Elsevier

Biochemical Pharmacology

Volume 68, Issue 4, 15 August 2004, Pages 747-752
Biochemical Pharmacology

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

https://doi.org/10.1016/j.bcp.2004.05.004Get rights and content

Abstract

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HOradical dot) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HOradical dot formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450. Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HOradical dot formation. HOradical dot formation in liver microsomes was inhibited by the HOradical dot radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HOradical dot. Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.

Section snippets

Reagents

All chemicals obtained commercially were of the highest purity grade and were used without additional purification. Pure 2-OH terephthalate was kindly provided by Prof. Lewis Kirschenbaum (University of Rhode Island, Kingston, RI). Potassium phosphate monohydrate, DMSO, water and organic solvents, all HPLC grade, were purchased from Fisher Scientific (Fairlawn, NJ). Terephthalate was obtained from Aldrich (Milwaukee, WI). Superoxide dismutase (SOD), catalase, EDTA (sodium salt), isoniazid,

Results and discussion

Fluorescence methods have been developed to determine, in real time, the 2-OH terephthalate formation directly in a cuvette [26], [27]. In our initial experiments the calibration curve obtained with the 2-OH terephthalate standard (added to pure 20 mM potassium phosphate buffer, pH 7.4) demonstrates high sensitivity i.e. lower limit of 1.0 nM 2-OH terephthalate. However, background fluorescence increased significantly after the addition of components required for microsomal-supported reactions,

Acknowledgements

We thank Professors Gisela Witz and Michael Iba for helpful support and critical comments. The authors acknowledge the kind donation of 2-OH terephthalate by Professor Lewis Kirschenbaum.

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