Tryptophan Pyrolysis Research Articles

Selenium and vitamin E inhibit radiogenic and chemically induced transformation in vitro via different mechanisms.

Results from in vivo and in vitro studies showing that antioxidants may act as anticarcinogens support the role of active oxygen in carcinogenesis and provide impetus for exploring the functions of dietary antioxidants in cancer prevention by using in vitro models. We examined the single and combined effects of selenium, a component of glutathione peroxidase, and vitamin E, a known antioxidant, on cell transformation induced in C3H/10T-1/2 cells by x-rays, benzo[a]pyrene, or tryptophan pyrolysate and on the levels of cellular scavenging systems and peroxide destruction. Incubation of C3H/10T-1/2 cells with 2.5 microM Na2SeO3 (selenium) or with 7 microM alpha-tocopherol succinate (vitamin E) 24 hr prior to exposure to x-rays or the chemical carcinogens resulted in an inhibition of transformation by each of the antioxidants with an additive-inhibitory action when the two nutrients were combined. Cellular pretreatment with selenium resulted in increased levels of cellular glutathione peroxidase, catalase, and nonprotein thiols (glutathione) and in an enhanced destruction of peroxide. Cells pretreated with vitamin E did not show these biochemical effects, and the combined pretreatment with vitamin E and selenium did not augment the effect of selenium on these parameters. The results support our earlier studies showing that free radical-mediated events play a role in radiation and chemically induced transformation. They indicate that selenium and vitamin E act alone and in additive fashion as radioprotecting and chemopreventing agents. The results further suggest that selenium confers protection in part by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown while vitamin E appears to confer its protection by an alternate complementary mechanism.

Metabolic, desmutagenic and anticarcinogenic effects of N-acetylcysteine.

N-acetylcysteine (NAC) is often administered to respiratory patients with histories of exposure to noxious agents (e.g. cigarette smoke and atmospheric pollutants), which are known to act as glutathione (GSH) depletors and as cancer initiators and/or promoters. Since NAC is a precursor of intracellular GSH, we investigated its effects on GSH metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds. In vitro, NAC induced a significant increase in oxidized glutathione (GSSG) reductase activity in rat liver preparations and counteracted the mutagenicity of direct-acting compounds (such as epichlorohydrin, hydrogen peroxide, 4-nitroquinoline-N-oxide and dichromate), as a result of its reducing and scavenging properties. At high concentrations, the drug completely inhibited the mutagenicity of procarcinogens (cigarette smoke condensate, tryptophan pyrolysate, cyclophosphamide, 2-aminofluorene, benzo(a)pyrene and aflatoxin B1) by binding their electrophilic metabolites. In contrast, their metabolic activation was stimulated by decreasing NAC concentrations, especially when liver preparations from enzyme-induced rats were used. Lung and liver subcellular preparations of rats treated in vivo with NAC, in various combinations with enzyme inducers and/or GSH depletors, also affected the mutagenicity of a number of compounds. NAC generally increased intracellular GSH and restored its levels following depletion. It did not affect the levels nor the spectral properties of cytochromes P-450 in pulmonary and hepatic microsomes, whereas it stimulated, especially in Aroclor-pretreated animals, cytosolic enzyme activities involved in NADP or GSSG reduction (G6PD, 6PGD and GSSG reductase) and in the reductive detoxification of xenobiotics (DT diaphorase). When administered with the diet, at a nontoxic posology (120 mg/kg b.w.), NAC markedly inhibited the induction of lung tumors in mice by a potent carcinogen (urethane).

Metabolic activation of hepatocarcinogens in chronic hepatitis B

S9 fraction pools of liver biopsy samples, collected from 129 patients in two consecutive studies, were comparatively assayed for their ability to activate aflatoxin B1 (AFB1) and a tryptophan pyrolysate product (Trp-P-2) in a miniaturized Salmonella mutagenicity test system. Metabolic activation was not affected to a significant extent by most of the monitored variability factors, such as sex, alcohol, cigarette smoking and liver histology (minimal changes, chronic persistent (CPH) or active (CAH) hepatitis, CAH steatosis, or cyrrhosis). Conversely, a significant enhancement of activation was observed for AFB1 in cases of mild CAH and especially for Trp-P-2 in hepatitis B virus carriers, irrespective of their histologic diagnosis.

Absorption of 3-amino-1-methyl-5H-pyrido[4,3-b]indole, a mutagen-carcinogen present in tryptophan pyrolysate, from the gastro-intestinal tract in the rat.

The absorption characteristics of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a mutagen-carcinogen present in tryptophan pyrolysate, in the gastrointestinal tract was investigated in Wistar male rats by an in situ loop technique. Trp-P-2, a weak base with a pKa of 8.2, was poorly lipophilic below pH 5 and therefore the gastric absorption was negligible at pH 1.1 and 3.0. On the other hand, this compound was rapidly absorbed from the small intestine even at pH 4.0 and the absorption was even faster at higher pH where the unionized fraction increases. The absorption from the large intestine was also rapid. Although the intestine is one of the major metabolic organs for ingested materials, metabolism of Trp-P-2 by the scraped mucosa of the small intestine was slow in comparison with that in the liver. Formation of direct-acting mutagens (towards S. typhimurium TA98) associated with the metabolism was also much slower in the small-intestinal mucosa than in the liver. These results suggest that this mutagen is activated mainly after being incorporated into the liver, and are consistent with the liver-specific carcinogenicity of Trp-P-2 in rats.

In vivo effects of N-acetylcysteine on glutathione metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds

N-acetylcysteine (NAC) was administered to rats in various combinations with an enzyme inducer (Aroclor 1254) and with depletors of reduced glutathione (GSH), i.e., diethyl maleate (DEM) and buthionine sulfoximine (BSO). NAC increased intracellular glutathione levels in erythrocytes and in liver and lung cells, and replenished its stores following depletion. It did not affect the concentrations nor the spectral properties of cytochromes P-450 in hepatic and pulmonary microsomes, whereas it stimulated, especially in Aroclor-pre-treated animals, cytosolic enzyme activities involved in NADP reduction (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), in glutathione reduction (GSSG-reductase) and in the reductive detoxication of xenobiotics by-passing formation of reactive oxygen species (DT-diaphorase). In vivo treatment with the drug enhanced detoxication by liver and lung S-12 fractions of direct-acting mutagens (ICR 191, epichlorohydrin, 4-nitroquinolino-N-oxide and dichromate) and counteracted opposite effects triggered by administration of GSH depletors. The metabolic activation of procarcinogens (aflatoxin B1, 2-aminofluorene, cyclophosphamide, benzo[a]pyrene, a tryptophan pyrolysate product and cigarette smoke condensate) was inhibited by NAC in uninduced rats, while it was further stimulated in Aroclor-pre-treated animals. Additional assays, performed also with other enzyme inducers (phenobarbital and 3-methylcholanthrene) suggested that the effect of NAC on the metabolic activation of procarcinogens depends on the balance between an increased production of mutagenic metabolites (prevailing in induced animals) and their binding by intracellular thiols (prevailing under normal conditions). Thus, due to its dual role as a nucleophile and as a SH donor, NAC appears to exert protective effects by modulating glutathione metabolism and the biotransformation of mutagenic/carcinogenic compounds. This may have clinical relevance, since NAC is administered to individuals, such as cigarette smokers, who are more heavily exposed to GSH depletors and to carcinogenic agents.

Examination of DNA lesion by the alkaline elution technique. II. DNA lesion induced by hydrogen peroxide

Sister-chromatid exchanges (SCEs) in a permanent cell line of human lymphoblastoid cells were induced by 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-6-methyldipyridol[1,2-a:3′,2′-d]imidazole (Glu-P-1) and 2-amino-9H-pyrido[2,3-b]indole (2-amino-α-carboline). The first two compounds were found in tryptophan pyrolysates, the third in a glutamic acid pyrolysate and the last in a globulin pyrolysate. All these compounds required the metabolic activation system (S9 mix) for induction of SCE. Trp-P-2 had the highest SCE-inducing activity of these chemicals (approximately equivalent to that of aflatoxin B1), followed by Trp-P-1, Glu-P-1 and then 2-amino-α-carboline.

Comparative induction of somatic eye-color mutations and sex-linked recessive lethals in Drosophila melanogaster by tryptophan pyrolysates

The mutagenecities of the products of pyrolysis of tryptophan, Trp-P-1 and Trp-P-2, on Drosophila melanogaster were examined by measuring the effects of these compounds in inducing recessive lethals and somatic eye-color mutations. Since negative results have already been obtained by the standard procedure in males, Trp--P-1 and Trp-P-2 (0.75 to 6 mg/ml) in sucrose solution were given to females for assay of recessive lethal mutations in X-chromosomes. These compounds caused a marginal increase above the control level in the mutation frequency. For the assay of effects on somatic eye-color mutations, Trp-P-1 (200 and 400 ppm) and Trp-P-2 (400 and 800 ppm) were fed to male larvae of a tester strain carrying a genetically unstable marker set of z and w + on the X-chromosome. These compounds caused dose-dependent increases above the control level in somatic eye-color mutattions in adults. It is concluded that, under the conditions used, the somatic eye-color mutation system was more sensitive than the recessive lethal system to the mutagenic effects of tryptophan pyrolysates.

Interactions between the active metabolite of tryptophan pyrolysate mutagen, N-hydroxy-Trp-P-2, and lipids: The role of lipid peroxides in the conversion of N-hydroxy-Trp-P-2 to non-reactive forms

The interactions between lipids and the mutagenic active metabolite of 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2) and 3-hydroxyamino-1-methyl-5 H-pyrido[4,3- b]indole ( N-hydroxy-Trp-P-2), were studied. Oleic acid showed an inhibitory effect on the formation of this active metabolite mainly by inhibition of hepatic microsomal oxidation systems. On the other hand, microsomal lipids from rat liver and commercial pig liver lecithin diminished the amount of N-hydroxy-Trp-P-2 without inhibiting the metabolism of Trp-P-2. The direct reaction of these lipids with N-hydroxy-Trp-P-2 was disclosed by experiments using N-hydroxy-Trp-P-2 and lipids without microsomes. Furthermore, the participation of lipid peroxides in this reaction was suggested by a linear relationship between the concentrations of the conjugated diene of lipids and the disappearance of N-hydroxy-Trp-P-2. When [ 3H] N-hydroxy-Trp-P-2 was incubated in the presence of pig liver lecithin, the polar products which were not formed in the incubation without lipids were newly detected by thin-layer chromatography (TLC) analysis.

The Identification of 3-Amino-9H-pyrido[3,4-b]indole Derivatives in l-Tryptophan Pyrolysates

Abstract The compounds, 3-amino-9H-pyrido[3,4-b]indole and 3-amino-1-methyl-9H-pyrido[3,4-b]indole, which are effectors in induction of sister chromatid exchanges in human cells, were isolated from pyrolysis products of l-tryptophan and characterized by HPLC and UV techniques.

Formation of mutagenic derivatives from tryptophan pyrolysis products (Trp P 1 and Trp P 2) by rat intestinal S9 fraction

The formation of mutagenic derivatives from TrpP 1 and TrpP 2 by rat intestinal enzymes was observed in the intestinal S9 fraction in the Ames test. TrpP 2 produced more mutagenic derivatives than TrpP 1. The intestinal mutagenic activation of both tryptophan pyrolysates was preferentially induced by 3-methylcholanthrene.

Defective repair of tryptophan pyrolysate (Trp P-1 and Trp P-2) and aflatoxin B1 damage in xeroderma pigmentosum cells.

Defective repair of tryptophan pyrolysate (Trp P-1 and Trp P-2) and aflatoxin B1 damage in xeroderma pigmentosum cells.

Induction of bladder cancer in mice by implanting pellets containing tryptophan pyrolysis products

A high incidence of transitional cell carcinomas were induced in the bladder of mice by bladder implantation of pellets containing crude tryptophan pyrolysate and the pyrolysis products, 3-amino-1,4-dimethyl-5 H-pyrido-[4,3- b]indole (Trp-P-1) and 3-amino-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2), which are highly mutagenic substances.

Covalent binding of N-hydroxy-Trp-P-2 to DNA by cytosolic proline-dependent system

A new enzymatic activation system for the covalent binding of a mutagenic metabolite of a tryptophan pyrolysate, N-hydroxy-Trp-P-2, is described. The system exists in hepatic cytosolic fraction of rats, requiring ATP and some amino acids as the cofactor. Proline was the most effective among amino acids examined. These results suggest that N-hydroxy-Trp-P-2 formed by microsomal cytochrome P-450 is activated by prolyl-tRNA synthetase or related enzyme(s). Possible roles of sulfation and acetylation in the formation of the covalent adducts were also discussed.

Mutagen and anti-mutagen in ginger, Zingiber officinale

When rhizome juice of ginger, Zingiber officinale, was added to a solution of 2(2-furyl)-3-(5-nitro-2-furyl)acryl amide (AF2) or N-methyl- N′-nitro- N-nitro-soguanidine (NTG), mutagenesis by these chemicals was markedly increased. As a result of the component fractionation of the ginger juice, it was found that [6]-gingerol was a potent mutagen. However, the ginger juice also contained anti-mutagenic component(s) against [6]-gingerol (CAS No. 58253-27-3) (present study) and tryptophan pyrolysates (Kada et al., 1978; Morita et al., 1978). It is suggested, therefore, that the [6]-gingerol component may be mutagenically activated by the presence of AF2 and NTG.

Liver cancer and precancerous changes in rats induced by the basic fraction of tryptophan pyrolysate

The basic fraction of a tryptophan pyrolysate (Trp-P-BF) was given orally to Wistar rats for about 2 years. In Experiment I, 5 male rats each were given 0.2, 0.4, 0.6 and 0.8% Trp-P-BF. Dose-dependent growth retardation was observed in these groups and neoplastic nodules were found in the liver of 1 rat given 0.2% Trp-P-BF and a hepatocellular carcinoma was found in 1 rat given 0.4% Trp-P-BF diet. In Experiment II, 25 rats of both sexes were fed 0.5% or 0.2% Trp-P-BF diet. Neoplastic nodules were induced in 2 of 22 males and 5 of 18 females given 0.2% Trp-P-BF diet. Mammary adenomas developed, but no neoplastic nodules were found in the liver of rats fed on 0.05% Trp-P-BF or control diet. Females were more sensitive to Trp-P-BF than males.

Synthesis of mutagens isolated from tryptophan pyrolysate and of some analogs, 3-amino-5H-pyrido(4,3-b)indoles.

A potent mutagen, 3-amino-1-methyl-5H-pyrido [4, 3-b] indole, isolated from tryptophan pyrolysate, and some analogs were synthesized. The key reaction was the AlCl3-catalyzed condensation of 2-cyanomethylindole and alkyl (or phenyl) cyanide.

Purification and properties of a plant desmutagenic factor for the mutagenic principle of tryptophan pyrolysate.

A desmutagenic factor for Trp-P-2 which is a potent mutagen discovered by Sugimura and his colleagues in pyrolysis products of tryptophan has been identified from extracts of cabbage (Bvassica oleracea) leaves. Analysis of this factor to determine its composition revealed that it had a molecular weight of 43, 000 and contained 45.2 μg of sugar per mgof protein. The purified factor exhibited a hemo-protein-like absorption spectrum with a Soret band of 404 nm and α and β bands at 640 and 497 nm, respectively. In addition to counteracting the mutagenicity of Trp-P-2, the mutagenic activity of Trp-P-1, which is 4-methyl derivative of Trp-P-2, was equally abolished by the factor. Other mutagens, such as ethidium bromide and 2-aminoanthracene, were found to be susceptible to the factor, but it had no effect on such mutagens as ICR-170 and AF-2. Furthermore, the factor greatly enhanced the mutagenicity of 1, 2-diamino-4-nitrobenzene. Several lines of investigations were pursued to determine the possible mode of action to explain the results obtained with the factor. These studies indicated that the factor was a peroxidase which possessed NADPH-oxidaseactivity. In this paper, the mechanism of the desmutagenic action of the factor is discussed with special reference to these enzymatic activities.

Myeloperoxidase-catalyzed binding of 3-amino-1-methyl-5 H-pyrido[4,3- b]indole, a tryptophan pyrolysis product, to protein

Incorporation of 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2), the principal mutagen in a tryptophan pyrolysate, into bovine serum albumin was catalyzed by myeloperoxidase. Hydrogen peroxide was essential for the incorporation reaction and albumin was required for optimal incorporation of Trp-P-2 into protein. Other various proteins, such as histone, lysozyme, cytochrome c, and γ-globulin could also incorporate Trp-P-2, but poly(L-Arg), poly(L-Lys), and poly(L-Glu) could not. The incorporation of Trp-P-2 into albumin was inhibited by L-tyrosine and L-tryptophan, but not by other amino acids. Trp-P-2 incorporated into albumin was not released from the protein by treatment with 0.3 N HCl, or 0.3 N NaOH for 2 h at 37°C, or with 1% sodium dodecylsulfate for 2.5 min at 100°C. On electrophoresis on polyacrylamide containing sodium dodecylsulfate or urea and on chromatography on Sepharose CL-6B in 6 M guanidine/HCl, Trp-P-2 incorporated into albumin or lysozyme migrated with these proteins. These findings indicate that Trp-P-2 is covalently bound to these acceptor proteins.

Differentiation of Fetal and Adult Bloodstains by Pyrolysis-Gas-Liquid Chromatography

A pyrolysis-gas-liquid chromatographic method has been developed for the differentiation of adult and fetal bloodstains. In a blind-coded study, five adult and three fetal bloodstains were correctly identified on the basis of the pyrograms of stain extracts. The differentiation between adult and fetal bloodstains is based on the peak height ratio of two long-retention-time peaks appearing in their pyrograms. The first of these peaks has been tentatively identified as indole derived from the pyrolysis of tryptophan, while the second peak is an as-yet unidentified molecular fragment produced by the pyrolysis of some component of the hemoglobin molecule other than the amino acids tryptophan, tyrosine, and phenylalanine.

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.