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ABSTRACT: An increased risk of breast cancer has been observed in women who consume "very well-done" meats. Heterocyclic amines are mutagenic and carcinogenic pyrolysis products formed during high temperature cooking of meats. In the present study, human milk samples were analyzed for PhIP, one of the most abundant dietary heterocyclic amine. A protocol was developed with a mixed-mode cation exchange sorbent for the extraction of heterocyclic amines from milk. Milk samples were acquired from healthy Canadian women. With LC/MS analysis and the method of isotope dilution for quantification, levels of PhIP were determined in human milk samples. PhIP was detected in 9 of the 11 milk samples, at levels as high as 59 pg/mL (ppt). No PhIP was detected in the milk of the vegetarian donor. Detection of PhIP in milk indicates that ductal mammary epithelial cells are directly exposed to this carcinogen, suggesting that heterocyclic amines are possible human mammary carcinogens.
Chemical Research in Toxicology 12/2001; 14(11):1523-8. · 3.78 Impact Factor
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ABSTRACT: We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/2001; 497(1-2):39-47. · 2.85 Impact Factor
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ABSTRACT: Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.
Environmental and Molecular Mutagenesis 02/2001; 38(1):12-8. · 3.71 Impact Factor
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P D Josephy
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ABSTRACT: The Escherichia coli lacZ reversion assay, based on the set of episomal lacZ alleles engineered by Miller et al., provides an attractive system for studies of mutagenesis and mutational specificity. Each strain in the lacZ set reverts by a specific base substitution or frameshift event. Revertants are selected by growth on lactose minimal medium. In this review, I describe the development of the assay and its subsequent modifications and improvements. Examples of its application are presented and detailed protocols for the implementation of the assay are given.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2000; 455(1-2):71-80. · 2.85 Impact Factor
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ABSTRACT: Understanding the relationships between the sequences and catalytic activities of P450 enzymes that catalyze the bioactivation of mutagens and carcinogens is an important goal in mutation research. Escherichia coli strain DJ4309 expresses recombinant human P450 1A2 and activates promutagens such as MeIQ (2-amino-3, 4-dimethylimidazo[4,5-f]quinoline), as measured by induction of reverse mutations detected as lacZ(+) colonies on minimal lactose (ML) plates. Pools of P450 1A2 mutants were constructed by polymerase chain reaction (PCR) mutagenesis of putative substrate recognition sites (SRSs). Cultures of individual clones were patched onto MeIQ/ML plates and the growth of revertant microcolonies within each patch was inspected after 2 days of incubation. Beginning with a pool of several thousand clones, we identified 25 distinct P450 1A2 SRS variants with altered activities. In this study, the MeIQ dose-responses of all the variants are reported. The implications of the results are considered with reference to published models of the protein's structure.
Environmental and Molecular Mutagenesis 02/2000; 35(4):328-35. · 3.71 Impact Factor
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ABSTRACT: The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions. Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins. Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101. Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309. This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens. We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo[4,5-f]quinoline (MeIQ), a typical heterocyclic amine mutagen. Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis. Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/1999; 429(2):199-208. · 2.85 Impact Factor
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ABSTRACT: Cytochrome P450 (P450) 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens. The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals. Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities. The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines. Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein. Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines. A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy. A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions. These results may be of relevance in consideration of genetic polymorphisms. Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others. Some dinitropyrenes are P450 1A2 substrates but others are not. 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/1999; 428(1-2):115-24. · 2.85 Impact Factor
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ABSTRACT: Heterocyclic aromatic amines (HAAs) are potent bacterial mutagens and potential human carcinogens formed in heat processed proteins. The Ames test (strain TA98) is a useful mutagenicity test system to screen food products for these compounds. HAAs require activation to their genotoxic forms, and in the Ames test, a rat liver S-9 preparation is normally used. In order to better understand the mechanisms of mutagen activation with respect to human metabolism, new bacterial strains containing human cytochrome P450s and other metabolic enzymes have recently been developed. We have investigated the capacity of one of these strains, DJ4309 [Josephy et al., Chem. Res. Toxicol. 11 (1998) 70-74] as a screening tool for mutagens in food products. DJ4309 expresses the human P450 1A2, human NADPH cytochrome reductase and the bacterial acetyl CoA:arylamine N-acetyltransferase. This strain is as sensitive as the Ames system to the mutagenic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline, but less sensitive to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. However, the mutagenicity of the arylamine 2-aminofluorene is considerably higher in DJ4309 than in the Ames test system. Meat extracts with a total HAA content ranging from less than 2 ng/g to 20 ng/g are efficiently detected by the Ames TA98 strain with rat liver S-9 activation. DJ4309 is less sensitive, with fewer revertants induced over the same dose range. Unknown compounds present in the meat extracts appear to inhibit the activity of the P450 1A2 enzyme in the DJ4309 strain. We have therefore demonstrated that although DJ4309 is a useful tool for mechanistic studies in chemical carcinogenesis, the screening of complex food matrices for HAAs by this bacterial strain must be conducted with caution.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/1999; 442(2):79-87. · 2.85 Impact Factor
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ABSTRACT: Random mutagenesis is an approach that has the potential to provide useful information about cytochrome P450 (P450) enzymes but has not been extensively used to date. We applied our previously developed systems for generation of random libraries of human P450 1A2 with the putative substrate recognition sequences mutated (individual residues) and an Escherichia coli genotoxity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). A total of 27 mutants were screened from 6000 clones, a small portion of the library. The sequence changes were identified, and E. coli membranes containing each P450 (with NADPH-P450 reductase expressed using a bicistronic vector) were used to determine kcat and Km values for 7-ethoxyresorufin and phenacetin O-deethylation and the (in vitro) activation of MeIQ with another bacterial genotoxicity system (Salmonella typhimurium umu). Within each assay, the values of kcat/Km varied by 2 orders of magnitude, and in some cases these parameters were 3-4-fold higher than for the native enzyme. The profiles of the mutants varied considerably for the three different reactions. Some of the mutants in the Asp-320 region may be compared with site-directed mutants of rat P450 1A2 already reported, with several differences noted. Other mutants have not been studied before in human P450 1A2 or homologues and are of interest; i.e., all Phe-226 mutants showed considerably reduced activity but Glu-225 mutants had increased catalytic activities. In principle, this approach may be applied to random mutagenesis of any enzyme that converts chemicals to mutagens and can be expressed in bacteria.
Biochemistry 05/1999; 38(17):5283-9. · 3.42 Impact Factor
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ABSTRACT: Environmental chemicals may play a role in the etiology of human breast cancer. Aromatic amines, industrial chemicals found as environmental pollutants, have been identified as rat mammary carcinogens. We have detected the monocyclic aromatic amines, aniline, o-toluidine, and N-methylaniline at parts per billion levels in human milk samples, by applying solid-phase microextraction coupled with gas chromatography/mass spectrometry. Our findings indicate that human breast ductal epithelial cells are directly exposed to aromatic amines, including o-toluidine, which is a mammary carcinogen in female rats.
Chemical Research in Toxicology 02/1999; 12(1):78-82. · 3.78 Impact Factor
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ABSTRACT: A solid-phase microextraction (SPME) protocol was developed for the quantitative analysis of monocyclic aromatic amines from biological fluids. The headspace SPME sampling technique was optimized for extraction and concentration of five target analytes (aniline, o-toluidine, 2-chloroaniline, 2,6-dimethylaniline, 2,4,6-trimethylaniline) from urine, blood, and milk. The temperature, pH, and ionic strength of the matrix sample were modified to allow maximum adsorption of the analytes onto the SPME fiber. This method is rapid yet sensitive and can be completed in 15 min on a 5-mL sample. SPME/GC/MS analysis yielded good reproducibility (RSD > 11%) for each analyte from urine, blood, and milk. Method detection limits for the various biological fluids were determined and ranged from 0.40 ppb for 2,4,6-trimethylaniline in urine to 7.7 ppb for aniline in blood. This SPME sampling protocol can be applied to the biomonitoring of monocyclic aromatic amines from occupational, environmental, and medical exposure.
Analytical Chemistry 06/1998; 70(9):1986-92. · 5.86 Impact Factor
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ABSTRACT: We describe the construction of a new strain of Escherichia coli designed to bioactivate aromatic amines and to detect their mutagenicity with high sensitivity. Strain DJ4309 bears two plasmids, a pACYC184-derived plasmid which expresses Salmonella typhimurium acetyl CoA:arylamine N-acetyltransferase (NAT) and a pBR322-derived plasmid which expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase. The combined actions of these enzymes convert aromatic amines into reactive, mutagenic N-acetoxy esters. The strain also carries a mutated copy of the lacZ gene (on an F' factor) which reverts to the wild-type gene by a -(GpC) frameshift mutation. Strain DJ4309 expresses high levels of NAT and cytochrome P450 1A2 and is very sensitive to mutagenesis induced by representative aromatic amines. Mutagenicity of 2-aminoanthracene in strain DJ4309 is higher than can be obtained by rat liver homogenate 9000g supernatant (S9) activation in the parent strain lacking the P450 expression vector. Strain DJ4309 provides a useful system for detecting mutagenic aromatic amines and for studying their metabolism by human P450 1A2.
Chemical Research in Toxicology 02/1998; 11(1):70-4. · 3.78 Impact Factor
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ABSTRACT: We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase was inhibited, in a concentration-dependent manner, by alpha-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) = 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.
Chemical Research in Toxicology 06/1997; 10(5):582-8. · 3.78 Impact Factor
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ABSTRACT: Bacterial mutagenicity assays have been widely used in genotoxicology research for two decades. We discuss the development of such assays, especially the Ames test, with particular attention to strain engineering. Genes encoding enzymes of mutagen bioactivation, including N-acetyltransferase, nitroreductase, and cytochrome P450, have been introduced into tester strains. The processing of DNA damage by the bacterial strains has also been modified in several ways, so as to enhance mutagenesis. These efforts have greatly increased the sensitivity of mutation assays and have illuminated the molecular mechanisms of mutagenesis. We also discuss the relationship between bacterial assays and in vivo mutation assays which use transgenic rodents.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/1997; 386(1):1-23. · 2.85 Impact Factor
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ABSTRACT: An international symposium entitled Chemical Carcinogenesis, Mutagenesis and Teratogenesis: a Tribute to James and Elizabeth Miller was held in Toronto, Ont., July 19, 1994. This symposium theme was discussed in the presence of James Miller, 79 years young, who with his wife, Elizabeth Miller (1920-1987), are considered to be the pioneers of this medical and environmental toxicology research field. It is generally believed that the susceptibility of an individual to chemical carcinogenesis or teratogenesis varies considerably depending upon their genetic makeup, diet, lifestyle, and their environmental exposure. One goal of the research discussed at this symposium was an examination of the role of the enzymes involved in the metabolic activation and detoxification of carcinogens and teratogens. The interindividual variabilities in the levels and activity of these enzymes could contribute to the susceptibility of the individual to chemical carcinogens or teratogens. At the symposium evidence was presented indicating that theta-class glutathione (GSH) S-transferase levels activate dihalomethanes and could therefore initiate the carcinogenic response to butadiene and 1,2-dibromo-3-chloropropane. The dramatic genetic polymorphism of this class of GSH S-transferase could thereby contribute to the individual's susceptibility to these carcinogens. Similarly, the GSH S-transferase and GSH levels in the embryo and yolk sac that are determined during organogenesis could also be important factors in determining the susceptibility of the embryo to teratogens. The levels of cytochrome P450 1A2, aromatic amine N-acetyltransferases, and sulfotransferases could also determine the susceptibility of the individual to carcinogenic arylamines. Accordingly, an Ames tester strain was described that was genetically engineered so as to express both aromatic amine N-acetyltransferase and human cytochrome P450 1A2. This should prove useful for predicting which arylamines are likely to be carcinogenic to humans. Nonsteroidal anti-inflammatory drugs may also prove useful in inhibiting the cytochrome P450s that activate the nitrosamines found in tobacco smoke suspected to cause lung cancer. Finally, the sulfotransferase isoforms involved in the metabolic activation of carcinogenic arylamines were identified.
Canadian Journal of Physiology and Pharmacology 06/1996; 74(5):565-71. · 1.95 Impact Factor
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P D Josephy
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ABSTRACT: Aromatic amines are mammary carcinogens in rodents and exposure to aromatic amines may be associated with increased risk of breast cancer in women. Peroxidases present in milk can oxidize aromatic amines to reactive electrophiles which bind to DNA and induce mutations. Hydrogen peroxide, required for peroxidase-dependent oxidations, is supplied by milk xanthine oxidase and by the respiratory burst of neutrophils, cells which are present in milk and activated by exposure to it. In this paper, I propose that lactoperoxidase and myeloperoxidase activate aromatic amines, within the breast ducts, and that these enzymes play a crucial role in the chemical induction of breast cancer.
Mutagenesis 02/1996; 11(1):3-7. · 3.18 Impact Factor
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ABSTRACT: Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions. We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity. Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems. The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX. NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2. (1332 vs. 37 nmol/min/U of immunoreactive NAT protein). Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations. Similar pathways were observed in human hepatic cytosolic fractions. In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs. 11.5% dead cells at a concentration of 300 microM). N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test. These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.
Journal of Pharmacology and Experimental Therapeutics 10/1995; 274(3):1099-104. · 3.83 Impact Factor
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ABSTRACT: Escherichia coli lacZ strains CC107-CC111, which detect specific frameshift mutations, were used to study the mutational specificities of 2-nitro-3-methylimidazo[4,5-f] quinoline (NO2-IQ) and rat hepatic S9-activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). New constructs were made in which UvrABC-dependent excision repair was eliminated (strains DJ3107-DJ3111), followed by introduction of plasmid pYG219 conferring acetyl CoA:arylamine N-acetyltransferase/acetyl CoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity (strains DJ3207-DJ3211). Sensitivity to mutagens was greatly enhanced. The mutational specificity of NO2-IQ was identical to that of the corresponding amine, IQ. The most prominent mutations caused by the two compounds were -2(CGGC) and 1(CG) frameshifts. +1(AT) Frameshifts play a minor role in the pattern of mutational specificity. Induction of all three mutations was similarly influenced by NAT/OAT activation and UvrABC-dependent excision repair. These new tester strains provide an effective tool for the study of aromatic amine mutational specificity and the influences of excision repair and NAT/OAT activation.
Carcinogenesis 10/1995; 16(9):2037-43. · 5.70 Impact Factor
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ABSTRACT: Heterocyclic aromatic amines formed during the cooking of meat and meat-derived products can be activated to reactive metabolites which bind to DNA, induce mutations and cause tumors in animals. A principal route of metabolic activation is N-oxidation to hydroxylamines, and their subsequent activation by acetyltransferase-catalyzed O-acetylation. We have used mutagenicity assays to study O-acetylation of heterocyclic arylhydroxylamines by the two isozymes of human N-acetyltransferase, NAT1 and NAT2, expressed in Salmonella typhimurium. N-Acetylation was also examined, using an HPLC method. In addition, Salmonella strains with endogenous acetyltransferase and lacking this activating activity were used. Hydroxylamines of nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx, NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situ by rat liver S9 mix. The strains expressing human NAT1 and lacking acetyltransferase activity showing little or no ability to activate these substrates. The strains expressing human NAT2 and Salmonella acetyltransferase supported to different extents the activation of all the compounds except PhIP and Trp-P-2. N-Acetylation of IQ, MeIQx and PhIP was slow or not detectable. In conclusion, human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines. NAT2 probably plays a key role in the genotoxic effects of the above heterocyclic amines except for PhIP and Trp-P-2, which have NAT2-independent mutagenic activity.
Carcinogenesis 04/1995; 16(3):643-8. · 5.70 Impact Factor
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ABSTRACT: The most widely used bioassay in genetic toxicology is the Ames test, which combines a bacterial mutagenicity assay (reversion of Salmonella typhimurium histidine-auxotrophic tester strains) with an exogenous bioactivation system (hepatic postmitochondrial supernatant or "S9"). The enzymatic activities of S9 prepared from the tissues of experimental animals are difficult to control. We show that the requirement for S9 can be obviated by the engineered expression of enzymes of bioactivation within the bacterial cell. With this strategy, reactive metabolites are produced inside the bacterial cell, proximate to the genetic target. Species boundaries can be crossed, and chimeric or mutant enzymes can be studied. We have constructed an Ames tester strain, expressing both aromatic amine N-acetyltransferase and human cytochrome P4501A2, which detects aromatic amine mutagenicity in the absence of S9.
Cancer Research 03/1995; 55(4):799-802. · 7.86 Impact Factor
