Metabolomic analysis of oxidative stress: Superoxide dismutase mutation and paraquat induced stress in Drosophila melanogaster.

Oxidative stress results in substantial biochemical and physiological perturbations in essentially all organisms. To determine the broad metabolic effects of oxidative stress, we have quantified the response in Drosophila melanogaster to both genetically and environmentally derived oxidative stress. Flies were challenged with loss of Superoxide dismutase activity or chronic or acute exposure to the oxidizing chemical paraquat. Metabolic changes were then quantified using a recently developed chemical isotope labeling (CIL) liquid chromatography - mass spectrometry (LC-MS) platform that targets the carboxylic acid and amine/phenol submetabolomes with high metabolic coverage. We discovered wide spread changes in both submetabolomes in response to all three types of stresses including: changes to the urea cycle, tryptophan metabolism, porphyrin metabolism, as well as a series of metabolic pathways involved in glutathione synthesis. Strikingly, while there are commonalities across the conditions, all three resulted in different metabolomic responses, with the greatest difference between the genetic and environmental responses. Genetic oxidative stress resulted in substantially more widespread effects, both in terms of the percent of the metabolome altered, and the magnitude of changes in individual metabolites. Chronic and acute environmental stress resulted in more similar responses although both were distinct from genetic stress. Overall, these results indicate that the metabolomic response to oxidative stress is complex, reaching across multiple metabolic pathways, with some shared features but with more features unique to different, specific stressors.

Sex and Genetic Background Influence Superoxide Dismutase (cSOD)-Related Phenotypic Variation in Drosophila melanogaster.

Mutations often have drastically different effects in different genetic backgrounds; understanding a gene's biological function then requires an understanding of its interaction with genetic diversity. The antioxidant enzyme cytosolic copper/zinc superoxide dismutase (cSOD) catalyzes the dismutation of the superoxide radical, a molecule that can induce oxidative stress if its concentration exceeds cellular control. Accordingly, Drosophila melanogaster lacking functional cSOD exhibit a suite of phenotypes including decreased longevity, hypersensitivity to oxidative stress, impaired locomotion, and reduced NADP(H) enzyme activity in males. To date, cSOD-null phenotypes have primarily been characterized using males carrying one allele, cSodn108red, in a single genetic background. We used ANOVA, and the effect size partial eta squared, to partition the amount of variation attributable to cSOD activity, sex, and genetic background across a series of life history, locomotor, and biochemical phenotypes associated with the cSOD-null condition. Overall, the results demonstrate that the cSOD-null syndrome is largely consistent across sex and genetic background, but also significantly influenced by both. The sex-specific effects are particularly striking and our results support the idea that phenotypes cannot be considered to be fully defined if they are examined in limited genetic contexts.

A model of oxidative stress management: moderation of carbohydrate metabolizing enzymes in SOD1-null Drosophila melanogaster.

The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP(+) in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP(+)-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain.

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress. To identify the product of the whd gene we used a sequence-based positional candidate approach and by this method we determined that whd encodes carnitine palmitoyltransferase I (CPT I), an enzyme of the outer mitochondrial membrane that is required for the import of long-chain fatty acids into the mitochondria for beta-oxidation. Although this function is not vital under laboratory conditions, whd adults were found to be highly sensitive to starvation and to heavy metal toxicity relative to controls. This work uncovers a novel relationship between fatty acid metabolism and reactive oxygen metabolism. Further, these results in conjunction with past research on whd and on mammalian CPT I support the hypothesis that CPT I serves a vital function in the response to thymine supplementation.