Escherichia coli BTC, a human cytochrome P450 competent tester strain with a high sensitivity towards alkylating agents: involvement of alkyltransferases in the repair of DNA damage induced by aromatic amines

Abstract

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.

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

“…A suitable E. coli host, the BTC strain, was applied for adequate coexpression of CYP1A2 allelic variants with human CYPOR, using a bi-plasmid system. 26 CYPOR expression levels were determined in the membrane protein fraction and were comparable among all strains (7.9-11.4 pmol mg À1 membrane protein). The CYPOR/CYP ratios for the different variants were found in the range of the normal values as observed in human liver microsomes (0.08-0.5) 36 except for variants I386F and R456H, which had no detectable CYP holoenzyme.…”

“…All variants, except I386F, were constructed using plasmid pCWh1A2 26 as DNA template, encoding human CYP1A2*1 (isoform 2) (NCBI NM_000761) with the N terminus modified as described by Fisher et al 25 In the case of the variant I386F, the template was pUC18_h1A2. This plasmid was constructed by cloning the cDNA of CYP1A2 from pCWh1A2 as an NdeI-XbaI fragment in pUC18.…”

“…26 Briefly, each strain was cultured in the TB (Terrific Broth) medium supplemented with peptone (2 g l À1 ), thiamine (1 mg ml À1 ), ampicillin (50 mg ml À1 ), kanamycin (15 mg ml À1 ), chloramphenicol (10 mg ml À1 ), trace elements solution 27 (4 ml ml À1 ) and 0.2 mM of isopropyl b-D-thiogalactoside (dioxane-free) (final concentrations). Cultures were started with 250 ml of À80 1C glycerol stocks, and cells were grown for 16 h at 28 1C with moderate agitation.…”

“…26 The present polymorphic CYP1A2 variants were further characterized with this cell model. Three premutagens (2AA, NNK and IQ) were used for this purpose (Table 1 and Supplementary Figures S5-S8).…”

“…For this purpose, the cDNA of the WT allele, present in a bacterial expression vector, 25 was adapted through genetic engineering to generate the eight different CYP1A2 forms. CYP1A2 alleles were coexpressed with human CYPOR in a bacterial cell model, 26 and the activity of the eight allelic variants and WT was investigated. The enzyme activity of these variants was analyzed together with WT and scrutinized through multivariate analysis.…”