Syntheses of hydroxyamino, nitroso and nitro derivatives of Trp-P-2 and Glu-P-1, amino acid pyrolysate mutagens, and their direct mutagenicities towards Salmonella typhimurium TA98 and TA98NR

Hydroxyamino, nitroso and nitro derivatives of 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole(Glu-P-1), mutagens-carcinogens produced on pyrolysis of aminoacids, were synthesized from Trp-P-2 and Glu-P-1. 3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole(N-OH-Trp-P-2) and 2-hydroxyamino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) were obtained with good yieldsby controlled catalytic reduction of 3-nitro-l-methyl-5H-pyrido[4,3-b]indoleand 2-nitro-6-methyldipyrido[1,2-a:3',2'-d]imidazole. Subsequentoxidation of N-OH-Trp-P-2 and N-OH-Glu-P-1 with -manganese dioxideyielded 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole and 2-nitroso-6-methyldipyrido[1,2-a:3',2'-d]imidazole.All six synthesized compounds were mutagenic to Salmonella typhimuriumTA98 without mammalian activation systems. The mutagenic activitiesof hydroxyamino and nitroso derivatives were identical for bothS. typhimurium TA98 and TA98NR, the nitroreductase deficientstrain. However, nitro derivatives were essentially mutageniconly towards S. typhimurium TA98.     

DNA binding of N-hydroxy-Trp-P-2 and N-hydroxy-Glu-P-1 by acetyl-CoA dependent enzyme in mammalian liver cytosol

3-Hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole and 2-hydroxyamino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole, active metabolites of protein pyrolysatecarcinogens, were further activated to DNA-binding species byan acetyl-CoA dependent enzyme in hepatic cytosol. This activitywas supported also by N-hydroxy-2-acetylaminofluorene insteadof acetyl-CoA, and it was inhibited by iodoacetamide and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) but not by diethyl-p-nitrophenylphosphate.The activity was observed in the cytosols of rats, mice andrapid acetylator rabbits, which possessed N-acetylation activityof 2-aminofluorene and Trp-P-2. On the other hand, the activitycould not be detected in the cytosols of a dog and a slow acetylatorrabbit.     

Generation of intracellular active oxygens in mouse FM3A cells by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, the activated Trp-P-2

Mouse FM3A cells in culture were treated with a reactive metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2-(NHOH]. When the treated cells, which were judged as viable on the basis of trypan-blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp-P-2. Single-strand breaks in the cellular DNA took place during this treatment with Trp-P-2(NHOH). Since Trp-P-2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp-P-2(NHOH) treatment produces intracellular active oxygens that can damage DNA.     

Carcinogenicity of heterocyclic amines for the pancreatic duct epithelium in hamsters.

Effects of eight heterocyclic amines (HCAs) on pancreatic duct carcinogenesis were investigated in a rapid production model in hamsters. N-Nitrosobis(2-oxopropyl)amine (BOP) was given to effect initiation, followed by augmentation pressure consisting of four daily i.p. injections of 500 mg/kg DL-ethionine, a choline-deficient (CD) diet, a single i.p. injection of 800 mg/kg L-methionine and a s.c. injection of 20 mg/kg BOP. After two cycles of augmentation pressure, the HCAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 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-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) or 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [4,8-DiMeIQx) were administered in the diet for 50 or 70 days. The numbers of pancreatic ductal hyperplasias (H) and a total lesions including, atypical hyperplasia (AH), carcinomas in situ (CIS) and invasive carcinomas, were increased in hamsters given the diet containing 0.02% Trp-P-1 for 50 days. This result was confirmed and extended by the finding of increased numbers of invasive carcinomas in hamsters given 0.02% Trp-P-1 for 70 days. The number and incidence of invasive carcinomas were also elevated in hamsters given the diet containing 0.06% 4,8-DiMeIQx for 50 days. These results suggest a possible involvement of Trp-P-1 and 4,8-DiMeIQx in pancreatic duct carcinogenesis.     

Synergistic enhancement of hepatic foci development by combined treatment of rats with 10 heterocyclic amines at low doses

Potential synergism between 10 carcinogenic heterocyclic amines[3-amino-l,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-6 methyldipyrido[l,2-a:3',2'-d]imidazole(Glu-P-1), 2-ammo-dipyrido[l,2-a:3',2'-d]imidazoIe (Glu-P-2),2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline(MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx),2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA     

Carcinogenic effect of the simultaneous administration of five heterocyclic amines to F344 rats

F344 rats were given five mutagenic and carcinogenic heterocyclic amines, i.e., 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-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AaC), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), mixed together in the diet each at one-fifth of the concentration used in the previous single-compound carcinogenesis experiment. Liver, colon and Zymbal gland tumors in both sexes, skin tumors in males and clitoral gland tumors in females were induced at significantly higher incidences than in control groups. Among them, the incidences of liver tumors in both sexes, skin tumors in males and clitoral gland tumors in females were significantly higher than those expected from the simple assumptions that the incidence of tumors induced by each compound would be one-fifth of that in the corresponding previous experiment and that the combined effect of the five chemicals would be additive.     

Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not of inpatients receiving parenteral alimentation

For estimation of human exposures to carcinogenic heterocyclic amines, the amounts of four compounds, 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-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), in human urine were measured. Twenty-four hour urine specimens were collected from ten healthy volunteers eating normal diet (five males and five females) and three inpatients (two males and a female) receiving parenteral alimentation, and the levels of the four heterocyclic amines were measured by HPLC after partial purification by treatment with blue cotton and ion exchange column chromatography. Trp-P-1, Trp-P-2, PhIP and MeIQx were detected in the 24 h urine samples of all healthy volunteers at levels of 0.04-1.43 ng, 0.03-0.68 ng, 0.12-1.97 ng and 11-47 ng respectively. As 1.8-4.9% of an oral dose of MeIQx is reported to be excreted unchanged in the urine, the daily exposure of humans to MeIQx was estimated to be 0.2-2.6 micrograms/person. The four heterocyclic amines were not detected in the urine of parenterally fed inpatients. These results indicate that humans are continually exposed to carcinogenic heterocyclic amines in food, and these compounds may not be formed endogenously.     

Tumor induction in mice administered neonatally with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole or 3-amino-1-methyl-5H-pyrido[4,3-b]indole

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) or 3-amino-1-methyl-5H-pyrido[4,3-b]mdole(Trp-P-2), which is a potent mutagen from pyrolysates of tyrptophan,was given subcutaneously to neonatal ICR mice, and all animalswere observed for 1 year. Tumors of the livers and lympho-retkulartissue were induced. In the mice given Trp-P-1, the incidencesof these tumors were as follows: liver tumors in 45% of themales; malignant lymphoma in 13% of the males and in 24% ofthe females. In the mice given Trp-P-2, the incidences of livertumors in the males were dose-dependent (12.5 mg/kg, 12%; 25mg/kg, 18%), while those of malignant lymphoma varied withina range from 5 to 19%. Statistical analysis revealed that theincidences of the liver tumor in the mke given Trp-P-1 or Trp-P-2and those of lymphoma in the mice given Trp-P-1 were significantlyhigher than those of the controls. In the control mice, theincidences of tumors were as follows: malignant lymphoma in5% of the females; lung tumor in 14% of both sexes.     

Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines

Heterocyclic aromatic amines, derived from the pyrolysis of amino acids and proteins, are potent mutagens in the Ames Salmonella assay with rodent liver activation. Additionally, heterocyclic aromatic amines are multipotent carcinogens. We report evidence that these compounds are substrates for the hydroperoxidase activity of prostaglandin H synthase, as measured by alterations in UV/visible spectra, and are bioactivated to macromolecule-reactive species by this enzyme. Indirect electron paramagnetic resonance studies indicate that this activation may occur via a one-electron mechanism. 2-Amino-3-methylimidazo[4,5f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) are direct-acting mutagens in TA98. The mutagenicity of IQ and MeIQ, but not Trp-P-2, were enhanced by activation with ram seminal vesicle microsomes (a rich source of prostaglandin H synthase). Subsequent experiments utilized the newly constructed tester strain TA1538/1,8-DNP6 (pYG 121), which has enhanced arylamine N-acetyltransferase activity. In this strain IQ, MeIQ and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) were mutagenic with ram seminal vesicle microsome activation. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was a weak direct-acting mutagen, and was not activated by the ram seminal vesicles (RSV) system. The responses of IQ and MeIQ were markedly enhanced in TA1538/1.8-DNP6 (pYG 121), relative to TA98. These data are consistent with the involvement of prostaglandin H synthase-catalyzed activation in heterocyclic aromatic amine-induced extrahepatic neoplasia.     

Metabolic activation of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole(TRP-P-2) by isolated rat liver nuclei

The metabolic activation of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2), by rat liver nuclei was studied. Nuclei from the livers of rats treated with polychlorinated biphenyl (PCB) or 3-methylcholanthrene (MC) showed high mutagenic activity with Trp-P-2 in the Ames test, but activities with nuclei of untreated or phenobarbital (PB)-treated rat livers were quite low. The formation of N-hydroxy-Trp-P-2 by nuclei of PCB- or MC-treated rat livers was greater than that by nuclei of untreated or PB-treated rat livers. Similar results were observed with microsomes, which suggests that Trp-P-2 is metabolized by the same type of monoxygenase system in nuclei as in microsomes.     

3-Amino-1-methyl-5H-pyrido[4,3-b]-indole initiates two-stage carcinogenesis in mouse skin but is not a complete carcinogen

In two separate experiments, 2.0 mg of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was applied to the back of female CD-1 mice twice weekly for 5 weeks followed by application of 2.5 micrograms of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) twice weekly for 47 weeks. Skin squamous cell papillomas and carcinomas developed in 6 of 20 mice (30%) in Experiment 1 and in 3 of 19 mice (16%) in Experiment 2. In contrast, application of 2.0 mg of Trp-P-2 twice weekly for 52 weeks did not produce any skin tumors. These data suggest that Trp-P-2 acts primarily as an initiator, not as a complete carcinogen, for mouse skin.     

Genotoxicity of heterocyclic amines in the Salmonella/hepatocyte system

The Salmonella/hepatocyte system was employed to determine the mutagenicity in bacteria as well as the DNA damage induced in mouse hepatocytes following exposure to heterocyclic amines. With hepatocytes from C57BL/6N mice, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) showed a clear mutagenic effect in the Salmonella, while weak mutagenic effects were observed with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-6-methyldipyrido[1,2-a:3',2'-b]imidazole (Glu-P-1), and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). All the compounds induced low levels of DNA damage in the hepatocytes. In vivo pretreatment of mice with the potent monooxygenase inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 50 micrograms/kg) clearly increased both the mutagenicity in the bacteria and the DNA damage induced in the hepatocytes in vitro. Glu-P-2 showed the lowest mutagenic effect but induced more DNA damage at low concentrations than the other compounds when TCDD-pretreated hepatocytes were used. These data indicate that the genotoxic potency of Glu-P-2 in the intact hepatocyte differs from that observed in the bacteria. Treatment of hepatocytes with a-naphthoflavone, a selective inhibitor of polycyclic hydrocarbon-inducible cytochrome P-450 form(s), prior to exposure to the heterocyclic amines completely inhibited the mutagenic effect in the bacteria. In vivo administration of all the heterocyclic amines 4 hr prior to isolating the hepatocytes resulted in DNA damage, and this effect was augmented by TCDD pretreatment of mice. Our data suggest that agents modulating the activity and composition of the cytochrome P-450 system may greatly influence both toxicity and carcinogenicity of these heterocyclic amines.     

Synergistic enhancement of glutathione S-transferase placental form-positive hepatic foci development in diethylnitrosamine-treated rats by combined administration of five heterocyclic amines at low doses.

Potential synergism among 5 heterocyclic amines at low doses in the induction of glutathione S-transferase placental form (GST-P)-positive liver cell foci was examined in an 8-week experiment using male rats initially given diethylnitrosamine (200 mg/kg, ip). The heterocyclic amines applied were 3-amino-1-methyl-5H-pyrido[4,3-b]indole (500 ppm), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (500 ppm), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (800 ppm), 2-amino-9H-pyrido[2,3-b]indole (800 ppm), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 400 ppm). Separate groups received each chemical at the dose used in earlier carcinogenicity assays (above doses), at 1/5 or 1/25 of these, or all 5 chemicals together, each at the 1/5 or 1/25 levels. The numbers and areas of GST-P-positive foci were significantly increased with all chemicals, except for PhIP, at the highest dose, the results being consistent with the reported liver carcinogenicity. In the combined treatment at the 1/5 dose levels, synergistic enhancement occurred; the numbers and areas of foci were significantly increased above the sums of individual data. However, this was not the case for the 1/25 dose groups. Although the synergism between pyrolysis products in liver carcinogenesis depended on the dose and combination of chemicals, the findings, together with those from a previous experiment using 5 different heterocyclic amines, are of particular significance since several heterocyclic amines might be simultaneously generated during cooking of foodstuffs.     

Carcinogenicity in rats of a mutagenic compound, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, from tryptophan pyrolysate

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a mutagenic principle isolated from a tryptophan pyrolysate. When Trp-P-1 was given to male and female F344 rats at concentrations of 0.015% and 0.02%, respectively, in the diet, it induced hepatocellular carcinomas in high incidence within a year. No tumor was found in control rats.     

Catalysis of the covalent binding of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole to DNA by a L-proline- and adenosine triphosphate-dependent enzyme in rat hepatic cytosol

An enzymatic mechanism involved in the activation of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), a mutagenic intermediate of a tryptophan pyrolysate, was studied in vitro. In hepatic cytosol supplemented with adenosine triphosphate and L-proline, N-hydroxy-Trp-P-2 was converted to a form which reacts readily with DNA. The enzyme responsible for the activation was partially purified and identified as prolyl transfer RNA synthetase as judged by their cofactor requirements, inhibition by pyrophosphate or adenosine monophosphate, and copurification of their activities. The prolyl transfer RNA-dependent covalent binding of N-hydroxy-Trp-P-2 to DNA of hepatic cytosol was highest in rats, followed by mice, hamsters, rabbits, and guinea pigs in that order. The capacity for the binding of N-hydroxy-Trp-P-2 was largely consistent with their prolyl transfer RNA synthetase activity. With regard to the ultimate form of N-hydroxy-Trp-P-2 for the covalent binding, a possible formation of N,O-prolyl-3-amino-1-methyl-5H-pyrido[4,3-b]indole was proposed.     

Acetyl coenzyme A dependent activation of N-hydroxy derivatives of carcinogenic arylamines: mechanism of activation, species difference, tissue distribution, and acetyl donor specificity

Acetyl coenzyme A dependent activation of 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) was investigated using cytosols from hepatic and extrahepatic tissues of various animal species in comparison with that of N-hydroxy-2-aminofluorene. N-OH-Glu-P-1 and N-OH-Trp-P-2 were metabolized to the reactive species capable of binding to transfer RNA through a putative O-acetylation process by liver cytosols. Kidney, small intestinal mucosa, lung, and bladder from hamsters and rats also mediated the reaction, although their activities were lower than that in the liver. Marked species differences in the enzymatic activities of livers were observed. Hamsters showed the highest ability in the activation for N-OH-Glu-P-1 and N-OH-Trp-P-2, followed by rats. Rabbits with a rapid acetylator phenotype, which showed a high activity in the N-acetylation of arylamines, activated N-OH-Glu-P-1 but scarcely N-OH-Trp-P-2. A rabbit with a slow acetylator phenotype, mice, guinea pigs, and a dog showed marginal or nondetectable activities with N-OH-Glu-P-1 and N-OH-Trp-P-2. A typical nonheterocyclic N-hydroxyarylamine, N-hydroxy-2-aminofluorene was also activated by the acetyl coenzyme A dependent system to an intermediate which bound to transfer RNA. However, the acetyl-CoA dependent binding of N-hydroxy-2-aminofluorene was markedly different from those observed with N-OH-Glu-P-1 and N-OH-Trp-P-2 concerning the order of activities among animal species used. In addition to short chain acyl coenzyme As, N-hydroxy-2-acetylaminofluorene also served as an acetyl donor for the activation of N-OH-Glu-P-1 and N-OH-Trp-P-2 in liver cytosol systems. The formation of N-acetyl-N-OH-Glu-P-1, however, was not detected in the cytosolic system of N-OH-Glu-P-1 with acetyl-CoA, suggesting the direct O-acetylation at the N-hydroxy group as a major pathway for the activation of N-hydroxyarylamines.     

Influence of amino acids on the formation of mutagenic/carcinogenic heterocydic amines in a model system

Mixtures of creatinine, glucose and various single amino acidswere heated at 180°C for 10 min in an aqueous model system.The heated mixtures all showed mutagenic activity, ranging from80 to 2400 TA98 revertant colonies/µmol creatinine withmetabolic activation. Testing of HPLC fractions for mutagenicactivity showed each mixture to contain several mutagenic components,some of which corresponded to known heterocyclic amines andothers to unknown compounds. The presence of 2-amino-3-methyl-imidazo[4,5-f]quinoxaline,2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxalinein most of the samples was established using HPLC with photodiodearray detection and liquid chromatography/mass spectrometrywith electrospray interface and single ion monitoring. In addition,2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine,3-amino-1,4-di-methyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indoleand the co-mutagenic compounds 9H-pyrido[3,4-bindole and 1-methyl-9H-pyrido[3,4-b]indolewere detected in some samples.     

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

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 beta-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.     

Inhibition of DNA adduct formation and mutagenic action of 3-amino-1-methyl-5h-pyrido[4,3-b]indole by chlorophyllin-chitosan in rpsL transgenic mice.

We have studied the inhibitory effect of chlorophyllin-chitosan (Chl-Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen-carcinogen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp-P-2, DNA adducts were formed in various tissues in a dose-dependent manner, with the maximum level observed in the liver. Addition of 3% Chl-Chi to the diet reduced the Trp-P-2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp-P-2 diet. Addition of Chl-Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl-Chi was detected during these experiments. The results show that Chl-Chi may be a candidate chemopreventive agent against the genotoxic action of Trp-P-2, and possibly also other aromatic carcinogens in the diet.     

Inhibitory effect of chlorophyll on the genotoxicity of 3-amino-1-methyl-5H-pyrido[4,3-b indole (Trp-P-2)

Genotoxicity of 3-amino-1-methyl-5H-pyrido [4,3-b indole(Trp-P-2)on Drosophila was suppressed by chlorophyll. Using the winghair spot test, we found that the formation of mutant hairsin adult files as a result of feeding them with Trp-P-2 in theirlarval stage was efficiently inhibited by coadininistrationof chlorophyll. The decrease in the spot frequencies was dependenton the dose of chlorophyll, and at the highest dose used, wherethe ratio in weight of Trp-P-2 to chlorophyll was 1:80, a completeprevention of the small single-spot formation was observed.A similar inhibitory effect was detected for chlorophyllin,the chromophore of chlorophyll. In the studies to investigatethe mechanism of inhibition, we observed that the mutagenicityof 3-hydroxyamino-1-methyl-5H-[4,3-bpyrido [Trp-P-2(NHOH)],the metabolically activated form of Trp-P-2, in Salmonella typhimuriumTA98 was suppressed effectively with chlorophyll and chlorophyllin.We also found that chlorophyll and chlorophyllin can producecomplexes with Trp-P-2 and Trp-P-2(NHOH). A straightforwardmechanism of these inhibitions is that Trp-P-2 [and Trp-P-2(NHOH)]becomes no longer available to organisms on forming the chlorophyllcomplex.