NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

We report here on strain BTC, a new Escherichia coli mutagenicity tester strain for the expression of human cytochrome P450 (CYP) with an enhanced sensitivity for the detection of alkylating agents. This strain was developed first through knocking out of the genes ada and ogt in our previously developed strain BMX100, resulting in PD1000. Strain PD1000 demonstrated a significantly higher detection sensitivity towards several alkylating agents such as N-nitrosodiethylamine (NNdEA), N-nitrosodi-n-propylamine (NNdPA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Unexpectedly, this strain also showed an enhanced sensitivity towards 2-aminoanthracene (2AA), 4-aminobiphenyl (4AbPh), 2-aminofluorene (2AF) and 2-nitroanthracene (2NA) mutagenicity. Subsequently, our previously developed bi-plasmid system for the co-expression of a specific human CYP form (CYP1A2, 2A6 or 2E1) with human NADPH-cytochrome P450 reductase (RED) was introduced in strain PD1000, resulting in strains BTC1A2, BTC2A6 and BTC2E1, respectively. The mutagenicity of NNdEA and NNK was successfully detected with strains BTC2A6 and BTC2E1 and with strains BTC1A2 and BTC2A6, respectively, in contrast to the corresponding MTC (ada+ ogt+) CYP strains. The (ada- ogt-) deficient strain BTC1A2 also showed an enhanced sensitivity towards the detection of 2AA mutagenicity, when compared with the proficient repair strain MTC1A2. This enhancement was much more pronounced with strain PD1000 using the rat liver S9 fraction than with strain BTC1A2.

Results indicate the ability of b5 to affect CYP1A2 variants to behave more like the wild-type variant, attenuating detrimental effects of structural mutations of these variants, seemingly through extensive allosteric effects.

Cytochrome b(5) (b(5)) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b(5)/CYP-competent mutagenicity tester bacteria to study the role of b(5) in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b(5), and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b(5)-1A2, BTC-b(5)-2A6 and BTC-b(5)-2E1, respectively. The relative content of b(5) with CYP and RED in these three BTC-b(5)-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b(5)-void strains to determine the effect of b(5) on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased ( approximately 1.5-fold) in the presence of b(5). The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased approximately 3- and 23-fold, respectively when the bacteria contained b(5). The CYP2A6-mediated mutagenicity of NNK increased approximately 9-fold when co-expressed with b(5). The stimulatory effect of b(5) on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b(5) with CYP2A6 or CYP2E1. This demonstrates the prominent role of b(5) in the bioactivation of this carcinogen.

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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).

Small, highly strained heterocycles are archetypical alkylating agents (oxiranes, beta-lactones, aziridinium, and thiirinium ions). Oxetanes, which are tetragonal ethers, are higher homologues of oxiranes and reduced counterparts of beta-lactones, and would therefore be expected to be active alkylating agents. Oxetanes are widely used in the manufacture of polymers, especially in organic light-emitting diodes (OLEDs), and are present, as a substructure, in compounds such as the widely used antimitotic taxol. Whereas the results of animal tests suggest that trimethylene oxide (TMO), the parent compound, and beta,beta-dimethyloxetane (DMOX) are active carcinogens at the site of injection, no studies have explored the alkylating ability and genotoxicity of oxetanes. This work addresses the issue using a mixed methodology: a kinetic study of the alkylation reaction of 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilicity similar to that of DNA bases, by three oxetanes (TMO, DMOX, and methyloxetanemethanol), and a mutagenicity, genotoxicity, and cell viability study (Salmonella microsome test, BTC E. coli test, alkaline comet assay, and MTT assay). The results suggest either that oxetanes lack genotoxic capacity or that their mode of action is very different from that of epoxides and beta-lactones.

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