Drosophila Melanogaster as a Model for Molybdo-Flavoenzyme Mediated Protection against Chemical and Physical Stress

Aldehyde oxidase (AO) and xanthine oxidoreductase (XOR) are molybdo-flavoenzymes (MFEs) involved in the oxidation of hundreds of endogenous and exogenous aldehydes and N-heterocyclic compounds many of which are drugs, vitamins and environmental pollutants. Mutations in the XOR and molybdenum cofactor sulfurase (MCS) genes result in a deficiency of XOR or dual AO/XOR deficiency respectively. At present despite AO and XOR being classed as detoxification enzymes the definitive experimental proof of this has not been assessed in any animals thus far.

The aim of this project was to evaluate ry and ma-l strains of Drosophila melanogaster as experimental models for XOR and dual AO/XOR deficiencies respectively and to determine if MFEs have a role in the protection against chemical and physical stress. An additional aim was to determine the molecular basis for deficiency in ma-l strains by cloning and sequencing the MCS gene.

Spectrophotometric and HPLC assays demonstrated that Drosophila AO and XOR were able to catalyse the biotransformation of numerous substrates of the well-scrutinised mammalian orthologs. These included several aromatic aldehydes and N-heterocyclic pollutants, drugs and endogenous vitamins. Investigation of the enzyme activity in ry strain revealed a compromised ability to biotransform several XOR substrates that reflected the situation in human hereditary xanthinuria type I. Both ma-l strains were found to be unable to biotransform all AO substrates tested. These results confirmed that Drosophila were good experimental models for testing the role of MFEs as detoxification enzymes.

In order to test the role of the enzymes in chemoprotection, MFE substrates were administered to Drosophila in media and survivorship monitored. It was demonstrated that several methylated xanthines were toxic to XOR-deficient strains. In addition a range of AO substrates including N-heterocycles and aldehydes were significantly more toxic to ma-l AO-null strains. This study therefore provides definitive proof that both AO and XOR are involved in detoxification.

Investigations to determine the effect of MFE deficiencies on lifespan revealed that ry and ma-l strains had significantly reduced lifespan when compared with the wild type strain with the mean lifespan being reduced approximately 60% and 30% respectively ii in these strains. The effect the MFE deficiencies have on heat and cold stress indicated that ry and ma-l strains had significantly reduced ability to survive, with the survivorship of the AO-null ma-l strain being most compromised in these conditions. The effect of the toxic chemicals, lifespan and temperature stress were rescued in ry mutant animals that had the normal ry (XOR) gene reinserted by transgenesis.

Cloning and DNA sequencing of the MCS gene in the ma-l strains revealed that the mal-1 and mal-f1 strains had a 6 bp insertion and a 23 bp deletion in exon 4 respectively that are predicted to lead to alterations of the deduced MCS protein structure, thus explaining the dual AO/XOR deficiency in these strains.