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Role of P450IIE1 in the Metabolism of 3-Hydroxypyridine, a Constituent of Tobacco Smoke: Redox Cycling and DNA Strand Scission by the Metabolite 2,5-Dihydroxypyridine¹

The Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201

The metabolism of 3-hydroxypyridine, a significant constituent of tobacco smoke, to 2,5-dihydroxypyridine has been characterized in hepatic microsomes and in the reconstituted enzyme system using purified forms of P450. The redox cycling activity of the metabolite and its ability to damage DNA in vitro have been examined. Pyridine-induced microsomes, which contain elevated levels of P450IIE1 (Kim et al., J. Pharmacol. Exp. Ther., 246: 1175–1182, 1988), catalyzed an 8-fold increase in the production of 2,5-dihydroxypyridine, relative to control, which showed biphasic kinetics. Pyridine-induced rabbit hepatic microsomes exhibited a V_max of 5.9 nmol 2,5-dihydroxypyridine/min/mg protein and a K_m value of 110 µM. In contrast, phenobarbital- and isosafrole-induced microsomes had V_max values of 2.5 and 1.2 nmol/min/mg protein and K_m values of 590 and 134 µM, respectively. Pyridine-induced rat hepatic microsomes also exhibited elevated catalytic activity toward the hydroxylation of 3-hydroxypyridine, with an 8-fold increase in V_max (2.74 nmol/min/mg protein) relative to uninduced rat hepatic microsomes (V_max = 0.34 nmol/min/mg protein). In the reconstituted system, cytochrome P450IIE1 displayed the greatest activity in the production of 2,5-dihydroxypyridine of the major forms of rabbit P450 examined. P450IIE1 was 34-fold more active than P450IIB1 and 12-fold more active than P450IA2 in the production of 2,5-dihydroxypyridine. The redox cycling activity of 2,5-dihydroxypyridine has been characterized. The rate of NADPH oxidation in the presence of 0.5 mM 2,5-dihydroxypyridine was stimulated 4-fold (69.2 nmol NADPH oxidized/min/mg protein), relative to control (16 nmol/min/mg protein). 2,5-Dihydroxypyridine at 0.5 and 1.0 mM produced a 12- and 17-fold increase, respectively, in the rate of superoxide anion production compared to control, as monitored by the SOD-inhibitable reduction of acetylated cytochrome c. 3-Hydroxypyridine alone failed to increase the rate of superoxide production. Inclusion of reduced glutathione in the incubation resulted in a pronounced decrease in the 2,5-dihydroxypyridine-stimulated rate of cofactor oxidation and superoxide production. The ability of 2,5-dihydroxypyridine to damage DNA was assessed by monitoring X-174 DNA strand scission. The band intensity of the supercoiled form of DNA, when incubated with 1 mM 2,5-dihydroxypyridine, decreased substantially, with a concomitant increase in intensity of the band associated with the open circular form of DNA. The change in X-174 DNA topology produced by 2,5-dihydroxypyridine was accelerated in a dose-dependent manner, with an estimated EC₅₀ of 60 µM. Neither pyridine nor 3-hydroxypyridine produced DNA damage. These results show that P450 catalyzes the metabolism of 3-hydroxypyridine to 2,5-dihydroxypyridine, that P450IIE1 is the principal catalyst of 3-hydroxypyridine metabolism, and that the metabolite 2,5-dihydroxypyridine redox cycles and causes DNA strand scission.

¹ Supported by National Institutes of Environmental Health Sciences Grant ES 03656. S. G. K. is the recipient of an Eli-Lilly Predoctoral Fellowship Award.

² To whom requests for reprints should be addressed, at The Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201.

Received 6/ 9/89. Revised 3/26/90.

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