The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450-mediated biotransformation (a corrigendum report on Duarte et al. (2005) Mutagenesis 20, 93-100)

Abstract

This corrigendum report describes the study of the comparison of human cytochrome b(5) (b(5)) with rat b(5) when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b(5) in the coupling with CYP. Indeed, the plasmid pLCM-b(5)-RED used in our former study on b(5) [Duarte et al. (2005) Mutagenesis, 20(2), 193-100] erroneously contained rat b(5). Plasmid pLCM-b(5)-RED was corrected with human b(5) and subsequently all experimental work was repeated as was described for the rat b(5) plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b(5) could be confirmed using human b(5). The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b(5) could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b(5) on the activity of CYP1A2, CYP2A6 and CYP2E1 was in general similar, when compared with rat b(5), though less quantitatively pronounced. This was both the case when using membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay. Human b(5) stimulated the bioactivation of all compounds as described for rat b(5), except for CYP1A2 mediated bioactivation of 2-aminoanthracene (2AA), which was not stimulated by human b(5). All other main findings of the effect of rat b(5) were confirmed with human b(5), i.e. for CYP2A6: N-nitrosodiethylamine (NNdEA): approximately 14-fold increase ( approximately 23-fold with rat b(5)) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): approximately 3-fold ( approximately 9-fold with rat b(5)); for CYP2E1: NNdEA: approximately 1.5-fold increase ( approximately 3-fold with rat b(5)); NNK: no mutagenicity with or without human b(5). Both CYP2A6 and CYP2E1 demonstrated total dependence on the presence of human b(5) for N-nitrosodi-n-propylamine (NNdPA) mutagenicity, as was shown before with rat b(5).

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding

“…As such, any difference detected in the CYP activities can be ascribed to differences in the CPR mut :CYP coupling. Indeed, the CPR:CYP stoichiometries measured in membrane fractions were similar to those described previously for the BTC system in our former studies [11,16,33,[36][37][38][39], and comparable with the ranges of those reported for human liver microsomes, which range from 1:2 to 1:13 [40,41]. These ratios evidence a submolar presence of CPR vs. CYP, which is recapitulated in the BTC system, as already demonstrated in our former studies [11,22,36,39].…”

“…The characterization of contents of heterologous expressed proteins found in the membrane fractions (isolated using the bacterial CPR/CYP co-expression system, BTC) [22,36] containing the wildtype CPR (CPR wt ) and mutant CPRs (CPR mut ) combined with CYP1A2, 2A6 or 3A4 is shown in Table 1. Co-expression of CPR mut with CYP1A2, 2A6, or 3A4 was achieved with a stoichiometry approximating the one obtained with CPR wt for each CYP, demonstrating no significant differences (p > 0.05).…”

“…The Escherichia coli cell model BTC was used for the heterologous co-expression of membrane-bound human CYP isoforms (CYP1A2, 2A6 or 3A4) together with human full-length CPR, using a biplasmid co-expression system, as previously reported [11,16,18,22,33,36,37]. CPR FD mutants P117H, G144C, G175D, and N151D, harbored in the pLCM_POR plasmid were used to transform E. coli PD301, already containing the expression vector (pCWori) for human CYP1A2, 2A6 or 3A4 (expressed in N-terminal modified forms), creating the following BTC strains: CPR P117H /CYP2A6, CPR P117H /CYP3A4, CPR G144C /CYP2A6, CPR G144C /CYP3A4, CPR G175D /CYP1A2, CPR G175D /CYP3A4, CPR N151D /CYP1A2, and CPR N151D /CYP2A6.…”

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley–Bixler syndrome variants of cytochrome P450 oxidoreductase

“…Previously, this simple cell-model demonstrated the co-expression of CYPOR with human CYPs (including CYP1A2), via a bi-plasmid system [15]. The two human proteins are correctly expressed, membraneanchored and are fully active, reflecting in vivo activities and representing the stoichiometry of the membrane-bound enzyme complex in the endoplasmic reticulum [16]. This is of importance, as exaggerated CYPOR:CYP ratios (≥ 2) lead to irrelevant enzyme kinetics and, thus, cell models containing such relative expression levels are not representative for studying the effects of ABS-CYPOR mutants on CYP activity.…”

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

“…δ-Aminolaevulinic Acid (5-ALA) is the first committed intermediate of the heme biosynthesis pathway in vivo , and ferric citrate can provide ferrous iron for this pathway as well as heme for the baculovirus/ sf9 system. POR should be taken into consideration for P450s heterologous expression because POR content is rate-limiting for CYP reactions[9]. Previous studies showed that functional CYP450s could be obtained by adding cofactors ( e.g.…”