Differences in charge and kinetic properties of alcohol dehydrogenase 4 from C57BL/6 mice compared to other inbred strains are associated with a cysteine120 to arginine120 substitution.

Alcohol dehydrogenase class IV (ADH4) participates in retinol metabolism and is expressed primarily in ocular, digestive, and reproductive tissues of the mouse. A naturally occurring genetic variant in C57BL/6J mice results in a faster migrating ADH4 enzyme during electrophoresis when compared to other non-C57BJ/6J strains. The C57BL/6 ADH4 gene coding sequence is found to have two nucleotide substitutions when compared to the gene from C3HeB/FeJ mice. The substitution in exon 5 encodes Arg120 instead of Cys120 in C57BL/6 ADH4 polypeptide; that would account for the protein electrophoretic phenotype. Arg120 is present in all published mammalian ADH4 sequences but is only in a limited number of mouse strains. The Arg120 residue is part of the outer loop of the substrate binding pocket and appears to have an effect on the affinity of the enzyme for several substrates.

Molecular analysis of genetic differences among inbred mouse strains controlling tissue expression pattern of alcohol dehydrogenase 4.

The ADH gene family in vertebrates is composed of at least seven distinct classes based upon sequence comparisons and enzyme properties. The Adh4 gene product may play an important role in differentiation and development because of its capacity to metabolize retinol to retinoic acid. Allelic gene differences exist among inbred mouse strains which control structure and tissue-specific regulation of Adh4. C57BL/6 mice are unique and have no detectable ADH4 enzyme activity in epididymis and low levels in seminal vesicle, ovary and uterus compared to other strains. C57BL/6 mice express Adh4 in stomach at levels similar to other strains. The goal of this research was to investigate this genetic variation at the molecular level. Northern analysis revealed that the content of ADH4 mRNA in tissues correlate with the enzyme expression pattern. Interestingly, C57BL/6 mice express an ADH4 mRNA in stomach which is smaller than expressed in C3H and other mice. An analysis of the 5'- and 3'-ends of the mRNA using RACE analysis determined that the ADH4 mRNA in C57BL/6 mice is truncated in the 3'-untranslated region. Sequence analysis of RACE products showed that the truncation is due to a single nucleotide mutation which produces an early polyadenylation signal. Additional RACE and Northern analysis revealed that at least five different polyadenylation sites are used in the Adh4 gene. Using 3'-end polymorphisms found between C57BL/6 and C3H strains and RT-PCR, it was shown that the lack of expression in epididymis in C57BL/6 mice is cis-acting in F(1) hybrid animals. The DNA sequence of the proximal promoter (-600/+42 nt) was determined in several mouse strains differing in tissue-specific expression patterns and did not reveal any nucleotide substitutions correlating with expression pattern suggesting further upstream or downstream sequences may be involved.

Cytochrome P450 2E1 (CYP2E1)-dependent production of a 37-kDa acetaldehyde-protein adduct in the rat liver.

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) has been shown to be involved in the metabolism of both ethanol and acetaldehyde. Acetaldehyde, produced from ethanol metabolism, is highly reactive and can form various protein adducts. In this study, we investigated the role of CYP2E1 in the production of a 37-kDa acetaldehyde-protein adduct. Rats were pairfed an isocaloric control or an alcohol liquid diet with and without cotreatment of YH439, an inhibitor of CYP2E1 gene transcription, for 4 weeks. The soluble proteins from rat livers of each group were separated on SDS-polyacrylamide gels followed by immunoblot analysis using specific antibodies against the 37-kDa protein acetaldehyde adduct. In addition, catalytic activities of the enzymes involved in alcohol and acetaldehyde metabolism were measured and compared with the adduct level. Immunoblot analysis revealed that the 37-kDa adduct, absent in the pair-fed control, was evident in alcohol-fed rats but markedly reduced by YH439 treatment. Immunohistochemical analysis also showed that the 37-kDa adduct is predominantly localized in the pericentral region of the liver where CYP2E1 protein is mainly expressed. This staining disappeared in the pericentral region after YH439 treatment. The levels of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase isozymes were unchanged after YH439 treatment. However, the level of the 37-kDa protein adduct positively correlated with the hepatic content of P4502E1. These data indicate that the 37-kDa adduct could be produced by CYP2E1-mediated ethanol metabolism in addition to the ADH-dependent formation.

Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

Genetic mapping of a possible new alcohol dehydrogenase sequence to mouse chromosome 3 at the Adh-1/Adh-3 complex.

Southern blot analysis of mouse genomic DNA reveals two EcoRI fragments which faintly hybridize to mouse Adh-1 cDNA and are not part of the Adh-1 gene. These fragments were isolated from agarose gels, cloned, and characterized. Sequence analysis of the 2.1-kb EcoRI fragment suggests that it is likely a pseudogene since it does not contain a long open reading frame. However; the 2.0-kb EcoRI fragment contains a coding sequence with a long open reading frame which corresponds to exon 6 of the mouse Adh-1 gene. Comparison of the coding sequence with other known ADHs suggests that the sequence has diverged sufficiently from any currently known class of ADH to be a possible distinct class. Further confirmation awaits analysis of currently available genomic clones. Using these sequences as probe, restriction fragment length polymorphisms were identified for each sequence between C57Bl/6J and DBA/2J inbred mouse strains. The strain distribution pattern for these allelic differences was determined among the B x D recombinant inbred strains. This analysis revealed that the 2.1-kb EcoRI sequence is located on chromosome 3 but at a distance from the Adh-1/Adh-3 complex as previously reported. However, the new polymorphism identified in the 2.0-kb EcoRI fragment enabled this sequence to be mapped at the Adh-1/Adh-3 complex.

Ten kilobases of 5'-flanking region confers proper regulation of the mouse alcohol dehydrogenase-1 (Adh-1) gene in kidney and adrenal of transgenic mice.

The expression profile of the mouse Adh-1 gene, which encodes class I alcohol dehydrogenase enzyme (ADH), is complex and includes tissue specificity and differential hormone responsiveness. Whereas kidney Adh-1 transcription rate is stimulated six- to sevenfold by testosterone treatment, adrenal gland ADH-1 mRNA is reduced to less than 5% of control level within 18 h following hormone administration. Androgen receptor is required for both responses since neither occurs in Tfm mutant mice lacking receptor. Hormonal and tissue-specific aspects of Adh-1 regulation were studied in transgenic mice harboring either of two constructs containing either -2.5 kb or -10 kb of 5'-flanking sequence attached to an Adh-1 minigene. The minigene transcript was expressed in kidney and adrenal tissues, but not liver, in five independent lines harboring a transgene with -2.5 kb of 5'-flanking sequence. Androgen treatment repressed the level of the minigene transcript in adrenal gland, but did not cause induction in kidney. In four lines of transgenic mice carrying the construct with -10 kb of 5'-flanking sequence, the minigene transcript was both repressed in adrenal and induced in kidney by testosterone. These lines have no detectable transgene expression in liver tissue. The -10 kb region in the mouse Adh-1 gene contains necessary controlling regions for proper tissue expression and hormonal regulation in kidney and adrenal; however, this region does not contain all essential elements necessary for expression in liver.

Molecular basis of the alcohol dehydrogenase-negative deer mouse. Evidence for deletion of the gene for class I enzyme and identification of a possible new enzyme class.

The molecular basis of the alcohol dehydrogenase (ADH)-negative deer mouse (Peromyscus maniculatus) has been investigated. Several classes of mammalian ADHs have been recognized based upon biochemical and structural properties. ADH cDNA clones identified by hybridization to a mouse class I ADH cDNA clone were obtained from a deer mouse ADH-positive liver cDNA library. This cDNA has been identified as being a class I sequence and represents the deer mouse Adh-1 gene. An additional cDNA sequence identified in both the ADH-positive and -negative deer mouse cDNA libraries was identified by weak cross-hybridization to the mouse cDNA. This cDNA encodes an amino acid sequence representing a new class of mammalian ADH, and the deer mouse gene for this ADH is named Adh-2. ADH-negative deer mice do not produce mRNA, that is detected by the Adh-1 cDNA probe. However, both stocks of deer mice produce high levels of Adh-2 mRNA in liver. Southern analysis using an essentially full-length Adh-1 cDNA probe has shown that the Adh-1 gene is deleted in the ADH-negative mice. Biochemical analysis of enzyme activity suggests at least three ADH polypeptides are expressed in different tissues and have somewhat different substrate specificities, as in the mouse.

Mouse ethanol-inducible cytochrome P-450 (P450IIE1). Characterization of cDNA clones and testosterone induction in kidney tissue.

A mouse cDNA clone for the ethanol-inducible cytochrome P-450 (P450IIE1) was obtained by screening a liver cDNA library with an oligonucleotide representing a consensus sequence found in the orthologous rat, human, and rabbit sequences. The protein sequence deduced from the cDNA sequence had an identity of 93% to rat, 81% to rabbit, and 76% to human orthologous sequences. The highest levels of P450IIE1 mRNA were found in liver of both sexes, and male kidney. Developmentally, C57BL/6 female liver P450IIE1 mRNA was detectable 1 day postpartum and reached steady-state levels in animals approximately 16-20 days of age. Kidney and adrenal gland P450IIE1 mRNA was found to be induced 25-50-fold and 4-fold by testosterone treatment, respectively, and the level in both tissues reached maximum levels between 12 h and 2 days after treatment. Nuclear run-on experiments demonstrated that testosterone treatment for 24-48 h resulted in a slight transcriptional activation of the P450IIE1 gene in the kidney. However, the increase in transcription rate was far below the increase in mRNA level, suggesting that much of the induction occurs by posttranscriptional mechanisms. This process requires the androgen receptor since mutant Tfm mice lacking receptor are not inducible.

Tissue-specific genetic variation in the level of mouse alcohol dehydrogenase is controlled transcriptionally in kidney and posttranscriptionally in liver.

Tissue-specific genetic variation in expression of the alcohol dehydrogenase, encoded by the Adh-1 gene, is found between C57BL/6J (B6) mice and B6.S congenic mice. B6.S mice contain a variant Adh-1 allele derived from a wild Danish strain in a B6 genetic background. B6 mice have nearly twice the alcohol dehydrogenase activity in liver but less than half the activity in kidney as B6.S mice. These tissue-specific genetic changes in alcohol dehydrogenase expression are manifest at the level of Adh-1-encoded mRNA. The regulatory site(s) involved act cis in both kidney and liver. These strains also differ in the extent to which androgen induces mRNA encoded by kidney Adh-1, with androgen increasing these levels 17-fold and 7.4-fold in the B6 and B6.S kidney, respectively. To identify the regulatory mechanism(s) underlying this strain variation in Adh-1 transcription in the B6 and B6.S kidney, liver, and androgen-induced kidney. For both uninduced and induced kidney, a difference in the transcription rate alone accounts for the strain difference in mRNA concentration. In contrast, because the Adh-1 transcription rate in liver does not differ significantly between B6 and B6.S mice, strain-specific variation in posttranscriptional regulation must be operative. Taken together these results indicate that the variation in Adh-1 expression between B6 and B6.S mice results from changes in both transcriptional and posttranscriptional control, and these controls are differentially operative in kidney and liver.

Mechanism of induction of mouse kidney alcohol dehydrogenase by androgen. Androgen-induced stimulation of transcription of the Adh-1 gene.

The three alcohol dehydrogenase genes in the mouse are subject to developmental, hormonal, and genetic control as revealed by variation in expression among inbred strains. The primary purpose of this study was to determine the mechanism by which androgen regulates the expression of the Adh-1 gene in kidney. In addition, the fold-induction in several inbred strains was examined in a search for possible genetic variation in the induction process, and Adh-1 expression in several tissues was studied. Testosterone treatment of female mice results in a 10-12-fold increase in alcohol dehydrogenase activity and a corresponding increase in the rate of enzyme synthesis accounts for this induction. The induction of Adh-1 mRNA after androgen treatment is sufficient to account for the induction in enzyme synthesis. An increase in Adh-1 transcription accounts for a substantial part of the increase in Adh-1 mRNA level following androgen simulation. This conclusion was reached using nuclear "run-on" assays, in vivo labeling, and a kinetic analysis of Adh-1 mRNA accumulation and loss in response to hormone. This induction requires androgen receptor. The fold induction by androgen of Adh-1 mRNA is similar in eight inbred mouse strains. There is almost a 100-fold variation in Adh-1 mRNA concentrations among various mouse tissues. Tissues with lowest level of expression are brain and heart, while liver and adrenals have the highest content of Adh-1 mRNA.

Molecular analysis of mouse alcohol dehydrogenase: nucleotide sequence of the Adh-1 gene and genetic mapping of a related nucleotide sequence to chromosome 3.

The mouse has three genes (Adh) encoding alcohol dehydrogenase (ADH) enzymes of different tissue specificity and catalytic properties. Identified regulatory loci are known to affect the expression of Adh-1 and Adh-3, which are closely linked on chromosome 3. The Adh-1 gene product is expressed predominantly in liver, and its mRNA product is androgen-inducible in kidney. In this study, genomic clones of Adh-1 were obtained from a Balb/cJ DNA library. The nucleotide sequences of all exons, intron/exon boundaries and 5'- and 3'-flanking regions were obtained. The gene spans nearly 13 kb and is divided into nine exons and eight introns. The transcription start point of this gene was determined by S1 nuclease mapping studies and presumptive regulatory regions in the 5'-flanking regions were identified, including a TATA box and a glucocorticoid-responsive element. A restriction fragment length polymorphism in the Adh-1 gene was identified among inbred strains and mapped at the [Adh-1, Adh-3] complex on chromosome 3. An additional 'Adh-like' sequence in the genome was also mapped to chromosome 3 approx. 9 centiMorgans from Adh-1.

Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis.

A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined.

Hepatotoxicity due to allyl alcohol in deermice depends on alcohol dehydrogenase.

The role of alcohol dehydrogenase in the hepatic necrosis due to allyl alcohol was studied in two strains of the deermouse, Peromyscus maniculatus. Mice of the alcohol dehydrogenase-negative (AdhN) strain which lack alcohol dehydrogenase activity were resistant to allyl alcohol toxicity. In contrast, dose-dependent necrosis of periportal regions of the liver and increases in plasma levels of lactate dehydrogenase, sorbitol dehydrogenase and SGOT were observed in plasma from alcohol dehydrogenase-positive deermice (AdhF) 24 hr following administration of allyl alcohol (21 to 84 mg per kg). Half-maximal damage to periportal areas was observed with about 52 mg per kg allyl alcohol. Thus, these data demonstrate that metabolism of allyl alcohol to acrolein by alcohol dehydrogenase is obligatory for the hepatotoxicity of allyl alcohol.

Role of alcohol dehydrogenase in the swift increase in alcohol metabolism (SIAM). Studies with deer mice deficient in alcohol dehydrogenase.

Previous studies have shown that rates of ethanol metabolism increase markedly 2-4 hr after the administration of ethanol in rats and in four inbred strains of mice. This phenomenon, called the swift increase in alcohol metabolism (SIAM), also exists in humans. To determine whether alcohol dehydrogenase (ADH) is necessary for the SIAM response, we compared ethanol metabolism in two strains of the deer mouse, Peromyscus maniculatus. One strain lacks alcohol dehydrogenase (ADH-negative), whereas the other strain has normal ADH levels (ADH-positive). Rates of ethanol elimination were determined after a single intraperitoneal injection of ethanol at different doses (0.5 to 3.0 g/kg) and also after both strains were exposed to various levels of ethanol vapor for 4 hr. The ADH-positive strain exhibited up to a 72% increase in the rate of ethanol elimination after exposure to ethanol vapor compared to the ethanol-injected controls. In contrast, treatment with ethanol vapor did not alter rates of ethanol elimination in the ADH-negative strain. These data demonstrate clearly that ADH is required for SIAM in the deer mouse. In addition, in both the ADH-positive and the ADH-negative strain, rates of ethanol elimination increased in both the ethanol-injected and vapor-treated groups 2- to 3-fold as the dose of ethanol was increased from 100 to 500 mg/100 ml. Thus, it is concluded that this "concentration effect" of ethanol on rates of ethanol metabolism does not involve ADH in the . deer mouse.

Non-alcohol dehydrogenase-mediated metabolism of methylazoxymethanol in the deer mouse, Peromyscus maniculatus.

The concept that alcohol dehydrogenase (ADH) is involved in the metabolism of methylazoxymethanol (MAM) was examined in a model consisting of two strains of the deer mouse, Peromyscus maniculatus, one of which has a normal complement of the enzyme [ADH(+)], and the other, which completely lacks it [ADH(-)]. Both the ADH(+) and the ADH(-) strains rapidly metabolized [14C]MAM, administered in the form of the acetic acid ester, [14C] MAMOAc , to 14CO2, and the rates and extents of metabolism were virtually identical. Determination of O6-methylguanine and 7-methylguanine in liver DNA 6 and 24 hr after MAMOAc (25 mg/kg) administration showed that the levels of DNA methylation induced by the carcinogen were not significantly different in the two strains, indicating that both are capable of the metabolic activation of MAM to methylating species. Pyrazole, a potent inhibitor of ADH, inhibited MAM metabolism as well as liver DNA methylation in the ADH(+) strain; however similar inhibition of these processes also occurred in the ADH(-) strain. 3-Methylpyrazole, a weak or noninhibitor of ADH, also decreased the levels of MAM metabolism in both the ADH(+) and the ADH(-) strains. From these results, we conclude that ADH is not obligatory either in the metabolism or in the metabolic activation of MAM. As a possible alternative to ADH, liver microsomes were examined for their ability to metabolize MAM. In the presence of a NADPH-generating system, liver microsomes from both strains converted [14C]MAM to 14CH3OH and 14CH2O , although liver microsomes from the ADH(-) strain were more active in this respect. The microsomal metabolism was sensitive to inhibition by CO as well as to inhibition by pyrazole and 3-methylpyrazole.

Ethanol metabolism in vivo by the microsomal ethanol-oxidizing system in deermice lacking alcohol dehydrogenase (ADH).

To assess the importance of non-ADH ethanol metabolism, ADH-negative and ADH-positive deermice were fed liquid diets containing ethanol or isocaloric carbohydrate for 2-4 weeks. Blood ethanol disappearance rate increased significantly after chronic ethanol feeding in both strains. Although at low ethanol concentrations (between 5 and 10 mM) there was no significant difference between ethanol-fed and pair-fed control animals, at high ethanol concentrations (between 40 and 70 mM) blood ethanol elimination rates were increased significantly after chronic ethanol feeding in both ADH-positive and ADH-negative animals. There was no significant effect of the catalase inhibitor 3-amino-1,2,4-triazole on the ethanol elimination/rates in both strains. Whereas catalase and ADH activities were not altered after chronic ethanol treatment, the activity of the microsomal ethanol-oxidizing system (MEOS) was enhanced three to four times in both strains, and microsomal cytochrome P-450 content was also increased significantly. When MEOS activity was expressed per cytochrome P-450 content, it was higher in ADH-negative than in ADH-positive animals, and it increased after ethanol administration. When microsomal proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ethanol-fed animals had a distinct band which reflected the increase in microsomal cytochrome P-450 content and seemed to reflect a unique form of cytochrome P-450 induced by ethanol. Thus, despite the absence of the ADH pathway, a large amount of ethanol was metabolized by MEOS in ADH-negative deermice; this was associated with increased blood ethanol elimination rates, enhanced MEOS activity, and quantitative and qualitative changes of cytochrome P-450.

Linkage relationships among eleven biochemical loci in Peromyscus.

Interspecific F1 hybrids of Peromyscus maniculatus (deermice) and P. polionotus (oldfield mice) were backcrossed to P. maniculatus. Backcross progeny were electrophoretically typed for 11 variant protein markers: albumin, transferrin, leucine aminopeptidase, amylase, 6-phosphogluconate dehydrogenase, nucleoside phosphorylase, dipeptidase, tripeptidase, glutamate oxaloacetate transaminase, alcohol dehydrogenase, and sorbitol dehydrogenase. Genetic variation for each protein was attributed to a single autosomal locus. The alcohol dehydrogenase (Adh), salivary amylase (Amy), and albumin (Alb) loci appeared to be linked in the sequence of Adh-11.5 cM-Amy-33.3 cM-Alb. The tripeptidase locus, Pep-2, also may be loosely linked to Alb in this group. Variants at all other loci assorted independently. These and other known linkage relationships in Peromyscus correspond closely to those of the house mouse, Mus musculus. The available evidence in Peromyscus further supports the concept of linkage conservation by natural selection.

Turnover of cytoplasmic and mitochondrial aspartate aminotransferase isozymes in mouse liver and transplantable hepatomas.

Activities of the cytoplasmic and mitochondrial isozymes of aspartate aminotransferase (aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, AAT) in transplantable mouse hepatomas BW7756 and H-4 are reduced when compared to normal adult liver. Both proteins have been purified to homogeneity from a single preparation of mouse liver and monospecific antibodies raised to each isozyme. By quantitative immunotitration analysis, the activity of each isozyme in liver and hepatoma has been shown to correlate with levels of immunoprecipitable protein. Furthermore, for each isozyme, the liver versus hepatoma species is indistinguishable by heat inactivation kinetics, Km's for substrates, and molecular weights. Thus, the reduction of mitochondrial and cytoplasmic AAT activities in hepatoma tissue is due not to alterations in the catalytic activity of the enzyme molecules, but to a decrease in the number of enzyme molecules present. Turnover of the isozymes was studied in liver and hepatoma tissue using in vivo radiolabeling and specific immunoprecipitation techniques. The cytoplasmic isozyme has a similar rate of degradation in liver and hepatoma, while the rate of synthesis of this isozyme in hepatoma is approximately tenfold less than in liver. The mitochondrial isozyme is also degraded at a similar rate in both tissues, but the rate of synthesis is sixfold greater in normal liver tissue than in hepatoma. It is concluded that decreased amounts of both isozymes in hepatoma as compared to liver are the result of a reduction in the rate of synthesis of each isozyme without any change in the rate of degradation.

Genetics, biochemistry, and developmental regulation of alcohol dehydrogenase in peromyscus and laboratory mice.

The ADH-negative deermouse and the strain-specific variation in level of ADH in liver tissue of inbred mice represent useful systems to investigate gene regulation at a molecular level. Few systems have advanced to the state where specific hybridization probes can be employed to determine if loci which control the amount of a protein in a tissue do so by controlling the concentration of a specific messenger RNA [Owerbach et al, 1981; Tukey et al, 1981]. The relatively high level of ADH in mouse liver and the tissue and developmental specificity of expression of this enzyme should provide useful tools for use in identifying specific ADH cDNA clones in a liver cDNA library Norgard et al, 1980]. In addition, the deermouse ADH is immunologically cross-reactive with the mouse enzyme [Felder, unpublished observation]; the availability of ADH-negative and ADH-positive deermice may also prove useful in developing successful cloning strategies.

Alcohol dehydrogenase (ADH) independent ethanol metabolism in deermice lacking ADH.

To assess the importance of non-ADH ethanol metabolism, ADH-negative (ADH-) and ADH-positive (ADH+) deermice were fed for 2-4 weeks liquid diets containing ethanol or isocaloric carbohydrate. They consumed progressively increasing amounts of ethanol. Blood ethanol clearance (BEC) increased significantly in both strains. It remained almost unchanged at low ethanol concentrations (5-10 mM), but at high levels (40-70 mM) BEC was strikingly increased with significant differences between ethanol-fed and control animals. Kinetics were consistent with the activity of a non-ADH high Km system such as the microsomal ethanol-oxidizing system (MEOS). Naive ADH- had a more active MEOS and more abundant SER than naive ADH+. After ethanol feeding, MEOS was increased 3-4 times in both strains. There was striking proliferation of SER and cytochrome P-450 was enhanced significantly. Expressed per P-450, MEOS activity was higher in ADH- than ADH+. Thus despite absence of ADH, ADH- deermice can consume large amounts of ethanol: this is associated with increased BEC, SER proliferation, enhanced MEOS activity and quantitative and qualitative changes of cytochrome P-450.