Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a:3',2-d)-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.     

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.     

Non-enzymatic glutathione conjugation of 2-nitroso-6-methyldipyrido [1,2-a: 3',2'-d] imidazole (NO-Glu-P-1) in vitro: N-hydroxy-sulfonamide, a new binding form of arylnitroso compounds and thiols

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.     

N-hydroxyarylamine O-acetyltransferase in hamster liver: identity with arylhydroxamic acid N,O-acetyltransferase and arylamine N-acetyltransferase

N-Hydroxyarylamine O-acetyltransferase, arylhydroxamic acid N,O-acetyltransferase, and arylamine N-acetyltransferase in hamster liver cytosol were co-purified almost to electrophoretical homogeneity by ion exchange chromatography on DEAE-cellulose, gel filtration on Cellulofine GCL-2000-sf and high-performance KB-hydroxyapatite chromatography. The molecular weight of the acetyltransferase was estimated to be 33,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The three acetyltransferase activities were inhibited by iodoacetamide, pentachlorophenol, and 1-nitro-2-naphthol. Furthermore, 2-aminofluorene, a substrate for arylamine N-acetyltransferase, inhibited the reactions of N-hydroxyarylamine O-acetyl transfer and arylhydroxamic acid N,O-acetyl transfer. These results suggest that the same enzyme catalyzes the three types of acetyl transfer reactions. The acetyltransferase could activate N-hydroxyarylamines, such as 2-hydroxyamino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, and N-hydroxy-2-aminofluorene, to the corresponding N-acetoxyarylamines, which are capable of binding to nucleic acid. Polyguanylic acid was most efficiently modified by the N-acetoxyarylamines formed by the acetyltransferase.     

Exposure level monitor of a carcinogenic glutamic acid pyrolysis product in rabbits

In order to determine a suitable indicator for estimating the exposure levels of the dietary carcinogen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), a carcinogenic glutamic acid pyrolysis product, the levels of Glu-P-1 bound to blood components were monitored for 8 weeks by a high-performance liquid chromatography method after the dietary carcinogen was administered as single oral doses (0.2-50 mg) to rabbits. In all rabbits dosed with Glu-P-1, Glu-P-1 in erythrocytes was detectable even on day 42 after administration. Glu-P-1 in plasma disappeared faster than did Glu-P-1 in erythrocytes. Glu-P-1 levels in rabbit hemoglobin were linearly related to administered doses at all points of time investigated. The results suggest that Glu-P-1 covalently bound to hemoglobin is very suitable for monitoring long-term exposure levels.     

Metabolic activation of glutamic acid pyrolysis products, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole, by purified cytochrome P-450

Metabolic activation by cytochrome P-450 of glutamic acid pyrolysis products, 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) and 2-amino-dipyrido(1,2,-a:3',2'-d)imidazole (Glu-P-2), to mutagenic metabolites was studied using Salmonella typhimurium TA98 as a tester strain. Cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH were essential requirements for the activation of these compounds. Of the four forms of cytochrome P-450 examined, polychlorinated biphenyls (PCB) P-448 and 3-methylcholanthrene (MC) P-448 purified from liver microsomes of rats treated with a PCB mixture and MC, respectively, showed high activity in the activation of both Glu-P-1 and Glu-P-2. The presence of three metabolites from Glu-P-1 or Glu-P-2 was demonstrated by high performance liquid chromatographic (HPLC) analysis. Among the metabolites of Glu-P-1, two metabolites were mutagenic without any further enzymatic activation. In accordance with the results of a mutation assay, PCB P-448 also exhibited higher activity to form the major mutagenic metabolite of Glu-P-1. The major active metabolite of Glu-P-1 was characterized as N-hydroxy-Glu-P-1 by chemical analysis using oxidizing and reducing reagents and by mass spectrometry.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

N-acetyl derivative as the major active metabolite of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole in rat bile

2-Amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) is a mutagen and carcinogen isolated from a glutamic acid pyrolysate. When this 14C-labeled compound was administered to male F344 rats at a dose of 0.3 mCi (20.8 mg)/kg b.w., 70% of the radioactivity was excreted into the bile in 24 h. On HPLC analysis of this bile, several metabolites of Glu-P-1 were found with unmetabolized Glu-P-1. One of the mutagenic metabolites was identified as N-acetyl-Glu-P-1. This metabolite had a specific mutagenic activity of about one quarter of that of Glu-P-1 and its amount in the bile corresponded to a few percent of the dose of Glu-P-1 administered.     

Metabolic activation of a mutagen, 2-amino-6-methyldipyrido-[1,2-a:3′,2′-d]imidazole. Identification of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and its reaction with DNA

A potent mutagen, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(Glu-P-1), isolated from pyrolysates of L-glutamic acid and casein, was metabolically activated and bound to DNA. An activated form was identified as 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(N-OH-Glu-P-1). Synthetic N-OH-Glu-P-1 reacted with DNA only after O-acetylation to give a modified DNA, which on hydrolysis gave 2-(C⁸-guanyl)amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(gua-Glu-P-1). The same adduct was isolated from DNA modified with Glu-P-1 by microsomes in vitro, as reported earlier.     

Detection of N-acetyl derivative of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) in Glu-P-1-injected rats

In order to confirm in vivo N-acetylation of 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) in rats, we developed a new high-performance liquid chromatography (HPLC) method for detecting the N-acetyl derivative of Glu-P-1 in animal organs. Using this method, N-acetyl-Glu-P-1 was detected in rat liver, kidney and intestinal contents 6 h after intraperitoneal injection of Glu-P-1. This fact provides evidence to support in vivo N-acetylation of Glu-P-1 in rats.     

Interaction of mutagens isolated from L-glutamic acid pyrolysate with DNA

The interaction of Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole) and Glu-P-2 (2-aminodipyrido[1,2-a:3′,2′-d]imidazole) with DNA were studied. Agarose gel electrophoresis of Closed-circular DNA treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of Glu-P-1 or Glu-P-2 revealed that Glu-P-1 and Glu-P-2 intercalated into DNA. Correlation with the binding parameters, measured by optical titrations, showed that Glu-P-1 and Glu-P-2 caused about 20° unwinding of the DNA double helix.     

Oxidation of the 2-hydroxyamino derivative of 2-amino-6-methyl-dipyrido[1,2-a: 3',2'-d] imidazole (Glu-P-1) to its 2-nitroso form, an ultimate form reacting with hemoglobin thiol groups

The binding to hemoglobin of synthetic 2-hydroxyamino-6-methyldipyrido[1,2-a: 3',2'-d] imidazole from the carcinogenic product of L-glutamic acid pyrolysis 2-amino-6-methyldipyrido[1,2-a: 3',2'-d] imidazole were investigated in vitro. The hydroxylamine required oxidation to its nitroso derivative to bind to rat hemoglobin through thiol groups. Oxidation of the hydroxylamine to the nitroso form was found to be enhanced by oxyhemoglobin and superoxide dismutase at pH 7.4 under aerobic conditions. Since these conditions might also enhance this oxidation in vivo, the conversion of the DNA-reactive arylhydroxylamines to the DNA-non-reactive nitroso compounds and their subsequent binding to highly abundant thiol groups of proteins could be considered as a process for detoxification of toxic arylhydroxylamines.     

Possibility of the involvement of 9H-pyrido[3,4-b]indole (norharman) in carcinogenesis via inhibition of cytochrome P450-related activities and intercalation to DNA

This study investigated the inhibitory effect of 9H-pyrido[3,4-b]indole (norharman), one of the naturally occurring beta-carbolines, on cytochrome P450 (CYP)-related activities and the relationship between its inhibitory effect, its intercalation to DNA, and its comutagenic effect. Norharman reduced the mutagenicities of heterocyclic amines (HCAs) containing 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), aflatoxin B1, benzo[a]pyrene (BP), and some nitrosamines in the presence of 10 microl liver S9 (20.9 microg protein/ml) from polychlorinated biphenyl-treated rats. Norharman inhibited microsomal CYP-related enzyme activities and CO-binding to the CYP heme (50% inhibitory concentration (IC50), 0.07-6.4 microg/ml). It also inhibited the formation of 3-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) and was a noncompetitive-inhibitor of CYP1A-related activities, while it enhanced the direct mutagenicity of N-OH-Glu-P-1 (50% effective concentration, 25.0 microg/ml) and inhibited topo I activity (IC50, 31.0 microg/ml). In the presence of norharman, S9 up to 100 microl incrementally enhanced the mutagenicities of HCAs, BP and dimethylnitrosamine. These data clarified that norharman acts as an inhibitor of the CYP-mediated biotransformation of Glu-P-1 via inhibition of O2-binding to CYP heme, and its inhibition of CYP enzymes occurs at much lower concentration than that for its intercalation to DNA. It is indicated that norharman's inhibitory effect on CYP results in the inhibition of excess metabolism by S9 and this is more likely the mechanism for comutagenic action than the intercalation. Norharman's inhibition of CYP and its enhancement of the N-OH-Glu-P-1 mutagenicity suggest that beta-carbolines modulate chemical carcinogenesis by controlling the xenobiotic metabolism and by intercalating to DNA.     

Effect of dipyridoimidazole pretreatment on mutagen activation in rats

Rats were pretreated with a single oral dose of different mutagenic fractions obtained from glutamic acid pyrolysate: Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole), Glu-P-3 (3-amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole), the tar residue and a basic extract (B2). The liver S9 fractions of these animals were used to investigate the mutagenic activation of 3 promutagens (2-aminoanthracene, Glu-P-2 and Glu-P-3) in Salmonella typhimurium strain TA1538. Different factors were analyzed; influence of the structure of the compounds administered, doses, time interval between pretreatment and sacrifice and sex of the rats. Interpretation of the hepatic induction effects was complicated, however, by the fact that simple oral pretreatment with the solvents (DMSO or ethanol) enhances the activation of the substrates tested for mutagenicity. A dose-effect relationship was found between 2-AA mutagenic activation and Glu-P-2 pretreatment. Glu-P-3 induced the activation of 2-AA more than did Glu-P-2, in the male as in the female. The mutagenicity of 2-AA activated with S9 from male rats was found to be optimal after 24 h pretreatment with 20 mg Glu-P-2/kg b.w. The mutagenicity of Glu-P-2 was poorly influenced by the different pretreatments applied to either the males or the females, whereas some dose effect was found in the autoinduction of Glu-P-2 mutagenicity. Compared to Glu-P-2, the mutagenicity of Glu-P-3 was increased at higher levels when tested with S9 from males pretreated with the same compound, but no differences were observed between males and females.     

Copper, zinc-superoxide dismutase enhances the mutagenicity in Salmonella typhimurium induced by 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

The mutagenicities of various carcinogens induced by liver microsomes are increased in the presence of liver cytosol in rodents. It still remains, however, to be clarified which factor or factors in the cytosol enhance(s) the microsome-mediated mutagenicities. In the present study, we sought to identify the enhancing factor in liver cytosol prepared from rats using the microsome-mediated Salmonella mutagenicity induced by 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole (Glu-P-1). By a series of chromatographic steps, we purified a 16-kDa protein on SDS-PAGE from the cytosol of rat livers. Partial amino acid sequences of this protein revealed that the 16-kDa protein was copper, zinc-superoxide dismutase (CuZn-SOD). The purified CuZn-SOD enhanced the microsome-mediated mutagenicities of several heterocyclic amines and aromatic amines. Furthermore, bovine and human CuZn-SOD also enhanced the microsome-mediated mutagenicity of Glu-P-1. The CuZn-SOD caused an increase in the mutagenicity of N-hydroxylated Glu-P-1 formed from Glu-P-1 by the microsomes, although CuZn-SOD did not affect either the formation or the stability of the N-hydroxylated derivative. These findings suggest that the enhancing cytosol factor for the mutagenicity of Glu-P-1 is CuZn-SOD, which stimulates the mutagenicity of N-hydroxylated Glu-P-1 without changing its metabolism.     

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.     

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase

The glutathione (GSH) conjugation reaction of the active metabolite of a potent protein-pyrolysate carcinogen, 2-nitroso-6-methyldipyrido[1,2-a:3',2'-d]imidazole (NO-Glu-P-1), occurred in glycerol matrix inside a fast atom bombardment (FAB) mass spectrometer. The short lived GSH-conjugates were detected by in situ FAB analysis. The precursor-product relationship between the conjugates was indicated by following the reaction by measuring [M + H]+ ions of the conjugates.     

In vivo studies of the inhbitory effect of various food components on aflatoxin B1 metabolism

Possible interferences with aflatoxin B1 metabolism, of some compounds naturally present in food (quercetin, beta-naphthoflavone), resulting from way of cooking method (2-aminodipyrido [1,2-a; 1',2'-d] imidazole (Glu-P-2), norharmane; NH) or used as food additives (butylated hydroxytoluene; BHT) have been studied in vivo by evaluating the production of adducts to glutathione and adducts to serum proteins in laboratory rats. Glu-P-2 and norharmane inhibit strongly the production of adducts to glutathione whereas quercetin and beta-naphthoflavone have only a low effect. BHT is completely ineffective. The adducts to proteins are inhibited by the five compounds, norharmane being the most efficient.     

Two bases are eliminated from DNA by treatment with bleomycin or with hemin-intercalators

A hemin-intercalator, H4G-His, which possesses 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) as an intercalator moiety and histidine as an intramolecular ligand of the ferrous ion of the hemin ring, cleaves DNA very efficiently, and acts at guanine-pyrimidine sequences preferentially. Bleomycin (BLM) also cleaves DNA with the same base-sequence selectivity shown by H4G-His. The 5'-terminal of the DNA fragments cleaved by H4G-His or by BLM bears a phosphoryl group, while the 3'-terminal of the cleaved DNA fragments does not possess a 3'-phosphoryl group. There are three (or more) kinds of structures of the 3'-terminals. One of the structures of the 3'-terminals of the cleaved DNA fragments is a free 3'-hydroxy group. We propose that there exist plural mechanisms for DNA cleavage by H4G-His or by BLM, one of which involves elimination of two bases from DNA.     

The formation of heart DNA adducts in F344 rat following dietary administration of heterocyclic amines

Most heterocyclic amines formed during the cooking of meat and fish have been shown to form adducts in the livers of rats. Recently, however, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), administered in the diet to Fischer 344 (F344) rats for 4 weeks, was shown to produce the highest levels of adducts in the heart. In the present study 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:1',2'-d]imidazole (Glu-P-1) were given to F344 rats at carcinogenic dose levels (IQ 0.03%, MeIQx 0.04%, Trp-P-1 0.015%, Glu-P-1 0.05%) in the diet for 4 weeks. DNA adducts in the liver and heart were analyzed by 32P-postlabeling. DNA adducts were demonstrated to appear in the hearts of all animals exposed to heterocyclic amines at the following levels: IQ, 1.8 adducts/10(7) nucleotides, MeIQx, 3.8/10(7) ntd, Trp-P-1, 20/10(7) ntd and Glu-P-1, 7.2/10(7) ntd. Values for the heart were 10-20% of the respective liver adduct levels. Heart adducts increased linearly throughout the observed period when MeIQx was administered for up to 40 weeks. When MeIQx feeding was discontinued after 20 weeks and the animals subsequently given the basal diet, the adduct level at 20 weeks did not change during the following 20 weeks. A possible role for heart DNA alterations caused by food-borne heterocyclic amines in the development of age-related myopathies and cardiovascular disease is not inconceivable.