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Biomedical subjects

Yukio Kodama

Publications and source records attributed to Yukio Kodama.

14 recordsLinked to original sources
PubMed2007

Increased susceptibility to hepatocarcinogenicity of Nrf2-deficient mice exposed to 2-amino-3-methylimidazo[4,5-f]quinoline.

To elucidate the roles of the transcription factor NF-E2-related factor (Nrf2) in hepatocarcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a mutagenic and carcinogenic heterocyclic amine, Nrf2-deficient mice were treated with 300 p.p.m. IQ in their diet for 1, 4 or 52 weeks. In the long-term experiment, the multiplicity and incidence of liver tumors in male and female IQ-treated Nrf2 deficient (-/-) mice were significantly higher than those in their counterpart wild-type (+/+) mice exposed to IQ. In the short-term experiment, although IQ exposure to Nrf2(+/+) mice of both sexes did not modify UDP-glucuronosyltransferase values, glutathione S-transferase values were significantly increased due to IQ treatment, in contrast to no alteration in male and female Nrf2(-/-) mice. Levels of oxidative stress markers such as 8-hydroxydeoxyguanosine and thiobarbituric acid reactive substances in the livers of all treated mice were not changed by IQ treatment. IQ-specific DNA adduct levels were elevated only in female Nrf2(-/-) mice, although the increase was not significant. IQ treatment caused an increase in proliferating cell nuclear antigen labeling indices only in male Nrf2(-/-) mice. The present data clearly show that Nrf2(-/-) mice of both sexes are susceptible to IQ hepatocarcinogenicity, which might result from IQ accumulation due to failure of metabolizing enzyme induction. In addition, inconsistent results concerning IQ-specific adducts and proliferating cell nuclear antigen labeling indices in male and female Nrf2(-/-) mice suggest the existence of different contributions of Nrf2 to IQ hepatocarcinogenesis between mice of the two sexes.

Animals↗
PubMed2006

Lack of in vivo mutagenicity and oxidative DNA damage by flumequine in the livers of gpt delta mice.

Flumequine (FLU), an anti-bacterial quinolone agent, has been recognized as a non-genotoxic carcinogen for the mouse liver, but recent reports have suggested that some genotoxic mechanism involving oxidative DNA damage may be responsible for its hepatocarcinogenesis. In the present study, we investigated this possibility in the mouse liver using male and female B6C3F1 gpt delta mice fed diet containing 0.4% FLU, a carcinogenic dose, for 13 weeks. Measurements of 8-hydroxydeoxyguanosine levels in liver DNA, and gpt point and deletion mutations revealed no significant increases in any of these parameters in either sex. Histopathologically, centrilobular swelling of hepatocytes with vacuolation was apparent, however, together with significant increase in bromodeoxyuridine-labeling indices in the treated males and females. These results suggest that genotoxicity, including oxidative DNA damage, is not involved in mouse hepatocarcinogenesis by FLU, which might rather solely exert tumor-promoting effects in the liver.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays.

BACKGROUND: Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell. RESULTS: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90 % concordance. CONCLUSION: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories.

Animals↗
PubMed2006

Nine-week detection of six genotoxic lung carcinogens using the rasH2/BHT mouse model.

A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

Thioredoxin overexpression in mice, model of attenuation of oxidative stress, prevents benzene-induced hemato-lymphoid toxicity and thymic lymphoma.

OBJECTIVE: Reactive oxygen species (ROS), generated following benzene exposure, are considered to trigger the development of hematopoietic neoplasms, although little supporting evidence has been found. In this study, we examined whether the experimental elimination of ROS generated following benzene exposure prevents the development of benzene-induced hematopoietic disorders to clarify the mechanism underlying the development of benzene-induced hematopoietic disorders. METHODS: C57BL/6 mice, overexpressing human thioredoxin (h-Trx-Tg), were used to examine the possible nullification of ROS induction following benzene exposure. The experimental group was exposed to 300 ppm benzene 6 hours/day, 5 days/week, for 26 weeks, and lifetime observation followed by molecular and histopathological examinations were carried out. RESULTS: The present study using h-Trx-Tg mice showed a complete suppression of the development of thymic lymphoma induced by benzene inhalation (0% in h-Trx-Tg vs 30% in wild-type (Wt) mice). This was associated with a 48% decrease in the incidence of clastogenic micronucleated reticulocyte induction in the h-Trx-Tg mice compared with the Wt control after 2 weeks of inhalation. As underlying mechanisms, the attenuation of oxidative stress was accompanied by a complete abrogation of hemato-lymphoid toxicity, as shown by the upregulation of the activity of superoxide-dismutase, and a consequently stable ROS level, as determined by cell sorting using 2', 7'-dichlorodihydrofluorescein diacetate, along with a significant attenuation of the overexpression of a cell cycle-dependent kinase inhibitor, p21. CONCLUSION: The attenuation of benzene-induced oxidative stress and that of the consequent lymphomagenesis were observed for the first time, and these indicate a role of oxidative stress in benzene-induced clastogenesis and lymphomagenesis. (These attenuations were not seen in nonthymic lymphomas, and no leukemias developed in C57BL/6 used in this study.) During the constitutive overexpression of h-Trx, the expression of aryl-hydrocarbon receptor in h-Trx-Tg mice was downregulated, which may also contribute to the attenuation.

Animals↗
PubMed2005

A crucial role of Nrf2 in in vivo defense against oxidative damage by an environmental pollutant, pentachlorophenol.

Our goal was to elucidate roles of Nrf2 in in vivo defense against pentachlorophenol (PCP), an environmental pollutant and hepatocarcinogen in mice. We examined oxidative stress and cell proliferation, along with other hepatotoxicological parameters, in the livers of nrf2-deficient (wild:+/+, heterozygous:+/-, homozygous:-/-) animals fed PCP in their diet at doses of 0, 150, 300, 600, or 1200 ppm for 4 weeks. For measurement of methoxyresorufin-O-demethylase (CYP 1A2), NAD(P):quinone oxidoreductase 1 (NQO1), and UDP-glucuronosyltransferase (UDP-GT), an additional study was performed with all but the 150-ppm dose. Significant elevation of 8-hydroxydeoxyguanosine (8-OH-dG) levels in the liver DNA was observed only in -/- mice treated with PCP at 1200 ppm. Levels of thiobarbituric-acid-reactive substances (TBARS) were also raised significantly compared to those of the relevant +/+ mice. Bromodeoxyuridine labeling indices (BrdU-LIs) of hepatocytes in -/- mice were significantly higher at all doses than those in the relevant +/+ mice. Relative liver weights were unchanged in mice lacking Nrf2, whereas liver weight in +/+ and +/- mice was increased. Significant elevations of serum ALP activity, but not ALT and AST activity, occurred at 600 ppm and above in -/- mice compared to the relevant +/+ mice. Histopathologically, centrilobular hepatocyte necrosis was severe in the -/- mice that received 600 ppm. Although CYP 1A2 activity was elevated in all treated mice, increases in NQO1 levels and UDP-GT activities did not occur only in -/- mice. These data suggest that Nrf2 plays a key role in prevention of PCP-induced oxidative stress and cell proliferation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2004

Exacerbation of benzene pneumotoxicity in connexin 32 knockout mice: enhanced proliferation of CYP2E1-immunoreactive alveolar epithelial cells.

The pulmonary pathogenesis triggered by benzene exposure was studied. Since the role of the connexin 32 (Cx32) gap junction protein in mouse pulmonary pathogenesis has been suggested, in the present study, we explored a possible role of Cx32 in benzene-induced pulmonary pathogenesis using the wild-type (WT) and Cx32 knockout (KO) mice. The mice were exposed to 300 ppm benzene by inhalation for 6 h per day, 5 days per week for a total of 26 weeks, and then sacrificed to evaluate the pneumotoxicity or allowed to live out their life span to evaluate the reversibility of the lesions and tumor incidence. Our results clearly revealed exacerbated pneumotoxicity in the benzene-exposed Cx32 KO mice, characterized by diffuse granulomatous interstitial pneumonia, markedly increased mucin secretion of bronchial/bronchiolar and alveolar epithelial cells, and hyperplastic alveolar epithelial cells positive for CYP2E1. But the results did not indicate any enhancement of pulmonary tumorigenesis in the Cx32 KO mice though the number of animals was small.

Administration, Inhalation↗
PubMed2003

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice.

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.

Alkylating Agents↗
PubMed2003

Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene.

Administration, Inhalation↗
PubMed2002

Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity.

Benzene can induce hematotoxicity and leukemia in humans and mice. Since a review of the literature shows that the CYP2E1 knockout mouse is not known to possess any benzene toxicity, the metabolism of benzene by CYP2E1 in the liver is regarded to be prerequisite for its cytotoxicity and genotoxicity, although the mechanism is not fully understood yet. Because it was found some years ago that benzene was also a substrate for CYP1A1, we investigated the involvement of the aryl hydrocarbon receptor (AhR) in benzene hematotoxicity using AhR wild-type (AhR(+/+)), heterozygous (AhR(+/-)), and homozygous (AhR(-/-)) male mice. Interestingly, following a 2-week inhalation of 300 ppm benzene (a potent dose for leukemogenicity), no hematotoxicity was induced in AhR(-/-) mice. Further, there were no changes in cellularity of peripheral blood and bone marrow (BM), nor in levels of granulocyte-macrophage colony-forming units in BM. This lack of hematotoxicity was associated with the lack of p21 overexpression, which was regularly seen in the wild-type mice following benzene inhalation. Combined treatment with two major benzene metabolites, phenol and hydroquinone, induced hemopoietic toxicity, although it was not known whether this happened due to a surprising lack of expression of CYP2E1 by AhR knockout, or due to a lack of other AhR-mediated CYP enzymes, including 1A1 (i.e., a possible alternative pathway of benzene metabolism). The former possibility, evaluated in the present study, failed to show a significant relationship between AhR and the expression of CYP2E1. Furthermore, a subsequent evaluation of AhR expression after benzene inhalation tended to show higher but less significant expression in the liver, and none in the BM, compared with sham control. Although this study failed to identify the more likely of the above-mentioned two possibilities, the study using AhR knockout mice on benzene inhalation presents the unique possibility that the benzene toxicity may be regulated by AhR signaling.

Administration, Inhalation↗
PubMed2002

The mouse rasH2/BHT model as an in vivo rapid assay for lung carcinogens.

We have demonstrated the utility of a 9-week in vivo two-stage assay for lung cancer initiating agents, using transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and butylhydroxytoluene (BHT) as a potent lung promoter (rasH2/BHT model). In the present study, to ascertain appropriate conditions for BHT administration in this model, the effects of exposure on proliferation of alveolar type II cells in male rasH2 mice were examined. Additionally, use of BHT was validated for promotion of urethane (UR) carcinogenesis in male and female rasH2 mice. In a time-course study of a single intragastric administration of BHT at a dose of 400 mg/kg, increased bromodeoxyuridine-labeling index (LI) reached a maximum 3 days after treatment and was still observed after 7 days. In a dose-response study, effects were dose-dependent, the dose of 400 mg/kg causing eight-fold elevation as compared to the control. With repeated administration, whereas the LI was increased dramatically at first, effects gradually diminished with further exposure, and finally six BHT treatments failed to induce cell proliferation. In a two-stage model using UR as the initiator, although up to five consecutive doses of BHT were able to exert continued enhancing effects in terms of adenoma yield, no increment was evident with further treatments. The data overall indicate that a rasH2/BHT model with five weekly administrations of BHT at a dose of 400 mg/kg is most efficacious.

Animals↗
PubMed2002

Serial transplantation of p53-deficient hemopoietic progenitor cells to assess their infinite growth potential.

Thirty-five years ago, Siminovitch et al. (Siminovitch L, Till JE, McCulloch EA. J Cell Com Physiol 64:23-32, 1964), using serially transplanted mouse spleens at 14-day intervals, observed a markedly progressive decline in the proliferative capacity of bone marrow (BM) cells, with the loss of clonogenicity by the fourth transplant generation. Using the same protocol, we assessed the proliferative capacity of p53-deficient mouse BM cells transplanted serially at the same 14-day intervals into lethally irradiated mice, which was a useful tool for understanding the characteristics of hemopoietic stem cells lacking solely the p53 gene function. BM cells from p53-deficient homozygous (p53(-/-)), p53-heterozygous (p53(+/-)), and wild-type (p53(+/+)) C57BL/6 mice were transplanted into lethally irradiated C57BL/6 recipients. Fourteen days later, the repopulated spleens were harvested, and 10(7)cells were retransplanted into secondary recipients. Serial transplantation was continued at 14-day intervals until hemopoietic repopulation failure. The number of heterozygous and homozygous p53-deficient spleen cells increased logarithmically up to the fourth and fifth passages, respectively, whereas wild-type spleen cells ceased to proliferate by the third passage. The number of macroscopic spleen colonies increased logarithmically until the third passage in recipients of heterozygous and homozygous p53-deficient cells, but ceased to grow by the second passage in recipients of wild-type cells. The numbers of heterozygous and homozygous p53-deficient colony forming units in spleen (CFUs-S) remained stable during the first four transplant generations, whereas that of wild-type CFUs-S decreased progressively from the first transplant generation onward. The clonogenicity of p53-deficient cells was lost when the number of CFUs-S per spleen decreased to below 10. This suggests that one out of 10 CFUs-S might be long-term repopulating cells (LTRCs), and that p53-deficient LTRCs may proliferate more rapidly than wild-type LTRCs. Longer passages that were possible in the p53-deficient groups were considered to be due to the faster cell cycle of the p53-deficient hemopoietic progenitor cells, as determined by bromodeoxyuridine incorporation with purging by UV light exposure, followed by hemopoietic colony assay (BUUV assay).

Animals↗
PubMedDate not supplied

Pentachlorophenol (but not phenobarbital) promotes intrahepatic biliary cysts induced by diethylnitrosamine to cholangio cystic neoplasms in B6C3F1 mice possibly due to oxidative stress.

Administration of diethylnitrosamine (DEN) to B6C3F1 mice at low dose (20 ppm) in drinking water for long duration resulted in formation of multifocal cystic biliary lesions in the liver. To investigate the potential of the lesions to be promoted to neoplasias by chemicals, we examined the effects of 2 different types of hepatocarcinogenesis promoters, pentachlorophenol (PCP) and phenobarbital (PB) in B6C3F1 mice. Two weeks' exposure to PCP at a concentration of 600 ppm in the diet increased 8-oxodeoxyguanosine (8-oxodG) levels in liver nuclear DNA, and cell proliferation quantified by bromodeoxyuridine (BrdU) incorporation in epithelial cells of intrahepatic bile ducts as well as hepatocytes. In mice initiated with DEN at 20 ppm in the drinking water for the first 13 weeks followed, after a 4-week recovery interval, by PCP at a concentration of 600 ppm in the diet for 25 weeks, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinomas were present at statistically significant higher incidences. In contrast, neoplasia did not occur in animals treated with 500 ppm PB, and there were no elevations in 8-oxodG levels or increases in the proliferation of biliary epithelium, although proliferation was increased in hepatocytes. These findings suggest that oxidative stress due to PCP might exert a promoting action on the biliary cystic lesions produced by DEN.

8-Hydroxy-2'-Deoxyguanosine↗