The effect of dietary ethanol on the composition of lipids of Drosophila melanogaster larvae

At a moderate concentration (2.5%, v/v) dietary ethanol reduced the chain length of total fatty acids (FA) and increased the desaturation of short-chain FA in Drosophila melanogaster larvae with a functional alcohol dehydrogenase (ADH). The changes in length in total FA were postulated to be due to the modulation of the termination specificity of fatty acid synthetase. Because the ethanol-stimulated reduction in the length of unsaturated FA was blocked by linoleic acid, it was thought to reflect the properties of FA 9-desaturase. Although the ethanol-stimulated reduction in chain length of unsaturated FA was also observed in ADH-null larvae, ethanol promoted an increase in the length of total FA of the mutant larvae. Thus, the ethanolstimulated change in FA length was ADH dependent but the ethanol effect on FA desaturation was not. Ethanol also stimulated a decrease in the relative amount of phosphatidylcholine and an increase in phosphatidylethanolamine. Because similar ethanol-induced changes have been found in membrane lipids of other animals, ethanol may alter the properties of membranes in larvae. It is proposed that ethanol tolerance in D. melanogaster may be dependent on genes that specify lipids that are resistant to the detrimental effects of ethanol.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Alcohol dehydrogenase and ethanol tolerance at the cellular level in Drosophila melanogaster

Exposure of early third instar larvae of Drosophila melanogaster to a nonlethal dose of ethanol was detrimental to larvae lacking alcohol dehydrogenase (ADH) but beneficial to wild-type larvae in terms of surviving a later ethanol tolerance test, indicating that one of the important functions of the ADH system is to supply derivatives of ethanol to larvae that in turn promote ethanol tolerance. High intracellular concentrations of ethanol in ADH-deficient (Adhn2) larvae fed ethanol were accompanied by a decrease in the cell membrane infoldings of fat body cells, suggesting that the capacities to absorb and release molecules were reduced. Marked effects of ethanol on the endoplasmic reticulum and mitochondria of ADH-deficient larvae were also evident. The absence of similar changes in wild-type larvae that were fed moderate levels of ethanol showed that the ADH system kept the intracellular level of ethanol at a concentration low enough to avoid cell damage. A cytometric analysis of electron micrographs showed that there were ethanol-induced reductions in glycogen, lipid, and protein stores in the fat body cells of ADH-deficient larvae fed 1.25% ethanol (v/v) compared with null larvae fed an ethanol-free diet. This finding implied that the capacities to synthesize or store these compounds may be limited by high intracellular concentrations of ethanol. The cytometric analysis also revealed that the consumption of diets containing 2.5% and 4.5% ethanol by Canton-S wild-type larvae for 3 days after 4 days of feeding on an ethanol-free diet resulted in decreases in glycogen and protein deposits in fat body cells, but increased the amount of lipid deposits compared to larvae fed an ethanol-free diet. This observation, coupled with the greater weight of wild-type adults that were fed a growth-limiting concentration of ethanol compared with control adults, suggested that a metabolic defense mechanism in larvae is to convert toxic ethanol to nontoxic storage products. Dietary ethanol alone and in combination with isopropanol stimulated an increase in the size of the NAD-pool in larvae, a condition that may favor the activity of ADH. A low dietary level of isopropanol (1%) completely blocked glycogen deposition in wild-type larvae, whereas ethanol did not. Thus ethanol and isopropanol exert some different toxic effects on larval fat bodies.     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

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.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.     

Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.     

Isolation ofCaenorhabditis elegans mutants lacking alcohol dehydrogenase activity

Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematodeCaenorhabiditis elegans. Extracts of wild-typeC. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols.C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression inC. elegans.     

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l⁻¹. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.     

Introduction of Alcohol Dehydrogenase in Lettuce Seedlings by Flooding Stress

Alcohol dehydrogenase (ADH), its isozyme profiles and ethanol concentration in lettuce (Lactuca sativa L.) seedlings subjected to flooding stress were determined. Flooding stress caused increases in ADH activity and ethanol concentration. By 48 h, ADH activity and ethanol concentration in the flooded seedlings increased 3.2- and 7.0-fold, respectively, in comparison with those in non-stressed seedlings. Five electrophoretically separable ADH bands were found in extract of the flooded seedlings, whereas only two or three ADH bands were found in extract of non-stressed seedlings. These results indicate that lettuce ADH may have a system of three-gene and six-isozyme, and the increase in ADH activity in the flooded seedlings may be due to increased synthesis of the enzyme.     

Respective roles of the microsomal ethanol oxidizing system and catalase in ethanol metabolism by deermice lacking alcohol dehydrogenase

To evaluate the roles of MEOS (microsomal ethanol oxidizing system) and catalase in non-alcohol dehydrogenase (ADH) ethanol metabolism, MEOS and catalase activities in vitro and ethanol oxidation rates in hepatocytes from ADH-negative deermice were measured after treatment with catalase inhibitors and/or a stimulator of H2O2 generation. Inhibition of ethanol peroxidation by 3-amino-1,2,4-triazole (aminotriazole) was found to be greater than 85% up to 3 h and 80% at 6 h in liver homogenates. Urate (1 mM) which stimulates H2O2 production in living systems, increased ethanol oxidation fourfold in control but not in cells from aminotriazole-treated animals, documenting effective inhibition of catalase-mediated ethanol peroxidation by aminotriazole. While aminotriazole slightly depressed (15%) basal ethanol oxidation in hepatocytes, in vitro experiments showed a similar decrease in MEOS activity after aminotriazole pretreatment. Azide (0.1 mM), a potent inhibitor of catalase in vitro, also did not affect ethanol oxidation in control cells. By contrast, 1-butanol, a competitive inhibitor of MEOS, but neither a substrate nor an inhibitor of catalase, decreased ethanol oxidation rates in a dose-dependent manner. These results show that, in deermice lacking ADH, catalase plays little if any role in hepatic ethanol oxidation, and that MEOS mediates non-ADH metabolism.     

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.     

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn²⁺ significantly inhibited activity. The K _m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.     

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. α,α-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.     

Genetic sexing in the mediterranean fruit fly,Ceratitis capitata,using the alcohol dehydrogenase locus

Summary Using the alcohol dehydrogenase (ADH) locus a genetic sexing system is being developed in the Mediterranean fruit fly Ceratitis capitata based on the sensitivity of ADH null mutations to environmental ethanol. A series of null mutants have been induced at this locus, however, none proved viable as homozygotes. One of these null mutants was translocated to the male determining chromosome and this line can be used for genetic sexing. When larvae from this line were reared on larval medium containing various concentrations of allyl alcohol, 97% of the emerging adults were males; in the absence of the allyl alcohol the sex ratio in the line is distorted in favour of the females. It is proposed that the higher ADH activity of the females (homozygous positive) in comparison with the males (heterozygous null) is responsible for their lower survival in larval medium containing allyl alcohol. ADH converts the allyl alcohol to the lethal ketone. The possible use of this line to sex large populations of medflies for use in sterile insect release programmes is discussed.     

Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels.