A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.     

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.     

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamineO-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAt activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxyacetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyridol[1,2-α:3′,2′-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.     

New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes

The nitroreductase and acetyltransferase genes of Salmonella typhimurium TA1538 have been cloned into pBR322. When transformed into TA1538 derivatives, these plasmids provided the basis for an extremely sensitive assay for the detection of mutagenic nitroarenes.     

Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possesing elevated levels of both nitroreductase and acetyltransferase activity

Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium. The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S. typhimurium TA98 and TA100. The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains. These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Mutagenicity assay for nitroarenes of air pollutants held in leaves of woody plants.

A new method was developed for the detection of mutagenic nitroarenes held in the leaves of woody plants using a small amount of leaves. This method consists of extraction of mutagen from fresh leaves (15-30 g) by ultrasonication with ethyl acetate and purification by elution with benzene on a silica gel column to remove such inhibitory components for mutagenesis as chlorophyll. The mutation assay uses the new Salmonella typhimurium strains YG1021 (a nitroreductase-overproducing strain of TA98) and YG1024 (an O-acetyltransferase-overproducing strain of TA98), which are very sensitive to some nitroarenes in the absence of S9, and standard strain YG1020 (TA98 containing pBR322-Aps) for comparison. This method was applied to woody plants growing on various sites in the Tokyo metropolitan area and the suburbs. It was shown that the leaves of all woody plants tested contained different amounts of mutagens, probably mutagenic nitroarenes, depending upon their growing sites.     

Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 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), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Organ selective induction of cytochrome P-448 isozymes in the rat by 2-methoxy-4-aminoazobenzene and 3-methylcholanthrene

Male Sprague Dawley rats were injected intraperitoneally with 2-methoxy-4-amino-azobenzene (2-MeO-AAB) or 3-methylcholanthrene (MC), and then the expression of microsomal cytochrome P-450 isozymes in liver and extrahepatic tissues was investigated by means of immunological methods and a bacterial mutation test. The results of protein A-enzyme-linked immunosorbent assaying and immunoblotting using anti-rat cytochrome P-448 monoclonal antibodies showed that MC induced at least two microsomal cytochrome P-448 isozymes, a high spin form (cytochrome P-448H) and a low spin form (cytochrome P-448L), in liver, but that it induced only cytochrome P-448L in extrahepatic tissues such as lung, kidney, small intestine, and colon. The results also indicated that, in contrast to MC, 2-MeO-AAB selectively induced microsomal cytochrome P-448H in liver but did not induce any cytochrome P-448 isozymes in extrahepatic tissues. The activities of 9,000 X g supernatants from the individual organs, as to the mutagenic conversion of 3 aromatic amines (3-amino-1-methyl-5H-pyrido(4,3-b)indole, 2-amino-6-methyldipyrido(1,2-a: 3',2'-d)-imidazole and 3-methoxy-4-aminoazobenzene), toward Salmonella typhimurium TA 98 bacteria were dependent upon the quantity and/or quality of the microsomal cytochrome P-448 isozymes in the organs.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 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), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

Phenotypic instability of Salmonella strain YG1024 during mutagenicity assays of arylamine promutagens

During spot tests using Salmonella TA98 derivatives (YG1021, YG1021) and TA100 derivatives (YG1026, YG1029), a unique response of O-acetyltransferase (OAT)-enhanced strains YG1024 and YG1029 to arylamines was observed. On plates containing rat-liver S9, these strains yielded revertant colonies induced in two separate concentric rings around the site of application, while the parent (TA98, TA100) and nitroreductase-enhanced strains (YG1021, YG1026) did not exhibit this response. The inner ring of revertants was accompanied by cytotoxicity and microcolony formation, with the outer ring in a region without background lawn toxicity. Addition of tetracycline to the top agar eliminated formation of the inner ring of YG1024 revertants in spot tests and reduced the revertant count in preincubation assays at cytotoxic dose levels of 2-aminoanthracene, 2-aminofluorene, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline. Tetracycline sensitivity indicates that mutant colonies developing at high concentration/toxicity arose, in effect, from TA98 regenerated by functional loss of the tetracycline-resistance plasmid (pYG219) from YG1024. Mutant colonies found at low concentration/toxicity arose from normal plasmid-bearing YG1024. These results indicate the need to consider coincidental toxicity-induced instability in YG1024 during quantitative mutagenicity assays of arylamines and uncharacterized complex mixtures.     

Mutagenicity of native cigarette mainstream smoke and its gas/vapour phase by use of different tester strains and cigarettes in a modified Ames assay

The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX1 system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.     

Mechanism of activation of proximate mutagens in Ames' tester strains: the acetyl-CoA dependent enzyme in Salmonella typhimurium TA98 deficient in TA98/1,8-DNP6 catalyzes DNA-binding as the cause of mutagenicity

The mechanism of activation of proximate mutagens in Ames' tester strains was described. 2-Hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) were activated to DNA-binding species in the presence of acetyl-CoA by the enzyme(s) in Salmonella typhimurium TA98, and this enzyme was deficient in TA98/1,8-DNP6. Mutagenicity of N-OH-Glu-P-1 to TA98/1,8-DNP6 was much lower than that to TA98. Therefore, it was demonstrated that the acetyl-CoA dependent enzyme(s) activated N-OH-Glu-P-1 to the active form which could covalently bind to DNA and subsequently caused mutagenicity.     

Mutagenic profile of rubber dust and fume exposure in two rubber tire companies

The aim of this study was to evaluate current mutagenic activity of ambient rubber dust and fume exposure in the mixing and curing departments of two rubber tire companies situated in The Netherlands and Sweden. Salmonella typhimurium strains YG1021, YG1024 and YG1041 were used to study the possible presence of mutagenic nitroarenes and aromatic amines. A large difference in mutagenic activity was found between the two companies. While the rubber tire company situated in The Netherlands revealed overall high mutagenic activity of rubber dust and fumes in the mixing and curing departments, respectively, 430 and 279 rev/m(3) (YG1041), the Swedish company showed almost no mutagenic activity, respectively, 18 and 54 rev/m(3) (YG1041). Further identification of the mutagenic profile showed that mutagenic activity was exclusively observed in S. typhimurium strains with elevated levels of O-acetyltransferase activity (YG1041 and YG1024) in the presence of a metabolic active liver S9 fraction, possibly indicating the presence of indirect mutagenic aromatic amines. These results show that although production processes and lay-out within rubber tire companies are comparable, differences in rubber chemicals used and overall level of control measures (e.g., good housekeeping, cleanliness) are likely to result in substantial differences in mutagenic exposure levels between companies.     

Mutagenic activation of aflatoxin B1 by P-450 HFLa in human fetal livers

The genotoxic and mutagenic activation of promutagens by human fetal livers was measured by the induction of umu gene expression in Salmonella typhimurium TA1535/pSk1002. Liver homogenates of human fetuses were the most active for the mutagenic activation of aflatoxin B1 (AFB1), followed by 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), and to a lesser extent by 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1). The amounts of P-450 HFLa immunochemically determined in human fetal livers correlated highly with the induction of umu gene expression by AFB1 (r = 0.98, n = 5). P-450 HFLa catalyzed the mutagenic activation of AFB1 in a reconstituted system: cytochrome b5 markedly stimulated the activation. Anti-P-450 HFLa antibodies inhibited the mutagenic activation of AFB1 in a dose-dependent manner. These results strongly support the idea that P-450 HFLa is responsible for the mutagenic activation of AFB1 in human fetal livers.     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

A new assay for N-hydroxyarylamine O-acetyltransferase: reduction of N-hydroxyarylamines through N-acetoxyarylamines

A simple fluorometric assay for N-hydroxyarylamine O-acetyltransferase is described and compared with a nucleic acid-binding assay. The assay method is based on the finding that the highly mutagenic 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) was reduced to the corresponding amine, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), through N-acetoxy-Glu-P-1 as the reactive intermediate in the presence of N-hydroxyarylamine O-acetyltransferase, acetyl-CoA, and a sulfhydryl compound. The formation of Glu-P-1 was determined by its characteristic fluorescence intensity at 445 nm with excitation wavelength at 376 nm. The reductive reaction was inhibited by the addition of tRNA, DNA, and poly(G), to which the enzymatic product, N-acetoxy Glu-P-1, bound effectively due to its electrophilic nature. Since the fluorometric assay for the O-acetyltransferase is rapid, simple, and sensitive as compared with the nucleic acid-binding method using radioisotope-labeled N-hydroxyarylamine, this method is applicable to the general assay for the formation of reactive N-acetoxy-Glu-P-1.