In Vivo ESR Spin Trapping Evidence for Hydroxyl Radical-Mediated Toxicity of Paraquat and Copper in Rats

Abstract

The electron spin resonance (ESR) spin trapping technique was employed to study the role of Cu(II) in the generation of hydroxyl radical during paraquat (PQ2+) intoxication in rats. A secondary radical trapping technique was used for the detection of hydroxyl radical generated in vivo during copper-mediated PQ2+ toxicity. According to this technique (Burkitt and Mason, Proc. Natl. Acad. Sci. USA88, 8440-8444, 1991), the hydroxyl radical generated reacts with dimethyl sulfoxide (DMSO) in vivo to form the methyl radical, which is then spin trapped by phenyl-N-tert-butylnitrone (PBN). The relatively stable methyl radical adduct of PBN was detected in the bile of rats 2 hr after treatment with simultaneous doses of CuSO4, PQ2+, PeN, and DMSO, whereas no radical adducts were detected in the absence of administered PQ2+. Bile samples were collected into Cu(I)- and Fe(II)-stabilizing agents in order to prevent the occurrence of radical adducts generated ex vivo in bile during its collection. The analysis of radical adducts excreted via the biliary route provide strong ESR evidence for the generation of the hydroxyl radical as a result of the known futile enzymatic redox cycling of PQ2+, with copper playing an essential mediatory role. No radical adducts were detected when either CuSO4 or PQ2+ was excluded. From a different perspective, in vivo copper-dependent hydroxyl radical generation could be said to be promoted by PQ2+. This is the first report of ESR evidence for this synergetic hydroxyl radical generation by copper and PQ2+ in a whole animal.