GENERATION OF REACTIVE OXYGEN SPECIES DURING INTERACTION OF DIESEL EXHAUST PARTICLE COMPONENTS WITH NADPH-CYTOCHROME P450 REDUCTASE AND INVOLVEMENT OF THE BIOACTIVATION IN THE DNA-DAMAGE

Since the toxicity of diesel exhaust particles (DEP) after intratrache al injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al, Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzym atically and continuously generated from diesel exhaust particles (DEP ), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extr act of DEP with the Triton N-101 treated microsomal preparation of mou se lung whereas the cytosolic fraction was less active, suggesting tha t DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6. 2.4, P450 reductase) rather than DT-diaphorase. When purified P450 red uctase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate fo r P450 reductase because reaction was inhibited by addition of glutath ione (GSH) to form those GSH adduct or pretreatment with NaBH4 to redu ce those to the hydroxy compounds although a possibility of nitroarene s as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 mu g) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P45 0 reductase caused DNA scission which was reduced in the presence of s uperoxide dismutase (SOD), catalase, or hydroxyl radical scavenging ag ents. Taken together, these results indicate that DEP components, prob ably quinoid or nitroaromatic structures, that appear to promote DNA d amage through the redox cycling based generation of superoxide. Copyri ght (C) 1996 Elsevier Science Inc.