Sakae Arimoto

Dietary inhibitors of mutagenesis and carcinogenesis

Dietary inhibitors of mutagenesis and carcinogenesis are of particular interest because they may be useful for human cancer prevention. Several mutagenesis inhibitors have been demonstrated to be carcinogenesis inhibitors also, e.g., ellagic acid, palmitoleic acid, and N-acetylcysteine. This means that the search for mutagenesis inhibitors may be useful for discovering anticarcinogenic agents. Many mutagenesis inhibitors have been discovered by the use of short-term assays, particularly the Ames Salmonella test. This simple in vitro system has provided opportunities to elucidate the mechanisms of inhibition. The elucidation of the mechanism may allow us to infer the possible anticarcinogenic activity of the reagent. In this chapter, inhibitors of mutagenesis and carcinogenesis that can arise as components of diet have been reviewed. Most of the inhibitors have been demonstrated to be effective against a specific class of mutagens or carcinogens. Therefore, it may be argued that these inhibitors are antagonistic only to those particular agents. Here again, understanding of the mechanisms of these inhibitions is necessary for the assessment. Dietary inhibitors reviewed in this article include: (1) as inhibitors of mutagenesis: porphyllins, fatty acids, vitamins, polyphenols, and sulfhydryl compounds, (2) as inhibitors of carcinogenesis: vitamins A, E and C, ellagic acid, sulfhydryl compounds, fats, selenium, calcium, and fiber. Further studies in this area of science appear to help establish the recipe of a healthy diet.

Adsorption of mutagens to cotton bearing covalently bound trisulfo-copper-phthalocyanine

A method for separating nonpolar mutagens from their dilute aqueous solutions is described. It utilizes the affinity of the mutagens to a phthalocyanine derivative attached to cotton through a covalent bond. For mutagens having 3 or more fused aromatic rings in their structures, efficient adsorption took place on soaking the cotton in their solutions. The mutagens adsorbed can be recovered by elution with ammoniacal methanol. Mutagenicity in smokers urine, cooked beef, and river water was detected by use of this method.

Binding of polycyclic planar mutagens to chlorophyllin resulting in inhibition of the mutagenic activity

Chlorophyllin is known to inhibit the mutagenicity of a variety of compounds. Using highly purified samples of chlorophyllin and its family compounds, we studied the mechanism of the inhibition. Since mutagens with polycyclic planar structures are particularly strongly inhibited, it seemed likely that the inhibition arises by trapping of the mutagens by chlorophyllin through complex formation at the planar surfaces of these molecules. To explore this possibility, we prepared a Sepharose bearing covalently linked chlorophyllin as ligand, and the adsorption of mutagens to this Sepharose was measured. Three different chlorophyllin derivatives were used, i.e., copper-chlorin, iron-chlorin and chlorin, to investigate the role of metal in the center of the chlorophyllin chromophore. Adsorption of 37 different compounds, mostly mutagens, in 0.02 M Tris-HCl buffer at pH 8.0 to these chlorophyllin-Sepharose preparations was studied in a quantitative manner. The results showed that most of the compounds having three or more fused rings were strongly adsorbed with apparent dissociation constants of 10(-5)-10(-6) M, whereas those having two fused rings or one ring were only poorly adsorbed. Since the three Sepharose adsorbents gave similar adsorption profiles, it appeared that the central metal in the chlorophyllin molecule does not play a crucial role in the adsorption. We also measured the inhibitory effect of copper-chlorin against the mutagenicity of some of these compounds using the Salmonella assay. The results showed that those mutagens that were strongly adsorbable to copper-chlorin-Sepharose were subject to efficient inhibition by copper-chlorin, whereas many of those only poorly adsorbed were inhibited only weakly. We concluded that trapping by complex formation plays a role in the antimutagenic actions of chlorophyllin against many mutagens, particularly notable being the actions against ICR-170, quinacrine, aflatoxin B1, Trp-P-1 and Trp-P-2. An unusual behavior of Trp-P-2 in the adsorption process, i.e., a very tight complex formation at an extremely low Trp-P-2 concentration, was found; the implication of this phenomenon in relation to the real environmental setting is discussed.

Parasite infection and cancer: with special emphasis on Schistosoma japonicum infections (Trematoda). A review

This article contains a review of current knowledge on the association of parasite infections and cancer formation, especially that of Schistosoma japonicum (Trematoda) in man and experimental animals. The association of S. haematobium infection and bladder cancer is well known and documented. However, S. japonicum infection has also been reported to be associated with cancer, in this case hepatocellular carcinoma and/or colorectal cancer. Pathological records and analyses have shown a correlation between this infection and cancer, and pathohistological descriptions have been numerous, together with clinical case reports. Epidemiological analyses have been conducted in China and Japan and support a role of S. japonicum infection as one of the risk factors in cancer formation, along with others, such as hepatitis virus infection and alcoholic intake. Experimental results have also shown that cancer appears early and in larger numbers in experimentally infected animals given a known carcinogen. In spite of these positive end-point associations, the mechanism of schistosome-mediated enhancement of carcinogenesis is obscure. A suggestive observation is that in S. japonicum-infected mice carcinogen-metabolizing hepatic activity including P-450 was decreased so that an administered carcinogen persisted for a longer period than in uninfected mice. Further studies, both epidemiological and experimental, are needed to firmly establish the relationship between schistosome infection and cancer.

Porphyrins as potential inhibitors against exposure to carcinogens and mutagens

Studies have shown that there are many substances that can interfere with the actions of carcinogens and mutagens. Porphyrins, which often are constituents of diet, are a class of such inhibitors. Hemin can inhibit selectively the activity of mutagens having polycyclic structures by forming complexes with them. These effects were found with the use of bacterial assays and also by in vitro chemical experiments. A survey of porphyrins for similar effects has been done in our laboratory and it was found that chlorophyll and chlorophyllin act like hemin. These green pigments are antimutagenic in Salmonella and in Drosophila. Work from other laboratories also has supported the antimutagenic character of chlorophyllin. The possibility of modifying human exposure to carcinogens by use of these porphyrins is discussed. A porphyrin-like molecule, copper phthalocyanine trisulfonate, has been shown to have strong affinity to polycyclic compounds. Blue cotton, a cotton preparation bearing this blue pigment as a covalently bound ligand, has been demonstrated to be an adsorbent useful for isolating heterocyclic amines from food and other materials.

Role of hemin in the inhibition of mutagenic activity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other aminoazaarenes

The mechanism of inhibition by hemin of the mutagenic activities of food pyrolysate aminoazaarenes, particularly that of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), was investigated. Hemin efficiently inhibited the metabolic activation by S9 of Trp-P-2, as demonstrated by high-performance liquid chromatographic analysis of the reaction mixtures in which Trp-P-2 had been treated with S9 in the presence or absence of hemin. N-Hydroxy-Trp-P-2, an activated form of Trp-P-2 having direct mutagenicity on Salmonella typhimurium TA98, undergoes spontaneous oxidative degradation in its aqueous solution, and the presence of hemin in the solution accelerated the degradation significantly. The presence of excess hemin with N-hydroxy-Trp-P-2 completely abolished the mutagenic activity of this mutagen towards Salmonella. A UV-visible spectroscopic study has suggested the formation of a complex between hemin and N-hydroxy-Trp-P-2/Trp-P-2. In support of this view, the fluorescence spectrum of a Trp-P-2 solution was quenched efficiently by the addition of hemin. These observations indicate that this complex formation plays a role in the observed multiple actions of hemin. Similar inhibitory actions of hemin on several other direct-acting aminoazaarene mutagens are also described, as well as the inhibition activities of protoporphyrin, chlorophyllin, biliverdin and bilirubin.

Inhibitory effect of hemin, chlorophyllin and related pyrrole pigments on the mutagenicity of benzo[a]pyrene and its metabolites.

Hemin and chlorophyllin are known to inhibit strongly the mutagenicity of benzo[a]pyrene in the Salmonella assay. To further investigate this phenomenon, a series of these pyrrole pigments including pure samples of Cu- and Fe-chlorins were tested for their potency to inhibit the mutagenicity of benzo[a]pyrene and its metabolites, benzo[a]pyrene-7,8-diol, benzo[a]pyrene-4,5-epoxide, and benzo[a]pyrene-7,8-diol-9,10-epoxide. Hemin was the most potent among the pigments tested for these inhibitions. Both hemin and Cu-chlorin accelerated efficiently the degradation of benzo[a]pyrene-7,8-diol-9,10-epoxide, and this acceleration seemed to be the predominant mechanism by which these pigments inhibit the overall mutagenicity of benzo[a]pyrene in Salmonella. Based on spectroscopic evidence, we speculate that a complex formation between hemin and benzo[a]pyrene-7,8-diol-9,10-epoxide takes place and that this complexing is the cause of the accelerated degradation.

A solvent effect on the mutagenicity of tryptophan-pyrolysate mutagens in the Salmonella/mammalian microsome assay.

The effect of adding organic solvents to the pre-incubation mixture in the Salmonella/mammalian microsome assay was examined in tests of the tryptophan-pyrolysate mutagens, 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). Acetonitrile enhanced the mutagenic activity of Trp-P-1 and Trp-P-2 by an order of magnitude under optimal conditions. Strong enhancement of mutagenicity was also observed for the addition of ethanol, methanol, tetrahydrofurfuryl alcohol, tetrahydrofuran, 1,2-dimethoxyethane and N,N-dimethylformamide. The enhancing effect of acetonitrile on the mutagenicity of Trp-P-2 was found to be result of enrichment of the active metabolite in the assay system.

Evaluation of the mutagenicity and the tumor-promoting activity of parasite extracts: Schistosoma japonicum and Clonorchis sinensis.

In relation to the observed association of carcinogenesis with parasitic infections, the mutagenicity of extracts of Schistosoma japonicum and Clonorchis sinensis was examined. In the bacterial mutagenicity tests using the Ames Salmonella typhimurium strains TA98, TA100, TA97 and TA102, and Escherichia coli WP2 and WP2 uvrA pKM101 Schistosoma soluble egg antigen and a homogenate of adult Schistosoma worms showed no positive responses either in the presence or in the absence of S9 mix. Likewise, adult worm extracts of Clonorchis showed no mutagenicity. The Schistosoma soluble egg antigen showed a weak but significant activity for the induction of Epstein-Barr virus expression in viral genome-carrying human lymphoblastoid cells in culture. This phenomenon suggests that the soluble egg antigen possesses tumor-promoting activity.

Inhibitory effect of myoglobin and hemoglobin on the direct-acting mutagenicity of protein pyrolysate heterocyclic amine derivatives

We have shown in our earlier reports (Arimoto et al., 1980a, b) that hemin and some other porphyrins can inhibit the mutagenicity in Salmonella of heterocyclic amines derived from cooking of proteins. A direct interaction between hemin and the mutagens was implicated on the basis of the observation that some of the mutagens were inactivated when they had been metabolically converted into direct-acting mutagens before the treatment with hemin. Hemin is a bound constituent of various proteins including hemoglobin and myoglobin, which are abundantly present in the blood and muscles, respectively. An interesting question is whether or not hemoglobin and myoglobin can inhibit the activities of the mutagenic heterocyclic amines.