Yutaka Kawazoe

Formation of 8-hydroxyguanine residues in cellular DNA exposed to the carcinogen 4-nitroquinoline 1-oxide

8-Hydroxyguanine (8-OH-Gua) residues were formed in DNA of Ehrlich ascites cells exposed to the carcinogen 4-nitroquinoline 1-oxide. Formation of 8-OH-Gua was confirmed by chemical treatment of calf thymus DNA with the proximate metabolite of this carcinogen, 4-hydroxyaminoquinoline 1-oxide, together with seryl-adenosine monophosphate. The ratio of the rates of formations of 8-OH-Gua and the quinoline-bound adducts was about 0.2-0.3. A conceivable mechanism of formation of 8-OH-Gua is proposed.

Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay

The alkaline comet assay, employing a single cell gel electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual mammalian cells. In this report, we describe a modified version of this assay which we used to assess DNA damage as a result of treating lysed cells with genotoxic and antimetabolic agents. By means of this modified assay, DNA is no longer held under the regulation of any metabolic pathway or membrane barrier. Using 3 direct-acting agents, hydrogen peroxide, N-methyl-N-nitrosourea, and bleomycin, we were able to induce increased DNA migration by both the standard and modified comet assays. In contrast, with 4-nitroquinoline 1-oxide, 5-fluorouracil, and methotrexate, which require cellular enzymatic activity to induce DNA damage, we succeeded in inducing increased DNA migration using the standard comet assay conditions only. In some cases, the modified comet assay might be helpful in analyzing chemical and biological characteristics of genotoxic agents when performed in combination with the standard comet assay.

Mutagenic characteristics of formaldehyde on bacterial systems

The mutagenic characteristics of formaldehyde on bacteria were examined. All the tester strains of Escherichia coli deficient in DNA-repair enzymes tested in the present study were significantly more sensitive to the killing effect of formaldehyde than the corresponding wild-type strain. Among the E. coli B strains, H/r30R (wild-type) and Hs30R (uvrA) were mutable, whereas NG30 (recA) and O16 (polA) were not. There is no appreciable difference in mutation frequency of E. coli B between the wild-type and the uvrA strains in a dose range below 4 mM. However, the mutation frequency of the wild-type strain started to decrease in a higher concentration range, whereas that of the uvrA strain continued to increase linearly. This was confirmed with the E. coli B/r tester strains. The decrease in mutation frequency may be produced by prolongation of the lag period before entering the S-phase so as to give the cells a greater chance for DNA repair through the excision mechanism. In fact, it was evidenced that formaldehyde retarded to a remarkable extent the initiation of DNA synthesis of the cells at the higher dose range used for mutation assay. Some discrepancies found between the results obtained in this study and those previously reported by Nishioka (1973) were pointed out.

Binding of quinoline to nucleic acid in a subcellular microsomal system

Quinoline, a hepatocarcinogen in rats and mutagen in Salmonella typhimurium, binds to RNA, DNA and certain polyribonucleotides in the presence of NADPH and rat liver microsomes. The binding was pronounced with the help of liver microsome preparations made from the rats pretreated with some inducers of the microsomal monooxygenase system. The binding reaction required NADPH and was inhibited by carbon monoxide or aniline, and also by 7,8-benzoflavone, methyrapone or SKF 525A. These results suggested that the cytochrome P-450-linked monooxygenase system is involved in this binding process. Quinoline bound preferably to poly(A), poly(C), poly(G) and poly(X), but negligibly to poly(U) and poly(I). Most of the quinoline residues of the adducts, regardless of the kind of polynucleotides used, were released by acid or alkali at 100 degrees C in the form of 3-hydroxyquinoline. This suggests that 2,3- or 3,4-epoxy derivative of quinoline is the reactive intermediate for nucleic acid modification.

In vivo mutagenesis by the hepatocarcinogen quinoline in the lacZ transgenic mouse: evidence for its in vivo genotoxicity

Quinoline is carcinogenic to the liver of rats and mice and mutagenic to bacterial tester strains in the presence of rat liver microsomal enzymes. The unscheduled DNA synthesis (UDS) study suggested that quinoline might be a non-genotoxic carcinogen because of the lack of UDS-inducing capacity. In order to determine whether or not cancer induction is initiated by mutagenic DNA lesions, the present study was undertaken to evaluate the mutagenicity of quinoline in an in vivo mutation assay system using the lac Z transgenic mouse (Muta Mouse). Mutation was only induced in the liver, the target organ of carcinogenesis by quinoline, but not in the other organs examined, i.e. lung, kidney and spleen. Mutant frequency in the liver was 4-fold higher than in the untreated control animals. Dimethylnitrosamine, used as a positive control, induced mutation at a frequency 5-fold higher in the liver and 3-fold higher in the spleen than in their respective control organs. It can be concluded that the genotoxicity of quinoline is responsible for its hepatocarcinogenesis, although UDS was not induced under the conditions previously reported.

Effects of oligofluorine substitution on the mutagenicity of quinoline: a study with twelve fluoroquinoline derivatives.

A total of 12 variously fluorinated derivatives of quinoline (Q) were tested for their mutagenicity in Salmonella typhimurium TA100 in the presence of S9 mix to investigate the structure-mutagenicity relationship in oligofluorinated quinolines. Nine of them, 3,7-di-, 5,6-di-, 6,7-di-, 6,8-di-, 7,8-di-, 3,5,7-tri-, 5,6,8-tri-, 6,7, 8-tri-, and 5,6,7,8-tetrafluoroquinolines (FQs), were newly synthesized for this purpose. Those fluorinated at position 3 were all non-mutagenic. Mutagenicity was enhanced by fluorine-substitution at position 5 or 7, but not in 3-FQs (i.e., 3, 5-di-, 3,7-di-, and 3,5,7-triFQs). Some of the 6-fluorinated derivatives showed less maximum induced-revertants with more mutagenic potencies in terms of induced-revertants per dose than quinoline. No marked change occurred by fluorine-substitution at position 8. These results show that the effect of di- and trifluoro-substitution on mutagenicity is generally additive, while that of tetrafluorination approaches the deactivating effect of perfluorination. Our study suggests that 3-fluorine-substitution in the pyridine moiety may be a useful means of antimutagenic structural modification in pyridine-fused aromatic chemicals for medicinal and agricultural use.

Possible involvement of lignin structure in anti-influenza virus activity

Commercial lignins suppressed the growth of influenza A virus infecting MDCK cells, and the RNA-dependent RNA synthesis, as efficiently as the high-molecular weight fractions extracted from pine cone of Pinus parviflora Sieb. et Zucc. The anti-influenza A virus activity of both pine cone extract and commercial alkali-lignin was considerably reduced by treatment with sodium chlorite, but was not affected by sulfuric acid or trifluoroacetic acid. The degraded components of lignin, various synthesized polyphenols unrelated to lignin, and natural and chemically modified glucans, were not appreciably inhibitory. The data suggest that the polymerized phenolic structure of lignified materials is responsible for the anti-influenza A virus activity.

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.

Formation of 8-hydroxyguanine residues in DNA treated with 4-hydroxyaminoquinoline 1-oxide and its related compounds in the presence of seryl-AMP

The mechanism whereby treatment of DNA with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP leads to the formation of 8-hydroxyguanine (8-OH-Gua) residues in DNA (Kohda et al., this journal, 139, 626, 1986) has been studied. In the survey of other N-arylhydroxylamines, only 4HAQO analogues which could bind to DNA produced 8-OH-Gua. The amount of 8-OH-Gua varied depending on the structure of 4HAQO analogues and that of DNA. The formation of 8-OH-Gua was not inhibited by active oxygen scavengers. Possible mechanisms are discussed.

Antimutagenic structural modification of quinoline assessed by an in vivo mutagenesis assay using lacZ-transgenic mice

Quinoline, a hepatocarcinogen, mutates the bacterial tester strains in the presence of the rat liver microsomal enzymes and induces GST-P (placental glutathione S-transferase)-positive foci in a medium-term bioassay system for hepatocarcinogenesis. On the other hand, 3-fluorinated quinoline was neither mutagenic nor carcinogenic in the same assay systems, whereas, 5-fluoroquinoline was mutagenic and carcinogenic. Quinoline was recently demonstrated to be mutagenic in an in vivo mutagenicity assay system using the lacZ-transgenic mouse (MutaMouse). The present study was undertaken to know whether 3-fluoroquinoline would be devoid of in vivo mutagenicity in MutaMouse. Quinoline and 5-fluoroquinoline were also tested in the same system. Mutagenicity was evaluated in the liver, the target organ of quinoline carcinogenesis, and also in the bone marrow and testis. The results strongly indicate that fluorine-substitution at the position-3 of quinoline could be an anti-genotoxic structural modification of quinoline in a wide range of its genotoxic end-points.