Hiroshi Kasai

Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA

The hOGG1 gene encodes a DNA glycosylase that excises 8-hydroxyguanine (oh8Gua) from damaged DNA. Structural analyses of the hOGG1 gene and its transcripts were performed in normal and lung cancer cells. Due to a genetic polymorphism at codon 326, hOGG1-Ser326 and hOGG1-Cys326 proteins were produced in human cells. Activity in the repair of oh8Gua was greater in hOGG1-Ser326 protein than in hOGG1-Cys326 protein in the complementation assay of an E. coli mutant defective in the repair of oh8Gua. Two isoforms of hOGG1 transcripts produced by alternative splicing encoded distinct hOGG1 proteins: one with and the other without a putative nuclear localization signal. Loss of heterozygosity at the hOGG1 locus was frequently (15/23, 62.2%) detected in lung cancer cells, and a cell line NCI-H526 had a mutation leading to the formation of the transcripts encoding a truncated hOGG1 protein. However, the oh8Gua levels in nuclear DNA were similar among lung cancer cells and leukocytes irrespective of the type of hOGG1 proteins expressed. These results suggest that the oh8Gua levels are maintained at a steady level, even though multiple hOGG1 proteins are produced due to genetic polymorphisms, mutations and alternative splicing of the hOGG1 gene.

The Oxidized Forms of dATP Are Substrates for the Human MutT Homologue, the hMTH1 Protein

The possibility that Escherichia coliMutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5′-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.

Chemistry-based studies on oxidative DNA damage: formation, repair, and mutagenesis,

Since the discovery of 8-OH-dG formation, various aspects of oxidative DNA damage have been studied. For example, 2-OH-dA and a glyoxal-dG adduct were discovered as new types of oxidative DNA damage; 2-OH-dATP was found to induce mutations and to be a good substrate of a nucleotide sanitization enzyme, the MTH1 protein; and efforts were continued to establish standard methodologies for 8-OH-dG analyses in urine and cellular DNA. By these studies, we found solid chemistry-based approaches were often useful to clarify the biological phenomena.Since the discovery of 8-OH-dG formation, various aspects of oxidative DNA damage have been studied. For example, 2-OH-dA and a glyoxal-dG adduct were discovered as new types of oxidative DNA damage; 2-OH-dATP was found to induce mutations and to be a good substrate of a nucleotide sanitization enzyme, the MTH1 protein; and efforts were continued to establish standard methodologies for 8-OH-dG analyses in urine and cellular DNA. By these studies, we found solid chemistry-based approaches were often useful to clarify the biological phenomena.

Life Style and Urinary 8‐Hydroxydeoxyguanosine, a Marker of Oxidative DNA Damage: Effects of Exercise, Working Conditions, Meat Intake, Body Mass Index, and Smoking

The urinary levels of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a marker of oxidative DNA damage, of 318 healthy men aged 18‐58 were measured with high resolution by a newly developed automated high‐pressure liquid chromatography (HPLC) system coupled to an electrochemical detector (ECD). The mean 8‐OH‐dG level (μMg/g creatinine) was 4.12±1.73 (SD). An eleven‐fold inter‐individual variation was observed. The accuracy of the measurement estimated from the recovery of an added 8‐OH‐dG standard was 90‐98%. By univariate analysis, it was found that moderate physical exercise (P=0.0023) and high body mass index (BMI) (P=0.0032) reduced the 8‐OH‐dG level, while physical labor (P=0.0097), smoking (P=0.032), and low meat intake (less than once/ week) (P=0.041) increased its level. Based on a multi‐regression analysis of the log‐transformed values, moderate physical exercise (P=0.0039), high BMI (P=0.0099), and age (P=0.021) showed significant reducing effects on the 8‐OH‐dG level, while low meat intake (P=0.010), smoking (P=0.013), and day‐night shift work (P=0.044) increased its level. These results suggest that many types of life‐style factors that either generate or scavenge oxygen radicals may affect the level of oxidative DNA damage of each individual.

Action of chlorogenic acid in vegetables and fruits as an inhibitor of 8-hydroxydeoxyguanosine formation in vitro and in a rat carcinogenesis model

Various plant extracts, such as carrot, burdock (gobou), apricot and prune, showed inhibitory effects in an in vitro assay of lipid peroxide-induced 8-hydroxydeoxyguanosine (8-OH-dG) formation. The major inhibitor purified from various plants extracts was identified as chlorogenic acid (CA), on the basis of UV- and mass-spectra and comparison with a standard sample. To examine whether CA also inhibits 8-OH-dG formation in animal organs, an oxygen radical-forming carcinogen, 4-nitroquinoline-1-oxide, was administered to rats, with or without CA. The 8-OH-dG level in the DNA of the rat tongue, the target organ, was significantly reduced in the CA-treated group.

Detection of potent mutagens, Trp-P-1 and Trp-P-2, in broiled fish☆

The potent mutagens Trp-P-1 (3-amino-1, 4-dimethyl-5H-pyrido-[4,3-b]-indole) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) are known to be produced by pyrolysis of tryptophan [8]. To determine whether such mutagens are produced by cooking foods, the fractions obtained from broiled sardines cooked in the ordinary way were analysed by gas chromatography/mass spectrometry. The results showed that 13.3 ng of Trp-P-1 and 13.1 ng of Trp-P-2 were, in fact, present per gram of broiled sardines.

Serial review: oxidative DNA damage and repairChemistry-based studies on oxidative DNA damage: formation, repair, and mutagenesis1,2

Since the discovery of 8-OH-dG formation, various aspects of oxidative DNA damage have been studied. For example, 2-OH-dA and a glyoxal-dG adduct were discovered as new types of oxidative DNA damage; 2-OH-dATP was found to induce mutations and to be a good substrate of a nucleotide sanitization enzyme, the MTH1 protein; and efforts were continued to establish standard methodologies for 8-OH-dG analyses in urine and cellular DNA. By these studies, we found solid chemistry-based approaches were often useful to clarify the biological phenomena.Since the discovery of 8-OH-dG formation, various aspects of oxidative DNA damage have been studied. For example, 2-OH-dA and a glyoxal-dG adduct were discovered as new types of oxidative DNA damage; 2-OH-dATP was found to induce mutations and to be a good substrate of a nucleotide sanitization enzyme, the MTH1 protein; and efforts were continued to establish standard methodologies for 8-OH-dG analyses in urine and cellular DNA. By these studies, we found solid chemistry-based approaches were often useful to clarify the biological phenomena.

Infrequent mutations of the hOGG1 gene, that is involved in the excision of 8-hydroxyguanine in damaged DNA, in human gastric cancer.

DNA glycosylase, encoded by the hOGG1 gene, repairs 8‐hydroxyguanine (oh8Gua), which is an oxidatively damaged mutagenic base. To clarify whether the DNA repair activity of hOGG1 protein is involved in gastric carcinogenesis, we examined 9 gastric cancer cell lines and 35 primary gastric cancers for mutations and genetic polymorphisms of the hOGG1 gene by polymerase chain reaction‐single strand conformation polymorphism analysis. A G‐to‐A transition was detected in a gastric cancer cell line, MKN1. This nucleotide change caused the conversion of the amino acid from Arg to His at codon 154, which is located in a domain highly conserved among human, mouse, and yeast OGG1 proteins. No mutation was detected in primary gastric cancers. We compared the distribution of the polymorphic alleles associated with enzymatic activity (hOGG1‐Ser326 vs. hOGG1‐Cys326) between 35 gastric cancer patients and 42 healthy individuals. Although the frequency of the Cys326 allele, associated with low enzymatic activity, in gastric cancer patients was a little higher than that in healthy individuals, the difference did not reach statistical significance. These results suggest that low hOGG1 activity due to mutations and genetic polymorphisms is involved in the development of only a small subset of gastric cancers.

Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo

We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5′-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides inducedlacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into anE. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A·T → C·G transversions, and the 2-OH-dATP elicited G·C → T·A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.

5-Formyldeoxyuridine: a new type of DNA damage induced by ionizing radiation and its mutagenicity to salmonella strain TA102.

An aqueous solution of calf thymus DNA was irradiated by 60Co gamma-rays and modified nucleosides produced in DNA were analyzed by high-pressure liquid chromatography coupled with a photodiode array UV detector. A new product with UV absorption maxima at 230 nm and 280 nm was observed. The structure of this compound was proposed to be 5-formyldeoxyuridine (f5dU) based on the mass spectrum of its trimethylsilyl derivative (M+, m/z472) and the structure was confirmed by chemical synthesis. The yield of f5dU (2.4/10(4) dT/krad) in DNA was of roughly the same order as that of 8-hydroxydeoxyguanosine and 5-hydroxymethyldeoxyuridine. Free f5dU was mutagenic to Salmonella typhimurium strain TA102: therefore f5dU incorporated into DNA may induce mutations.