Biochemical Genetics, Vol. 14, Nos. 11/12, 1976 Biological Role of Alcohol Dehydrogenase in the Tolerance of Drosophila melanogaster to Aliphatic Alcohols: Utilization of an ADH-Null Mutant Jean R. David, 1 Charles Bocquet, 2 Marie-Fran~oise Arens, 1 and Pierre Fouillet 1 Received 6 May 1976--Final 24 May 1976 The toxicity of the first eight primary alcohols and of four secondary alcohols was compared in a wild-type strain (having active A D H ) and an ADH-negative mutant. Differences between LCso measured in the two strains allowed an evaluation of the biological activity of the enzyme. In vitro, A D H is mainly active on secondary alcohols, while in vivo its main role is the detoxification and metabolism o f ethanol. These observations suggest that originally A D H was involved in unknown metabolic pathways and that its utilization in ethanol metabolism could be a recent event. KEY WORDS: Drosophila melanogaster; alcohol dehydrogenase; enzyme biological activity; toxicity of alcohols. INTRODUCTION In Drosophila melanogaster, alcohol dehydrogenase (ADH) is an abundant enzyme whose genetic variability (e.g., Ursprung and Leone, 1965; Ward and Herbert, 1972; Birley and Barnes, 1973; Clarke, 1975; Van Delden et al., 1975) and biochemical activity (Vigue and Johnson, 1973; Day et al., 1974) have received much attention. Several authors (McKenzie and Parsons, 1972, 1974; David and Becquet, 1974, 1975; Briscoe et al., 1975) have demonstrated the physiological and t Laboratoire d'Entomologie Exp6rimentale et de G6n6tique (Associ6 au CNRS), Universit6 Claude Bernard, Villeurbanne, France. Laboratoire de G6n6tique Evolutive du CNRS, Gif sur Yvette, France. 989 .~ 1976 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher. 990 David, Bocquet, Arens, and Fouillet ecological importance of ethanol tolerance for this species. This tolerance appears, at least in some cases, to be related to the presence of active ADH (Grell et al., 1968; Clarke, 1975; David and Bocquet, 1976a; Vigue and Sorer, 1976). However, a contradiction exists when biochemical and biological results are compared. In vitro, ADH is inactive on methanol, slightly active on ethanol, and very active on secondary alcohols (Sieber et al., 1972; Vigue and Johnson, 1973; Day et al., 1974)..Live Drosophila, on the other hand, are highly tolerant to ethanol and very sensitive to secondary alcohols (David and Bocquet, 1976b). It therefore appeared interesting to measure the biological role of ADH in the tolerance to various alcohols. Such a study is possible by comparing the sensitivities of a normal wild-type strain and homozygous ADH-negative mutant. MATERIAL AND METHODS The wild-type strain used is a French strain homozygous for the Adh v natural allele and having a high level of enzyme activity (David and Bocquet, 1976a). The mutant strain not producing ADH was homozygous for the Adh n4 allele (O'Donnell et al., 1975). This strain was also homozygous for the second chromosome marker black. Larvae of these strains were grown in low density on an axenic killedyeast medium (David and Clavel, 1965) containing no alcohol. Alcohol toxicity was measured on adults 2-3 days old by calculating the concentration killing 50% of the flies (LCso) after 2 days of treatment. For each determination, 560 adult flies were used. Males were usually a little more sensitive than females, but the difference between sexes was always small and so sex was ignored. More detailed information on these bioassay techniques can be found in previous articles (David et al., 1974; David and Bocquet, 1976b). RESULTS Toxicity data (LC 50) for the first eight primary alcohols and for four secondary alcohols are given in Fig. 1. In the ADH-negative strain, the tolerance to primary alcohols decreases regularly with molecule length: the LCso varies from about 3 ~ for methanol to 0.2% for octanol. With secondary alcohols, a similar observation is made and the toxicity increases with the length of the molecule. For a given number of carbons, the toxicities of primary and secondary alcohols are usually similar, as shown by the ratios in Table I. In the presence of ADH (wild-type strain), the results are greatly different Biological Activity of ADH in Drosophila 991 ® 10. a o u c d .A ® e o ~ O o • number of c a r b o n s of alcohols Fig. 1. Variation of the tolerance to various aliphatic alcohols (LCso, lethal concentration 50) as a function of the number of carbons. A: Toxicity of primary alcohols. B: Secondary alcohols. 0, Wild strain with active ADH; ©, mutant strain without ADH activity. In most cases, the tolerance o f the wild strain is higher than that o f the null m u t a n t and the greatest difference is observed for ethanol. A n o t h e r peculiarity o f the wild strain is that it is always more sensitive to secondary alcohols than to primary ones (see Table I). The differences between the wild-type and the A D H - n u l l strain are to be attributed mainly to the presence o f the enzyme. In other words, they measure 992 David, Bocquet, Arens, and Fouillet Table I. Comparison of the Toxicities of Primary and Secondary Alcohols: Ratios of the LCso of the Primary Alcohol to the LCso of the Secondary One Alcohol ADH null Wildtype Propanol Butanol Pentanol Hexanol 0.56 2.17 1.20 0.78 2.12 4.93 2.19 3.22 the biological activity of ADH. For a better evaluation of this phenomenon, the differences between the LCso of the two strains were calculated for each alcohol; data are given in Fig. 2. For primary alcohols, the difference is zero for methanol and more than 14% for ethanol, but then it decreases rapidly from propanol to octanol, where the difference is less than 0.3%. For secondary alcohols, a positive difference is also seen which decreases with the length of the molecule. The biological activity is, however, much lower than for the corresponding primary alcohols. This observation corresponds with the higher toxicity of secondary alcohols to wild flies (Table I). In a previous article (David and Bocquet, 1976b), it was demonstrated that D. simulans was generally more sensitive to various alcohols than its sibling species D. melanogaster. On the other hand, it is known that D. simulans has a much lower A D H activity than D. melanogaster (Pipkin and Hewitt, 1972). It seemed interesting therefore to calculate the differences between the L¢5o values of the two species. The results are shown in Fig. 2B. There is a striking similarity between these curves and those observed for wild and mutant strains of D. melanogaster. These observations leave very little doubt that the physiological differences between the two species are due to the low A D H activity level found in D. simulans. Previous authors (Vigue and Johnson, 1973; Day et al., 1974) have studied the biochemical substrate specificities of A D H for various alcohols. Our results allow the estimation of the biological activity of the enzyme for the same alcohols. Comparison of these results is made in Table II, where, in all cases, the activity on ethanol is taken as 100. There are some differences between authors concerning the biochemical activity on various substrates. Such differences may be explained by the utilization of different strains and techniques. However, results agree in showing no activity with methanol and a higher activity on alcohols with Biological Activity of ADH in Drosophila 993 ® ~ 10. ® g i _ . n u m b e r of c a r b o n s of alcohols Fig. 2. Biological activity of A D H for the tolerance to various alcohols. A: Difference between wild-type and ADH-null mutant of D. melanogaster. B: Difference between wild D. melanogaster and wild D. simulans, m, Primary alcohols; ~, secondary alcohols. David, Bocquet, Arens, and Fouillet 994 Table II. Comparison of Biological Activity and in Vitro Biochemical Activity of ADH ° Biochemical activity Alcohol Methanol Ethanol n-Propanol 2-Propanol n-Butanol 2-Butanol Biological Vigueand activity Johnson (1973) Day et al. (1974) 0 100 20 4 20 3 0 100 213 580 213 775 0 100 110 350 230 120 "All results are expressed relative to an activity on ethanol of 100. three or four carbons. Moreover, A D H seems, at least for propanol, more active on secondary than on primary alcohols. This conclusion was confirmed by Sieber et aI. (1972) and many others, and 2-propanol is now the classical substrate for staining A D H in electrophoretic studies. When the biochemical properties are compared with the biological activity, a concordance is observed for methanol only. For propanol and butanol, the biological activity is low while the biochemical activity is high. The discordance is particularly apparent with 2-propanol. DISCUSSION In the absence of A D H (negative mutants), alcohols are supposedly not metabolized so that their deleterious effects on the organism can be directly measured. In fact, this is not absolutely true because Drosophila contains another enzyme, octanol dehydrogenase (ODH), which is active on primary alcohols including ethanol (Sieber et al., 1972). O D H was present in both wild and mutant flies, but our results prove that this enzyme is not important in alcohol tolerance. The nature of the toxic effect of alcohols on Drosophila adults is not known, nor is the cause of death known. Probably all cells are affected by alcohol ingestion or by respiration of alcoholic vapors. Some cells are likely more sensitive than others and their dysfunction is lethal. Curiously, the toxic effect increases with length of the molecule. The shorter molecules, which are more volatile and more water soluble and hence should penetrate more readily into the organism, are less toxic. Penetration of the alcohols into lipidic cell structures may be involved in the toxic effects. The difference between the wild-type and the mutant strain may be Biological Activity of ADH in Drosophila 995 explained by the role of ADH in the metabolic utilization of alcohols. The inactivity of ADH on methanol explains the similar toxicity in the two strains. For ethanol, the higher tolerance of wild flies (and also of D. melanogaster as compared to D. simulans) is most likely due to the presence of an active ADH which uses alcohol as a substrate. In that case, the physiological picture becomes more complex. When a toxic product is taken into an organism, the overall reaction and toxic effect are the result of several interacting factors: the direct toxicity of the initial product, the toxicity of the transformed product(s), the rate of the enzymatic transformation, and further metabolism and utilization of these products. Such interactions are summarized in Fig. 3. In the case of ethanol, the alcohol is probably transformed by ADH into acetaldehyde. However, this product, in a direct test, is highly toxic (Los0 of about 0.50~). The transformation of ethanol by ADH should therefore increase the overall toxicity. Such is obviously not the case. This can be explained by a coupled reaction which transforms the aldehyde into nontoxic, useful products. This transformation, probably mediated by aldehyde oxidase (Day et al., 1974; Clarke, 1975), is demonstrated by the "feeding value" of ethanol (Van Herrewege and David, 1974; LibionMannaert et aL, 1976). This reasoning applies also to other alcohols. In all cases here studied, the presence of ADH decreases the toxicity of primary alcohols. ADH most likely transforms these alcohols into very toxic aldehydes. Since the overall toxicity is decreased, further metabolism probably transforms the long-chain aldehydes into acids of low toxicity. Such transformations may be made by aldehyde oxidase, but the activity of this enzyme on long-chain aldehydes is apparently not known. The primary toxicity of secondary alcohols is not higher than that of the corresponding primary ones (see Table I). But a relative increase of toxicity is observed when ADH is present. Such a result can be explained by the toxicity of the intermediate products which are ketones: either such ketones are more toxic than the corresponding aldehydes, or their further utilization is questionable. ALCOHOL 1 primary toxicity metabolism further bY ADH I I intermediate porduct " I utilizcti°n= I end (energetics} pd°rucst 1 secondary tgxicity Fig. 3. Relationship between alcohol metabolismand toxicityto the flies. H 996 David, Bocquet, Arens, and Fouillet Considering the diagram of Fig. 3, it is conceivable that, in some cases, enzymatic action would produce a very toxic product which, if not further transformed, would kill the flies. Such a process, in the case of ADH, has actually been demonstrated for some unsaturated secondary alcohols (Sofer and Hatkoff, 1972; O'Donnell et al., 1975) Finally, the biological activity of an enzyme can either increase or decrease the toxicity of a substrate. This explains the lack of correlation between the biochemical efficiency and the biological utilization. A D H is mostly active on secondary alcohols, while, in the living fly, it is mostly useful in the detoxification of ethanol. As pointed out previously, high ethanol concentrations are encountered by the flies in natural conditions and high ethanol tolerance clearly has an ecologically adaptive significance. If the normal substrate of the enzyme were environmental ethanol, it could be assumed logically that natural selection, acting for a long time, would have produced an enzyme with its maximum activity on ethanol. As this is clearly not the case, it can be considered that the primary physiological role of A D H in Drosophila metabolism was not ethanol use but some other, unknown processes involving secondary alcohols (Pipkin et al., 1975). In that case, the clinal ethanol adaptation observed in D. melanogaster wild populations could be a recent event, as supposed previously (David and Bocquet, 1975). Other examples in various organisms now exist that previously existing enzymes are often used when an adaptation occurs to a new substrate (Bari-Kolata, 1975). ACKNOWLEDGMENTS We thank Professor W. Sofer for providing the Adh n* strain and Dr. R. Grantham for help with the manuscript. REFERENCES Bari-Kolata, G. (1975). Evolution of DNA: Changes in gene regulation. Science 189:446. Birley, A. J., and Barnes, B. W. (1973). Genetical variation for enzyme activity in a population of Drosophila melanogaster. I. Extent of the variation for alcohol dehydrogenase activity. Heredity 31:413. Briscoe, D. 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