Cytochrome P450 2S1 ELISA Kit

Cigarette smoking is the predominant risk factor for bladder cancer. Aromatic amines such as 4-aminobiphenyl (ABP) is the major carcinogens found in tobacco smoke. Although it is generally accepted that ABP is metabolically activated via N-hydroxylation by CYP1A2 in human liver, previous studies using Cyp1a2-null mice indicated the involvement of other enzyme(s). Here we found that CYP2A13 can metabolically activate ABP to show genotoxicity by Umu assay. The K-m and V-max values for ABP N-hydroxylation by recombinant CYP2A13 in E. coli were 38.5 +/- 0.6 mu M and 7.8 +/- 0.0 pmol/min/pmol CYP, respectively. The K-m and V-max values by recombinant CYP1A2 were 9.9 +/- 0.9 mu M and 39.6 +/- 0.9 pmol/min/pmol CYP, respectively, showing 20-fold higher intrinsic clearance than CYP2A13. In human bladder, CYP2A13 mRNA, but not CYP1A2, is expressed at a relatively high level. Human bladder microsomes showed ABP N-hydroxylase activity (K-m = 34.9 +/- 4.7 mu M and V-max = 57.5 +/- 1.9 pmol/min/mg protein), although the intrinsic clearance was 5-fold lower than that in human liver microsomes (K-m = 33.2 +/- 2.0 mu M and V-max = 293.9 +/- 5.8 pmol/min/mg protein). The activity in human bladder microsomes was prominently inhibited by 8-methoxypsoralen, but not by fluvoxamine, anti-CYP1A2 or anti-CYP2A6 antibodies. CYP2S1, which is expressed in human bladder and has relatively high amino acid identities with CYP2As, did not show detectable ABP N-hydroxylase activity. In conclusion, although the enzyme responsible for ABP N-hydroxylation in human bladder microsomes could not be determined, we found that CYP2A13 metabolically activates ABP. (c) 2006 Wiley-Liss, Inc.

The physiological role of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix PER-ARNT-SIM (PAS) transcription factor family is not known. We have suggested that the AhR is involved in light signaling through binding of photoproducts with high AhR affinity. This suggestion is based on (i) the high AhR affinity of the tryptophan photoproduct formylindolo[3,2-b]carbazole (FICZ), (ii) the induction of rapid and transient expression of AhR-regulated genes by FICZ and by extracts of UV-irradiated tryptophan as well as (iii) the fact that light induces the AhR-regulated cytochrome P450s CYP1A1. CYP1B1 and CYP2S1. The transient mRNA expression caused by light and tryptophan photoproducts suggests that the biotransformation enzymes induced by AhR activation take part in a metabolic degradation of the natural AhR ligand. This study aimed at identifying the involvement of phase I and phase 11 enzymes in the metabolic degradation of FICZ. A cytochrome P450-dependent metabolism of FICZ giving rise to preferentially mono- and di-hydroxylated derivatives has earlier been reported. In the present Study, rat and human hepatic S9 mixes were employed together with specific enzyme inhibitors and cofactors. Compared to the Aroclor-induced rat liver S9, the non-induced rat liver S9 and the human liver S9 caused a more complex metabolite profile of FICZ. The CYP1A1 enzyme was confirmed to be the most important enzyme for the first step in the metabolism. CYP1A2 was found to have overlapping specificity with CYP1A1 being able to form the same major metabolites although with different kinetics. CYP1B1 turned out to be preferentially involved in the further metabolism of dihydroxylated metabolites. Microsomal epoxide hydrolase, and as yet not identified forms of sulphotransferases and glucuronosyltransferases were also found to take part in the metabolic degradation of FICZ. Thus, tryptophan photoproducts fit into a model in which the ligand-activated AhR signaling is autoregulated by the induced metabolic enzymes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.