Cytochromes P450 reconstituted with NADPH: P450 reductase mimic the activating and detoxicating metabolism of the anticancer drug ellipticine in microsomes.
OBJECTIVE
Ellipticine is a potent antineoplastic agent exhibiting multiple action mechanisms. Recently, we found that after cytochrome P450 (CYP)-mediated oxidation ellipticine forms covalent DNA adducts. Ellipticine oxidation by isolated CYP and its binding to DNA is the target of this study.


METHODS
High performance liquid chromatography (HPLC) was employed for separation and characterization of ellipticine metabolites generated by CYPs. The (32)P-postlabeling technique was utilized to determine ellipticine-DNA adducts.


RESULTS
Purified CYP enzymes reconstituted with NADPH:CYP reductase oxidized ellipticine to up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only CYP1A1 was capable to form all metabolites. Using the reconstituted enzymatic system, we demonstrated that the detoxication ellipticine metabolites, 7-hydroxyellipticine and 9-hydroxyellipticine, are mainly generated by CYP1A1 and 1A2, while those responsible for DNA binding, 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, by CYP3A1 and 2C3. Likewise, the most efficient CYPs forming DNA adducts from ellipticine were CYP3A1 and 2C3.


CONCLUSIONS
The results showed that the system of purified CYPs reconstituted with NADPH: CYP reductase proved for ellipticine oxidation provide a true reflection of the situation in the microsomal membrane.
Kotrbová
Vera
V
Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Aimová
Dagmar
D
Brezinová
Anna
A
Janouchová
Katerina
K
Poljaková
Jitka
J
Frei
Eva
E
Stiborová
Marie
M
D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D004611:Ellipticines / Q000493:pharmacokinetics*; D050216:Metabolic Detoxication, Phase I; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D000970:Antineoplastic Agents; D018736:DNA Adducts; D004611:Ellipticines; C034192:ellipticine; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase