The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster.

Oxygen free radicals and hydroperoxides have been postulated to play a causal role in the aging process, implying that antioxidant enzymes may act as longevity determinants. Catalase (H2O2:H2O2 oxidoreductase; EC1.11.1.6) is the sole enzyme involved in the elimination of H2O2 in Drosophila melanogaster; glutathione peroxidase being absent. A genomic fragment containing the Drosophila catalase gene was used to construct transgenic Drosophila lines by means of P element-mediated transformation. Enhanced levels of catalase (up to 80%) did not prolong the life span of flies, nor did they provide improved protection against oxidative stress induced by hyperoxia or paraquat treatment. However, enhanced resistance to hydrogen peroxide was observed in the overexpressors.     

Control of oxidative stress resistance by IP3 kinase in Drosophila melanogaster

Oxidative damage is thought to be a major causal factor of aging, and is implicated in several human pathologies such as Alzheimer's and Parkinson's diseases. Nevertheless the genetical determinants of in vivo oxidative stress response are still poorly understood. To identify cellular components whose deregulation leads to oxidative stress resistance, we performed a genetic screen in Drosophila melanogaster. We thus identified in this screen Drosophila Inositol 1,4,5-triphosphate kinase I (D-IP3K1), a Drosophila gene homologous to mammalian IP3Ks. In vertebrates, IP3Ks phosphorylate the second messenger Inositol 1,4,5-triphosphate (IP3) to produce Inositol 1,3,4,5 tetrakiphosphate (IP4). IP3 binding to its receptor (IP3R) triggers Ca(2+) release from the endoplasmic reticulum (ER) to the cytosol, whereas IP4 physiological role remains elusive. We show here that ubiquitous overexpression of D-IP3K1 confers resistance of flies to H(2)O(2)- but not to paraquat-induced oxidative stress. Additional genetic analysis with other members of IP3 and IP4 signaling pathways led us to propose that the D-IP3K1 protective effect is mainly mediated through the reduction of IP3 level (which probably results in reduced Ca(2+) release from internal stores), rather than through the rise of IP4 level.     

Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster

Background

Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.

Methods and Findings

We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79% and 56–66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.

Conclusions

We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.     

Mating increases starvation resistance and decreases oxidative stress resistance in Drosophila melanogaster females

Mating stimulates complex physiological changes in females of Drosophila melanogaster. Long-term effects of mating are manifested in increased fecundity and shortened lifespan. It is not clear how mating affects stress resistance in fly females. We addressed this question here and found that mated and highly fecund wild-type D. melanogaster females have significantly higher resistance to starvation throughout their lifetime than age-matched virgin females. Mean survival time under starvation was age dependent with maximum survival time observed in 15-day-old mated females. Mating-induced increase in starvation resistance was associated with significantly higher fat reserves stored as triacylglycerols. While mated females had higher resistance to starvation, their resistance to oxidative stress was significantly lower than in age-matched virgins. Our study revealed that mating leads to an opposing relationship between resistance to starvation and resistance to oxidative stress in Drosophila females. Thus, shortened lifespan of mated females is associated with their high-fat content and greater susceptibility to oxidative stress.     

Flavonoids and oxidative stress in Drosophila melanogaster

Flavonoids are a family of antioxidants that are widely represented in fruits, vegetables, dry legumes, and chocolate, as well as in popular beverages, such as red wine, coffee, and tea. The flavonoids chlorogenic acid, kaempferol, quercetin and quercetin 3β-d-glycoside were investigated for genotoxicity using the wing somatic mutation and recombination test (SMART). This test makes use of two recessive wing cell markers: multiple wing hairs (mwh) and flare (flr(3)), which are mutations located on the left arm of chromosome 3 of Drosophila melanogaster and are indicative of both mitotic recombination and various types of mutational events. In order to test the antioxidant capacities of the flavonoids, experiments were conducted with various combinations of oxidants and polyphenols. Oxidative stress was induced using hydrogen peroxide, the Fenton reaction and paraquat. Third-instar transheterozygous larvae were chronically treated for all experiments. The data obtained in this study showed that, at the concentrations tested, the flavonoids did not induce somatic mutations or recombination in D. melanogaster with the exception of quercetin, which proved to be genotoxic at only one concentration. The oxidants hydrogen peroxide and the Fenton reaction did not induce mutations in the wing somatic assay of D. melanogaster, while paraquat and combinations of flavonoids produced significant numbers of small single spots. Quercetin 3β-d-glycoside mixed with paraquat was shown to be desmutagenic. Combinations of the oxidants with the other flavonoids did not show any antioxidant activity.     

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

Halving the selenophosphate synthetase gene dose confers hypersensitivity to oxidative stress in Drosophila melanogaster

Several lines of evidence indicate that selenoproteins mainly act as cellular antioxidants. Here, we test this idea comparing the sensitivity to oxidative stress (paraquat and hydrogen peroxide) between wild type and heterozygous flies for the selenophosphate synthetase selD(ptuf) mutation. Whereas under normal laboratory conditions no difference in life span is observed, a significant decrease is seen in heterozygous flies treated with oxidant agents. In contrast, overexpression of the selD gene in motoneurons did not extend longevity. Our results strongly suggest that selD haploinsufficiency makes heterozygous flies more sensitive to oxidative stress and add further evidence to the role of selenoproteins as cellular antioxidants.     

Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.     

Genome-wide association for sensitivity to chronic oxidative stress in Drosophila melanogaster

Reactive oxygen species (ROS) are a common byproduct of mitochondrial energy metabolism, and can also be induced by exogenous sources, including UV light, radiation, and environmental toxins. ROS generation is essential for maintaining homeostasis by triggering cellular signaling pathways and host defense mechanisms. However, an imbalance of ROS induces oxidative stress and cellular death and is associated with human disease, including age-related locomotor impairment. To identify genes affecting sensitivity and resistance to ROS-induced locomotor decline, we assessed locomotion of aged flies of the sequenced, wild-derived lines from the Drosophila melanogaster Genetics Reference Panel on standard medium and following chronic exposure to medium supplemented with 3 mM menadione sodium bisulfite (MSB). We found substantial genetic variation in sensitivity to oxidative stress with respect to locomotor phenotypes. We performed genome-wide association analyses to identify candidate genes associated with variation in sensitivity to ROS-induced decline in locomotor performance, and confirmed the effects for 13 of 16 mutations tested in these candidate genes. Candidate genes associated with variation in sensitivity to MSB-induced oxidative stress form networks of genes involved in neural development, immunity, and signal transduction. Many of these genes have human orthologs, highlighting the utility of genome-wide association in Drosophila for studying complex human disease.     

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster

Objectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na₂MoO₄).

Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food.

Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration.

Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.     

Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and < 8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3-5 fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interventions in invertebrates.     

Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and < 8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3-5 fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interventions in invertebrates.     

Ether resistance in Drosophila melanogaster

Summary Strains set up from single inseminated females of D. melanogaster from the wild differ in their resistance to the anaesthetics, ether and chloroform. The main differences between four selected extreme strains could be explained by additive genes, which in the case of ether resistance were located to regions of chromosomes 2 and 3. The lack of correspondence between ether and chloroform resistance between strains indicates that although the type of genetic architecture controlling the traits is similar, the actual genes differ, which is reasonable in view of their differing chemical structures. Quite high heritabilities were found for resistance to ether based on five inbred strains. No significant associations between resistance to ether and body weight, developmental rate or longevity were found.It is clear that resistance to both anaesthetics would be amenable to more detailed genetic analyses. It is pointed out that the general conclusions reached from such studies will have implications with respect to the effect of chemicals such as insecticides, not naturally present in nature.     

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.     

Phenotypic consequences of copper-zinc superoxide dismutase overexpression in Drosophila melanogaster

We report here the isolation of a tandem duplication of a small region of the third chromosome of Drosophila melanogaster containing the Cu-Zn superoxide dismutase (cSOD) gene. This duplication is associated with a dosage-dependent increase in cSOD activity. The biological consequences of hypermorphic levels of cSOD in genotypes carrying this duplication have been investigated under diverse conditions of oxygen stress imposed by acute exposure to ionizing radiation, chronic exposure to paraquat, and the normoxia of standard laboratory culture. We find that a 50% increase in cSOD activity above the normal diploid level confers increased resistance to ionizing radiation and, in contrast, confers decreased resistance to the superoxide-generating agent paraquat. The duplication is associated with a minor increase in adult life-span under conditions of normoxia. These results reveal important features of the biological function of cSOD within the context of the overall oxygen defense system of Drosophila.     

Over-expression of human clusterin increases stress resistance and extends lifespan in Drosophila melanogaster

Clusterin is a disulfide-linked heterodimeric glycoprotein that has been implicated in a variety of biological processes. Its expression has been shown to be elevated during cellular senescence and normal aging, but it is uncertain whether clusterin protects against aging or whether its expression is a consequence of aging. To investigate the functions of clusterin during organismal aging, we established transgenic Drosophila alleles to induce the expression of the secretory form of human clusterin (hClu(S)) using the Gal4/UAS system. hClu(S) protein (~60 kDa) was detected in both adult homogenates and larval hemolymphs of flies ubiquitously overexpressing hClu(S) (da-Gal4>UAS-hClu(S)) and in motoneurons (D42-Gal4>UAS-hClu(S)). Interestingly, the mean lifespans of these hClu(S)-overexpressing flies were significantly greater than those of control flies that exhibited no hClu(S) induction. hClu(S)-overexpressing flies also showed significantly greater tolerance to heat shock, wet starvation, and oxidative stress. Furthermore, amounts of reactive oxygen species (ROS) in whole bodies were significantly lower in hClu(S)-overexpressing flies. In addition, clusterin was found to prevent the inactivation of glutamine synthetase (GS) by metal-catalyzed oxidation (MCO) in vitro, and this protection was only supported by thiol-reducing equivalents, such as, DTT or GSH, and not by ascorbate (a non-thiol MCO system). Furthermore, this protection against GS inactivation by clusterin was abolished by reacting clusterin with N-ethylmaleimide, a sulfhydryl group-modifying agent. Taken together, these results suggest that a disulfide-linked form of clusterin functions as an antioxidant protein via its cysteine sulfhydryl groups to reduce ROS levels and delay the organismal aging in fruit flies.     

Regulation of the oxidative NADP-enzyme tissue levels in Drosophila melanogaster. I. Modulation by dietary carbohydrate and lipid.

Wild-type third instar larvae of Drosophilia melanogaster fed a casein-sucrose synthetic diet supplemented with phosphatidylcholine (4 mg/ml) possessed 33% more tissue lipid and a modified fatty acid profile compared to larvae fed a fat free-sucrose diet. The rates of lipid synthesis and pentose shunt activity were 2.1 and 2.2 times greater respectively in larvae fed the fat free-sucrose diet than in fat-sucrose fed animals. The tissue concentrations of acetyl-CoA and acyl-CoA were 80 and 61% higher respectively, CoA 49% lower, and the NADPH/NADP+ ratio greater in fat-sucrose fed larvae than in larvae fed a fat free-sucrose diet. Thus, larvae effectively utilized dietary lipid for lipid synthesis and as a supplementary energy source to carbohydrate.