldehyde dehydrogenase isozymes have been observed among inbred strains of mice. Electrophoretic variants have been reported for a liver mitochondrial isozyme (AHD-I); a liver cytosolic isozyme (AHD-2) (for which one of the variant forms (AHD-2B) exists as a high activity phenotype as well); and the stomach-specific aldehyde dehydrogenase (AHD-4) [12, 25, 37]. Genetic analyses, using these variants as biochemical markers for the corresponding loci, have provided evidence that the genes encoding these enzymes (designated Ahd-l ; Ahd-2; and Ahd-4) are unlinked. Variation in the inducibility of the liver microsomal isozyme (AHD-3) by phenobarbital administration has been also described and a regulatory locus (Ahd-3r) proposed [36]. Moreover, activity variants for the testis specific form of aldehyde dehydrogenase (AHD-6) have been found among mouse inbred strains [25]. Genetic analyses using this activity variant as a biochemical marker for the proposed gene controlling this differential phenotype (designated Ahd-6) have indicated that this locus is inherited in a normal Mendelian fashion and is distinct from the other Ahd loci, although evidence for close linkage withAhd-4 was reported [25]. Thus, in contrast to the presently mapped loci encoding alcohol dehydrogenase isozymes in the mouse, which exist as a gene cluster [13], aldehyde dehydrogenase loci are separately localized on the mouse genome. Biochemical studies on purified preparations of mouse liver mitochondrial aldehyde dehydrogenases have demonstrated that AHD-5 is the most likely catalyst of acetaldehyde oxidation within mouse liver mitochondria [2]. It should be noted, however, that in contrast to rat liver, mouse liver aldehyde dehydrogenase activity is predominantly localized in the cytosol [35]. Studies by Smolen and coworkers [34] have suggested that the mouse cytosolic enzyme is important in acetaldehyde metabolism. In addition, a report at this meeting of the International Society for Biomedical Research on Alcoholism by Little and Peterson [21] supports this view and has indicated that there exists in both liver mitochondria and cytosol, "low-Km" aldehyde dehydrogenases, capable of oxidizing physiological concentrations of acetaldehyde. The relative metabolic capacities of these aldehyde dehydrogenases, under in vivo conditions remain to be determined.
ACKNOWLEDGEMENTS
This research was supported by grants from the Australian Associated Brewers, the National Health and Medical Research Council of Australia and the Australian Research Grants Scheme. We are grateful to the following for supplying mice used in these studies: Dr. J. Hilgers and Dr. J. Hilkens, Netherlands Cancer Institute, Amsterdam; Dr. S. Imal, Nara Medical University, Japan; Dr. V. Chapman, Roswell Memorial Institute, Buffalo; Dr. M. Lyon, M.R.C. Radiobiology Unit, Harwell, England; Dr. E. Eicher. The Jackson Laboratory, Maine, USA.
MOUSE ALDEHYDE DEHYDROGENASES
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