The 5th International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis.
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Animals↗
Biomedical subjects
H Hayatsu
Publications and source records attributed to H Hayatsu.
Use of activated heterocyclic amines in the screening of dietary antimutagens.
Experimental methods suitable for detecting antimutagenic substances against heterocyclic amines are described. The proposed methodology will allow identification of direct interactions between the metabolically activated form of a heterocyclic amine and an antimutagenic compound.
Amines↗
Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative.
Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.
Aerobiosis↗
Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout.
The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an 'interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.
Aflatoxin B1↗
Inhibition of N-nitrosation of secondary amines in vitro by tea extracts and catechins.
Inhibition of nitrite-mediated N-nitrosation of dimethylamine, morpholine and N-methylaniline by tea extracts and by 6 individual catechins in the extracts was studied. The inhibitions were detected by quantifying the nitrosamines formed. Eight different kinds of teas (5 green teas, a roasted green tea, an oolong tea, and a black tea) were examined for their inhibitory abilities and for their catechin contents, with an attempt to correlate the inhibitory activities to the catechin contents. The results showed that (1) the green tea extracts inhibit strongly the N-nitrosation of the three secondary amines tested, (2) the 6 catechins, notably epigallocatechin, are capable of blocking the N-nitrosations very efficiently, even more efficiently than ascorbic acid, and (3) the inhibition activities of green tea extracts are mostly ascribable to the catechins present in the extracts. These inhibitions occur by rapid reactions between nitrite and the catechins. It was observed that no mutagenicity results from the reaction between the tea extracts and nitrite.
Amines↗
Effects of treatment with nimustine (ACNU), a bifunctional alkylating anticancer agent, on cultured resting L1210 cells.
Resting L1210 cells were treated with nimustine (ACNU), a bifunctional alkylating anticancer agent, for 2 h in a nutrient-depleted medium. The cells were then transferred to a fresh medium and incubated for a further 48 h. Functions of the cells thus prepared were examined in terms of the dye-exclusion of the membrane, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H-tetrazolium hydroxide, inner salt, sodium salt (XTT)-reducing ability of the mitochondria, and heat generation due to vital metabolism as the measure of cell viability. The cells treated with ACNU were functioning normally in all the cell functions examined but were completely devoid of proliferating capacity. These results suggest the possibility that ACNU might impair the proliferative capacity of the resting cell population inside a solid tumor without causing such impairment to the cells of normal organs and tissues composed of intrinsically non-proliferative cells.
Animals↗
Formation of direct-acting mutagens from mixtures of N-nitrosomorpholine and carboxylates by UVA irradiation.
Previously, we found that a directly mutagenic compound is produced from N-nitrosopiperidine (NPIP) in phosphate buffer on exposure to near-ultraviolet light (UVA) and we identified its structure as alpha-hydroxy-N-nitrosopiperidine phosphate ester. In the present study, we show that a similar photoactivation of an N-nitrosamine can take place with carboxylates in place of phosphate. When a neutral solution of a mixture of N-nitrosomorpholine (NMOR) and sodium acetate was irradiated with UVA, the solution became directly mutagenic towards Salmonella typhimurium TA1535. O6-Alkylguanine-DNA alkyltransferase-deficient strains of S. typhimurium showed remarkably higher mutagenesis responses to this mutagen than the proficient strains. Citrate, succinate, and several other biological carboxylates were also effective in producing the mutagens. Since a treatment of the "NMOR plus acetate" photoproduct with carboxylic ester hydrolase resulted in a loss of the mutagenicity, the active principle is suggested to be an acetate-esterified derivative of NMOR. The role of the esters as intermediates in the photomutagenesis of nitrosamines is discussed.
Carboxylic Acids↗
Adsorption of mutagens to chlorophyllin-chitosan, an insoluble form of chlorophyllin.
Chlorophyllin, a water soluble derivative of chlorophyll is known to suppress the mutagenic and carcinogenic actions of compounds having polycyclic structures, e.g. heterocyclic amines and aflatoxin B1. There is evidence that this suppressing effect arises, at least in part, by a complex formation between the porphyrin-like structure of chlorophyllin and the planar molecular surfaces of these compounds. We report here that chlorophyllin can form an insoluble salt-like material when mixed with chitosan, a polyglucosamine, and that the solid chlorophyllin-chitosan thus prepared can efficiently trap polycyclic mutagenic compounds. The adsorbed polycyclic mutagens were elutable with buffers of acidic pH, but only to small extents. Chlorophyllin-chitosan may be expected to be useful as an intercepting agent against polycyclic mutagens and carcinogens.
Adsorption↗
Antigenotoxic activity of natural chlorophylls.
Chlorophyllin, a man-made water-soluble form of chlorophyll, is a focus of intensive studies from many laboratories for its antimutagenic and anticarcinogenic properties. Natural chlorophylls, in contrast, have been little studied in this regard. Since yellow-green vegetables are implicated to be protective against human cancers by epidemiological studies, it is important to explore the antigenotoxic properties of natural chlorophylls. Previously, we reported that a chlorophyll sample prepared from Chlorella vulgaris inhibited the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, a direct-acting mutagen, in Salmonella, and that the chlorophyll also showed inhibition of wing spot formation in Drosophila induced by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). We have now prepared several samples of chlorophyll from spinach and chlorella, and studied their effect on the genotoxicity of 4-nitroquinoline 1-oxide (4NQO) in Drosophila. The results showed that the genotoxicity of orally given 4NQO was suppressed by simultaneous administration of the chlorophylls. The mechanisms of this inhibition are discussed.
4-Nitroquinoline-1-oxide↗
The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.
The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.
Base Sequence↗
Heavy cigarette smokers show higher mutagenicity in urine.
We examined the mutagenicity of cigarette smoker's urine in 32 healthy male cigarette smoker and 37 healthy male non-smoker. Twenty-four-hour urine specimens were subjected to blue rayon extraction which selectively adsorb polycyclic compounds, after which the elutions were fractionated by carboxymethyl cellulose column chromatography for removing antimutagenic compounds. The mutagens were measured by using an S9-mediated Salmonella mutagenicity test on strain TA98. Compared with those with non-smokers, smokers' urine showed a significantly higher urinary level of mutagens in the acid-elutable and in the sum of all chromatography fractions. A similar tendency was also seen in the alkali-elutable fraction. The subjects were classified into three groups according to the number of smoked cigarettes. Heavy smokers, who smoked more than 20 cigarettes per day, showed a significantly higher urinary level of mutagens than both non-smokers and light smokers especially in the acid-elutable and in the sum of all chromatography fractions. Our findings suggest that smokers are exposed to a great amount of polycyclic carcinogens and mutagens by cigarette smoking. These results also suggest that urinary level of mutagens measured by using blue rayon extraction combined with carboxymethyl cellulose chromatography could be a good index for estimating the exposure to carcinogens and mutagens such as polycyclic compounds.
Adult↗
Enhancement of the clastogenicity of N-nitrosodialkylamines plus near-ultraviolet irradiation by ethanol in Chinese hamster lung fibroblasts.
Early work from our laboratory showed a synergistic action of N-nitrosodialkylamines and near-ultraviolet light (UVA, 320-400 nm) to cause mutations in bacteria and phages. Recently we reported that N-nitrosodialkylamines + UVA induces chromosome aberrations in Chinese hamster lung cells in culture. We have now found that ethanol can potentiate this clastogenic action of N-nitrosodialkylamines + UVA. When the cells were treated with N-nitrosopyrrolidine (NPYR) or N-nitrosodiethylamine (NDEA) + UVA for 2 hours in the presence of 1% ethanol, approximately 2-fold increase in the numbers of cells with aberrant chromosomes was observed, compared to those found without the ethanol. NPYR/NDEA only ethanol only, or ethanol + UVA did not cause the aberrations. The enhancement was dependent on the concentration of ethanol. Treatment of cells with ethanol before the NPYR + UVA was ineffective. By contrast, treatment of cells with NPYR-UVA and then with ethanol was as effective as with the simultaneous treatment. Methanol showed synergistic effects similar to those of ethanol, but mannitol did not. Intracellular hydrogen peroxide was found to be increased twofold over that in the background by a treatment with ethanol + UVA. The alcohol-mediated enhancement of the clastogenic action of N-nitrosodialkylamines + UVA may be a consequence of an increase in intracellular oxidative stress, or simply due to increased membrane permeability.
Animals↗
Improved blue rayon hanging technique that can measure a time-weighted average concentration of benzo(a)pyrene in sea water.
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Benzo(a)pyrene↗
Mutation and formation of methyl- and hydroxylguanine adducts in DNA caused by N-nitrosodimethylamine and N-nitrosodiethylamine with UVA irradiation.
Previously we reported that when Escherichia coli was treated with N-nitrosodialkylamine under irradiation with near UV light, mutagenesis of the bacteria took place: there was no requirement for metabolic activation. We have now studied the spectra of mutations caused by N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) with UVA (320-400 nm) irradiation, using standard tester strains for identifying types of mutations. Induced mutations by NDMA + UVA were the transition GC-->AT and transversions GC-->CG, GC-->TA and AT-->TA. NDEA + UVA induced mainly the GC-->CG transversion. In both cases no frameshift mutations were observed. When O6-alkylguanine-DNA alkyltransferase-deficient strains of E. coli and Salmonella typhimurium were used, the mutation levels with both NDMA + UVA and NDEA + UVA became remarkably higher than those observed with the proficient strains. We measured the O6-methylguanine (O6-meG) level in calf thymus DNA treated with NDMA + UVA. The O6-meG level was increased as a function of NDMA concentration and irradiation time. We also detected N7-methylguanine in DNA treated with NDMA + UVA. In our previous work we found formation of 8-oxodeoxyguanosine (8-oxodG) in DNA treated with N-nitrosomorpholine + UVA. The 8-oxodG/dG ratio in DNA treated with NDMA + UVA increased up to 42-fold over that of the untreated control and that in DNA treated with NDEA + UVA increased up to 67-fold. 8-OxodG formation was not affected by replacing H2O in the reaction mixture with D2O, suggesting that singlet oxygen is not the rate limiting factor in this photoactivation. We conclude that both alkylation and oxidation are involved in mutations induced by NDMA + UVA and NDEA + UVA.
8-Hydroxy-2'-Deoxyguanosine↗
Polynucleotide-chitosan complex, an insoluble but reactive form of polynucleotide.
DNA formed an insoluble complex on mixing with chitosan (poly-D-glucosamine) in solution. The DNA content of the complex was about 50% and the DNA remained insoluble in aqueous media of pH 2-7%; e.g., on treatment of the DNA-chitosan complex with phosphate-buffered saline at pH 7 and 37 degrees C for 26 h, the DNA released into the aqueous phase was less than 0.05%. Obviously, DNA and chitosan formed a tight complex due to ionic interactions. The DNA can be solubilized by treatment with 0.1 N NaOH. RNA and other polynucleotides formed similar insoluble complexes with chitosan. The DNA attached to chitosan can be digested with a mixture of DNase I and phosphodiesterase. Cytosine residues in the DNA (denatured DNA) can be deaminated by treatment with sodium bisulfite, forming uracil DNA-chitosan. The uracil DNA-chitosan served as a substrate for uracil DNA glycosylase. Using polynucleotide-chitosan as an adsorbent, the affinities of reagents for polynucleotides can be determined directly. With this technique it was found that carcinogenic heterocyclic amines have an affinity for RNA as well as DNA. The results with homopolyribonucleotide-chitosans as adsorbents for 4 heterocyclic amines indicated that the binding occurs in a purine nucleotide-specific manner. These results suggest that the polynucleotides in the chitosan complex are accessible to enzymes and reagents. This new derivative may be useful in chemical and biological studies of polynucleotides and substances interacting with polynucleotides.
Chitin↗
Preparation of DNA-chitosan columns and their applications: binding of carcinogens to the column.
DNA, either native or denatured, can be immobilized by complexing with chitosan. The DNA-chitosan, which is insoluble in aqueous media, contains DNA at about 50% in weight. The DNA in the complex is accessible by enzymes and reagents. Carcinogenic heterocyclic amines, having affinity to DNA, can be adsorbed to the DNA-chitosan. The DNA-chitosan complex can be homogeneously dispersed in celite powder, and the resultant DNA-chitosan-celite is a material suitable to construct an immobilized DNA-column. The column was shown to be effective in concentrating heterocyclic amines.
Adsorption↗
Yeast oligonucleotide transformation: its mechanism and application to analysis of mutations induced by defined DNA lesions.
We have studied mutagenic specificity of an abasic site by the yeast-transformation procedure using an oligonucleotide containing a single furan-type abasic site. The recipient yeast used was deficient in the major AP endonuclease (apn1). Sequence analysis of the transformants suggested that dATP was incorporated most frequently opposite the abasic site, while dGTP seemed to be incorporated opposite the abasic site in the recipient proficient in apn1. To explore the mechanism of this oligonucleotide transformation, we have also analyzed the transformation with phosphorothioate oligonucleotides with mismatched 3'-end. The results are discussed.
Carbon-Oxygen Lyases↗
Evaluation of blue-chitin column, blue-rayon hanging, and XAD-resin column techniques for concentrating mutagens from two Japanese rivers.
Highly mutagenic water of the Katsura River, Kyoto, and moderately mutagenic water of the Asahi River, Okayama, were used to evaluate the efficacy of three concentration techniques, the blue-chitin column, the blue-rayon hanging, and the XAD-2 column. These two river waters have been shown to exhibit high mutagenicity in the assay with Salmonella typhimurium TA98 with metabolic activation. With this assay as a measure, two water samples from the Katsura, collected on different dates, and a sample from the Asahi were submitted to the column concentration techniques, blue-chitin and XAD-2. Blue-chitin was more efficient than XAD-2 for all of these samples: e.g., for one Katsura sample, the mutagenicity found was 913 +/- 53 (mean +/- SD, n = 3) revertants/500 ml with blue-chitin, and 419 +/- 129 (n = 3)/500 ml with XAD-2. Blue rayon (0.5 g) hung in the Asahi for 24 h gave 563 +/- 74 (n = 3) revertants, while the water spot-sampled at the start of the hanging showed 253 +/- 10 (n = 3) revertants per 5 liter with the blue-chitin column technique. We conclude that for quantitative measurement of the "Salmonella TA98 +/- S9' mutagens in these rivers, the blue-chitin column is more efficient and accurate than the XAD-2 column and that for judging the presence of mutagens, the blue-rayon hanging is the most sensitive and convenient among the three methods examined.
Biotransformation↗