65] Oxygen radicals and drugs: In vitro measurements

Abstract

This chapter discusses a method to detect and quantify the drug-dependent generation of reactive oxygen species catalyzed by relatively crude tissue preparations. Drugs that can generate oxygen radicals include many compounds that can transfer one or two electrons to molecular oxygen via enzyme-catalyzed reactions. The most toxic xenobiotics in this category are compounds beating quinone, dipyridyl, nitro, or azo groups. Xenobiotics with these functional groups are particularly susceptible to enzymatic one-electron reduction to form intermediate radical anions that are rapidly reoxidized by O2. Xenobiotic-mediated one-electron transfer from cellular metabolites to molecular oxygen can generate damaging quantities of reactive oxygen species in tissues containing reductases capable of sustaining the reaction. However, two-electron reduction pathways also exist in tissues and may preempt or terminate this process by producing stable, nontoxic xenobiotic metabolites. The superoxide anion is usually the precursor in the metabolic generation of reactive oxygen species. Velocities of the drug-dependent production of reactive oxygen species in vitro are usually based on rates of superoxide anion generation. Direct measurements of superoxide anion in crude tissue preparations are complicated by endogenous superoxide dismutase (SOD), and most procedures simply determine drug-dependent generation of its dismutation product, H2O2. Many different methods for measuring H2O2 are available, but virtually all procedures couple H2O2 formed to the peroxidation of a compound that can be measured spectrophotometrically or fluorimetrically. Fluorimetric methods are usually more sensitive and may permit analyses in single cells.