Pieter W. H. Heinstra

A dual function of alcohol dehydrogenase in Drosophila

Alcohol dehydrogenase (ADH) of Drosophila not only catalyzes the oxidation of ethanol to acetaldehyde, but additionally catalyzes the conversion of this highly toxic product into acetate. This mechanism is demonstrated by using three different methods. After electrophoresis the oxidation of acetaldehyde is shown in an NAD-dependent reaction revealing bands coinciding with the bands likewise produced by a conventional ADH staining procedure. In spectrophotometric measurements acetaldehyde is oxidized in an NAD-dependent reaction. This activity is effectively inhibited by pyrazole, as specific inhibitor of ADH. By means of gas chromatographic analysis a quick generation of acetate from ethanol could be demonstrated. Our conclusion is further supported by experimental results obtained with either purified ADHF enzyme or genotypes with or without ADH, aldehyde-oxidase, pyridoxal-oxidase and xanthine-dehydrogenase activity. These results are discussed in relation to ethanol tolerance in the living organism in particular with respect to differences found between ADH in Drosophila melanogaster and D. simulans, and in relation to the possible implications for the selective forces acting on ADH-polymorphism.

Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).

Alcohol Dehydrogenase and Alcohol Tolerance in Drosophila melanogaster

Drosophila melanogaster has often been used as a model system for investigations of the genetic factors that underlie ethanol tolerance. The species not only is very tolerant to environmental ethanol (McKenzie and Parsons, 1972; David et al., 1976), but it is able to use ethanol as a food source (McKechnie and Geer, 1984; Geer et al., 1985). Judgement of the degree of ethanol tolerance in D. melanogaster is complicated by the complex nature of the trait, and by the diversity of the tests that have been used to measure tolerance. Ethanol tolerance is a composite of the abilities to grow and to survive in the presence of ethanol, and, as shown in this study, to utilize dietary ethanol as a foodstuff. The conditions for the tolerance test and the diagnostic traits in previous studies have varied. Many tolerance tests have been performed by adding ethanol to the medium (Gibson, 1970; McDonald et al., 1977; Cavener and Clegg, 1978), but different life stages have been tested. Ethanol tolerance in adult D. melanogaster has been equated to the concentration of alcohol in sealed tubes that kills 50% of the individuals in a treatment of fixed duration (David et al., 1978, 1984).

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.

Alcohol dehydrogenase of Drosophila: Conversion and retroconversion of isozyme patterns

Abstract 1. 1. In vitro and in vivo effects involved in the isozyme formation of homodimeric Drosophila alcohol dehydrogenase (ADH) have been studied. 2. 2. Total ADH activity of the three isozymes together as found after electrophoresis and MTT-formazan staining is often a poor predictor of total ADH activity in vivo . 3. 3. When ADH isozymes in zymograms are visualized by NADH fluorescence they do represent their in vivo activities. 4. 4. Various naturally-occurring β-keto metabolites caused conversion patterns of the ADH-isozymes in D. melanogaster and other Drosophila species, but not in two blowfly species. 5. 5. Retroconversion of ADH-isozymes in vivo has been achieved during exposure of flies to ethanol. 6. 6. The significance of these processes is discussed.

DIFFERENCES BETWEEN LARVAL AND ADULT DROSOPHILA IN METABOLIC DEGRADATION OF ETHANOL

The alcohol dehydrogenase polymorphism in Drosophila melanogaster is a widely cited paradigm ofthe occurrence ofnatural selection (van Delden 1982). However, the fruit flys life stages seem to vary substantially in sensitivity to selective forces of alcohols (e.g., Middleton and Kaeser 1983; Heinstra et al. 1987; Hoffmann and McKechnie 1991). Drosophila uses mainly alcohol dehydrogenase (ADH, EC 1.1.1.1) to detoxify dietary ethanol (for a recent review see Geer et al. 1991). Alcohol dehydrogenase has a dual function in larvae; dehydrogenation of ethanol into acetaldehyde and subsequently of acetaldehyde into acetic acid (Heinstra et al. 1983, 1989). Recently two independent investigations suggested that the enzyme aldehyde dehydrogenase (ALDH, EC 1.2.1.3) is more important than ADH for the dehydrogenation of acetaldehyde in adults (Anderson and Barnett 1991; Leal and Barbancho 1992). This points to differences in alcohol elimination between these life stages. Ethanol-derived (end)products, like fatty acids, a,a-trehalose, alanine, lactate, glutamate, glutamine, and proline have been detected in lar-

Metabolic physiology of alcohol degradation and adaptation in Drosophila larvae as studied by means of carbon-13 nuclear magnetic resonance spectroscopy

Alcohol dehydrogenase-mediated degradation of [2-13C]ethanol was followed in third instar larvae of Drosophila by means of 13C NMR. The tricarboxylic acid (TCA) cycle intermediates, citrate-C(2),4 and succinate-C2,3; the amino acids, glutamate-C4,3,2, glutamine-C4,3,2, proline-C4, alanine-C2,3 and the carbon nuclei of the glucosyl units of the disaccharide, α,α-trehalose, were intensely labeled in perchloric acid extracts of whole larvae. A model of the intermediary metabolism of ethanol degradation in larvae was formulated from these observations. The C2 atom of ethanol enters the mitochondrial TCA cycle as C2-acetyl-CoA and is converted into the TCA cycle intermediates. The TCA cycle intermediate 2-oxoglutarate(-C4) apparently is readily converted into glutamate(-C4) and subsequently to glutamine(-C4) and proline(-C4). Dietary ethanol is also a substrate for trehalose synthesis. This may occur by an exchange of malate(-C2,3) between its mitochondrial and cytosolic pools. Part of the cytosolic malate(-C2,3) may be diverted into pyruvate then generating alanine(-C2,3) as another product. The other part may be converted into glucose and subsequently into α,α-trehalose by the gluconeogenic pathway. 13C natural abundance signals of stored fatty acids and glycerol were readily detectable in chloroform extracts of control larvae. De novo synthesis of fatty acids from labeled ethanol also occurred after a lag period. Our findings show the coordinated nature of metabolic pathways, and we point to its consequences in understanding the dynamics in evolutionary processes.

Alcohol dehydrogenase polymorphism in Drosophila: enzyme kinetics of product inhibition.

SummaryBecause natural populations ofDrosophila melanogaster are polymorphic for different allozymes of alcohol dehydrogenase (ADH) and becauseD. melanogaster is more tolerant to the toxic effects of ethanol than its sibling speciesD. simulans, information regarding the sensitivities of the different forms of ADH to the products of ethanol degradation are of ecological importance. ADH-F, ADH-S, ADH-71k ofD. melanogaster and the ADH ofD. simulans were inhibited by NADH, but the inhibition was relieved by NAD+. The order of sensitivity of NADH was ADH-F<ADH-71k, ADH-S<ADH-simulans with ADH-F being about four times less sensitive than theD. melanogaster enzymes and 12 times less sensitive than theD. simulans enzyme. Acetaldehyde inhibited the ethanolto-acetaldehyde activity of the ADHs, but at low acetaldehyde concentrations ethanol and NAD+ reduced the inhibition. ADH-71k and ADH-F were more subject to the inhibitory action of acetaldehyde than ADH-S and ADH-simulans, with ADH-71k being seven times more sensitive than ADH-S. The pattern of product inhibition of ADH-71k suggests a rapid equilibrium random mechanism for ethanol oxidation. Thus, although the ADH variants only differ by a few amino acids, these differences exert a far larger impact on their intrinsic properties than previously thought. How differences in product inhibition may be of significance in the evolution of the ADHs is discussed.

A MULTILEVEL APPROACH TO THE SIGNIFICANCE OF GENETIC VARIATION IN ALCOHOL DEHYDROGENASE OF DROSOPHILA

Prior studies showed that differences in alcohol dehydrogenase (ADH) activity across genotypes of Drosophila are decisive for the outcome of selection by ethanol. In the present paper, the effect on ADH activity and egg‐to‐adult survival of combinations of ethanol, propan‐2‐ol, and acetone in naturally occurring concentrations is examined. Propan‐2‐ol is converted into acetone by ADH in vitro. Acetone is considered a competitive inhibitor of ethanol for the ADH enzymes. The melanogaster‐ADH‐S allozyme is two times more sensitive towards inhibition by acetone than either simulans‐ADH or melanogaster‐ADH‐F. The physiological implications of these in vitro differences for larvae were studied in short‐term in vivo and long‐term exposure experiments. No major differences in acetone accumulation or fitness parameters were found between the strains in response to ecologically relevant concentrations of acetone or propan‐2‐ol. Ethanol, however, strongly decreased egg‐to‐pupal survival in both Drosophila simulans strains and increased developmental time in four out of the five strains tested. Therefore, under physiological conditions only ethanol was shown to act as a selective agent on the ADH polymorphism during egg‐to‐pupa development in Drosophila.

Genetic and Dietary Control of Alcohol Degradation in Drosophila

Because of the consumption of ethanol-containing beverages by humans, information about the metabolism of ethanol is important. To be an alcoholic, a person need only consume enough ethanol to interfere with one of his or her important activities within our society. The decrease in efficiency of workers, the loss of life in alcohol-related accidents, and the personal grief are the result of the physiological and biochemical effects of ethanol. Chronic alcoholism is a health hazard to many individuals and, as such, poses a variety of social and economic problems.