Mutagenesis Advance Access originally published online on April 20, 2005
Mutagenesis 2005 20(3):199-208; doi:10.1093/mutage/gei028
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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
1Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal and 2Faculty of Sciences and Technology, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal
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Abstract |
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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.
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Introduction |
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Biotransformation plays an important role in the carcinogenic activity and organ specificity of chemical carcinogens (1
). In fact, a large proportion of carcinogens is chemically inert and requires metabolic activation to exert their detrimental effects (2). This metabolic activation is carried out, predominantly, by cytochrome P450 (CYP) (2). The success of detecting carcinogens as mutagens by mutagenicity assays is, in a large part, owing to the incorporation of metabolic systems, usually a rat liver extract (the so-called S9 fraction), to mimic the mammalian xenobiotic metabolism. This method, however, has several inherent limitations, in particular, the extracellular generation of reactive metabolites relative to the target cell, biotransformation differences between man and rodents and the high variability of enzyme activities of these extract preparations (3–5). Heterologous expression of human biotransformation enzymes in bacteria, yeast, insect- or mammalian-cells, led to the development of a new generation of mutagenicity assays without the limitations of exogenous metabolic systems. The ease in genetic manipulation and heterologous expression of mammalian (human) biotransformation enzymes, as well as the lack of (or low) endogenous biotransformation activities, are some of the advantages of using bacterial cells (5).
During the last decade, several human biotransformation enzymes, such as N-acetyltransferase, glutathione S-transferase, sulfotransferases and CYP have been expressed in mutagenicity tester bacteria (3,6). These specialized bacteria allow the study of the role of a specific human enzyme in the bioactivation of mutagens. Recently, new projects were set up (e.g. Environmental Genome Project) to stimulate research into the role of genetic variation to environmental exposure (7,8). The observation that many human CYP genes are polymorphic provides a rationale for interindividual differences in responses to xenobiotics (9). Variation in CYP activities may influence the adverse toxic effects, including carcinogenesis (1). In vitro assays, employing cell systems, which contain a discernible genotoxic parameter and simultaneously allow the expression of human biotransformation enzymes are considered to be a valuable tool to evaluate the role of polymorphisms encountered in CYPs and in other biotransformation enzymes, on their specificity and thus, on their role in genotoxicity of chemicals (3,5,7).
We reported previously on the development of a bi-plasmid system for the co-expression of human NADPH cytochrome P450 reductase with a particular human CYP form in the Escherichia coli tester strain MTC (10,11). This strain was derived from tester strain BMX100 (12), which detects efficiently chemical mutagenicity, monitored by the reversion to L-arginine auxotrophy. However, BMX100 and its derivatives, such as MTC, demonstrated a low sensitivity in the detection of DNA alkylating agents. This low sensitivity is a consequence of two very efficient alkyl-DNA repair enzymes, present in E.coli K12 bacteria. These two repair enzymes are encoded by the inducible ada gene and the constitutively expressed ogt gene (13). These enzymes mediate the irreversible transfer of methyl, ethyl, propyl or butyl groups from DNA to a specific cysteine residue in the methyltransferase itself. As the size of the adducts is increased, however, other cellular repair systems play increasing roles in the removal of the adducts from DNA (14). Goodtzova et al. (15) showed that both Ada and Ogt proteins, but also human O6-alkylguanine-DNA alkyltransferase are able to repair O6-benzylguanine adducts contained within an oligodeoxyribonucleotide and that Ogt protein can repair O6-methylguanine and O6-benzylguanine adducts with comparable rates. Human O6-alkylguanine-DNA alkyltransferase has also been shown to repair other large groups such as pyridyloxobutyl adducts (16). Although early studies suggested that O6-methylguanine was the ‘normal’ substrate for O6-alkylguanine-DNA alkyltransferase and that the repair rates declined with the size of the adduct, it is now apparent that this is an oversimplification and that the size of the adduct is not the only factor determining the repair of O6-derivatives in DNA by O6-alkylguanine-DNA alkyltransferase (16). Interestingly, recent studies have described the mutagenic repair of a human alkyltransferase. The human O6-alkylguanine-DNA alkyltransferase increases the mutagenicity and the cytotoxicity of 1,2-dibromoethane and dibromomethane, when expressed in E.coli (17,18).
In this work, we describe the development of a novel strain, namely BTC, a human CYP competent tester strain with increased sensitivity towards the detection of alkylating agents. This new strain was derived from strain PD1000 developed by knocking-out of both ada and ogt in strain BMX100. We demonstrate results with three human forms of CYP, in particular, CYP2A6 and CYP2E1, which are known to be involved in the bioactivation of alkylating promutagens.
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Reagents
L-Arginine,
-aminolevulinic acid, ampicillin, kanamycin sulfate, chloramphenicol, tetracycline HCl (Tet), coumarin, 7-hydroxycoumarin, cytochrome C, isopropyl ß-D-thiogalactoside (dioxane-free) (IPTG), thiamine, chlorzoxazone, 6-hydroxychlorzoxazone, N-nitrosodiethylamine (NNdEA), N-nitrosodi-n-propylamine (NNdPA), 2-aminoanthracene (2AA), 1-aminopyrene (1AP), 6-aminochrysene (6AC), 4-nitroquinoline-1-oxide (4NQO), benzo[a]pyrene (B[a]P), aflatoxin B1 (AfB1), cumene hydroperoxide (CHP), t-butyl-hydroperoxide (t-BHP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminofluorene (2AF), 4-aminobiphenyl (4AbPh), glucose-6-phosphate, NADPH, NADP+ and fusaric acid were obtained from Sigma Chemical Co. (St Louis, MO). 2-Amino-3-methylimidazo(4,5-f)quinoline (IQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were obtained from Toronto Research Chemicals Inc. (North York, Ontario). 2-Nitroanthracene (2NA) was obtained from Chiron AS (Trondheim, Norway). Bacto agar, bacto peptone, bacto tryptone and bacto yeast extract were obtained from Difco (Detroit, MI). All other chemicals were of the highest quality. Rat liver frozen S9 fraction from rats induced with phenobarbital and methylcholantren mixture with a dose of 50–20 mg/kg respectively was purchased from IFFA CREDO, Charles River Company (Spain).
Strains, plasmids and cultures
Strains and plasmids used in this study are presented in Table I. Strain PD1000 was obtained by the inactivation of both ada and ogt genes in strain BMX100. These inactivations were achieved by 
tranductions (19), using GW5352 and KT233 as donor strains (Table I). The ada– Tets mutant strain was isolated through selection for fusaric acid resistance (20). To confirm the ada/ogt double knockout a spot test with MNNG and strains FP400 (ada+ogt+), PD200 (ada–ogt+) and PD300 (ada–ogt–) was carried out. Strains were plated as for mutagenicity assays on VB plates and 0.1 and 0.3 µg of MNNG were applied on each plate. A stable mutant, deficient in the lipopolysaccharide core (LPSd) demonstrating a high permeability towards bulky compounds, was isolated by C21 infection as described previously (21). Subsequently, the mutator plasmid pLCM carrying the mucAB operon was introduced, obtaining strain PD1000 (Table I). For the development of strains BTC0, BTC1A2, BTC2A6 and BTC2E1, strain PD301 was transfected first with plasmid pLCMhOR (Table I) and subsequently with the different CYP expression vectors. All strains were verified for the correct phenotype for mutagenicity testing, as described previously (24). All transfections to tester strain PD301 and derivatives were carried out by the CaCl2 method (23). Plasmid pCW2E1 was obtained by courtesy of Dr R.W.Estabrook of Southwestern Medical Centre, University of Texas and was constructed from the human CYP2E1 containing plasmid from Todd Porter (21) with modifications as described by Gillam et al. (25) (construct no. 18). For mutagenicity assays with strains BMX100 and PD1000 bacteria were grown for 15 h in LB medium and appropriate antibiotics at 37°C and 210 r.p.m. For heterologous expression bacteria were grown at 28°C for 15.5 h in TB medium supplemented with peptone (2 g/l), ampicillin (50 µg/ml), kanamycin (15 µg/ml), chloramphenicol (10 µg/ml) (only BTC), -aminolevulinic acid [0.1 mM, except for strains with CYP1A2 for which no -aminolevulinic acid was added (10,11)], thiamine (1 µg/ml), a mixture of trace elements (0.4 ml/l) (12) and IPTG (0.2 mM). The cultures were analysed for their RED- and CYP-expression, CYP activities and applied in mutagenicity assays.
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Analysis of RED, CYP expression and CYP activities
Preparation of bacterial membranes, determination of protein content, CYP- and RED-expression, ethoxyresourfin deethylation, methoxyresourfin demethylation and coumarin 7-hydroxylation activities were performed as described previously (11
). Chlorzoxazone 6-hydroxylation was measured by reverse phase HPLC according to Pearce et al. (26).
Mutagenicity assays
Mutagenicity assays with strains PD1000 and BMX100 were performed using the liquid pre-incubation assay technique as described previously (22). The S9 mix (10%) was prepared according to Maron and Ames (27). The mutagenicity assays with BTC strains were performed using the liquid pre-incubation assay technique, as described previously (12). Experiments were performed al least in triplicate. Revertant colonies were counted after the usual 48 h of incubation at 37°C. Mutagenic activities (in revertants/nmol or revertants/µmol) were determined from the slope of the linear portion of the dose–response curve.
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Mutagenicity testing with PD1000
The mother strain of the MTC-CYP strains, BMX100 (Table I), demonstrated low sensitivity in detecting alkylating mutagens. We decided to develop a new strain with an enhanced sensitivity for the detection of alkylating agents. This new strain, PD1000 was obtained by inactivation of the DNA repair genes ada and ogt. Figure 1 shows a spot test with MNNG and strains FP400, PD200 and PD300. The increase of the zone of revertant colonies surrounding the application area of MNNG, in particular the 0.1 µg dose, showed an increased sensitivity (both in reversion as well as growth inhibition) to this alkylating mutagen confirming the single (PD200, ada–) and the double (PD300 ada–ogt–) knockout of these DNA repair genes.
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Several known alkylating agents were tested with strains PD1000 and BMX100. The spontaneous reversion rates were similar in both strains. The new strain was
3-fold more sensitive towards NNdPA and 9-fold more sensitive towards MNNG (Figure 2). The improvement in sensitivity for detecting alkylating agents by PD1000 is best demonstrated with NNK and NNdEA inducing 298 and 112 revertants per µmol, respectively in comparison with strain BMX100, which did not demonstrate any mutagenicity when these two compounds were tested up to 1 (NNK) and 3 µmol/plate (NNdEA) (Figure 2).
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Strain PD1000 was also checked for its capacity in detecting non-alkylating mutagens compared with strain BMX100. The sensitivity for the mutagenicity of 4NQO, B[a]P, AfB1, CHP and t-BHP was not significantly altered (Figure 3). However, strain PD1000 was more sensitive towards the mutagenicity of 2AA (2.5-fold), 2AF (1.5-fold), 4AbPh (2.5-fold) and 2NA (1.5-fold) than strain BMX100 (Figure 4). This higher sensitivity was not observed with other aromatic (1AP and 6AC) or heterocyclic amines (IQ) (Figure 4).
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Development of BTC-tester strains
With strain MTC, RED was expressed by means of a mutator-plasmid pLCMhOR containing both the cDNA of human RED and the SOS mutagenesis operon mucAB for increased sensitivity in the L-arginine reversion assay (10
,11). The different human CYP forms were expressed by means of the pCWori-vector containing the different CYP cDNA (Table I). This bi-plasmid approach was also used to obtain the new BTC-CYP tester strains, namely BTC1A2, BTC2A6 and BTC2E1, through introduction of the expression vectors of human CYP1A2, 2A6 or 2E1 in strain PD301/pLCMhOR, respectively (Table I).
Characterization of RED and CYP expression and CYP activities
Before employment in mutagenicity testing, BTC-CYP strains were characterized in what concerns expression levels and activities of the heterologous proteins CYP and RED (Figure 5, Table II). RED was expressed similarly in strains expressing CYP1A2 and CYP2A6 (12 pmol/mg protein). Strain MTC2E1 presented a slightly higher level (16 pmol/mg protein) and strain BTC2E1 presented a RED content, which was 2-fold higher compared with the other strains (33 pmol/mg protein). CYP1A2 was expressed similarly in strains MTC1A2 and BTC1A2 (168 versus 213 pmol/mg, respectively). Strain MTC2A6 presented a higher CYP2A6 expression than strain BTC2A6 (267 versus 125 pmol/mg, respectively) and the new BTC2E1 strain presented a higher CYP2E1 expression than the former MTC2E1 strain (124 versus 66 pmol/mg, respectively).
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The enzymatic activities of the different CYPs, using membranes derived from cells cultured under the exact same conditions as for mutagenicity tests, were determined using their specific chemical probes. CYP activities were determined as ethoxyresorufin deethylase (EROD) and methoxyresorufin demethylase (MROD) for the CYP1A2 expressing strains, as coumarin 7-hydroxylase for the CYP2A6 expressing strains and as chlorzoxazone 6-hydroxylase for the CYP2E1 expressing strains (Table III). EROD and MROD activities were similar in strains MTC1A2 and BTC1A2. Both strains demonstrated a higher MROD activity compared with EROD activity. Membranes derived from both CYP2A6 and CYP2E1 expressing strains presented similar coumarin 7-hydroxylase activity and chlorzoxazone 6-hydroxylase activity, respectively. No CYP specific activity could be detected for the control strains MTC0 and BTC0 (Table III).
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Mutagenicity testing with BTC-CYP strains
Strains expressing CYP1A2, CYP2A6 and CYP2E1 were tested with several pro-carcinogens. The spontaneous reversion rates were similar in MTC-CYP and BTC-CYP strains.
The mutagenicity of NNdEA was successfully detected with the new BTC2A6 and BTC2E1 tester strains, in contrast with the BMX100 derivatives MTC2A6 and MTC2E1 (Figure 6). The mutagenic activities were 447 and 1228 revertants/µmol for strain BTC2A6 and BTC2E1, respectively. No mutagenicity could be detected for NNdPA with MTC2A6, BTC2A6, MTC2E1 or BTC2E1. NNK demonstrated mutagenicity in strains BTC1A2 (1165 revertants/µmol) and BTC2A6 (290 revertants/µmol) (Figure 7). Several aromatic and heterocyclic amines, namely 2AA, 2AF, 4AbPh, IQ, 6AC and 1AP were tested with strains MTC1A2 and BTC1A2. BTC1A2 seems to be more efficient in detecting the 2AA mutagenicity (Figure 8). The mutagenic activities were 5786 revertants/µmol for strain MTC1A2 and 7860 revertants/µmol for BTC1A2. MTC1A2 and BTC1A2 showed similar sensitivities towards the mutagenicity of 1AP, IQ and 6AC. No mutagenicity could be detected for 2AF and 4AbPh with these strains.
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The development of metabolic competent mutagenicity test cell system is of interest, in the study of the role of human enzymes in the bioactivation of promutagens, in particular, for its potential use in functional analysis of the polymorphic human metabolizing enzymes, in chemical carcinogenesis.
We reported previously on a bi-plasmid co-expression system with the E.coli tester strain MTC, combining the expression of human RED with a specific human CYP form (10,11). E.coli K12 bacteria are void of CYPs and thus, appropriate for human CYP expression without the interference of endogenous CYPs, which is the case of several other cell systems (28). However, MTC strain failed in the detection of alkylating mutagens owing to the presence of two very efficient alkyl-DNA repair enzymes encoded by ada and ogt genes in E.coli K12. To overcome this limitation, we knocked out the genes ada and ogt in strain BMX100, the mother strain of MTC, thus generating strain PD1000 (Table I). The relevance of these two DNA repair enzymes can be seen by comparing the mutagenic effects of the alkylating mutagens NNK, NNdEA, NNdPA and MNNG in the repair proficient strain BMX100 and repair deficient strain PD1000 (Figure 2). Strain PD1000 was 3-fold more sensitive towards NNdPA and 9-fold more sensitive towards MNNG. The improvement in sensitivity for detecting alkylating agents by PD1000 is best demonstrated with NNK and NNdEA inducing 298 and 112 revertants/µmol, respectively in comparison with strain BMX100, which did not demonstrate any mutagenicity when these two compounds were tested up to 1 (NNK) and 3 µmol/plate (NNdEA) (Figure 2). Yamada et al. (29) also showed that deletion of ogtST or ogtST plus adaST genes substantially increased the sensitivity of the Salmonella typhimurium strains TA1535 and TA100 to the mutagenicity of several alkylating mutagens.
Strain PD1000 was transformed with the bi-plasmid system for the co-expression of human CYP1A2, 2A6 or 2E1 with RED, resulting in BTC-CYP strains. The expression of CYP and RED could be quantified in all strains (Figure 5; Table II). The lack of the two DNA methyltransferases seems to have an influence in the yield of CYP2A6 and CYP2E1. The expression level of CYP2A6 decreases and the expression level of CYP2E1 increases in the new BTC strains compared with the corresponding MTC strains. Moreover, the co-expression of CYP2E1 with RED in an ada and ogt deficient strain seems to increase RED expression level (Table II), as observed before (30).
The enzymatic properties of CYPs expressed in MTC and BTC strains were evaluated using their specific chemical probes. The results showed that each form efficiently catalyzed the oxidation of each specific substrate (Table III). The determined activities could be clearly attributed to the heterologous expression of CYPs as no such activities could be determined in the CYP-void strains MTC0 and BTC0 (Table III). The turnover rates obtained for MTC-CYP and BTC-CYP strains were similar.
We tested several N-nitrosamines to evaluate the improvement in mutagenicity detecting of the new BTC strains in comparison with the corresponding MTC strains. This class of mutagens is recognized as among the most potent chemical carcinogens widely present in the environment, as evidenced by the fact that they induce cancer in various organs in experimental animals and are probably implicated in the aetiology of several human cancers (31). These compounds are metabolized by different CYP forms, resulting in the formation of highly reactive alkylating mutagens (32).
The mutagenicity of NNdEA was successfully detected with BTC2A6 and BTC2E1 in contrast to the corresponding MTC strains, which did not demonstrate any mutagenic response to this alkylating agent (Figure 6). To compare the sensitivity of different strains, we calculated the minimal detectable dose (MDD), which is the ratio between the mutagenic activity and the spontaneous revertants. MDD represents the dose level of the compound necessary to induce the doubling of the spontaneous revertants, indicating the minimal dose level for a positive result. For NNdEA the MDD for BTC-CYP strains were substantially lower compared with the one obtained with the strain PD1000 in combination with the rat liver S9 fraction (Table IV). Thus, the detection of NNdEA occurred more efficiently with BTC2A6 and BTC2E1 strains compared with PD1000 plus the S9 fraction (Table IV). This enhancement in sensitivity could be the result of the intracellular metabolism of NNdEA in contrast to the extracellular bioactivation of S9 or could be the result of differences in species. The results presented here showed the prominent role of CYP2E1 in the bioactivation of this particular compound (Table IV). Strains BTC2A6 and BTC2E1 failed in the detection of NNdPA mutagenicity. Activation of NNdEA and NNK by CYP2A6 was also reported by others using an 
S.typhimurium tester strain, co-expressing human CYP2A6 and RED (33–37). In contrast to the present study, the mutagenicity of NNdPA could be detected with those S.typhimurium tester strains co-expressing CYP2A6 or CYP2E1 with RED (32,34–37). The results reported in these studies also demonstrated a larger contribution of CYP2A6 in the bioactivation of NNdEA compared with CYP2E1. The RED:CYP ratios presented by these S.typhimurium strains are much higher (>2.5) than the same ratio presented by BTC strains [(0.23–0.31) to BTC2E1 and (0.08–0.15) to BTC2A6] (30). These differences in RED:CYP ratios could be responsible for the difference in results. The molar RED:CYP ratio is a critical factor for the catalytic activities of a particular CYP form in a particular tissue, subcellular fraction or expression system and is determined not only by the abundance of the CYP, but also its electron transport partners (38). The RED:CYP ratios presented by the new BTC strains presented here are closer to the RED:CYP ratios in human liver microsomes, which range from 0.08 to 0.5 (39,40), and may therefore be more significant for the physiological situation (30).
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The tobacco mutagen NNK could clearly be identified as an alkylating agent, demonstrated mutagenicity in the ada– ogt– strain PD1000 (Figure 2) and its BTC-CYP derivatives, in comparison with BMX100 and the MTC-CYP (Figure 7). The prominent role of CYP2A6 but particularly that of CYP1A2 in the bioactivation of NNK is demonstrated, corroborating the results obtained by Fujita and Kamataki (36
). The MDD for NNK with strains BTC1A2, BTC2A6 and PD1000 plus S9 mix are presented in Table V. In contrast to the results obtained with NNdEA, strains BTC1A2 and BTC2A6 did not show any enhancement in sensitivity towards NNK mutagenicity compared with PD1000 plus the S9 fraction. This can, in part, be explained by the presence of multiple CYPs and/or other enzymes in rat liver S9 fraction, which could be involved in the biotransformation of NNK.
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When testing a specific class of carcinogens, namely aromatic amines, using the rat liver S9 fraction, an unexpected higher sensitivity was observed with strain PD1000 for 2AA (2.5-fold), 2AF (1.5-fold) and 4AbPh (2.5-fold) in comparison with the ada+ogt+ strain BMX100 (Figure 4). However, this higher sensitivity was not observed with other aromatic (1AP and 6AC) or heterocyclic (IQ) amines (Figure 4). The dose–response curves for 2AA, 2AF and 4AbPh with strains PD1000 and BMX100 did not demonstrate different shapes (Figure 4), which did not suggest different mechanisms of mutagenic/toxic action between the two strains. Moreover, 2AA, 2AF and 4AbPh did not demonstrate any mutagenicity with strains BMX100 and PD1000, directly or using S9-without co-factors (data not shown). This indicates that the enhancement of their mutagenicity with strain PD1000 was S9-dependent. This also excludes the hypothesis that direct acting impurities of the mutagen batches would be responsible for the observed increased sensitivity. The major bioactivation route of aromatic and heterocyclic amines involve two subsequent steps catalyzed by CYP1A2 (N-hydroxylation) and N-acetyltransferases (N-acetylation) or sulfotransferases (sulfonation) with formation of N-acetoxy or N-sulfoxy esters, which form highly reactive bulky nitrenium/carbonium ions that bind to DNA (41
,42). The higher sensitivity observed with PD1000 relative to BMX100, indicates that the products of ada and/or ogt genes can repair, at least in part, the 2AA, 2AF and 4AbPh induced DNA damage. This implicates either (i) the formation of a small alkylating product by rat liver S9 enzymes or (ii) one or both alkyltransferases may participate in the repair of some particular bulky DNA adducts, although the ada ogt mediated repair is considered to be particularly efficient with small alkylating groups (43). If the observed difference in sensitivity with the two strains was related to the hydroxylamine metabolites formed by N-hydroxylation catalyzed by CYP1A2, then the difference in sensitivity should also be observed with the nitro analogues of these aromatic amines, which do not require S9 activation owing to endogenous bacterial nitroreductases. In fact, strain PD1000 was more sensitive towards 2NA mutagenicity than strain BMX100 (Figure 4). Our results suggest the involvement of Ada and/or Ogt proteins in the repair of specific bulky DNA adducts, as the enhancement in sensitivity with strain PD1000 was only observed with 2AA, 2AF, 4AbPh and 2NA and not with other mutagens that generate bulky DNA adducts, such as 6AC, IQ, B[a]P or AfB1.
The same aromatic and heterocyclic amines were tested in MTC1A2 and BTC1A2 strains (Figure 8). The MDD for 1AP, 2AA and IQ were much lower in the human CYP1A2/RED expressing strains compared with the use of rat liver S9 fraction (Table VI). These differences could be related not only with the intracellular metabolism of the compounds, but also with the different activities between human CYP1A2 and the rat CYP1A2 orthologues. It is known that human CYP1A2 is considerably more active in N-hydroxylation of several aromatic amines than rat CYP1A2 (12). However, BTC1A2 and MTC1A2 failed to detect the 2AF and 4AbPh mutagenicity and both showed a lower sensitivity towards 6AC mutagenicity compared with PD1000 plus the S9 fraction (Table VI). It is likely that the bioactivation of these compounds involve other CYP forms and/or other enzymes present in rat liver S9 fraction, not present in BTC1A2 or MTC1A2 bacteria. Kawabe et al. (44), using in vitro studies with CYP1A2-null and wild-type mouse liver microsomes showed that CYP1A2 is not the primary enzyme responsible for 4AbPh carcinogenesis. Another study, using a N-acetyltransferase over-expressing S.typhimurium strain showed that the rates of 6AC activation by 18 human liver samples have good correlations to the contents of CYP2B6, as well as CYP3A4 (45). The deficient repair strain BTC1A2 was more sensitive towards 2AA mutagenicity than the proficient repair strain MTC1A2. However, this difference was more pronounced with rat liver S9 fraction than with human CYP1A2.
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In conclusion, this study describes a new ada–ogt– tester strain, namely PD1000 with increased sensitivity through the detection of alkylating mutagens. This strain was used as starting point for three new human CYP competent E.coli mutagenicity tester strains. These new strains co-express human RED with functional human CYP1A2, 2A6 or 2E1 and can efficiently detect the mutagenicity of several known CYP-dependent alkylating and non-alkylating promutagens. The application of genetically engineered E.coli cells, expressing functional human CYPs for bacterial mutagenesis assays, provides a useful approach for the evaluation of agents potentially harmful to humans. The intracellular generation of reactive metabolites permits the detection of both short-lived and long-lived intermediates without transmembrane migration. Furthermore, the expression of human CYPs avoids the differences in patterns of metabolism between species. Strains BTC seems to be appropriate for the study of the role of human CYPs in the bioactivation of promutagens and can be an efficient mean for the functional studies of CYP allelic variants in chemical mutagenesis, particularly in what concerns CYP2A6 and CYP2E1, which are known to be involved in the bioactivation of alkylating promutagens, but also for other CYPs as has been shown recently for CYP1A2 (46
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Acknowledgments |
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We are grateful to Dr Leona Samson and Dr Yusaku Nakabeppu for the gift of strain GW5253 and strain KT233, respectively. The financial support of the Fundação Programa Operacional Ciência, Tecnologia e Inovação (POCTI Medida 1.2) for MK at the Faculty of Medical Sciences of the Universidade Nova de Lisboa is acknowledged.
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Notes |
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* To whom correspondence should be addressed. Tel: +351 21 3610297; Fax: +351 21 3622018; Email: mkranendonk.gene{at}fcm.unl.pt
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References |
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Received on September 22, 2004; revised on March 11, 2005; accepted on March 17, 2005.
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