Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Variation in amount of enzyme protein in natural populations

Among strains of Drosophila melanogaster each derived from a single fertilized female taken from natural populations, there is variation in both alcohol dehydrogenase (ADH) activity and the amount of ADH protein. The correlation between ADH activity and number of molecules over all strains examined is 0.87 or 0.96 in late third instar larvae depending on whether the substrate is 2-propanol or ethanol. With respect to the two common electrophoretic allozymic forms, F and S, segregating in these populations, the FF strains on the whole have higher ADH activities and numbers of ADH molecules than the SS strains. Over all strains examined, enzyme extracts from FF strains have a mean catalytic efficiency per enzyme molecule higher than that of enzyme extracts from SS strains when ethanol is the substrate, and much higher when 2-propanol is the substrate. One FF strain had an ADH activity/ADH protein ratio characteristic of SS strains.     

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA^r) was selected from 6 × 10⁸ suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA^r cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA^r cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Genetic considerations in the effects of ethanol in mice. I. Genotype-dependent alterations in alcohol dehydrogenase activity

Most genetic studies on individual and racial differences in sensitivity to alcohol intoxication have concentrated on genetic variations associated with structural genes for the enzymes involved in alcohol metabolism, including alcohol dehydrogenase (ADH; E.C. 1.1.1.1). We studied the ethanol-induced regulation of ADH following chronic administration of ethanol in mice. Newly weaned males from six inbred strains (BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, S.W., and 129/ReJ) were subjected to ethanol administration. Alterations in the level of liver ADH activity, relative to matched littermate controls, were evaluated. The change in ADH activity was found to be strain (genotype) specific, which may explain the contradictory results in the literature. Strains which showed induction of ADH activity, in general, reflected a strain-specific time-dependent profile. Strains which showed repression, however, were independent in the degree of repression to the duration of ethanol exposure. Such variable, ethanol-induced regulatory responses (induction/repression) in ADH activity of different genotypes may account for individual and population variations in response to alcohol. Additional work, however, is needed to establish the molecular bases of ADH inducibility and its specific role in relative susceptibility to alcohols.     

NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.     

Transcriptional repression of ADH2-regulated beta-xylanase production by ethanol in recombinant strains of Saccharomyces cerevisiae

The regulation of endo-beta-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l(-1). Pulses of 190-630 mmol ethanol l(-1) into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l(-1).     

Alcohol dehydrogenase thermostability variants in Drosophila melanogaster: Comparison of activity ratios and enzyme levels

Representatives of five allozymic classes of Drosophila alcohol dehydrogenase have been compared with respect to their activity levels on two alcohol substrates, quantities of ADH protein, and stability in crude extracts. Within each allozymic class, strains from widely diverse geographic locations differ in their enzyme activity levels but are identical for a measure known as "activity ratio," which is obtained by dividing the average activity reading on isopropanol by that obtained with ethanol. They are also similar in the rate at which ADH activity declines in crude extracts held at 25 degrees C. For several of the fast-resistant and fast-moderate strains, differences in ADH activity are associated with differences in the amount of enzyme present. The catalytic efficiencies of the fast-resistant forms are considerably lower than those of the fast-moderate allozymes. The origin and persistence of the rare but ubiquitous fast-resistant allozyme is discussed.     

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems.     

Alcohol dehydrogenase and adaptation to ethanol in Drosophila

The subject of this research is activity and allozyme spectra of alcohol dehydrogenase (ADH), and survival of mutant strains of Drosophila kept in standard nutrient medium with added ethanol. In all experiments the ADH of flies revealed greater affinity to isopropanol than ethanol. The mutant strains considerably differed from one another and from the wild type of flies in the level of enzyme activity, which may be connected with genotypic properties in the mutants studied. The ADH variability in mutant strains seems to be caused by different alleles of the structural ADH gene, which was established as a result of investigation of activity, electrophoretic mobility and thermostability of corresponding allozymes. As follows from experiments on the genotypical structure of populations in the conditions of fly selection in the medium containing ethanol (10%), the adaptation of flies to exogenous ethanol takes place via mechanisms of allele control of the ADH activity. Phenotypical manifestation of the ADH locus and its effect on the resistance of Drosophila to alcohol are supposed to depend on complex gene interactions determined by the genotype as a whole.     

Influence of expression of the pet operon on intracellular metabolic fluxes of Escherichia coli

Fermentation patterns of Escherichia coli HB101 carrying plasmids expressing cloned genes of Zymomonas mobilis pyruvate decarboxylase (PDC) and alcohol dehydrogenase li (ADH) were determined in glucose-limited complex medium in pH-controlled anaerobic batch cultivations. Time profiles of glucose, dry cell weight, succinate, formate, acetate, and ethanol were determined, as were the activities of ADH and PDC. Fluxes through the central carbon pathways were calculated for each construct utilizing exponential phase data on extracellular components and assuming quasi-steady state for intermediate metabolites. Overall biomass yields were greatest for cells expressing both PDC and ADH activities. Yields of carbon catabolite end products were similar for all PDC-expressing strains and different from those for other strains. Relative to its glucose uptake rate, the strain with greatest PDC and ADH activities produces formate and acetate more slowly and ethanol more rapidly than other strains. Strong influences of plasmid presence and metabolic coupling complicate detailed interpretations of the data.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.     

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase.

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.     

Expression, localization and potential physiological significance of alcohol dehydrogenase in the gastrointestinal tract.

ADH1 and ADH4 are the major alcohol dehydrogenases (ADH) in ethanol and retinol oxidation. ADH activity and protein expression were investigated in rat gastrointestinal tissue homogenates by enzymatic and Western blot analyses. In addition, sections of adult rat gastrointestinal tract were examined by in situ hybridization and immunohistochemistry. ADH1 and ADH4 were detected along the whole tract, changing their localization and relative content as a function of the area studied. While ADH4 was more abundant in the upper (esophagus and stomach) and lower (colorectal) regions, ADH1 was predominant in the intestine but also present in stomach. Both enzymes were detected in mucosa but, in general, ADH4 was found in outer cell layers, lining the lumen, while ADH1 was detected in the inner cell layers. Of interest were the sharp discontinuities in the expression found in the pyloric region (ADH1) and the gastroduodenal junction (ADH4), reflecting functional changes. The precise localization of ADH in the gut reveals the cell types where active alcohol oxidation occurs during ethanol ingestion, providing a molecular basis for the gastrointestinal alcohol pathology. Localization of ADH, acting as retinol dehydrogenase/retinal reductase, also indicates sites of active retinoid metabolism in the gut, essential for mucosa function and vitamin A absorption.     

Alcohol dehydrogenase (ADH). Influence of homo- and heterologous ADH administration on albino rats craving for alcohol and on ADH isozyme activity in the liver.

The effects of homo- and heterologous alcohol dehydrogenase (ADH) administration into albino rats were investigated. It was found that homologous ADH increases and heterologous ADH decreases the craving for ethanol. The latter effect was accompanied by the appearance of anti-ADH-3 antibodies and by a decrease in ADH-3 activity in the liver. Craving for alcohol decreased after both active and passive immunization against ADH.     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols.

A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.