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 metabolism of 3-hydroxyp)ridine, a significant constituent of tobacco smoke, to 2,5-dihydroxypyridine has been characterized in he patic 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 P4SOIIE1 (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 )'„,„ of 5.9 nmol 2,5-dihydroxypyridine/min/mg protein and a A,,,value of 110 UM.In contrast, phénobarbitaland isosafrole-induced microsomes had >',„., values of 2.5 and 1.2 nmol/min/mg protein and A',,, values of 590 and 134 /JM. respectively. Pyridine-induced rat hepatic microsomes also exhibited elevated catalytic activity toward the hydroxylation of 3-hydroxypyridine, with an 8-fold increase in t „,(̂2.74 nmol/ min/inn protein) relative to uninduced rat hepatic microsomes ( rrm,v= 0.34 nmol/min/mg protein). In the reconstituted system, cytochrome P450IIE1 displayed the greatest activity in the production of 2,5-dihy droxypyridine of the major forms of rabbit P450 examined. P450HE1 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 HIM2,5-dihydroxypyridine was stimulated -4-fold (69.2 nmol NADPH oxidized/min/mg protein), rela tive to control (16 nmol/min/mg protein). 2,5-Dihydroxypyridine at 0.5 and 1.0 HIMproduced a 12and 17-fold increase, respectively, in the rate of Superoxide aniÃ3nproduction compared to control, as monitored by the SOD-inhibitable reduction of acetylated cytochrome c. 3-Hydroxypyri dine 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 4>X-174DNA strand scission. The band intensity of the supercoiled form of DNA, when incubated with 1 HIM2,5-dihydroxypyridine, decreased substantially, with a concomitant increase in intensity of the band associated with the open circular form of DNA. The change in ¿X-174DNA topology produced by 2,5-dihy droxypyridine was accelerated in a dose-dependent manner, with an estimated EQo of ~60 MM. 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.