Ewa Kukiełka

NADH-dependent microsomal interaction with ferric complexes and production of reactive oxygen intermediates.

The interaction of NADPH with ferric complexes to catalyze microsomal generation of reactive oxygen intermediates has been well studied. Experiments were carried out to characterize the ability of NADH to interact with various ferric chelates to promote microsomal lipid peroxidation and generation of .OH-like species. In the presence of NADH and iron, microsomes produced .OH as assessed by the oxidation of a variety of .OH scavenging agents. Rates of NADH-dependent .OH production were 50 to 80% those of the NADPH-catalyzed reaction. The oxidation of dimethyl sulfoxide or t-butyl alcohol was inhibited by catalase and competitive .OH scavengers but not by superoxide dismutase or carbon monoxide. NADH-dependent .OH production was effectively catalyzed by ferric-EDTA and ferric-diethylenetriaminepentaacetic acid (DTPA), whereas ferric-ATP and ferric-citrate were poor catalysts. All these ferric chelates were reduced by microsomes in the presence of NADH (and NADPH). H2O2 was produced in the presence of NADH in a reaction stimulated by the addition of ferric-EDTA, consistent with the increase in .OH production. The latter appeared to be limited by the rate of H2O2 generation rather than the rate of reduction of the ferric chelate. NADH-dependent lipid peroxidation was much lower than the NADPH-catalyzed reaction and showed an opposite response to catalysis by ferric complexes compared to .OH generation as production of thiobarbituric acid-reactive material was increased with ferric-ATP and -citrate, but not with ferric-EDTA or- DTPA, and was not affected by catalase, SOD, or .OH scavengers. These results indicate that NADH can support microsomal reduction of ferric chelates, with the subsequent production of .OH-like species and peroxidation of lipids. The pattern of response of the NADH-dependent reactions with respect to catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with NADPH. Many of the metabolic actions of ethanol have been ascribed to production of NADH as a consequence of oxidation by alcohol dehydrogenase. Since the cytosol normally maintains a highly oxidized NAD+/NADH redox ratio, it is interesting to speculate that increased availability of NADH from the oxidation of ethanol may support microsomal reduction of iron complexes, with the subsequent generation of reactive oxygen intermediates.

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of .OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for .OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of .OH scavengers was sensitive to catalase and competitive .OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H2O2; stimulation of H2O2 production may play a role in the increase in .OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or .OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize .OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.

Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals.

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.

Increased oxidation of ethylene glycol to formaldehyde by microsomes after ethanol treatment: role of oxygen radicals and cytochrome P450

The production of ferryl-type oxidants by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for the production of formaldehyde from ethylene glycol. Microsomes from the ethanol-fed rats were more reactive than controls in oxidizing ethylene glycol. Catalase was a powerful inhibitor for this reaction, superoxide dismutase was slightly inhibitory and hydroxyl radical scavengers had no effect. These results suggest an important role for H2O2, but not O2-. or .OH in the overall pathway for oxidizing ethylene glycol to formaldehyde. The production of H2O2 by microsomes was increased after ethanol treatment, the extent of increase corresponding to the increase in oxidation of ethylene glycol. A variety of inhibitors and ligands of cytochrome P450, including miconazole, diethyldithiocarbamate, tryptamine, and 4-methylpyrazole, inhibited formaldehyde production by both microsomal preparations. Anti-cytochrome P4502E1 IgG also inhibited the reaction with both microsomal preparations and prevented the increase caused by ethanol treatment. These results indicate that microsomes from ethanol-treated rats are more reactive than pair-fed controls in generating ferryl-type oxidants and that increased production of H2O2 by cytochrome P4502E1 plays a role in the elevated oxidation of ethylene glycol to formaldehyde.