Search

Early third instar larvae of wild-type Drosophila melanogaster were transferred to medium supplemented with 2-methoxyethanol (2-ME) or ethylene glycol monomethyl ether. 2-ME produced terata in adult flies as wing notches and duplications of macrochaetae similar to feeding with methoxyacetic acid (MAA), the oxidation product of 2-ME. Larval feeding with 2-ME also affected the fertility of both females and males. 2-ME or more likely its intermediate oxidation product, methoxyacetaldehyde (MAALD), concurrently generated mutations in the premeiotic stages of the oocytes in the early third instar larvae. The mutagenicity of 2-ME has been confirmed in subsequent small scale experiments. The mutation frequency ranged from 4 x 10(-4) to 1 x 10(-2). Although terata were not supposed to be heritable, 1.1 to 8.7% of the affected females produced offspring with phenotypic similarity to the female parent. This phenomenon looked like a classical example of inheritance of an acquired character. The question is addressed why a Notch-like phenocopy, generated by larval 2-ME treatment, could bring forth Notch and rudimentary mutants in particular. Administration of 2-ME to larvae, containing the highly active alcohol dehydrogenase variant ADH-71k, exposed the mitotic germ cells and the mitotic somatic cells of the imaginal discs simultaneously to the mutagen MAALD and the teratogen MAA, respectively. The chances for specific gene mutations, though non-adaptive, were likely increased by a feedback mechanism: gene-products that were inhibited or disturbed by the teratogen demanded increased transcription of their encoding genes. Transcribed genes are more susceptible to mutagens.

Drosophila melanogaster strains, homozygous for the alcohol dehydrogenase alleles AdhF, AdhS, and Adhn4 respectively, were tested for oviposition site preference with a Multiple Choice Device consisting of 18 patches per choice disk. Equal numbers of patches with ethanol-, acetic acid-, and water-supplemented medium were offered simultaneously. Patches with acetic acid-supplemented medium were chosen predominantly as oviposition sites. Pretreatment of flies with increasing concentrations of 2-propanol to inhibit alcohol dehydrogenase (ADH) activity resulted not only in a decreasing choice of acetic acid patches, but also in the laying of a decreasing number of eggs. Adh-null mutant flies showed a similar change in behavior pattern after 2-propanol treatment. Therefore it was concluded that ADH activity is not involved primarily in oviposition site preference behavior. A complicating factor is acetone, the oxidation product of 2-propanol, which had an even larger impact on egg production. However, differences in ADH allozymes with respect to biochemical oxidation capacity of secondary alcohols will not necessarily lead to differences between the Adh genotypes in oviposition rates or apparent changes in preferences, due to additional biochemical differences in inhibition rates by acetone of the various allozymes and other enzyme systems.

Pyrazole and 4-methylpyrazole (4-MP) are effective inhibitors of alcohol dehydrogenase (ADH) activity in mammals both in vivo and in vitro. 4-MP has a tenfold higher inhibition specificity compared with pyrazole. Pyrazole proved a teratogenic compound in Drosophila melanogaster. Treatment of third instar larvae of D. melanogaster with pyrazole, in contrast with 4-MP, produced an increase in the number of dorsocentral and scutellar macrochaetae and wing-notches in the adult fly. A large difference in the penetrance of terata in males and females was observed. Similar effects were observed in flies lacking ADH molecules. The teratogenicity of pyrazole must be due to disturbance of processes other than ADH inhibition. Synergistic effects were observed between pyrazole and methoxyacetic acid (MAA), an in vitro inhibitor of sarcosine dehydrogenase activity. Each of these compounds, when fed to early third instar larvae, produced terata resembling the Notch mutant of D. melanogaster.

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. of Drosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics and Km values, due to retrograde evolution at site 214, Pro-->Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.

Pyrazole and 4-methylpyrazole (4-MP) are in vivo and in vitro inhibitors of alcohol dehydrogenase activity in mammals. The fruitfly Drosophila melanogaster has been used to demonstrate the influence of genetic variation in alcohol dehydrogenase alleles on the results of larval treatment with pyrazole and 4-MP. Genetic polymorphism of organisms involved in experiments with teratogenic and toxic agents is not often considered. Administration of pyrazole to larvae of isogenic D. melanogaster strains, differing mainly in their Adh alleles, caused large Notch-like teratogenic aberrations, macrochaetae multiplication, and pupal mortality. The level of teratogenicity and developmental-toxicity of pyrazole was both concentration and Adh-genotype-dependent. The strain with the highest ADH activity showed smaller effects after the treatments with the two concentrations used. 4-MP does not cause morphological aberrations, although treatment of larvae with an isogenic background caused a high pupal mortality due to non-differentiated material in the pupal case.

Alcohol dehydrogenase of Drosophila melanogaster catalyzes the oxidation of many primary and secondary alcohols. We show that sarcosine, choline and dihydroorotate are substrates of ADH in vitro. The first two substrates are regular substrates of the choline shunt, and the latter of the de novo pyrimidine synthesis. Differences in oxidative ability towards sarcosine and dihydroorotate between two ADH allozymes, ADH71k and ADHF, are observed. The catalytic activity of ADH71k towards sarcosine and dihydroorotate might be responsible for its allelic fixation in Notch8 mutant stocks, in which Notch females have a decreased level of the regular enzymes for these substrates. Their oxidation by ADH71k might act as a bypass, which restores at least part of the decreased activity of enzymes encoded by the Notch locus.

Ethylene glycol monomethyl ether (EGME) or 2-methoxyethanol (2-ME) is a toxic compound that has teratogenic effects in developing organisms similar to its oxidation product methoxyacetic acid (MAA). Alcohol dehydrogenase (ADH) is supposed to be involved with the oxidation of EGME into MAA. It has been shown here that Drosophila ADH dehydrogenates EGME in an in vitro test system. Three strains of Drosophila melanogaster, genetically different from each other with regard to ADH activities (qualitatively and quantitatively), were used to test the in vivo involvement of ADH with the oxidation of EGME into MAA. The toxicity of EGME in egg-to-adult and third instar larval-to-adult development is concentration and strain dependent. Higher detoxification occurs in the strain with increased ADH activity. EGME is much more toxic than its oxidation product MAA at the level of adult eclosion. Teratogenic effects were observed in the ADH-negative strain in spite of lacking ADH activity. EGME is apparently a teratogenic compound by itself.

Until recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process. A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t 1/2 at 35 degrees C, being 45 min and 130 min for ADH-F and ADH-71k respectively. The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase; the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F. Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors. From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%-40%) than ADH-71k. The physiological consequences of these differences are discussed.