Biomedical subjects
Publications and source records attributed to H Hayatsu.
N4-Aminocytidine was readily formed on treatment of cytidine with hydrazine-bisulfite under mild conditions. This reaction can be carried out for cytosines in polynucleotids as well. The nucleoside N4-aminocytidine was strongly mutagenic to bacteria and phage. The transformation of cytosines in polynucleotides into N4-aminocytosines is a convenient method for generating reactive groups in the polymer.
When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.
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.
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By carrying out the pre-incubation of bacteria with N-nitrosamines under acidic conditions, the mutagenicity of N-nitrosodimethylamine on Salmonella TA100 and E. coli WP2 try, hcr was detected with increased sensitivity, compared to usual assays in neutral buffers. Using this modified assay system, compounds have been screened for their modulating effects on N-nitrosamine-mediated mutagenesis and the following were found to be inhibitors: cysteine, cysteamine, bisulfite, n-propylamine, n-butylamine, n-hexylamine, n-octylamine, n-nonylamine, serotonine, tryptamine, epinephrine, norepinephrine and oleic acid. The mechanisms of inhibition are discussed.
An ether extract of normal human feces showed inhibitory effects on the activities of several mutagens in the Ames tests. By addition of the ether extract at an amount equivalent to 0.5 g of a sample of feces, the mutagenicity of 1.5 nmole of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) on Salmonella typhimurium TA98 was completely inhibited. No killing of the bacteria was detected during this treatment. Other mutagens also subject to the inhibition were 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-9H-pyrido[2,3-b]indole (Glob-P-2), 2-amino-3-methylimidazo[4,5-d]quinoline (IQ), benzo[a]pyrene and aflatoxin B1. Apart from these mutagens, which require S9 for their activation, the direct mutagen prepared from Trp-P-1 by treatment with S9 was also inhibited by the fecal extract. The inhibitory principles in the fecal extract were fractionated by thin-layer chromatography on silica gel and were identified as oleic and linoleic acids. Whereas these unsaturated fatty acids showed strong inhibitory activities, saturated fatty acids, i.e, stearic and palmitic acids, did not exhibit any inhibition. Although the physiological significance of these effects of oleate and linoleate is yet to be elucidated, this finding has indicated that care must be taken in screening mutagens by the Ames tests to avoid false negatives resulting from the presence of unsaturated fatty acids in the system.
By using the Salmonella/microsome system, it was found that the activity of mutagens present in the basic fraction of cooked-ground-beef was completely suppressed by addition of the acidic fraction obtained from the cooked-beef. The suppression was ascribable to the presence of oleic acid in the acidic fraction. This finding indicates that no, or diminished, mutagenicity would be found in materials containing fat.
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The mutagenicity of some N-nitrosodialkylamines, i.e. N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-butylamine, N-nitrosomorpholine and N-nitrosopyrrolidine, was assayed on Salmonella typhimurium TA100 by the pre-incubation method, and the effect of changing the pH of the pre-incubation mixture was examined. Markedly higher mutagenicities were observed when the pre-incubation of bacteria with nitrosamine and S9 mix was done at pH 5.2, compared with mutagenicities observable after the pre-incubations at conventional pH 7. Pre-incubations at pH 6.2 resulted in responses of intermediate strength. With phenobarbital-induced rat S9, the ratios of mutagenic potency found by the pH 5.2 pre-incubation to that found by the pH 7.2 preincubation were 15-30 for N-nitrosodimethylamine, 5-10 for N-nitrosodiethylamine, 10-20 for N-nitrosodi-n-butylamine, 2-3 for N-nitrosomorpholine and 4-6 for N-nitrosopyrrolidine. The mutagenic potency of each nitrosamine varied with the change of S9 source. The S9 sources examined were PCB-induced rat and mouse livers, and uninduced rat and mouse livers. No exceptions were observed for these S9 preparations regarding the higher mutagenicity at pH 5 than at pH 7. It is speculated that the higher mutagenicity observed by the pH 5 pre-incubation was due to the stability of the active intermediate, alpha-hydroxynitrosodialkylamines, in weakly acidic media.
An inhibitory effect of hemin on mutagenicities of a range of carcinogens was found by adding hemin to the preincubation mixture of the Ames' test. Strong inhibitions were observed for benzo[alpha]pyrene, 3-methylcholanthrene, 7,10-dimethylbenz[alpha]anthracene, chrysene, 2-acetylaminofluorene, 2-nitrofluorene and aflatoxin B1. Generally, 50% inhibition was caused by an amount of hemin 1--2 equivalents to the mutagen. Excess of hemin caused complete inhibitions. Hemin did not affect the mutagenicities of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 4-nitroquinoline-1-oxide, nitromin, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosodi-n-butyl-amine, quinoxaline-1,4-dioxide and carbadox. Biliverdin, bilirubin and chlorophyllin were also effective as inhibitors for the mutagenicity of benzo[alpha]pyrene.
The direct action of bisulfite on cultured mammalian cells was studied. Chinese hamster cells precultured in plastic dishes for 2 hr were treated for 1 hr with balanced salt solutions containing bisulfite. Then they were washed, and their colony-forming ability in normal medium was determined. Significant decrease in the colony formation was observed upon treatment with bisulfite solutions over 30 mM concentration. The decrease in the colony number was found to be due to detachment of the cells from the dishes during treatment with bisulfite and subsequent washing. When the preculture time was prolonged to allow the cells to adhere to the dish more firmly, the effect of bisulfite was no longer observable. These results indicate that factors required for cell-substratum adhesion are susceptible to bisulfite.
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When Bacillus subtilis transforming DNA was irradiated with near-ultraviolet light in the presence of quinoxaline-1,4-dioxide, the activity of DNA decreased rapidly. This loss of activity was ascribed to damages on DNA that lead to chain cleavages of the molecule. A feature of this phototoxic action of the reagent is that it takes place in nitrogen atmosphere and is inhibited by oxygen.
alpha-Secondary isotope effect provides a useful tool for detection of transient 5,6-dihydropyrimidine intermediates which have been indirectly implicated in a number of chemical and enzymic conversions of pyrimidine heterocycles. The method involves the use of kinetic secondary alpha-hydrogen isotope effects which are expected to accompany sp2 to sp3 rehybridization of C-6 of the pyrimidine ring if they occur prior to or at the rate determining step. Thus, using 6-tritiated pyrimidines, and measurement of the isotopic ratio of reactant and products, kT/kH values of 1.15 or greater would be indicative of rehybridization. Using this method, we have studied the inhibition of thymidylate synthetase by 5-fluorodeoxyuridylate and 5-nitro-deoxyuridylate as well as the chemical and enzymic dehalogenation of 5-bromodeoxyuridine and 5-bromodeoxyuridylate. Large secondary isotope effects observed in all of these reactions provided strong evidence for the formation of 5,6-dihydropyrimidine intermediates.
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5-Hydroxymethylcytosine reacted with bisulfite and, instead of undergoing usual deamination process, gave cytosine 5-methylenesulfonate as the product. The conversion was rapid and quantitative, and the optimum pH was 4.5. The product was isolated as crystals and characterized. Cytosine 5-methylenesulfonate was only very slowly deaminated by treatment with bisulfite. 5-Hydroxymethyl-2'-deoxycytidine 5'-phosphate reacted with bisulfite in the same way as 5-hydroxymethylcytosine. Residues of 5-hydroxymethylcytosine in native as well as denatured T2 DNA were convertible to those of cytosine 5-methylenesulfonate by treatment of the DNA with bisulfite. While it is known that the 5-hydroxy-methyl groups of T-even bacteriophage DNA can be enzymatically glucosylated, this observation offers chemical evidence that the 5-hydrozymethyl groups in DNA are situated in such a way that they can readily react with external agents. 5-Hydroxymethyluracil gave uracil 5-methylenesulfonate on treatment with bisulfite. This reaction was much slower than that of 5-hydroxymethylcytosine, and the optimum pH was between 6 and 7.
Chemical reactivity of cytosines in 32P-labeled E. coli tRNA1Leu, E. coli tRNAPhe and yeast tRNAPhe before and after aminoacylation was examined by use of a cytosine-specific reagent, semicarbazide-bisulfite mixture. In all the three tRNA species examined, the cytosine residues that were susceptible to the modification were the same in the aminoacylated tRNA and the unacylated tRNA. Only a limited number of the cytosine residues were modifiable: those that occur in the anticodon, the 3'-CCA terminus, the D-loop, and the extra loop. The sites accessible by the reagent are in good agreement with the general three-dimensional structure of tRNA proposed in literature. These results indicate that the gross conformation of these tRNAs does not change on aminoacylation, and consequently favor the view that the T psi C(G) sequence could become exposed in later steps of protein synthesis in order to achieve the binding of aminoacyl tRNA to ribosomes.
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