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Makoto Asamoto

Publications and source records attributed to Makoto Asamoto.

At least 19 recordsLinked to original sources
PubMed2006

Prediction of carcinogenic potential by a toxicogenomic approach using rat hepatoma cells.

The long-term rodent bioassay is the standard method to predict the carcinogenic hazard of chemicals for humans. However, this assay is costly, and the results take at least two years to produce. In the present study, we conducted gene expression profiling of cultured cells exposed to carcinogenic chemicals with the aim of providing a basis for rapid and reliable prediction of carcinogenicity using microarray technology. We selected 39 chemicals, including 17 rat hepatocarcinogens and eight compounds demonstrating carcinogenicity in organs other than the liver. The remaining 14 were non-carcinogens. When rat hepatoma cells (MH1C1) were treated with the chemicals for 3 days at a non-toxic dose, analysis of gene expression changes with our in-house microarray allowed a set of genes to be identified differentiating hepatocarcinogens from non-carcinogens, and all carcinogens from non-carcinogens, by statistical methods. Moreover, optimization of the two gene sets for classification with an SVM and LOO-CV resulted in selection of 39 genes. The highest predictivity was achieved with 207 genes for differentiation between non-hepatocarcinogens and non-carcinogens. The overlap between the two selected gene sets encompassed 26 genes. This gene set contained significant genes for prediction of carcinogenicity, with a concordance of 84.6% by LOO-CV SVM. Using nine external samples, correct prediction of carcinogenicity by SVM was 88.9%. These results indicate that short-term bioassay systems for carcinogenicity using gene expression profiling in hepatoma cells have great promise.

Animals↗
PubMed2006

Characterization and application of polyclonal antibodies that specifically recognize JC virus large T antigen.

Polyomavirus JC virus (JCV) is the causative agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease of the central nervous system. Similar to other polyomaviruses, such as simian vacuolating virus 40 (SV40) and BK virus (BKV), JCV is also associated with human tumours. The Polyomavirus early protein large T antigen (TAg) plays a crucial role in tumour pathogenesis. An antibody to SV40 TAg (PAb416), which cross-reacts with TAgs of both JCV and BKV, has been used widely for the detection of JCV and BKV TAgs. As a consequence, it is difficult to discriminate between the TAgs of SV40, BKV and JCV by immunohistochemical analyses. In the present study, we generated JCV TAg-specific polyclonal antibodies (JCT629 and JCT652) by immunization of New Zealand white rabbits with synthetic peptides reproducing the JCV TAg carboxyl-terminal region as immunogens. Immunoblotting analyses indicated that the new antibodies bind specifically to JCV TAg, and not to those of SV40 or BKV. We also demonstrated that these antibodies can be used for immunoprecipitation, immunocytochemical analyses and immunohistochemical staining of routinely processed specimens. In conclusion, the newly generated JCV-specific TAg antibodies may be useful both in the investigation of the pathophysiological function of JCV TAg and in discriminating between Polyomavirus-related clinical samples.

Adult↗
PubMed2006

Differences between latent and clinical prostate carcinomas: lower cell proliferation activity in latent cases.

BACKGROUND: Biological significance of prostate latent cancers as early phase of clinical cancers has been controversial. The characterization of the latent cancer may be important to investigate differences between latent and clinical prostate cancers. METHODS: Latent cancers of the prostate, discovered at autopsy in men with no prior treatment for prostate disease and clinical prostate cancers, were compared for cell proliferation activities with parameters such as markers Ag-nucleolar organizer regions (AgNOR), topoisomerase II-alpha, and Ki-67. We also immunohistochemically studied alpha-methylacyl-CoA racemase (AMACR) expression that was recently identified as a possible positive marker of the prostate cancers. We analyzed 50 latent cancers and 50 clinical cancers, and samples were analyzed with Gleason grades or tumor volume. RESULTS: In the latent cancers, Gleason grades 1-4 were observed, but in the clinical cancers Gleason grades 2-5 were recognized. Cell proliferation activities were significantly lower in the latent cancers in Gleason grade 3, and similar results were obtained but without statistical significance in Gleason grades 2 and 4. When analysis was performed according to the tumor size, it was shown that the growth activities of the tumor of the clinical cancer were higher than the latent cancer. CONCLUSIONS: These results indicate that proliferation activities of the latent cancers were lower than the clinical cancers at the same Gleason grades. The data also suggest that latent cancers are just of preclinical stage and there is a possibility to progress to clinical ones by changing life style and longer life expectancy.

Antigens, Neoplasm↗
PubMed2006

Inhibition of prostate carcinogenesis in probasin/SV40 T antigen transgenic rats by leuprorelin, a luteinizing hormone-releasing hormone agonist.

The effects of leuprorelin acetate, a luteinizing hormone-releasing hormone agonist (LHRH-A), on prostate carcinogenesis in probasin/SV40 Tag transgenic rat was investigated. Fifteen weeks after administration of 0.28 and 2.8 mg/kg leuprorelin, prostate weights and serum testosterone levels were significantly decreased compared to values for transgenic controls. Histopathological findings revealed that the incidence of prostatic adenocarcinomas was significantly reduced in ventral, dorsal and lateral lobes of the prostate, correlating with decreased expression of SV40 Tag oncoprotein as well as inhibition of DNA synthesis and proliferation of epithelial cells in neoplastic lesions of the ventral prostate. Microarray analysis further showed leuprorelin acetate to significantly inhibit testicular steroidogenesis, suppressing the expression of SV40 Tag oncoprotein and altering the expression of a large number of genes which might be involved in the inhibition of prostate cancer progression in this rat model.

Adenocarcinoma↗
PubMed2005

Lack of modification of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) rat hepatocarcinogenesis by caffeine, a CYP1A2 inducer, points to complex counteracting influences.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. To test the hypothesis that it is metabolically activated by CYP1A2, we here investigated the effects of caffeine as a CYP1A2 inducer on MeIQx induced rat hepatocarcinogenesis in a medium-term liver bioassay system. Unexpectedly, no modifying effects of caffeine on MeIQx-induced hepatocarcinogenesis were evident, although up-regulation of CYP1A2 and NAT2 were detected. Therefore, mRNAs extracted from GST-P positive foci and the surrounding liver tissue in each group were analyzed to explore mechanisms in detail. The results suggest that suppression of syndecan-2 (Sdc2) and induction of cell cycle arrest through a p21-dependent pathway might have counter-acted any promotion effects of up-regulation of CYP1A2.

Acetyltransferases↗
PubMed2005

Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells.

In the present study, Tbx3, a member of the T-box family of transcription factors, was identified as an up-regulated gene by mRNA differential display in the regression (apoptosis) stage after uracil-induction of papillomatosis in the rat urinary bladder. Immunohistochemical analysis revealed that apoptosis cells are negative and apoptosis resistant cells are positive for Tbx3 expression. That suggests that Tbx3 is an apoptosis resistant gene rather than an apoptosis induced gene. We have found the rat bladder carcinoma cell line, BC31, to feature Tbx3 expression detectable by RT-PCR. To investigate its role in such cancer cells, they were transfected with an anti-sense Tbx3 expression vector. The obtained stable transfectant clones showed reduced expression of Tbx3 and much slower cell growth, as compared with mock transfectants, and many apoptotic cells were observed under normal culture conditions. These results indicate that Tbx3 is a negative regulator of apoptosis in bladder normal epithelial cells and suppression of Tbx3 expression causes inhibition of cell proliferation and induction of apoptosis in BC31, a rat bladder carcinoma cells.

Animals↗
PubMed2005

Chronic exposure to a 1.439 GHz electromagnetic field used for cellular phones does not promote N-ethylnitrosourea induced central nervous system tumors in F344 rats.

The present study was designed to evaluate whether a 2 year exposure to an electromagnetic field (EMF) equivalent to that generated by cellular phones can accelerate tumor development in the central nervous system (CNS) of rats. Brain tumorigenesis was initiated by an intrauterine exposure to N-ethylnitrosourea (ENU) on gestational day 18. A total of 500 pups were divided into five groups, each composed of 50 males and 50 females: Group 1, untreated control; Group 2, ENU alone; Groups 3-5, ENU + EMF (sham exposure and 2 exposure levels). A 1.439 GHz time division multiple access (TDMA) signal for the Personal Digital Cellular (PDC), Japanese standard cellular system was used for the exposure of the rat head starting from 5 weeks of age, 90 min a day, 5 days a week, for 104 weeks. Brain average specific absorption rate (SAR) was 0.67 and 2.0 W/kg for low and high exposures, respectively: whole body average SAR was less than 0.4 W/kg. There were no inter-group differences in body weights, food consumption, and survival rates. No increase in the incidences or numbers per group of brain and/or spinal cord tumors, either in the males or females, was detected in the EMF exposed groups. In addition, no clear changes in tumor types were evident. Thus, under the present experimental conditions, 1.439 GHz EMF exposure to the heads of rats for a 2 year period was not demonstrated to accelerate or affect ENU initiated brain tumorigenesis.

Cell Phone↗
PubMed2005

Lack of effects of 1439 MHz electromagnetic near field exposure on the blood-brain barrier in immature and young rats.

Possible effects of 1439 MHz electromagnetic near field (EMF) exposure on the blood-brain barrier (BBB) were investigated using immature (4 weeks old) and young (10 weeks old) rats, equivalent in age to the time when the BBB development is completed and the young adult, respectively. Alteration of BBB related genes, such as those encoding p-glycoprotein, aquaporin-4, and claudin-5, was assessed at the protein and mRNA levels in the brain after local exposure of the head to EMF at 0, 2, and 6 W/kg specific energy absorption rates (SARs) for 90 min/day for 1 or 2 weeks. Although expression of the 3 genes was clearly decreased after administration of 1,3-dinitrobenzene (DNB) as a positive control, when compared with the control values, there were no pathologically relevant differences with the EMF at any exposure levels at either age. Vascular permeability, monitored with reference to transfer of FITC-dextran, FD20, was not affected by EMF exposure. Thus, these findings suggest that local exposure of the head to 1439 MHz EMF exerts no adverse effects on the BBB in immature and young rats.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Downregulation of apoptosis revealed by laser microdissection and cDNA microarray analysis of related genes in rat liver preneoplastic lesions.

Regulation of cell proliferation and apoptosis plays a key role in tumor development. To elucidate mechanisms underlying hepatocarcinogenesis, patterns of gene expression, including apoptosis-related genes, were compared between glutathione S-transferase placental form (GST-P)-positive preneoplastic foci and surrounding tissue in the rat. Lesions were induced with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg bw) and then 100 ppm 2-acetylaminofluorene (2-AAF)-containing diet combined with two-thirds partial hepatectomy. Frozen sections of the livers were applied for immunohistochemical staining of GST-P, and both positive foci and surrounding negative areas were harvested by laser microdissection. Total RNAs were extracted and amplified with T7 polymerase to allow gene expression analysis by cDNA microarray assays. In the GST-P-positive foci, altered levels were observed for many genes, mostly related to metabolism or catalysis, with increased expression of testosterone-repressive prostate message-2 (TRPM-2), which is reported to act as a protective factor against apoptosis, and decreased expression of thymus-expressed acidic protein (TEAP), which is considered to promote apoptosis. The results indicate that rat liver preneoplastic lesions might be protected against apoptosis and that the approach adopted is useful for clarification of mechanisms underlying hepatocarcinogenesis.

Alkylating Agents↗
PubMed2005

Transgenic disruption of gap junctional intercellular communication enhances early but not late stage hepatocarcinogenesis in the rat.

Much experimental evidence supports the conclusion that loss of gap junctional intercellular communication (GJIC) contributes to carcinogenesis. Transgenic rats featuring a dominant negative mutant of the connexin 32 gene under albumin promoter control (Cx32Delta Tg-High and Cx32Delta Tg-Low lines, respectively with high and low copy numbers of the transgene) have disrupted GJIC, as demonstrated by scrape dye-transfer assay in vivo as previous report by Asamoto et al. (2004). In the present study, we investigated the susceptibility of these transgenic rats to a single intraperitoneal administration of diethylnitrosamine (DEN), and found a significant increase in preneoplastic glutathione S-transferase placental form (GST-P) positive lesions in the livers of Cx32Delta Tg-High but not Cx32Delta Tg-Low rats. However, incidences of adenomas and hepatocellular carcinomas were not elevated at the end of the experiment (52 weeks). In addition, we investigated the promotional effect of phenobarbital (PB) on Cx32Delta Tg-High rats pretreated with DEN and found enhanced formation of GST-P positive lesions, in contrast to the lack of promoting effects reported for Cx32 deficient mice. The results indicate that although both high and low expression of the dominant negative connexin 32 mutant gene in our rats is able to inhibit gap junctional capacity, only high expression is effective at enhancing susceptibility to early stage DEN-induced liver carcinogenesis.

Alkylating Agents↗
PubMed2005

Influence of atrazine administration and reduction of calorie intake on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats.

Atrazine, which has been used worldwide as a pesticide, is now known to exert endocrine disrupting (antiandrogenic) effects in mammals. In this study, modifying effects of dietary feeding of 500 and 1000 p.p.m. atrazine on the development of androgen-dependent prostate cancer were investigated using male probasin/SV40 T antigen transgenic (TG) rats. As administration of atrazine has now been identified as causing a decrease in bodyweight, a dietary-restricted TG rat group was also included in order to elucidate the influence of reduction of calorie intake per se on the development of prostate cancer. At week 13, almost the entire lobes of the prostate were occupied with tumor lesions, with no clear intergroup differences in the incidences and multiplicities. Therefore, morphometrical assessment ratios of the prostate epithelial area to the whole prostate tissue area were evaluated. The ratio in the lateral lobe of the 1000 p.p.m. atrazine-treated group was significantly decreased, and there was a tendency to decrease in the ratios in the dorsal lobe of the atrazine-treated groups. However, dietary restriction itself without atrazine treatment caused the same reduction to a similar or greater extent. Testosterone levels were not affected by atrazine administration or dietary restriction. Our results indicate that the observed atrazine-related suppression of prostate carcinogenesis was probably caused by the decrease in calorie intake, rather than by atrazine-related endocrine disruption.

Adenocarcinoma↗
PubMed2005

Lack of combination hepatocarcinogenicity of harman, norharman and amitrole when given with NaNO2 in the rat.

N-nitrosocompounds, which induce cancers in various organs, may be formed endogenously with intake of amino compounds such as secondary amines and sodium nitrite (NaNO(2)) in combination. The present study was performed to investigate whether three amino compounds, 1-methyl-9H-pyrido[3,4-b]indole (harman), 9H-pyrido[3,4-b]indole (norharman) and 2-amino-1,3,4-triazole (amitrole), might be converted in vivo to compounds capable of promoting hepatocarcinogenesis when given with NaNO(2). However, in an 8-week model, no modifying potential was evident in terms of numbers and areas of putative preneoplastic glutathione S-transferase placental form (GST-P)-positive foci in any of the groups receiving paired treatments. These results demonstrate that combinations of harman, norharman and amitrole with NaNO(2) lack promoting effects for liver carcinogenesis in our medium-term bioassay system.

Amitrole↗
PubMed2004

Equivocal impact of transplacental and lactational exposure to a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, on prostate and colon lesion development in F344 rats.

The carcinogenic potential of maternal dietary exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated with the focus on the prostate and mammary glands and colons of the offspring. PhIP-DNA adducts were immunohistochemically detected in all three organs of 3-week-old animals after dams had received 200 ppm PhIP in the diet from 4 weeks before mating to weaning. The sites were essentially the same as in adult rats, suggesting that the distribution of bioactivating enzymes for PhIP do not differ greatly. Ki-67 labeling indices were increased only in the colons of the female offspring at 3-weeks of age. Development of preneoplastic and neoplastic lesions in the prostate and colon was not enhanced when the rats were maintained on PhIP-free diet until 63 weeks of age, except for significant increase in multiplicity of aberrant crypt foci in the females. The present findings indicate that PhIP exposure via placenta and breast milk may be important but that high doses and long periods may be necessary for tumor development.

Animals↗
PubMed2004

Connexin 32 dominant-negative mutant transgenic rats are resistant to hepatic damage by chemicals.

Connexins are subunits of gap junction channels, which allow direct transfer of ions, secondary messenger molecules, and other metabolites between contacting cells. Gap junctions are believed to be involved in tissue homeostasis, embryonic development, and control of cell proliferation. Several studies have shown that cell damage signals are transmitted through gap junctions when cells are irradiated or when cells bearing the herpes simplex virus-thymidine kinase (HSV-TK) gene are treated with ganciclovir. We established 2 lines of transgenic rats with a dominant-negative mutant of connexin 32 gene under control of the albumin promoter. In the livers of transgenic rats, membrane localization of normal endogenous connexin 32 protein is disturbed, and gap junction capacity measured by scrape dye-transfer assay in vivo is markedly decreased when compared with wild-type rats. The present investigation concerned susceptibility to the liver-toxic substances D-galactosamine and carbon tetrachloride. These toxicants induced massive liver cell death and elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the wild-type rats; however, much fewer liver cells were damaged and serum enzyme elevation was much lower in the transgenic rats. In conclusion, gap junctional intercellular communication (GJIC) plays an important role in toxic effects of chemicals; damage or death signals may pass through gap junctions in the rat liver in vivo.

Alanine Transaminase↗
PubMed2004

Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan.

It has been reported that Simian virus 40 (SV40) is linked to human beings by inoculation of contaminated poliovaccines and may have a role in the etiology of malignant mesothelioma. However, there have been no reports describing the relationship between SV40 and malignant mesothelioma in Japan. A study was undertaken to investigate whether SV40 was related to patients of malignant mesothelioma in Japan by the polymerase chain reaction (PCR) assay, DNA sequence analysis, and immunohistochemical methods. Paraffin-embedded samples of the 18 autopsied patients with pleural malignant mesothelioma were collected from five hospitals in Japan. After isolation of DNA from paraffin blocks, PCR analyses followed by sequencing were performed using three different sets of primers for detection of SV40 large T antigen (TAg) gene. All 18 malignant mesothelioma samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 TAg antibodies. SV40 TAg genome was detected in eight malignant mesothelioma cases. Only one of three primer pairs successfully amplified SV40 genome in the samples, whereas all pairs yielded a PCR product in the controls, suggesting a low content of virus DNA. No immunopositive staining for SV40 TAg was found in any of the samples. This study shows that SV40 genome was present in a subset of Japanese malignant mesothelioma patients who were unlikely to have received a contaminated polio vaccine based on their age.

Antigens, Neoplasm↗
PubMed2004

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo.

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), beta-cryptoxanthin- and hesperidine-rich pulp (CHRP) and 1,1'-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

Animals↗
PubMed2004

Rapid analysis of gene expression changes caused by liver carcinogens and chemopreventive agents using a newly developed three-dimensional microarray system.

We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three-dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six-week-old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N-diethylnitrosamine (DEN), 0.01% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1'-acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip(\xa8) microarray A spotted for 28 genes of drug-metabolizing enzymes in duplicate, and PamChip microarray B spotted for 131 genes which are known to be up- or down-regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three-dimensional microarrays.

Animals↗