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-dihydroxypyridine.

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 Vmax of 5.9 nmol 2,5-dihydroxypyridine/min/mg protein and a Km value of 110 microM. In contrast, phenobarbital- and isosafrole-induced microsomes had Vmax values of 2.5 and 1.2 nmol/min/mg protein and Km values of 590 and 134 microM, respectively. Pyridine-induced rat hepatic microsomes also exhibited elevated catalytic activity toward the hydroxylation of 3-hydroxypyridine, with an 8-fold increase in Vmax (2.74 nmol/min/mg protein) relative to uninduced rat hepatic microsomes (Vmax = 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 approximately 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 phi 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 phi X-174 DNA topology produced by 2,5-dihydroxypyridine was accelerated in a dose-dependent manner, with an estimated EC50 of approximately 60 microM.(ABSTRACT TRUNCATED AT 400 WORDS)