Emily Vass

COMPARATIVE STUDIES OF ENHANCED IRON-MEDIATED PRODUCTION OF HYDROXYL RADICAL BY GLUTATHIONE, CYSTEINE, ASCORBIC ACID, AND SELECTED CATECHOLS

A sensitive electrochemical detection system was employed together with a specific salicylate hydroxylation assay to comparatively assess the effects of various substances on the iron-mediated generation of the hydroxyl radical (.OH). Hydroxyl radical production was found to be enhanced significantly by reduced glutathione, cysteine, ascorbic acid, and selected catechols, but not by mannitol, melatonin or tyramine. The data showed that over the range of concentrations examined, the augmented effects were linearly proportional to the amount of added reductant for a given amount of iron in the system. The pro-oxidant activity of thiols and ascorbate reduced and recycled iron providing both hydrogen peroxide (H2O2) and catalytic ferrous ions for augmented .OH production by the Fenton reaction. The enhanced production of .OH by catechols resulted from their oxidation either by molecular oxygen or ferric ions, with the accompanying formation of semiquinones, superoxide anion and H2O2. These data caution against therapeutic applications of thiols and ascorbate for ameliorating oxy-radical-induced tissue damage in environments where free redox-active metal ions may be present to function both as foci for site-specific peroxidative activity, and as catalysts to promote the pro-oxidant properties of certain endogenous reductants, thereby elevating rather than diminishing .OH levels.

HYDROXYL RADICAL FORMATION RESULTING FROM THE INTERACTION OF NITRIC OXIDE AND HYDROGEN PEROXIDE

The highly reactive and cytotoxic hydroxyl radical (OH) was found by electrochemical detection to be produced in reactions involving hydrogen peroxide (H2O2) and the nitric oxide (NO) donor diethylamine- NO complex. Using aromatic hydroxylation of salicylate as a specific indicator of OH, three salicylate hydroxylation products were identified; catechol, 2,3- and 2,5-dihydroxybenzoic acid. Four additional compounds were detected but not identified. The interactions of H2O2 and NO represent a biologically feasible reaction mechanism that can account for OH-induced damage in cellular environments where transition metal ions are unavailable for participation in the superoxide-mediated Fenton reaction. The ability of the NO/H2O2 complex to generate OH independently of iron or other transition metals provides a new focus for studies concerned with the origin of tissue-specific damage caused by oxygen-derived species.

Dexamethasone Inhibition of the Cellular Immune Response of Drosophila melanogaster Against a Parasitoid

Host larvae of Drosophila melanogaster injected with the eicosanoid biosynthesis inhibitor, dexamethasone, prior to parasitization by the wasp Leptopilina boulardi, exhibited significantly reduced rates of melanotic encapsulation in comparison with control and saline-injected larvae. The results of this investigation suggest that prostaglandins and other eicosanoids are involved as cell-signaling molecules in the hemocytic encapsulation reaction of D. melanogaster larvae.

Developmental and immunological aspects of Drosophila-parasitoid relationships.

The Drosophila–parasitic wasp (parasitoid) associations involve integrating adaptations of considerable complexity. This review focuses on some of the factors that influence these interactions including host immunity, nutrition and hormonal changes, and parasitoid virulence and mechanisms of immune suppression.

Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi

1. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant strain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.

Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols

The differing effects of O-methylated catecholamines and their dihydroxyphenyl precursors on the production of *OH were quantified using a previously established specific salicylate hydroxylation assay in conjunction with a sensitive electrochemical detection system. The production of *OH by the Fenton reaction was diminished significantly by O-methylated catecholamines (O-methyldopa, O-methyldopamine, O-methyltyrosine, and N-acetyl-O-methyldopamine), whereas radical production was augmented by dihydroxyphenyls (DOPA, dopamine, and N-acetyldopamine), including those with methylated side chains (N-methyldopamine and alpha-methyldopa). Monohydroxyphenyls such as octopamine, tyramine, tyrosine, and alpha-methyltyrosine had little or no effect on radical production. These data show that a methyl group positioned on the side chain of a catecholamine does not alter its pro-oxidant behavior, while a methyl group positioned on the aromatic ring renders the catecholamine sterically or kinetically unfavorable for coordination with transition metals, thus preventing the promotion of Fenton chemistry. These results highlight the importance of O-methylation in forming catechols that are less reactive than their dihydroxyphenyl precursors. Thus, factors regulating the methylation of brain catecholamines may play a crucial role in mediating neuronal integrity during aging and in the pathogenesis of certain neurodegenerative disorders. Competitive side-chain methylation reactions may sustain or perpetuate some dihydroxyphenyls, creating an oxidatively less favorable environment for cells than would result from compounds formed by O-methylation.

COMPARATIVE STUDY OF IMMUNE COMPETENCE AND HOST SUSCEPTIBILITY IN DROSOPHILA MELANOGASTER PARASITIZED BY LEPTOPILINA BOULARDI AND ASOBARA TABIDA

Larvae of an immune-reactive (R) strain of Drosophila melanogaster readily encapsulated and killed the eggs of 2 species of parasitic wasp, Leptopilina boulardi and Asobara tabida. High pressure liquid chroma- tography with electrochemical detection identified 5,6-dihydroxyindole (DHI) and N-acetylarterenone (NAA) in the hemolymph during parasite encapsulation, indicating that the capsules enveloping the parasites are comprised of both eumelanin and sclerotin. In larvae of a host strain susceptible (S) to L. boulardi, DHI and NAA were absent, and hemolymph catecholamine profiles from these hosts resembled those of nonparasitized controls. Susceptibility was determined to be species specific for L. boulardi, because S-strain larvae were highly immune reactive against A. tabida. As with reactive R-strain hosts, DHI and NAA were detected in the he- molymph from immune-reactive S-strain larvae when they were encapsulated by A. tabida. These observations, together with previous studies, indicate that the immune response initiated by larvae of D. melanogaster against different parasites involves similar cellular and biochemical responses. The virtual absence of immune reactivity in the S strain against L. boulardi suggests these otherwise immune competent hosts are unable to recognize this parasite as a foreign entity, or that the wasp actively suppresses the cellular encapsulation response of this host strain. These investigations highlight the complexity of insect host-parasite relationships that involve the co- evolution of varied reciprocal cellular, molecular, and biochemical strategies.

The effects of glutathione and ascorbic acid on the oxidations of 6-hydroxydopa and 6-hydroxydopamine

The interactions of ascorbic acid (AA) and reduced glutathione (GSH) in the oxidations of the catecholaminergic neurotoxins 6-hydroxydopa (TOPA) and 6-hydroxydopamine (6-OHDA) were investigated by both high performance liquid chromatography with electrochemical detection (HPLC-ED) and spectrometric methods. These comparative studies showed TOPA and 6-OHDA to be extremely unstable, with 100% of the trihydroxyphenyls oxidized within 0.5 min at physiological pH in potassium phosphate buffer. Neither AA nor GSH was found capable of significantly impeding the oxidations of these trihydroxyphenyls, or of regenerating these substances by reducing back their oxidation products, even though such a redox exchange mechanism was demonstrated for AA and the dihydroxyphenyl dopamine. Although ineffective in keeping TOPA and 6-OHDA as reduced molecules, GSH may nevertheless influence the neurotoxicity of trihydroxyphenyls by interacting with their oxidation products forming glutathionyl conjugates, thereby switching the reaction pathway away from potentially toxic eumelanin precursors and toward the production of pheomelanin. Electrochemical analyses established the formation of two oxidation products derived from each trihydroxyphenyl, one detected at -100 mV and the other at +700 mV. AA had no effect on either oxidation product, whereas GSH significantly decreased the levels of both oxidation products. The component detected at +700 mV is the cyclized, reduced leukochrome. The identity of the component detected at -100 mV was not established, but it is considered to be either the p-quinone or the cyclized, oxidized aminochrome.

Alterations in the activities of tyrosinase, N-acetyltransferase, and tyrosine aminotransferase in immune reactive larvae of Drosophila melanogaster

The activities of three enzymes, tyrosinase (monophenol oxidase, MPO), N-acetyltransferase (NAT), and tyrosine aminotransferase (TAT), were studied during eumelanotic encapsulation in host larvae of Drosophila melanogaster parasitized by the wasp, Leptopilina boulardi. At 24 h postinfection there was a tenfold increase in the MPO, whereas the activities of NAT and TAT were lower than those of nonparasitized controls. The data suggest that certain developmental processes are temporarily interrupted and alterations made in the metabolism of tyrosine to provide the metabolites necessary for a successful immune response. Two strains of D. melanogaster, R and Tyr-1, were parasitized and found to be immune reactive. The Tyr-1 strain is deficient in tyrosinase during the adult stage, but this mutation was found not to affect the immune capacity of the larvae. This is the first study to document concurrent alterations in the activities of various catecholamine-metabolizing enzymes during an immune response in an insect.

Hydroxyl radical production by ascorbate and hydrogen peroxide

Ascorbate (AH-) and certain other biological reductants have long been known to produce the cytotoxic hydroxyl radical (OH) when oxidized by hydrogen peroxide (H2O2) in the presence of copper or iron catalysts. The present study documents the in vitro production of the OH solely from the oxidation of AH- by H2O2, independent of mediation by transition metals. Hydroxyl radical generation resulting from the AH-/H2O2 system was quantitatively documented by the specific radical-mediated hydroxylation of salicylic acid, a reaction that was readily assayed with HPLC coupled with electrochemical detection. Two ascorbate-copper complexes (e.g., AH-/Cu2+-EDTA/H2O2 and AH-/Cu2+-EDTA) and a copper/H2O2 system also generated OH, but less effectively than the AH-/H2O2 system. The ability of AH- and H2O2 to generate cytotoxic OH documents a reaction mechanism that may account for cytotoxic activity in some cellular environments where metal catalysts are lacking.