Tomonari Matsuda

Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo assay systems

Abstract The genotoxic effects of multi-walled carbon nanotubes (MWCNTs) were examined by using in vitro and in vivo assays. MWCNTs significantly induced micronuclei in A549 cells and enhanced the frequency of sister chromatid exchange (SCE) in CHO AA8 cells. When ICR mice were intratracheally instilled with a single dose (0.05 or 0.2 mg/animal) of MWCNTs, DNA damage of the lungs, analysed by comet assay, increased in a dose-dependent manner. Moreover, DNA oxidative damage, indicated by 8-oxo-7,8-dihydro-2′-deoxyguanosine and heptanone etheno-deoxyribonucleosides, occurred in the lungs of MWCNT-exposed mice. The gpt mutation frequencies significantly increased in the lungs of MWCNT-treated gpt delta transgenic mice. Transversions were predominant, and G:C to C:G was clearly increased by MWCNTs. Moreover, many regions immunohistochemically stained for inducible NO synthase and nitrotyrosine were observed in the lungs of MWCNT-exposed mice. Overall, MWCNTs were shown to be genotoxic both in in vitro and in vivo tests; the mechanisms probably involve oxidative stress and inflammatory responses.

Molecular analysis of mutations induced by acrolein in human fibroblast cells using supF shuttle vector plasmids

Types of mutations induced by acrolein in the supF gene on the shuttle vector plasmid pMY189 replicated in normal human fibroblast cells were examined. Base sequence analysis of 92 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (76%) and the others were deletions and insertions (24%). Single base substitutions were most frequently found (46%), while multiple base substitutions were 18% and tandem (two adjacent) base substitutions were 12% of the mutations. Of the base substitution mutations, G:C to T:A transversions were 44% and G:C to A:T transitions were 24%. The mutations were distributed not randomly but located at several hotspots. Acrolein produced DNA intra-strand cross-links between guanine residues, which might be responsible for rather high induction of the tandem base substitution mutations.

Aryl hydrocarbon receptor ligand activity of polycyclic aromatic ketones and polycyclic aromatic quinones

Polycyclic aromatic ketones (PAKs) and polycyclic aromatic quinones (PAQs) are oxygenated polycyclic aromatic hydrocarbons (PAHs), and reports about the aryl hydrocarbon receptor (AhR) ligand activities of these compounds are few. In this study, activation of AhR by 41 polycyclic aromatic compounds (PACs), focusing especially on PAKs and PAQs, was determined by measuring beta-galactosidase activity from a reporter plasmid in yeast engineered to express human AhR and the AhR nuclear translocator proteins and by measuring luciferase activity from mouse hepatoma (H1L1) cells (chemical-activated luciferase expression [CALUX] assay). The PACs used in these experiments included 11 PAKs, seven PAQs, and 21 PAHs. In this study, the PAKs 11H-benzo[a]fluoren-11-one (B[a]FO), 11H-benzo[b]fluoren-11-one (B[b]FO) and 7H-benzo[c]fluoren-7-one and the PAQs 5,12-naphthacenequinone, 1,4-chrysenequinone, and 7,12-benz[a]anthracenequinone showed high AhR activities in H1L1 cells, although these values were not as high as that for benzo[a]pyrene (B[a]P). These PAKs and PAQs showed significantly stronger activities in yeast cells relative to B[a]P. It was predicted that PAKs such as B[a]FO and B[b]FO occupied 0.06% to 1.3% of the total induction equivalents, and each contribution matched the contribution of PAHs such as B[a]P, chrysene, and benz[a]anthracene in gasoline exhaust particulates and airborne particulates using data of CALUX assay.

Reduced expression of MUTYH with suppressive activity against mutations caused by 8-hydroxyguanine is a novel predictor of a poor prognosis in human gastric cancer

The MUTYH gene encodes a DNA glycosylase that can initiate the excision repair of adenine mispaired with 8‐hydroxyguanine (8OHG) and is responsible for a susceptibility to multiple colorectal adenomas and carcinomas. To determine whether the MUTYH gene is involved in gastric carcinogenesis, we first examined the expression level of MUTYH in gastric cancer. The reduced expression of MUTYH mRNA transcript was detected in both gastric cancer cell lines and primary gastric cancers using qRT–PCR analysis. Immunohistochemical analysis also showed a significant reduction in MUTYH protein expression in gastric cancer, compared with non‐cancerous gastric epithelium (immunohistochemical score, 175.5 ± 43.0 versus 281.5 ± 24.8; p < 0.0001). Among the gastric cancers, the MUTYH expression level was significantly associated with the histopathology (p < 0.0001) and the pT stage (p < 0.001). The outcome of patients with gastric cancer exhibiting low MUTYH expression was significantly worse than the outcome of patients with gastric cancer exhibiting high MUTYH expression (p = 0.0007, log‐rank test) and a multivariate analysis revealed that reduced MUTYH expression was an independent predictor of a poor survival outcome among the gastric cancer patients (hazard ratio, 1.865; 95% confidence interval, 1.028–3.529; p = 0.0401). We next compared the functional effects of MUTYH on gastric cancer cells, based on their MUTYH expression levels. MUTYH‐over‐expressing stable clones of the gastric cancer cell line AGS showed: (a) higher DNA cleavage activity towards adenine:8OHG mispair‐containing substrates; (b) higher suppressive activity against mutations caused by 8OHG in a supF forward mutation assay; and (c) higher suppressive activity for cellular proliferation than empty vector‐transfected AGS clones. These results suggested that MUTYH is a suppressor of mutations caused by 8OHG in gastric cells and that its reduced expression is associated with a poor prognosis in gastric cancer. Copyright

Aberrant expression and mutation-inducing activity of AID in human lung cancer

BackgroundActivation-induced cytidine deaminase (AID) is expressed in B lymphocytes and triggers antibody diversification. Recent reports have indicated that the constitutive expression of AID in mice causes not only lymphomas, but also cancers of some organs including the lung, prompting us to investigate the expression and effect of AID on human lung cancer.Materials and MethodsWe examined AID mRNA expression in 17 lung cancer cell lines and 51 primary lung cancers using a quantitative RT-PCR analysis. Next, we established H1299 lung cancer cells stably overexpressing AID and performed a supF forward mutation assay. We then examined AID protein expression and p53 mutation in 129 primary lung cancers by an immunohistochemical analysis and PCR-SSCP and sequencing analyses, respectively.ResultsAberrant mRNA expression of AID was detected in 29% (5 of 17) of the lung cancer cell lines and 31% (16 of 51) of the primary lung cancers. AID-overexpressing H1299 clones showed a 5.0- to 6.1-fold higher mutation frequency than an empty vector-transfected H1299 clone, and about half of the AID-induced mutations were base substitutions, indicating that AID induces gene mutations in lung cancer cells. Furthermore, an association was found between the AID protein expression level and the p53 mutation status in an analysis of 129 primary lung cancers. A further expression analysis revealed that a portion of AID is localized at the centrosomes.ConclusionOur current findings suggest that the aberrant expression of AID may be involved in a subset of human lung cancers as a result of its mutation-inducing activity.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

Combination of ADH1B*2/ALDH2*2 polymorphisms alters acetaldehyde‐derived DNA damage in the blood of Japanese alcoholics

The acetaldehyde associated with alcoholic beverages is an evident carcinogen for the esophagus. Genetic polymorphisms of the alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes are associated with the risk of esophageal cancer. However, the exact mechanism via which these genetic polymorphisms affect esophageal carcinogenesis has not been elucidated. ADH1B*2 is involved in overproduction of acetaldehyde due to increased ethanol metabolism into acetaldehyde, and ALDH2*2 is involved in accumulation of acetaldehyde due to the deficiency of acetaldehyde metabolism. Acetaldehyde can interact with DNA and form DNA adducts, resulting in DNA damage. N2‐ethylidene‐2′‐deoxyguanosine (N2‐ethylidene‐dG) is the most abundant DNA adduct derived from acetaldehyde. Therefore, we quantified N2‐ethylidene‐dG levels in blood samples from 66 Japanese alcoholic patients using liquid chromatography/electrospray tandem mass spectrometry, and investigated the relationship between N2‐ethylidene‐dG levels and ADH1B and ALDH2 genotypes. The median N2‐ethylidene‐dG levels (25th percentile, 75th percentile) in patients with ADH1B*1/*1 plus ALDH2*1/*1, ADH1B*2 carrier plus ALDH2*1/*1, ADH1B*1/*1 plus ALDH2*1/*2, and ADH1B*2 carrier plus ALDH2*1/*2 were 2.14 (0.97, 2.37)/107 bases, 2.38 (1.18, 2.98)/107 bases, 5.38 (3.19, 6.52)/107 bases, and 21.04 (12.75, 34.80)/107 bases, respectively. In the ALDH2*1/*2 group, N2‐ethylidene‐dG levels were significantly higher in ADH1B*2 carriers than in the ADH1B*1/*1 group (P < 0.01). N2‐ethylidene‐dG levels were significantly higher in the ALDH2*1/*2 group than in the ALDH2*1/*1 group, regardless of ADH1B genotype (ADH1B*1/*1, P < 0.05; ADH1B*2 carriers, P < 0.01) N2‐ethylidene‐dG levels in blood DNA of the alcoholics was remarkably higher in individuals with a combination of the ADH1B*2 and ALDH2*2 alleles. These results provide a new perspective on the carcinogenicity of the acetaldehyde associated with alcoholic beverages, from the aspect of DNA damage.

The Bacillus subtilis rec-assay: a powerful tool for the detection of genotoxic substances in the water environment. Prospect for assessing potential impact of pollutants from stabilized wastes

The Bacillus subtilis rec-assay has been specially developed to detect genotoxicity in environmental water samples. The rationale of the B. subtilis rec-assay is based on the relative difference of survival of a DNA repair-recombination proficient strain and its deficient strain, which is interpreted as genotoxicity. This assay method can be very powerful in that it has higher sensitivity for the detection of mutagens in highly polluted waters than other bacterial mutation assays. Hydrophobic fractions from various environmental waters were fractionated by using XAD-2 resins and assayed, targeting the detection of organic genotoxic substances. Genotoxic response was detected in most of them, which revealed that many unknown micropollutants with genotoxicity occur in public water bodies. Positive response was also detected from a treated municipal solid waste (MSW) landfill leachate. Genotoxicity remaining in the treated effluent suggests that genotoxic micropollutants may pass through conventional water treatment processes such as activated sludge treatment process. Without proper control of waste quality and landfill facilities, waste landfill could be a heavy pollution source.

Benzotriazole ultraviolet stabilizers show potent activities as human aryl hydrocarbon receptor ligands.

Benzotriazole ultraviolet stabilizers (BUVSs) used in consumer products are raising concerns as new pollutants in the aquatic environment. We determined the agonistic activities of eight BUVSs and a chemically distinct UV absorber (4-methylbenzylidinecamphor) toward the human aryl hydrocarbon receptor (AhR) and thyroid hormone receptors alpha and beta. Although none of the BUVSs showed ligand activity against the thyroid hormone receptors, four of them (UV-P, UV-9, UV-326, and UV-090) showed significant AhR ligand activity. Their half-maximal effective concentrations (EC50) were 130 nM for UV-P, 460 nM for UV-9, and 5.1 μM for UV-090 (a value for UV-326 could not be determined). Of the numerous AhR ligands, it is well-known that those considered nontoxic are quickly metabolized by enzymes such as CYP1A1, which destroys their ability to function as ligands. Accordingly, we established a new yeast assay for simultaneous monitoring of both the strength of AhR ligand activity and ligand degradation by CYP1A1. We found the AhR ligand activities of the above four BUVSs to be stable in the presence of CYP1A1; therefore, they have the potential to accumulate and exert potent physiological effects in humans, analogous to polycyclic aromatic hydrocarbons and dioxins, which are known stable and toxic ligands.

Lipid Peroxidation Generates Body Odor Component trans-2-Nonenal Covalently Bound to Protein in Vivo

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-Nϵ-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.