Bioactivation of 4-Ipomeanol by CYP4B1: Adduct Characterization and Evidence for an Enedial Intermediate
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- 作者：Brian R. Baer ; Allan E. Rettie ; and Kirk R. Henne
- 刊名：Chemical Research in Toxicology
- 出版年：2005
- 出版时间：May 2005
- 年：2005
- 卷：18
- 期：5
- 页码：855 - 864
- 全文大小：185K
- 年卷期：v.18,no.5(May 2005)
- ISSN：1520-5010
文摘
4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that bindsto DNA and other cellular macromolecules. Despite over 30 years of research in this area,detailed structural information on the nature of the IPO reactive intermediate is still lacking.In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenousnucleophiles and analyzed the products by liquid chromatography/electrospray ionization-massspectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multipleproducts due likely to the presence of both sulfur and nitrogen nucleophiles in the same trappingmolecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine(NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolationof the biologically derived material provided evidence for an N-substituted cysteinyl pyrrolederivative of IPO, analogous to that characterized previously in model chemical studiesconducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purifiedrecombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant humanCYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min)were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidencefor the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation,identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that thesame reactive intermediate is formed by both rabbit CYP4B1 and human P450s.