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H Hayatsu

Publications and source records attributed to H Hayatsu.

At least 145 records · Page 8Linked to original sources
PubMed1985

Mutagenesis by N4-aminocytidine: induction of AT to GC transition and its molecular mechanism.

N4-Aminocytidine is a potent mutagen toward Escherichia coli and Salmonella typhimurium. It induced reversion of an amber mutant of phi X174 phage (am3) to the wild type. This reversion was shown to be exclusively due to the AT to GC transition. It is likely that N4-aminocytidine is metabolized within the bacterial cells into N4-aminodeoxycytidine 5'-triphosphate and this nucleotide is incorporated into DNA during the multiplication of the cells and the phages, thereby causing base-pair transitions. The molecular basis for this erroneous replication was obtained in studies of in vitro incorporation of N4-aminodeoxycytidine 5'-triphosphate into polynucleotides catalyzed by the E. coli DNA polymerase I large fragment. The results have shown that this cytosine analogue can be efficiently incorporated as a substitute of cytosine and that it can also be incorporated as a substitute of thymine. The ratio in the rate of the N4-aminocytosine nucleotide incorporation to that of natural nucleotide incorporation was 1/2 to cytosine and 1/30 to thymine. Furthermore, the N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite to them.

Bacteriophage phi X 174↗
PubMed1985

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate.

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.

Bacteriophage phi X 174↗
PubMed1985

Fecal mutagenicity arising from ingestion of fried ground beef in the human.

Fried ground beef has been shown to contain mutagens, and the major mutagenic component has been identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Mutagens in feces of 3 adult volunteers were fractionated by treatment of the feces with blue cotton followed by chromatography on a carboxymethyl cellulose column. The chromatographic fraction, corresponding to MeIQx in terms of the position of elution, was examined for mutagenicity in S. typhimurium TA98 with metabolic activation. When meals containing no heated meat were eaten, this fraction of feces showed little or no mutagenicity. On eating fried ground beef, the feces excreted in the next two days showed greatly increased mutagenicity in this fraction. By eating no-meat meal subsequent to the meat meal, the mutagenicity resumed the original low level on the fourth day after the meat meal. The components in the mutagenic fraction were probably metabolites of the mutagens present in cooked meat, since analysis by high pressure liquid chromatography of the mutagenic fraction showed that the active components in the feces were different from the mutagens in cooked meat.

Animals↗
PubMed1985

Mutagenicity arising from near-ultraviolet irradiation of N-nitrosomorpholine.

When a mixture of N-nitrosomorpholine and S. typhimurium TA100 in saline was irradiated with near-ultraviolet light, mutagenesis of the bacteria took place. The same observation was made with S. typhimurium TA1535, E. coli WP2 uvrA, pKM101 and uvrA/pKM101. Several other nitrosamines showed ed the same, but weaker, effect. Evidence is presented to indicate that the mutagenicity arises from the cellular phosphate-mediated photochemical formation of direct-acting mutagen from the nitrosamine.

Escherichia coli↗
PubMed1985

Contamination by ATP of commercial dATP samples causing erroneous results in studies of DNA replication in isolated HeLa cell nuclei.

dATP at high concentrations was capable of replacing ATP required in the synthesis of Okazaki pieces in isolated HeLa cell nuclei. In addition, the levels of synthesis of high molecular weight DNA were observed to vary depending on the lot of dATP used. Analysis by HPLC revealed that dATP samples of a particular source contained ATP in the range of 0.25-0.43 mol%. With ATP-free dATP, almost no synthesis of high molecular weight DNA was observed, while with impure dATP, a small but significant amount of high molecular weight DNA was synthesized. While this observation confirmed our previous finding that dATP can replace ATP in the synthesis of Okazaki pieces but not in the joining of the pieces, it is also a warning to users of commercial dATP in biochemical and biological studies.

Adenosine Triphosphate↗
PubMed1985

Ambiguous incorporation of N4-aminodeoxycytidine 5'-triphosphate to DNA synthesized in vitro.

Molecular mechanism of the mutation induced by N4-aminocytidine was studied. The specificity of in vitro incorporation of N4-aminodeoxycytidine 5'-triphophate catalyzed by E. coli DNA polymerase large fragment was analyzed. The results have shown that this cytosine analog can be efficiently incorporated as a substitute of cytosine, and that it can also be incorporated with a low efficiency as a substitute of thymine. We have also shown that the N4-aminocytosine incorporated opposite adenine can be excised as its monophosphate at a high frequency. The N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite them.

DNA, Bacterial↗
PubMed1985

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.

Animals↗
PubMed1985

Mutagenicity of human urine caused by ingestion of fried ground beef.

On ingestion of fried ground beef by humans, the urinary mutagenicity, as examined by the Ames test, increased rapidly and then decreased during a period of 12 hr to resume the original low level. The excreted mutagens were shown to differ from 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, the major mutagen in the cooked beef.

Animals↗
PubMed1985

DNA strand cleavage in vitro by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]-indole, a direct-acting mutagen formed in the metabolism of carcinogenic 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) is a direct-acting mutagenic compound derived by metabolic activation from 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a strongly mutagenic carcinogen. The action of N-OH-Trp-P-2 on DNA in vitro was investigated. N-OH-Trp-P-2 inactivated Bacillus subtilis transforming DNA and produced single-strand cuts in a supercoiled circular DNA (phi X174RFI) under neutral conditions. When mouse FM3A cells in culture were treated with a noncytotoxic dose of N-OH-Trp-P-2 and then the cellular DNA was examined by the alkaline elution technique, chain cleavages of the DNA were observed. Cysteamine inhibited the spontaneous degradation of N-OH-Trp-P-2 and enhanced the covalent binding of [3H]N-OH-Trp-P-2 to DNA. This finding offered an explanation for the previously observed enhancement of Trp-P-2 mutagenicity by cysteamine. In contrast cysteamine inhibited the N-OH-Trp-P-2-mediated inactivation of B. subtilis DNA as well as the strand cleavage in phi X174RFI DNA. The cleavage in phi X174RFI DNA was also inhibited by catalase. These observations indicate that the mutagenicity and DNA-cleaving activity of N-OH-Trp-P-2 are distinct from each other, that the inactivation of transforming DNA was caused mainly by strand cleavage, and that the DNA cleavage was probably caused by active oxygen radicals produced in the oxidative degradation of N-OH-Trp-P-2.

Animals↗
PubMed1984

A new reaction useful for chemical cross-linking between nucleic acids and proteins.

Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.

Cross-Linking Reagents↗
PubMed1984

Inhibitory effect of saturated fatty acids on the mutagenicity of N-nitrosodimethylamine.

Saturated fatty acids, C5-C12, inhibited the mutagenic activity of N-nitrosodimethylamine (NDMA) in E. coli WP2 uvrA/pKM101. The inhibition by laurate (C12) was due to the suppression of the enzymatic demethylation of NDMA, whereas that by caprate (C10) was simply due to the bactericidal effect of the fatty acid. Caproate (C6) did not affect the NDMA-demethylase, and evidence is presented to show that the inhibition of mutagenesis by caproate was a result of its interference with the uptake of NDMA metabolites into bacterial cells. Possible biological significance of the inhibition is discussed.

Animals↗
PubMed1984

Activation of promutagenic N-nitrosomorpholine and other N-nitrosoalkylamines by near-ultraviolet irradiation in the presence of phosphates.

When a neutral solution of N-nitrosomorpholine, N-nitrosopyrrolidine, or N-nitrosopiperidine was irradiated with near-ultraviolet light, the solution became mutagenic to Salmonella typhimurium TA100 in the absence of metabolic activation. The formation of the active compounds required the presence of phosphate or its esters such as adenosine 5'-triphosphate during the irradiation.

Mutagenicity Tests↗
PubMed1984

A time-course study on the mutagenicity of smoker's urine.

A short-term study was made for the urinary mutagenicity caused by cigarette smoking in several volunteers. In order to concentrate the mutagens from urine, cotton linked to a copper-phthalocyanine derivative (blue cotton) was used as an adsorbent. The mutagenicity as measured by the Ames test on Salmonella typhimurium TA98 in the presence of S9 increased rapidly after the start of smoking. When smoking was stopped, the activity began to decrease, and after 6 to 13 hr reached the non-smoking level. There was no great difference among individuals regarding this time-dependent response. These studies have shown that the appearance and disappearance of mutagenicity in urine in relation to cigarette smoking are rapid processes.

Adult↗
PubMed1983

N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity.

The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.

Bacteriophage phi X 174↗
PubMed1983

Mutagenicity of chalks. A case in which the test results led to the improvement of the quality of commercial goods.

Mutagenicity testing, of methanolic extracts of chalks, by the Salmonella/mammalian-microsome system revealed that the blue and the green chalks contained mutagens. A positive mutagenic response was observed on Salmonella typhimurium strain TA98, both in the presence and absence of the microsome system (S9). The source of the mutagenicity was traced to the blue pigment used for manufacturing these chalks. The pigment, copper phthalocyanine, a product of a Japanese chemical industrial company, was found to contain impurities that were mutagenic. The mutagenic principle giving positive response in the TA98 in the presence of S9 was purified 10(5)-fold from the original pigment. Although its structure is yet to be elucidated, this indirect frame-shift mutagen had a strong activity: 5700 His+ revertants per microgram. This information, delivered in the beginning of 1981, prompted the manufacturer to start supplying a mutagen-free product. As a result, the blue chalks on the market became no longer mutagenic in the summer of 1982.

Chromatography, High Pressure Liquid↗
PubMed1983

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 smoker's urine, cooked beef, and river water was detected by use of this method.

Adsorption↗
PubMed1983

Characterization of mutagenic fractions in beef extract and in cooked ground beef. Use of blue-cotton for efficient extraction.

Mutagenic components in commercial beef extract and in cooked ground beef were adsorbed from their aqueous solutions on cotton bearing covalently linked trisulfo-copper-phthalocyanine residues (blue-cotton). By repeating the adsorption and elution, efficient concentration of the mutagenic components with a satisfactory overall recovery was achieved. Carboxymethyl cellulose column chromatography was found to be an excellent means to separate 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), two strong mutagens that have previously been found in heated beef samples. Chromatography of the mutagenic components of beef extract on this column gave two mutagenic fractions which corresponded to MeIQx and IQ in elution profile. In reversed-phase high performance liquid chromatography, the major active component of the MeIQx fraction and that of the IQ fraction behaved identically with standard samples of MeIQx and IQ, respectively. The contents of these mutagens in a sample of Difco beef extract were estimated at 200-300 ng of MeIQx and 20-40 ng of IQ per gram. By the same fractionation procedures, mutagenic substances in the cooked beef were fractionated into MeIQx-type and IQ-type components. The activity distribution among these two fractions was similar to that found for beef extract.

Adsorption↗