Mutagens formed from beta-carbolines with aromatic amines

Norharman, widely distributed in our environment such as cigarette smoke and cooked foods, is not mutagenic to Salmonella strains, but becomes mutagenic to Salmonella typhimurium TA98 and YG1024 with S9 mix in the presence of aromatic amines, including aniline and o-toluidine. Therefore, we have designated norharman as a "co-mutagen". Since, humans are simultaneously exposed to norharman and aromatic amines in daily life, it is important to clarify the mechanisms of its co-mutagenic action to further understanding of the potential genotoxic effects in humans. Regarding the mechanisms of this action of norharman with aniline, a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole[aminophenylnorharman (APNH)] is produced by their interaction, and converted to the hydroxyamino derivative which eventually forms the DNA adduct, dG-C8-APNH through possible ultimate reactive forms with esterification, and this induces mutations. Also other aminophenyl-beta-carboline compounds, such as 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole[amino-3'-methylphenylnorharman (3'-AMPNH)], 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole [amino-2'-methylphenylnorharman (2'-AMPNH)], 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole[aminophenylharman (APH)] and 9-(4'-amino-3'-methylphenyl)-1-methyl-9H-pyrido[3,4-b]indole[amino-3'-methylphenylharman (AMPH)], have been found on reaction of norharman or harman with aniline or toluidine isomers. These compounds showed mutagenic and clastogenic actions in bacterial and mammalian cells. Among them, APNH demonstrated the most potent activity, and it was most extensively studied. When APNH was administered as a single dose to F344 rats, severe testicular toxicity was observed after 6 days. Moreover, liver preneoplastic lesions (GST-P-positive foci) in the liver clearly developed in animals fed 10-50 ppm of APNH in the diet for 4 weeks. Since, APNH was detected in 24 h urine of rats upon simultaneous administration with norharman and aniline by gavage, it is likely to be also produced from norharman and aniline in the human body. From these findings, it is suggested that aminophenyl-beta-carboline derivatives may be classified as one of the novel types of endogenous mutagens and carcinogens.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

Identification of co-mutagenic chlorinated harmans in final effluent from a sewage treatment plant

Harman (1-methyl-9H-pyrido[3,4-b]indole) reacted readily with sodium hypochlorite in an aqueous medium to give the mono-chlorinated derivatives, which reportedly have greater co-mutagenic activity than harman in the presence of o-toluidine toward Salmonella typhimurium TA 98 with S9 mix. Mono-chlorinated harmans were detected by concentration using blue rayon (BR) and GC/MS analysis in the final effluent from a sewage treatment plant in Shizuoka, Japan. The amounts adsorbed for 24h were 1-45ng/gBR for mono-chlorinated harman and 110-730ng/gBR for harman.     

Effects of the comutagens, harman and norharman, on the interaction of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido (4,3-b) indole with DNA.

Harman and norharman, known as comutagens of many chemicals, were tested for their effect on the binding to DNA of 3-amino-1-methyl-5H-pyrido(4,3-b)indole, (Trp-P-2), a potent mutagen found with harman and norharman in the pyrolysate of tryptophan (1). We demonstrated that the alteration of the DNA helix by intercalation of these comutagens to DNA does not affect the affinity of this potent mutagen for DNA. Covalent binding, however, was inhibited by the comutagens.     

Effects of harman and norharman on the metabolism and genotoxicity of 2-acetylaminofluorene in cultured rat hepatocytes

Monolayers of rat hepatocytes metabolize 0.25 m M 2-acetylaminofluorene (AAF) to various ether-extractable, water-soluble as well as covalently bound products. The major ether-extractable metabolite formed is 2-aminofuorene (AF), followed by 7-OH-AAF and 9-OH-AAF. Pretreatment of rats with the inducer Aroclor 1254 (PCB) increased the metabolism of AAF and caused an increased DNA repair synthesis in hepatocytes exposed to AAF or AF. With N-OH-AAF, a decreased genotoxic response in PCB-treated cells compared to control cells was seen. The addition of harman and norharman decreased the metabolism of AAF to ether-extractable metabolites, water-soluble metabolites and metabolites covalently bound to macromolecules. In contrast, the DNA-repair synthesis caused by the same concentrations of AAF was increased by harman. One explanation for this apparent discrepancy could be that the aromatic amines changed the metabolism of harman and norharman in such a way that these compounds were converted into genotoxic metabolites.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-ydroxy-2-acetylaminofluorene - PCB polychlorinated biphenyls, Aroclor 1254 - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TdR thymidine - Trp-P-1 3-amino-1,4dimethyl-5H-pyrido(4,3b)indole - Trp-P-2 3-amino-l-methyl-5H-pyrido(4,3b)indole - UDS unscheduled DNA synthesis     

Structure-activity relationship for a series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles: potent subtype-selective inhibitors of N-methyl-D-aspartate (NMDA) receptors.

A series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles was synthesized as potential antagonists for the NR1A/2B subtype of N-methyl-D-aspartate (NMDA) receptors. Assayed by electrical recording under steady-state conditions, 7-hydroxy-2-(4-phenylbutyl)- 1,2,3,4-tetrahydropyrido-[3,4-b]indole (30) was the most potent compound in the series having an IC50 value of 50 nM at the NR1A/2B receptors.     

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.     

Activation of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole and its reaction with DNA

A potent mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), isolated from a tryptophan pyrolysate, was activated metabolically by rat liver microsomes and bound to DNA. An active metabolite formed by rat liver microsomes was identified as 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2). Synthetic N-OH-Trp-P-2 reacted with DNA efficiently after O-acetylation or to a lesser extent under acidic conditions (pH 5.5), but did not react appreciably under neutral conditions. Acid hydrolysis of DNA modified by O-acetylated N-OH-Trp-P-2 (N-OAc-Trp-P-2) gave 3-(8-guanyl)amino-1-methyl-5H-pyrido[4,3-b]indole (Gua-Trp-P-2), which is the main modified base of DNA formed by Trp-P-2 in the presence of microsomes. The glycoside bond of the modified base was found to be cleaved by heating at 100° for 1 hr at pH 7.0. In this way, the modified base was liberated from DNA modified by N-OAc-Trp-P-2 in good yield. N-OAc-Trp-P-2 bound to guanyl cytidine more effectively than to guanylic acid, suggesting that covalent binding with guanyl moiety of DNA involves intercalation of the ultimate mutagen into a base pair.     

Potent 1,3-disubstituted-9H-pyrido[3,4-b]indoles as new lead compounds in antifilarial chemotherapy.

Substituted 9H-pyrido[3,4-b]indoles (beta-carbolines) identified in our laboratory as potential pharmacophore for designing macrofilaricidal agents, have been explored further for identifying the pharmacophore responsible for high order of adulticidal activity. This has led to syntheses and macrofilaricidal evaluations of a number of 1-aryl-9H-pyrido[3,4-b]indole-3-carboxylate derivatives (3-7). The macrofilarical activity was initially evaluated in vivo against Acanthoeilonema viteae. Amongst all the synthesized compounds, only twelve compounds namely 3a, 3c, 3d, 3f, 4c, 4d, 4f, 5a, 6f, 6h, 6i and 7h have exhibited either > 90% micro- or macrofilaricidal activity or sterilization of female worms. These compounds have also been screened against Litomosoides carinii and of these only 3f and 5a have also been found to be active. Finally these two compounds have been evaluated against Brugia malayi. The structure activity relationship (SAR) associated with position-1 and 3 substituents in beta-carbolines have been discussed. It has been observed that the presence of carbomethoxy at position-3 and an aryl substituent at position- in beta-carbolines effectively enhance antifilarial activity particularly against A. viteae. Amongst the various compounds screened, methyl 1-(4-methylphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c) has shown highest adulticidal activity and methyl 1-(4-chlorophenyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxyla te (3a) has shown highest microfilaricidal action against A. viteae at 50 mg/ kg x 5 days (i.p.). Another derivative of this compound namely 1-(4-chlorophenyl)-3-hydroxymethyl-9H-pyrido[3,4-b]indole (5a) exhibited highest activity against L. carinii at 30 mg/kg x 5 days (i.p.) and against B. malayi at 50 mg/kg x 5 days (i.p.) or at 200 mg/kg x 5 days (p.o.).     

Dicorynamine and harmalan-N-oxide,two new β-carboline alkaloids from Dicorynia guianensis Amsh heartwood

The chemical investigations of Dicorynia guianensis heartwood led to the isolation of four new indole alkaloids for the first time in this plant. Compound (1) identified as spiroindolone 2′,3′,4′,9′-tetrahydrospiro [indoline-3,1′pyrido[3,4-b]-indol]-2-one, and compound (3) described as nitrone 1-methyl-4,9-dihydro-3H-pyrido [3,4-b] indole 2-oxide and were isolated for the first time as natural products. ABTS antioxidant activity guided their isolation.     

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.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC.

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by 32P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AalphaC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AalphaC. The highest AalphaC-DNA adduct levels were found in liver (137 adducts/10(8) normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N2-(deoxyguanin-8-yl)-2-amino-9H-pyrido [2,3-b]indole. Considering the abundance of AalphaC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AalphaC might be underestimated.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AαC), analysed by 32 P-HPLC

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by ³²P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AαC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AαC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AαC. The highest AαC–DNA adduct levels were found in liver (137 adducts/10⁸ normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N²-(deoxyguanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole. Considering the abundance of AαC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AαC might be underestimated.     

UDP-glucuronosyltransferase-mediated metabolic activation of the tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by (1)H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N(2)-(β-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Gl) and N(2)-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON(2)-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN(2)-O-Gl). AαC-HN(2)-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20-50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (V(max)/K(m)) at forming AαC-N(2)-Gl (0.67 μl·min(-1)·mg of protein(-1)), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl·min(-1)·mg of protein(-1)), whereas UGT1A1 was most efficient at forming AαC-HON(2)-Gl (5.0 μl·min(-1)·mg of protein(-1)). Human hepatocytes produced AαC-N(2)-Gl and AαC-HN(2)-O-Gl in abundant quantities, but AαC-HON(2)-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC.     

Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol.

Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 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) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s).     

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.     

Photoacoustic and luminescence characterization of nitrogen heterocyclic aromatic UV-MALDI matrices in solution

A group of nitrogen heterocyclic aromatic compounds used, among others, as UV-MALDI matrices was studied. By using spectroscopic, luminescence and photoacoustic techniques, as well as time resolved phosphorescence for singlet oxygen production determination, the behaviour of 9-aminoacridine (9AA), 3-aminoquinoline (3AQ), 2-(2-aminoethylamino)-5-nitropyridine (AAN) and 3,4-dihydro-7-methoxy-1-methyl-9H-pyrido[3,4-b] indole (harmaline, HLA) in acetonitrile solutions is described. The results show that for these compounds radiationless processes that release prompt heat to the media are a quite important deactivation mechanism.     

Analysis of human plasma as an exposure level monitor for carcinogenic tryptophan pyrolysis products

A high-performance liquid chromatography method for detecting 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) in human plasma was developed. Plasma samples of 10 normal subjects were examined. Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, were detected in all specimens, and the concentrations of Trp-P-1 and Trp-P-2 in plasma were 68.31 +/- 24.03 fmoles/ml (mean +/- S.D., n = 10) and 18.79 +/- 4.99 fmoles/ml, respectively. Our results suggest that plasma levels of carcinogenic tryptophan pyrolysis products may be useful indicators for estimating the exposure levels of the dietary carcinogens.     

Contribution of pi-electrons to the inhibition of S-adenosyl-L-methionine decarboxylase by aromatic tricyclic compounds

Eleven structural analogs with norharmane (9H-pyrido [3, 4-b] indole) as the basic structure were used to study structure-activity relations of adenosyl-methionine decarboxylase inhibition by this type of compounds. All analogs with an intact or 3,4-dihydrogenated pyrido ring inhibited the enzyme competitively with apparent Kis at the micromolar range, but complete hydrogenation of the pyrido moiety abolished the inhibition. This indicates that a suitable arrangement of pi-electrons is an essential structural feature in the process. Strongest inhibitors (Ki approximately 10-5M) were compounds methoxy or hydroxy groups in positions 6 or 7 of the indole moiety, suggesting that polarizing groups containing atoms with free electron pairs also are important in the interaction. The results offer a new viewpoint when action mechanisms of the known adenosylmethionine decarboxylase inhibitors are evaluated and they may prove useful when new inhibitors to the enzyme are designed.     

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.