Role of human N-acetyltransferases, NAT1 or NAT2, in genotoxicity of nitroarenes and aromatic amines in Salmonella typhimurium NM6001 and NM6002

Yoshimitsu Oda1, Hiroshi Yamazaki and Tsutomu Shimada

Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537-0025, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human NAT1 and NAT2 genes were subcloned into pACYC184 vector and the plasmids thus obtained were introduced into Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1,8-DNP/pSK1002), establishing new strains NM6001 and NM6002, respectively. We compared the sensitivities of these two strains with those of NM6000 towards carcinogenic nitroarenes and aromatic amines in the SOS/umu response. The induction of umuC gene expression by these chemicals in the presence and absence of the S9 fraction was assayed by measuring the cellular ß-galactosidase activity expressed by the umuC"lacZ fusion gene in the tester strains. 2-Nitrofluorene and 2-aminofluorene induced umuC gene expression more strongly in the NM6001 strain than in the NM6002 strain. In contrast, induction of umuC gene expression by 1,8-dinitropyrene, 6-aminochrysene and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline was weaker in the NM6001 strain than in the NM6002 strain. 1-Nitropyrene, 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were found to induce umuC gene expression at similar extents in both strains. These results suggest that the newly developed strains can be employed for the studies on mechanisms of genotoxicity of a variety of nitroarenes and aromatic amines, along with the assessment of cancer risk to humans.

Abbreviations: 1,8-DNP, 1,8-dinitropyrene; 1-NP, 1-nitropyrene; 2-AF, 2-aminofluorene; 2-NF, 2-nitrofluorene; 6-AC, 6-aminochrysene; Glu-P-1, 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole; MeA{alpha}C, 2-amino-3-methyl-9H-pyrido[2,3-b]indole; MeIQ, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline; NAT, N-acetyltransferase; O-AT, O-acetyltransferase; PABA, p-aminobenzoic acid; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SMZ, sulfamethazine; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino-1-methyl-5H-pyrido[4,3-b]indole


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Numerous studies have suggested that carcinogenic nitroarenes and aromatic amines are present in environmental or occupational places (1,2) and particularly, dinitropyrenes, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA{alpha}C) are shown to be strong mutagens in bacteria, and carcinogenic in rodents (36). Aromatic amines are activated to N-hydroxy metabolites by cytochrome P450 enzymes, and subsequently acetylated by O-acetyltransferase (O-AT) to give acetoxy esters, which form an arylnitrenium ion which binds to DNA. Thus, O-AT plays an important role as a phase II enzyme in metabolic activation of aromatic amines.

Human N-acetyltransferase (NAT) enzymes, NAT1 and NAT2, are known to be polymorphic (710) with rapid, intermediate and slow acetylator phenotypes (11). Epidemiological studies have suggested that there is a relationship between colorectal cancer and rapid acetylator (12,13), and between urinary bladder cancer and slow acetylator phenotypes (14,15). However, it is not clear whether human NAT1 and/or NAT2 contribute to the genotoxicity of aromatic amines and nitroarenes.

We have established previously an O-AT-overexpressing Salmonella typhimurium NM2009 strain and showed that the strain is extremely sensitive at detecting the genotoxicity of carcinogenic aromatic amines and nitroarene compounds (16,17). Recently, the strain has been used widely for monitoring environmental pollutions in river wastes, and for the study of roles of P4501A2 in metabolic activation of aromatic amines (1821).

This study was undertaken to investigate the roles of human NAT enzymes in the genotoxicity of aromatic amines and nitroarenes using two new tester strains, which were established by introducing human NAT1 or NAT2 cDNAs into the parent strain, S.typhimurium NM6000. We demonstrated that 2-nitrofluorene (2-NF) and 2-aminofluorene (2-AF) are metabolically activated primarily by NAT1, whereas 1,8-dinitoropyrene (1,8-DNP), 6-aminochrysene (6-AC) and MeIQ are catalyzed more significantly by NAT2.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and enzymes
1-Nitropyrene (1-NP) and 2-NF were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). 2-AF and 6-AC were purchased from Aldrich Chemical Co. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) were from Wako (Osaka, Japan). 1,8-DNP was a gift from Dr S. Sato (Kobe University, Kobe, Japan). MeIQ, MeA{alpha}C and PhIP were gifts from Dr K. Wakabayashi (National Cancer Center Research Institute, Tokyo, Japan). The restriction enzymes, DraI, BamHI, SphI and SalI were purchased from Takara Shuzo Co. (Kyoto, Japan). All other chemicals and reagents used were of the highest purity commercially available. Rat liver S9 fractions and the cofactors were obtained from Oriental Yeast Co. (Tokyo, Japan).

Bacterial strains and plasmids
The bacterial strains and plasmids used in this work are described in Table IGo. Salmonella typhimurium SJ10002 (restriction, modification+) was used for the modification of plasmid pACYC184 in Escherichia coli HB101.


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Table I. Bacterial strains and plasmids
 
Construction of plasmids and strains for expression of human NAT1 and NAT2 genes
The plasmids pNM63 and pNM64 were prepared by subcloning the recombinant inserts pNAT1 and pNAT2 (kindly provided by D.M.Grant, Hospital for Sick Children, University of Toronto, Canada), respectively, into the vector plasmid pACYC184. pNAT1 and pNAT2 encoding the human NAT gene were digested initially with SphI, followed by further digestion with EcoRI. The respective SphI–EcoRI DNA fragments were purified by agarose gel electrophoresis. After the cohesive ends were changed to blunt ends, the fragments were ligated into the DraI region of a plasmid vector pACYC184. Escherichia coli HB101 strain was used for the transformation with each plasmid using the CaCl2 methods (27). The resultant transformants were initially selected for tetracycline resistance, then the plasmid DNA isolated from transformants was digested completely with restriction enzymes SalI (in the case of NAT1) and BamHI (in the case of NAT2) for the direction of respective NAT gene transcription, and subjected to agarose gel electrophoresis. The plasmid DNA molecules with desired structures were obtained as verified by restriction analysis. After modifying each plasmid with S.typhimurium SJ10002 strain, each plasmid was again used to transform the O-AT-deficient S.typhimurium strain NM6000 (TA1538/1,8-DNP/pSK1002) and ampicillin/tetracycline-resistant transformants were selected. The resulting strains were designated as NM6001 (TA1538/1,8-DNP/pNM63/pSK1002) and NM6002 (TA1538/1,8-DNP/pNM64/pSK1002), respectively.

Enzyme assays
NAT activities were determined in bacterial lysates (10 000 g supernatant fraction) by a method described previously (16). NAT1 and NAT2 activities were measured using as substrates p-aminobenzoic acid (PABA) and sulfamethazine (SMZ), respectively, by the method of Hein et al. (28) and Andres et al. (29). The protein content was assayed by the method of Lowry et al. (30).

umu assay
umu assay was carried out according to the procedure described previously by Oda and co-workers (16,17,21). Briefly, the bacterial cells were grown for overnight at 37°C in LB broth containing tetracycline HCl (1 µg/ml) and/or ampicillin (25 µg/ml). The cultures were diluted 50-fold with TGA medium (1% Bactotryptone, 0.5% NaCl, 0.2% glucose, 20 µg/ml ampicillin) and further incubated at 37°C until the bacterial density reached an absorbance of ~0.3 at 600 nm. For the chemicals (nitroarenes) that did not require activation by S9 mix, the culture aliquot was subdivided into 2 ml in test tubes, and then test chemicals (dissolved in 20 µl dimethyl sulfoxide) were added to the tubes. For the chemicals (aromatic amines) that required activation by S9 mix, the cultures were subdivided into 1.7 ml aliquots in the test tubes, to which 0.3 ml of S9 mix and 20 µl of a test chemical were added. These mixtures were incubated at 37°C for 2 h with vigorous shaking, and then the bacterial density and the ß-galactosidase activity were measured by the method of Miller (31) with slight modification by Oda et al. (32). The effect of chemicals on cell growth was determined in the reaction mixture by measuring the absorbance at 600 nm. The results are presented as means of two tubes from two or three independent experiments.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Establishment of new S.typhimurium strains expressing the human NAT1 and NAT 2 genes
In order to express the NAT gene in S.typhimurium, we subcloned the 0.87 kb fragments of the phagemid vector pKEN2 containing human NAT1 and NAT2 genes into the DraI site in the multicopy plasmid vector pACYC184 (26). By subcloning into this site, the chloramphenicol gene was disrupted and the resulting plasmids (4.79 kb) containing tetracycline gene were designated as pNM63 and pNM64, respectively (Figure 1Go). These newly constructed plasmids were able to co-exist with plasmid pSK1002 containing the umuC''lacZ fusion gene that confers ampicillin resistance.



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Fig. 1. Plasmids constructed for the expression of human NAT1 and NAT2 genes. (A) Plasmid pNM63 for the expression of human NAT1 gene; (B) plasmid pNM64 for the expression of human NAT2 gene. The cleavage sites of some restriction enzymes are shown with italic letters. Tc, tetracycline gene; ori, the origin of replication from plasmid p15A. The sizes of plasmids pNM63 and pNM64 are 4.79 kb.

 
Table IIGo shows the results of enzyme activity catalyzed by two substrates in six different strains. As expected, the NAT-deficient strain NM6000 did not exhibit any detectable NAT activity. Bacterial O-AT-overexpressing strain NM2008 exhibited very low NAT activity when PABA was used as a substrate. Strains NM6001 and DJ400, which express human NAT1, exhibited higher NAT activities with PABA than with SMZ. In contrast, when SMZ was used as a substrate, NM6002 and DJ460, expressing human NAT2, showed almost similar activities with SMZ substrate.


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Table II. Rates of N-acetylation catalyzed by lysates of S.typhimurium strains
 
The background levels of umuC gene expression in S.typhimurium NM6001 and NM6002 strains were found to be >2-fold higher than that in the parent strain NM6000.

Effects of nitroarene and aromatic amine compounds on the cell growth rates and umuC induction in the Salmonella tester strain
We first determined the effects of 2-NF and 1,8-DNP on the cell growth rates in the newly developed tester strains. These chemicals exhibited different responses towards NM6000, NM6001 and NM6002 strains (Figure 2Go). The cell growth rates in the parent strain NM6000 and NAT1-expressing strain NM6001 were not affected by 1,8-DNP. In contrast, the cell growth rates in NAT2-expressing strain NM6002, were retarded in a dose-dependent fashion in the presence of 1,8-DNP, and the LD37 (37% lethal dose) value was calculated to be 1 nM. 2-NF was found to be not so cytotoxic in NM6000 and NM6002 strains up to 4.7 µM concentration, whereas inhibition of the cell growth in NM6001 was observed at 4.7 µM and the LD37 value was found to be 2.1 µM.



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Fig. 2. Effect of concentrations of chemicals on cell growth in S.typhimurium tester strains. ({circ}) NM6000 strain; (•) NM6001 strain expressing NAT1; ({blacktriangleup}) NM6002 strain expressing NAT2.

 
Next, we compared the sensitivities of two strains expressing different acetyltransferases and the parent strain towards the effects of three nitroarenes and eight types of aromatic amines on induction of umuC gene expression. As shown in Table IIIGo, NAT1-expressing strain NM6001 exhibited the highest sensitivity towards the induction of umuC gene expression by 2-NF and 2-AF, and the sensitivities were decreased with NM6002, followed by the parent strain NM6000. On the other hand, NAT2-expressing strain NM6002 exhibited the highest sensitivity to the induction of umuC gene expression by 1,8-DNP, MeIQ and 6-AC, and the sensitivities to the umu-inducing effects of these chemicals were decreased with NM6001 and NM6000 strains (Table IIIGo). When we compared the sensitivities of three strains towards chemicals in terms of fold induction relative to background, NM6001 and NM6002 exhibited the same sensitivity towards Glu-P-1, whereas NM6000 showed relatively low sensitivity towards this chemical (Table IIIGo). In the cases of 1-NP, Trp-P-1, Trp-P-2, MeA{alpha}C and PhIP, although NM6000 exhibited apparent insensitivity towards these chemicals because of its low background levels, NM6000, NM6001 and NM6002 exhibited a similar extent of sensitivity in the responses in inducing umuC gene expression (Table IIIGo).


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Table III. Genotoxic activity of nitroarenes and aromatic amines in S.typhimurium tester strains NM6000, NM6001 and NM6002
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we developed new umu test systems to investigate the roles of two human NATs, NAT1 and NAT2, in the genotoxicity of nitroarenes and aromatic amines. These two tester strains NM6001 and NM6002 were constructed by introducing human NAT1 and NAT2 cDNAs, respectively, into NM6000 (TA1538/1,8-DNP/pSK1002), which is a derivative of TA1538 devoid of endogenous Salmonella O-AT and harbors the umuC''lacZ fusion gene. These new strains, expressing human NAT1 and NAT2 genes, were used for assessing the genotoxicity of nitroarenes and aromatic amines. Nitroarene compounds are known to be activated by nitroreductase followed by O-AT (3,16), while aromatic amines are proposed to be activated by N-oxidation catalyzed by cytochrome P450s or prostaglandin H synthase, followed by acetylation (34,35). The results of this study suggest that these newly developed strains can detect the genotoxicity of nitroarenes and aromatic amines with high sensitivity. Our data showed that the genotoxicity of nitroarenes and aromatic amines depends on the levels of expression of acetyltransferase activities, except for 1-NP, Trp-P-2 and PhIP.

Among the two strains examined, the human NAT1-expressing NM6001 strain showed much higher sensitivity than the NM6002 strain to the cytotoxic and genotoxic effects of 2-NF and 2-AF. These results are in good agreement with those reported by Grant et al. (24) who showed that 2-AF exhibited the mutagenic response in a S.typhimurium strain expressing human NAT1 at much lower doses in the presence of rat liver S-9 fraction. Minchin et al. (33) also reported that NAT1 efficiently catalyzed O-acetylation of N-hydroxy-2-aminofluorene in vitro. Furthermore, the present results are quite similar to the previous results that S.typhimurium NM2009, overproducing O-AT activity of S.typhimurium, has higher sensitivity to the genotoxic effects of 2-NF and 2-AF than the parent strain (16,17). Therefore, O-acetylation seems to be one of the prevalent phase II pathways involved in the metabolic activation of 2-NF and 2-AF leading to genotoxicity. Since NAT1 has recently been reported to be polymorphic in humans (36), it would be most interesting to investigate whether the polymorphism will be associated with cancer caused by 2-NF and other nitroarenes.

In contrast, the NM6002 strain, which expressed human NAT2 acetyltransferase, showed higher sensitivity than the NM6001 strain to the cytotoxicity and genotoxicity of 1,8-DNP, MeIQ and 6-AC. 1,8-DNP has been shown to be catalyzed by nitroreductase to form 1-hydroxyamino-8-nitropyrene, and subsequent activation is suggested to be catalyzed by O-AT, liberating the ultimate electrophilic DNA-binding species, nitrenium cation. As a major DNA adduct, 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene has been identified (37, 38). Previously, we have reported that the genotoxicity of 1,8-DNP is dependent on the S.typhimurium nitroreductase/O-AT enzymes (16). Similarly, Watanabe et al. (39) observed that CHL cells expressing human NAT2 were much more sensitive to the clastogenicity of 1,8-DNP in the in vitro micronucleus test. Thus, the present results supported the view that 1,8-DNP is activated through nitroreduction and O-acetylation in the NM6002 strain. Our results also supported the view that MeIQ and 6-AC are activated by rat S9 as well as a liver microsomal fraction in S.typhimurium NM2009 overexpressing O-AT (17,20). In addition, the genotoxic activation of these chemicals has also been reported by other investigators who introduced human NAT1 or NAT2 genes into a bacterial system. By using the Salmonella strain expressing human NAT enzymes, Grant et al. (24) and Wild et al. (40) have reported that NAT2, but not NAT1, plays major roles in the mutagenic activation of MeIQ. Taken together, our results again suggested that NAT2, to esterify the N-hydroxy metabolites to ultimate genotoxic metabolites, is necessary for metabolic activation of MeIQ.

PhIP and Trp-P-2 showed similar genotoxicity in all three strains (NM6000, NM6001 and NM6002) (Table IIIGo), indicating that their metabolic activation seems to be fully independent of NAT. In previous studies we also showed that PhIP and Trp-P-2 do not require the O-AT enzyme derived from S.typhimurium (17). Using the S.typhimurium strains expressing either the human NAT1 or NAT2 gene, Wild et al. (40) reported that neither NAT enzyme is necessary for induction of the mutagenicity of PhIP and Trp-P-2. More recently, Wu et al. (41) reported that PhIP does not require esterification for metabolic activation leading to genotoxicity in Chinese hamster ovary cells. Therefore, these results suggest that other metabolic pathway may be involved in the bioactivation of PhIP and Trp-P-2. In the case of PhIP, Bounarati et al. (42) reported that sulfation of N-hydroxy-PhIP was more important than acetylation to produce DNA adducts in murine hepatic cytosol preparation. Additionally, with rat and human liver preparations, Malfatti et al. (43) and Ozawa et al. (44) showed that rat and human sulfotransferase can metabolically activate N-hydroxy-PhIP to a reactive sulfuric acid ester intermediate that binds covalently to DNA. Thus, the sulfation of N-hydroxy-PhIP appears to be a major biotransformation pathway for exerting the genotoxicity. Further research may be required to evaluate the possible role of sulfation in the activation of PhIP.

In conclusion, we established the new S.typhimurium NM6001 and NM6002 strains expressing human NAT1 and NAT2 genes, respectively. By using these strains for the umu assay, our results demonstrated that NAT1 plays the major role in the phase II activation of 2-NP and 2-AF, whereas NAT2 plays a key role in the genotoxicity of nitroarenes such as 1,8-DNP and aromatic amines including MeIQ and 6-AC. These two tester strains would be facilitated for the study of mechanisms of genotoxicity of nitroarenes and aromatic amines and of the role of human NAT in human cancer risk such as liver, colon, lung and bladder cancers.


    Acknowledgments
 
We are grateful to Drs D.M.Grant and P.D.Josephy for providing plasmids and strains in this study. This work was supported by a grant-in-aid from the Japan Health Sciences Foundation.


    Notes
 
1 To whom correspondence should be addressed Email: ysoda{at}iph.pref.osaka.jp Back


    References
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 Abstract
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 Materials and methods
 Results
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 References
 

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Received September 15, 1998; revised December 29, 1998; accepted January 25, 1999.