Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice.

BACKGROUND: Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2-/-::gpt/0; Msh2-KO). RESULTS: Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3'-side of 5'-GpG-3'), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. CONCLUSIONS: Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

Mutant Frequency is not Increased in Mice Orally Exposed to Sodium Dichromate.

The in vivo mutagenicity of hexavalent chromium in the small intestine, the target organ of tumorgenicity, was examined by means of a transgenic mouse gene mutation assay. Sodium dichromate dihydrate was administered orally in drinking water to male gpt delta mice at a dose of 85.7 or 257.4 mg/L for 28 days or at a dose of 8.6, 28.6 or 85.7 mg/L for 90 days. No significant increase in gpt mutant frequency relative to that in control mice was observed in the small intestine in either the 28- or 90-day study, whereas 28-day oral administration of potassium bromate, a positive control substance, increased mutant frequency.

Change over time of the mutagenicity in the lungs of gpt delta transgenic mice by extract of airborne particles collected from ambient air in the Tokyo metropolitan area.

BACKGROUND: Previously we found that DNA adducts were accumulated in the lungs of the rats exposed to ambient air in the Tokyo metropolitan area. To examine chronological change in in vivo mutagenicity of airborne particles, extracts produced from samples of total suspended particulates (TSP) collected from urban air in 1980, 1990, and 2010 in the Tokyo metropolitan area were intratracheally administered into the lungs of gpt delta mice, and differences in mutation and mutant frequency were determined by using the gpt assay. In vivo mutations induced by the extracts were characterized and mutation hotspots were identified by DNA sequencing of the mutated gpt gene. RESULTS: Administration of the 1990 extract at a dose of 0.3 mg/animal significantly elevated total mutant frequency to 3.3-times that in vehicle control, and the in vivo mutagenicity of the extract (induced mutation frequency per milligram extract) was estimated to be 2.0- and 2.4-times higher than that of the 2010 and 1980 extract, respectively. G-to-A transition was the most common base substitution in the vehicle control mice. However, administration of the 1990 extract increased the frequency of G-to-T transversion, which is a landmark base substitution induced by oxidative stress; furthermore, when the extract was administered at a dose of 0.15 mg, the mutant and mutation frequencies of G-to-T transversion were significantly increased to frequencies comparable with those of G-to-A transition. Similar increases in the mutant and mutation frequencies of G-to-T transversion were observed after administration of the 2010 extract. Hotspots (mutation foci identified in three or more mice) of G-to-A transition mutations at nucleotides 64 and 110 were induced by the 1980, 1990, and 2010 extracts; a hotspot of G-to-T transversions at nucleotide 406 was also induced by the 2010 extract. Previously, we showed that diesel exhaust particles or their extract, as well as 1,6-dinitropyrene, administered to mice induced these hotspots of G-to-A transitions. CONCLUSIONS: The results of the present study suggested that mutagenesis induced by extracts produced from TSP collected in the Tokyo metropolitan area induced in vivo mutagenicity via the same mechanism underlying the induction of in vivo mutagenicity by components of diesel exhaust.

Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner.

Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.

Successful application of atelocollagen for treatment of perforated teeth.

OBJECTIVE: Cervical or furcal root perforation is a serious clinical problem and one of its treatment modalities is perforation repair with composite resin. However, many cases still progress in inevitable extraction. When primary teeth are affected, early tooth loss can cause problems related to the eruption space for the permanent successors. The aim of the present study was to evaluate a novel clinical treatment method for perforated teeth. STUDY DESIGN: Atelocollagen was applied to perforated furcal and cervical areas of 13 primary teeth in 13 children aged 4-9 years and 8 permanent teeth in 8 adults aged 35-69 years after debridement with an electric knife. Thereafter the final restorations were performed after confirming good tooth conditions. Clinical evaluations were performed at follow-up examinations at approximately 3-month intervals. RESULTS: None of the treated primary teeth showed any clinical problems throughout the observation period, with eruption of the permanent successors noted in 7 cases. In the permanent teeth, no clinical problems were identified in any of the cases during follow-up periods of 10-60 months. CONCLUSION: This novel method may enable preservation of perforated primary teeth for a longer duration.

Detection of oral streptococci with collagen-binding properties in saliva specimens from mothers and their children.

BACKGROUND: Approximately 10-20% of Streptococcus mutans strains have been reported to possess collagen-binding properties, whereas other species in the oral cavity with those properties remain to be elucidated. Aim. To identify strains with collagen-binding properties and analyse their characteristics in comparison with S. mutans. DESIGN: A total of 110 expectorated saliva specimens were collected from 55 pairs of mothers and their children. Bacterial strains with collagen-binding properties were isolated and the species specified. In addition, strains with collagen-binding properties isolated from mother-child pairs were analysed using molecular biological approaches. RESULTS: The detection frequency of strains with collagen-binding properties was shown to be 40.9%, among which S. salivarius was the most frequently detected, followed by S. mutans. The collagen-binding activity of the S. mutans group was the highest, followed by S. salivarius. In addition, S. mutans and S. salivarius strains from 3 and 1 mother-child pairs, respectively, were shown to be the same clones. CONCLUSIONS: Our results indicate that S. mutans and S. salivarius are major species with collagen-binding properties in the oral cavity, and that strains with such properties may be related to mother-child transmission.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence.

Streptococcus mutans is generally known as a pathogen of dental caries, and it is also considered to cause bacteremia and infective endocarditis (IE). S. mutans was previously classified into 3 serotypes, c, e, and f, due to the different chemical compositions of the serotype-specific polysaccharides, which are composed of a rhamnose backbone and glucose side chains. We recently designated non-c/e/f serotype S. mutans strains as novel serotype k, which is characterized by a drastic reduction in the amount of the glucose side chain. A common biological feature of novel serotype-k strains is a lower level of cariogenicity due to alterations of several major cell surface protein antigens. As for virulence in blood, these strains survive in blood for a longer duration due to lower antigenicity, while the detection rate of all strains carrying the gene encoding collagen-binding adhesin has been shown to be high. Furthermore, molecular biological analyses of infected heart valve specimens obtained from IE patients revealed a high detection rate of serotype-k S. mutans. Together, these findings suggest that serotype-k S. mutans strains show low cariogenicity but high virulence in blood as compared to the other serotypes, due to alterations of several cell surface structures.

Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities.

BACKGROUND: Recent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers. METHODS: A total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species. RESULTS: The most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species. CONCLUSION: We found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.

Bacterial profiles of oral streptococcal and periodontal bacterial species in saliva specimens from Japanese subjects.

OBJECTIVE: Recent developments in molecular biological techniques have increased understanding of the distribution of oral bacterial species in clinical specimens, though few investigations have been conducted to simultaneously detect oral streptococcal and periodontal species in the same specimens. The purpose of the present study was to investigate the distribution and correlation of 6 oral streptococcal and 6 periodontal species in saliva specimens taken from children and their mothers. DESIGN: Seventy-four pairs of children and their mothers were approved to participate in this study. Saliva specimens were collected and bacterial DNA extracted, which was subjected to PCR analyses using species-specific sets of primers. The combinations of species able to be detected simultaneously were determined by statistical analyses. RESULTS: Streptococcus sobrinus and Porphyromonas gingivalis were detected more often in the mothers than the children. Streptococcus mutans, Streptococcus sanguinis, and Streptococcus oralis were detected simultaneously in a significant number of specimens, while the presence of Campylobacter rectus was correlated with the presence of at least one of the red complex species (P. gingivalis, Treponema denticola and Tannerella forsythensis). On the other hand, no correlation was shown between the rates of detection of oral streptococcal and periodontal species. CONCLUSIONS: Our results indicate that among streptococcal and periodontal species, several are able to coexist in saliva, while the presence of both does not have an influence on each other.

Comparison of immunotoxicity among tetrachloro-, pentachloro-, tetrabromo- and pentabromo-dibenzo-p-dioxins in mice.

There is concern about the growing environmental levels of brominated dioxins. Brominated dioxins are known to bind and activate the transcription factor aryl hydrocarbon receptor (AhR), as their chlorinated congeners do. However, data on the potency of brominated dioxins for immunotoxicity in vivo is largely lacking, even though the immune system is a vulnerable target for dioxins. In this study, we investigated the immunotoxic effects on mice of the brominated dioxins, 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) and 1,2,3,7,8-pentabromodibenzo-p-dioxin (PeBDD), in comparison with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), the two most toxic chlorinated dioxins, to gain insight into the potency of brominated dioxins for immunotoxicity. C57BL/6 mice were dosed with the dioxins and immunized with ovalbumin (OVA), and several endpoints that sensitively detect immunotoxicity were investigated, including IL-5 production by the splenocytes. The results of the present study demonstrated that TCDD and TBDD show identical effects on a per weight basis at 1.0-10mug/kg for all the endpoints examined. PeCDD also showed effects similar to those of TCDD. On the other hand, PeBDD showed somewhat dose-independent effects and was more potent at a lower dose and less potent at a higher dose than PeCDD. Dose-dependent linearity of PeBDD-induced induction of CYP1A1, an AhR target gene, was also less clear in the spleen than in the liver. These results have provided valuable data for estimating the potency of brominated dioxins for immunotoxicity.

Inhibitory effects of cacao bean husk extract on plaque formation in vitro and in vivo.

Cacao bean husk extract (CBH) has been shown to possess antibacterial and antiglucosyltransferase activities through its unsaturated fatty acids and epicatechin polymers, respectively. In the present study, the antiplaque activities of CBH were examined in vitro and in vivo. The extract inhibited the adherence of Streptococcus mutans MT8148 to saliva-coated hydroxyapatite and reduced the accumulation of artificial dental plaque by S. mutans MT8148 on orthodontic wire. The number of mutans streptococci in dental plaque was also significantly reduced when human dental plaque was exposed to CBH from 21 children at 37 degrees C for 1 h. For the in vivo study, 28 volunteers aged 19-29 yr old rinsed their mouth with CBH, before and after each intake of food and before sleeping at night for 4 d without using other oral hygiene procedures. Plaque depositions and the numbers of mutans streptococci were reduced in the subjects, compared with rinsing with 1% ethanol alone. These results indicate that CBH possesses significant antiplaque activity in vitro and in vivo.

The role of glucan-binding proteins in the cariogenicity of Streptococcus mutans.

Streptococcus mutans produces glucan-binding proteins (Gbps), which appear to contribute to the virulence of S. mutans. GbpA and GbpC genes were inactivated by the insertion of antibiotic-resistant genes into each gbp gene of S. mutans MT8148 to generate Gbp-defective mutants. Sucrose dependent adherences of the GbpA- and GbpC-defective mutants were found to be significantly lower than those of their parent strains MT8148. Caries inducing activity of the mutants in rats was significantly lower than that of strain MT8148R (streptomycin-resistant strain of MT8148). These results suggest that GbpA and GbpC participate in cellular adherence to tooth surfaces and contribute to the cariogenicity of S. mutans.