Short Communication FORMATION OF N-ALKYLPROTOPORPHYRIN IX FROM METABOLISM OF DIALLYL SULFONE IN LUNG AND LIVER

Diallyl sulfone (DASO2) is a garlic derivative formed during cooking or after ingestion. Bioactivation of DASO2 in murine lung and liver results in formation of an epoxide that inactivates CYP2E1 and significantly decreases cytochrome P450 and heme levels. In this study, we tested the hypothesis that DASO2 metabolism leads to production of the heme adduct, N-alkylprotoporphyrin IX (N-alkylPP). Formation of N-alkylPP in vivo and in vitro was determined by spectrophotometric and fluorometric methods, respectively. In in vivo studies, N-alkylPP was generated in the livers of male and female mice treated with DASO2, but was not detectable in the lungs of DASO2-treated mice. In in vitro studies, rates of formation of N-alkylPP in liver and lung microsomes incubated with DASO2 and NADPH were dependent on time and protein concentrations, but were negligible in control incubations performed in the absence of NADPH or DASO2 or with boiled microsomes. The rates of N-alkylPP formation generated in murine liver were higher than those in either murine lung or human liver. Kinetic analysis revealed that murine liver microsomes metabolized DASO2 to NalkylPP with higher affinity and catalytic efficiency than did murine lung or human liver microsomes. Recombinant rat CYP2E1 also metabolized DASO2 to N-alkylPP; however, rates of formation of the heme adduct was minimal in incubations of recombinant human CYP2E1 with DASO2. These findings demonstrated that the N-alkylPP adduct was produced via metabolism of DASO2 in murine liver and lung microsomes, in human liver microsomes, in recombinant CYP2E1, and in vivo in murine liver. In 1892, Semmler, a German chemist, applied steam distillation to cloves of garlic (Allium sativum) and produced a strong-smelling oil that on further purification produced diallyl sulfides (Block, 1985). The odoriferous constituent of garlic is allicin, which is formed by enzymatic conversion of S-allylcysteine sulfoxide (alliin) by alliinase to allicin. Allicin is an unstable component that can be further transformed to other garlic compounds including diallyl sulfide (DAS). In addition to being a component of garlic oil, DAS can be produced during cooking or after ingestion of garlic (Hayes et al., 1987). It has been estimated that 1 g of garlic yields approximately 30 to 100 g of DAS (Sparnins et al., 1988). Previous studies have identified both diallyl sulfoxide (DASO) and diallyl sulfone (DASO2) in extracts of liver, blood, and urine from rats treated with DAS, suggesting that DASO and DASO2 are derived from DAS (Brady et al., 1991b). Studies with rat liver microsomes indicated that DAS undergoes sequential metabolism to DASO and DASO2. Further studies confirmed that CYP2E1 catalyzes oxidation of the sulfur atom of DAS to yield DASO and subsequently DASO2 (Jin and Baillie, 1997). Although these garlic derivatives are all competitive inhibitors of CYP2E1, the inhibitory effect of DASO2 on CYP2E1 is more pronounced and is manifested more rapidly than by either DAS or DASO (Brady et al., 1991a). The efficacy of DASO2 as a CYP2E1 inhibitor has been ascribed to mechanism-based inactivation, and it is the metabolic event involving DASO2 that leads to CYP2E1 inactivation, which mediates the chemoprotective effects of DAS (Jin and Baillie, 1997). Previous studies have investigated the mechanisms responsible for the inactivation of CYP2E1 and the protective effective of DASO2 against lung cytotoxicity induced by 1,1-dichloroethylene (Forkert et al., 1996a,b, 2000; Premdas et al., 2000). The results showed that DASO2 undergoes P450-dependent oxidation at one of its terminal double bonds to form diallyl sulfone monoepoxide (1,2-epoxypropyl3,3 -sulfonyl-1 -propene; DASO3) (Fig. 1), a reactive metabolite believed to be responsible for CYP2E1 inactivation. Levels of immunodetectable CYP2E1, total cytochrome P450, and heme were all reduced (Premdas et al., 2000). Incubation of murine liver microsomes with DASO2 (1.0 mM) decreased total cytochrome P450 and heme levels by about 30% and 70%, respectively. Immunodetectable CYP2E1 was reduced and correlated with a 70% decrease in pnitrophenol hydroxylation 2 h after treatment of mice with DASO2 (100 mg/kg p.o.). These findings suggested the possibility that bioactivation of DASO2 produces DASO3, which alkylates the heme moiety at one of the four pyrrole nitrogens within the active site of P450, yielding the heme adduct N-alkylprotoporphyrin IX (N-alkylPP; Fig. 2) (for review, see Ortiz de Montellano and Correia, 1983, 1995). Here, we have undertaken studies to test the hypothesis that metabolism of DASO2 leads to the formation of N-alkylPP, an event that is likely associated with loss of heme and inactivation of cytochrome P450 reported previously (Premdas et al., 2000). Our results confirmed that the N-alkylPP adduct is produced from oxidative metabolism of DASO2 in murine liver and lung microsomes, in human liver microsomes, in recombinant rat CYP2E1, and in vivo in murine liver. This study was funded by Grant # 014061 from the National Cancer Institute of