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Drosophila and Antioxidant Therapy F. Missirlis1, J.P. Phillips2, H. Jäckle3 and T.A. Rouault1 1Cell biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland, U.S.A. 2Molecular Biology and Genetics, University of Guelph, Ontario, Canada. 3Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany Antioxidant intervention is a potential therapeutic approach to miti- gate the oxidative stress component of various human diseases. Genetic studies in the model organism Drosophila melanogaster indicate that the major antioxidant defense systems protect from oxidative stress in a compartment-specific manner. They also suggest that compensation for the loss of an antioxidant enzyme by experimentally inducing ex- pression of a second, distinct defense enzyme is possible in a few cases.
However, in other examples, overexpression of an antioxidant enzyme may also exacerbate an oxidant-sensitive phenotype. Thus, a successful antioxidant therapy should target the affected tissues, act in the appro- priate sub-cellular compartments and specifically rectify the underlying Oxidative stress is characterized by the generation of reactive oxy- gen species (ROS), primarily including the superoxide anion (O × –), hydrogen peroxide (H O ) and the hydroxyl radical, which damage proteins, lipids and nucleic acids. A variety of human pathologies are associated with oxidative stress, including cancer (Mantovani, et al. ’02; Lincoln, et al. ’03), cardiovascular (Levine, et al. ’96; Meagher and Rader ’01), inflammatory (Cuzzocrea, et al. ’01), viral (Peterhans ’97) and neuro- logical (Lodi, et al. ’01; Pitchumoni and Doraiswamy ’98; Halliwell ’01) diseases. Nevertheless, despite a variety of antioxidants used for therapeutic purposes, the response of patients has not always been positive2003 by MEDIMOND S.r.l.
148 Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications (Ebadi, et al. ’96; Brown, et al. ’01). Here we discuss results from Drosophila, which underscore the importance of considering ROS compartmentalization and ROS specificity in the design of such treat- Many studies have assessed the effects of various antioxidants and of antioxidant enzyme genetic manipulations on flies (for reviews see Le Bourg ’01 and Missirlis ’03). The recent development of fly disease models permits testing of drugs (Chan and Bonini ’00). Drosophila contains all major antioxidant defense enzymes that are employed by mammals, with the notable exception of glutathione reductase (Kanzok, et al. ’01). Two different superoxide dismutase (Sod) genes encode cytosolic and mitochondrial proteins (Phillips, et al. ’89; Kirby, et al.
’02). A thioredoxin reductase (TrxR-1) gene encodes two transcripts that give rise to cytosolic and mitochondrial isoforms (Missirlis, et al.
’02). Thioredoxin peroxidases (TPxs) are likewise localized in both these compartments (Radyuk, et al. ’01), whereas a glutathione peroxi- dase homologue with thioredoxin peroxidase activity (GTPx1) is de- tected in the secretory pathway (Missirlis, et al. ’03b).
The compartmentalization of antioxidant defense systems might have a redundant role in oxidative stress physiology, or it might reflect the existence of independent redox environments within the different com- partments of the same cell. We investigated the question of independent redox environments by selectively silencing Sod in the cytoplasmic or mitochondrial compartments and concurrently assaying the activity of cytosolic and mitochondrial aconitases, as markers of O ⋅ – reactivity (Missirlis, et al. ’03a). We showed that diminution of Sod2 results in mitochondrial, but not cytoplasmic aconitase inactivation and conversely diminution of Sod1 inactivates cytoplasmic, but not mitochondrial aco- nitase. In addition, overexpression of Sod2 could not ameliorate the Sod1 mutant phenotype, implying that enhancing the mitochondrial O ⋅2 – scavenging potential of cells is not beneficial if excess O ⋅ – is present in the cytosol. Furthermore, overexpression of Sod2 did not protect from paraquat, a O ⋅ – generating drug, because paraquat acted in the cytosolic compartment (Missirlis, et al. ’03a). In contrast, overexpression of Sod1 greatly protects from paraquat toxicity (Parkes, et al. ’98).
These results suggest that O ⋅ – is confined in vivo to the subcellular compartment in which it is formed and antioxidant treatment needs to be targeted to that compartment.
We have also generated transcript-specific mutants in the TrxR-1 gene, impairing the thioredoxin-dependent defense system in mitochon- dria or cytosol, respectively (Missirlis, et al. ’02). Loss of either activity resulted in lethality, suggesting that both isoenzymes are required for Ioannina, Greece, June 26-29, 2003 ROS detoxification. Transgene-dependent rescue experiments indicated that cytosolic TrxR-1 can compensate only for the lack of cytosolic TrxR-1 activity (as expected), but cannot substitute for the lack of mitochondrial TrxR-1 (although the two TrxR-1 isoforms have very similar biochemical properties) and vice versa. We conclude that sepa- rate compartmentalized ROS defense systems operate in the cytoplasm and mitochondria to sustain cell viability and propose that compromise of either the Sod-dependent or the thioredoxin-dependent antioxidant defenses cannot be compensated by isoforms that reside in the wrong ROS specificity is a second determinant for the appropriate selection of antioxidants used in different pathological conditions. We have pre- viously assessed the impact of Sod1 and Cat overexpression in the mutant for cytosolic TrxR-1 (Missirlis, et al. ’01). The results were surprisingly contrasting, as Cat offered a substantial rescue of viability and lifespan, whereas Sod1 impacted further the TrxR-1 phenotype (Missirlis, et al. ’01). We also investigated whether overexpression of GTPx1 is beneficial in flies lacking Sod1 or Cat. Biochemical charac- terization of GTPx1 shows that this enzyme is highly active with H O as a substrate, in vitro (Missirlis, et al. ’03b). We generated transgenic Drosophila that overexpress GTPx1, either from its endogenous or from an inducible promoter. Transgenic flies were resistant to paraquat tox- icity, implying an important role for GTPx1 in antioxidant defense.
However, GTPx1 overexpression did not enhance the viability of the Cat mutant (Missirlis, et al. ’03b). A similar function between two enzymes in vitro does not necessarily reflect the actual situation in vivo. We then generated flies heterozygous for the genomic GTPx1+ transgene on their second chromosome and heterozygous for two different Sod1 null-activity alleles on the third. Only 5-8% of Sod1 homozygous flies successfully eclose from their pupal case as adults and GTPx1+ segre- gates at the expected Mendelian ratio in wild type flies. We performed sibling analysis and counted how many of the viable Sod1 homozygous mutants contained zero, one or two copies of the GTPx1+ transgene.
Table 1 shows that a GTPx1+-heterozygote, Sod1 mutant fly is much less likely to eclose than a sibling Sod1 mutant with wild type second chromosomes (d”50%; expected ratio is 1:2:1). Two extra copies of GTPx1 are almost incompatible with viable Sod1 mutants. This interac- tion is specific to Sod1; Cat mutants are not affected by the presence or absence of the transgene (see also Missirlis, et al. ’03b). The finding that GTPx1 overexpression negatively impacts Sod1 mutants is even more surprising when one considers that the same genetic manipulation confers resistance to the O ⋅ – generating herbicide, paraquat. Although paraquat administration is often considered a pharmacological parallel to the genetic ablation of Sod1 (Missirlis, et al. ’03a), its mechanism of action also implicates O ⋅ – independent pathways (Berisha, et al. ’94).
150 Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications Table 1. Genetic interaction between GTPx1 and Sod1: Overexpression of GTPx1 enhances the Sod1 mutant phenotype, contrary to the prediction that increasing an antioxidant enzyme would be beneficial to an oxidatively-stressed individual. GTPx1+ heterozygote GTPx1+ homozygote Further experiments are required to explain these observations, however they underscore the importance of understanding the substrate specificity, subcellular location and cofactor requirements for each of the proteins in question.
By studying the antioxidant defense systems of Drosophila, we have shown that oxidative stress in one subcellular compartment is fairly insensitive to the redox status of adjacent compartments. We have also observed that, although antioxidant defense systems cooperate to pro- tect from ROS, we cannot assume that enhancing one system will al- ways be protective, especially if different antioxidant systems are fail- ing. Thus, before designing an antioxidant therapy, the source (tissue, intracellular location) of oxidative stress and the type of oxidative insult should be clarified. The need for development of antioxidant drugs that target different aspects of the machinery in different compartments is crucial. Complementing current efforts of generating drugs that have multi-enzyme properties or therapies that use antioxidant cocktails, we should also invest on issues of specificity.
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