Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes.

Human pluripotent stem cell-derived hepatocytes have the potential to provide in vitro model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major CYP genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of CYP genes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, and CYP2E1, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of CYP genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. Our findings suggest that limited expression of CYP genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.

Anti-inflammatory effects of the Zingiber officinale roscoe constituent 12-dehydrogingerdione in lipopolysaccharide-stimulated Raw 264.7 cells.

Ginger has long been used worldwide as a spice, seasoning, and wine and is also used as a traditional medicine. There have been no previous studies of the potential beneficial effects of the ginger constituent 12-dehydrogingerdione (12-DHGD). We investigated the anti-inflammatory effect of 12-DHGD on lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. The cytotoxicity of 12-DHGD was measured using the MTT assay, and production of prostaglandin E2 (PGE2 ) and the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was measured by ELISA. Production of nitric oxide (NO) was measured using Griess reagent and expression of cyclooxygenase-2 (COX-2) and inducible NO (iNOS) enzymes was assessed by reverse transcriptase-polymerase chain reaction. Treatment of Raw 264.7 cells with 12-DHGD significantly inhibited LPS-stimulated production of NO (at 12-DHGD concentrations of 150 and 200 ng/ml), IL-6 (at 50, 100, 150, and 200 ng/ml), and PGE2 (at 200 ng/ml). Consistent with the effects on NO and PGE2 production, 12-DHGD treatment also inhibited the LPS-stimulated increase in iNOS and COX-2 mRNA levels. However, 12-DHGD did not affect production of IL-1ß or TNF-α in response to LPS. 12-DHGD, a constituent of ginger, is a potent inhibitor of proinflammatory mediator production in Raw 264.7 macrophage cells.

Inflammatory response in rat lungs with recurrent exposure to welding fumes: a transcriptomic approach.

As chronic exposure to welding fumes causes pulmonary diseases, such as pneumoconiosis, public concern has increased regarding continued exposure to these hazardous gases in the workplace. In a previous study, the inflammatory response to welding fume exposure was analysed in rat lungs in the case of recurrent exposure and recovery periods. Thus using lung samples, well-annotated by histological observation and biochemical analysis, this study examines the gene expression profiles to identify phenotype-anchored genes corresponding to lung inflammation and the repair phenomenon after recurrent welding fume exposure. Seven genes (Mmp12, Cd5l, LOC50101, LOC69183, Spp1, and Slc26a4) were found to be significantly up-regulated according to the severity of the lung injury. In addition, the transcription and translation of Trem2, which was up-regulated in response to the repair process, were validated using a real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The differentially expressed genes in the exposure and recovery groups were also classified using k-means and hierarchical clustering, plus their toxicological function and canonical pathways were further analysed using Ingenuity Pathways Analysis Software. As a result, this comprehensive and integrative analysis of the transcriptional changes that occur during repeated exposure provides important information on the inflammation and repair processes after welding-fume-induced lung injury.

Differential gene expression profiles of radioresistant non-small-cell lung cancer cell lines established by fractionated irradiation: tumor protein p53-inducible protein 3 confers sensitivity to ionizing radiation.

PURPOSE: Despite the widespread use of radiotherapy as a local and regional modality for the treatment of cancer, some non-small-cell lung cancers commonly develop resistance to radiation. We thus sought to clarify the molecular mechanisms underlying resistance to radiation. METHODS AND MATERIALS: We established the radioresistant cell line H460R from radiosensitive parental H460 cells. To identify the radioresistance-related genes, we performed microarray analysis and selected several candidate genes. RESULTS: Clonogenic and MTT assays showed that H460R was 10-fold more resistant to radiation than H460. Microarray analysis indicated that the expression levels of 1,463 genes were altered more than 1.5-fold in H460R compared with parental H460. To evaluate the putative functional role, we selected one interesting gene tumor protein p53-inducible protein 3 (TP53I3), because that this gene was significantly downregulated in radioresistant H460R cells and that it was predicted to link p53-dependent cell death signaling. Interestingly, messenger ribonucleic acid expression of TP53I3 differed in X-ray-irradiated H460 and H460R cells, and overexpression of TP53I3 significantly affected the cellular radiosensitivity of H460R cells. CONCLUSIONS: These results show that H460R may be useful in searching for candidate genes that are responsible for radioresistance and elucidating the molecular mechanism of radioresistance.

Influence of p53 expression on sensitivity of cancer cells to bleomycin.

In this study, we determined whether p53 expression affected the sensitivity of non-small cell lung cancer (NSCLC) and colon cancer cells to bleomycin (BLM). Two human NSCLC cell lines (A549 expressing wild-type p53 and p53-null H1299) and two colon cancer cell lines (HCT116 p53+/+ and its p53 deficient subline HCT116 p53-/-) were subjected to treatment with BLM. Cells were treated with various concentrations of BLM, and cellular viability was assessed by formazan assay. To investigate the role of p53 in BLM sensitivity, we transduced cells with adenovirus with wild-type p53, dominant-negative p53, and GFP control, and analyzed the effect on cellular viability. Cells expressing wild-type p53 were more sensitive to BLM than p53-null cells in both NSCLC and colon cancer cells. Sensitivity to BLM of the cells with wild-type p53 was reduced by overexpression of dominant-negative p53, while BLM sensitivity of p53-null cells was increased by wild-type p53 in both NSCLC cells and colon cancer cells. In conclusion, our data show that p53 sensitizes all four cancer cells lines tested to BLM toxicity and overexpression of p53 confers sensitivity to the cytotoxic activity of the anticancer agent in p53-null cells.

Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice.

Many studies have reported that bleomycin, anti-cancer drug, induces pulmonary fibrosis as a side effect. However, few investigations have focused on the dose-response effects of bleomycin on pulmonary fibrosis. Therefore, in the present study, we investigated the effects of different doses of bleomycin in male mice. ICR mice were given 3 consecutive doses of bleomycin: 1, 2, or 4 mg/kg in bleomycin-treated (BT) groups and saline only in vehicle control (VC) groups. The animals were sacrificed at 7 and 24 days postinstillation. The severity of pulmonary fibrosis was evaluated according to inflammatory cell count and lactate dehydrogenase (LDH) activity in the broncho alveolar lavage fluid (BALF) , and lung tissues were histologically evaluated after hematoxylin and eosin (H&E) , and Masson's trichrome staining. BT groups exhibited changed cellular profiles in BAL fluid compared to the VC group, which had an increased number of total cells, neutrophils, and lymphocytes and a modest increase in the number of macrophages at 7 days post-bleomycin instillation. Moreover, BT groups showed a dose-dependent increase in LDH levels and inflammatory cell counts. However, at 24 days after treatment, collagen deposition, interstitial thickening, and granulomatous lesions were observed in the alveolar spaces in addition to a decrease in inflammatory cells. These results indicate that pulmonary fibrosis induced by 4 mg/kg bleomycin was more severe than that induced by 1 or 2 mg/kg. These data will be utilized in experimental animal models and as basic data to evaluate therapeutic candidates through non-invasive monitoring using the pulmonary fibrosis mouse model established in this study.

Inhibitor of differentiation 1 (Id1) expression attenuates the degree of TiO2-induced cytotoxicity in H1299 non-small cell lung cancer cells.

The inhibitor of differentiation (Id) family of genes, which encodes negative regulators of basic helix-loop-helix transcription factors, has been implicated in diverse cellular processes such as proliferation, apoptosis, differentiation, and migration. However, the specific role of Id1 in titanium dioxide (TiO2)-induced lung injury has not been investigated. In the present study, we investigated whether TiO2 induces apoptosis in H1299 lung cancer cells and by which pathways. Based on the results of the LDH assay, dual staining with Annexin V-FITC and propidium iodide (PI), and RT-PCR analysis of apoptosis-related gene expression, TiO2 caused a dose- and time-dependent decrease in cell viability and appeared to involve both necrosis and apoptosis. Furthermore, Id1 expression was significantly reduced in TiO2-treated cells compared with control cells. To further investigate the functional role of Id1, cells were transduced with a recombinant adenovirus expressing Id1, and the effects on sensitivity to TiO2 were analyzed. Id1 overexpression led to the enhancement of cellular proliferation and reduced the sensitivity of H1299 cells to TiO2. Our results indicate that Id1 expression attenuates the degree of TiO2-induced cytotoxicity in lung cells.

A novel mutation in Hr causes abnormal hair follicle morphogenesis in hairpoor mouse, an animal model for Marie Unna Hereditary Hypotrichosis.

Hairpoor mice (Hr(Hp)) were derived through N-ethyl-N-nitrosourea (ENU) mutagenesis. These mice display sparse and short hair in the Hr(Hp)/+ heterozygous state and complete baldness in the Hr(Hp)/Hr(Hp) homozygous state. This phenotype was irreversible and was inherited in an autosomal semidominant manner. Hair follicles (HFs) of Hr(Hp)/+ mice underwent normal cycling and appeared normal, although smaller than those of the wild-type mice. In contrast, HFs of Hr(Hp)/Hr(Hp) mice became cyst-like structures by postnatal day (P) 21. The number and length of vibrissae decreased in a dose-dependent manner as the number of mutant alleles increased. A positional candidate gene approach was used to identify the gene responsible for the hairpoor phenotype. Genetic linkage analysis determined that the hairpoor locus is 2 cm from D14Mit34 on chromosome 14. Sequence analysis of the exons of the candidate gene hairless revealed a T-to-A transversion mutation at nucleotide position 403 (exon 2), presumably resulting in abolishment of an upstream open reading frame (uORF). In addition, we also found that the near-naked mouse (Hr(N)), a spontaneously arising mutant, harbors a A402G transition in its genome. Both mutations were in the uATG codon of the second uORF in the 5' UTR and corresponded to the mutations identified in Marie Unna Hereditary Hypotrichosis (MUHH) patients. In the present study we describe the phenotype, histological morphology, and molecular etiology of an animal model of MUHH, the hairpoor mouse.

Microarray-based analysis of the lung recovery process after stainless-steel welding fume exposure in Sprague-Dawley rats.

Repeated exposure to welding fumes promotes a reversible increase in pulmonary disease risk, but the molecular mechanisms by which welding fumes induce lung injury and how the lung recovers from such insults are unclear. In the present study, pulmonary function and gene-expression profiles in the lung were analyzed by Affymetrix GeneChip microarray after 30 days of consecutive exposure to manual metal arc welding combined with stainless-steel (MMA-SS) welding fumes, and again after 30 days of recovery from MMA-SS fume exposure. In total, 577 genes were identified as being either up-regulated or down-regulated (over twofold changes, p < 0.05) in the lungs of low-dose or high-dose groups. Differentially expressed genes were classified based on a k-means clustering algorithm and biological functions and molecular networks were further analyzed using Ingenuity Pathways Analysis. Among the genes affected by exposure to or recovery from MMA-SS fumes, the transcriptional changes of 13 genes that were highly altered by treatment were confirmed by quantitative real-time PCR. Notably, Mmp12, Cd5l, Ccl7, Cxcl5, and Spp1 related to the immune response were up-regulated only in the exposure group, whereas Trem2, IgG-2a, Igh-1a, and Igh were persistently up-regulated in both the exposure and recovery groups. In addition, several genes that might play a role in the repair process of the lung were up-regulated exclusively in the recovery group. Collectively, these data may help elucidate the molecular mechanism of the recovery process of the lung after welding fume exposure.

Implications for tooth development on ENU-induced ectodermal dysplasia mice.

BACKGROUND: In this study, the mutated phenotypes were produced by treatment of chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed the mutated mice showing the specific phenotype of ectodermal dysplasia (ED) and examined the affected gene. METHODS: Phenotypes, including size, bone formation, and craniofacial morphology of ENU-induced ED mice, were focused. Tooth development and expression of several molecules were analyzed by histologic observations and immunohistochemistry. We carried out genome-wide screening and quantitative real-time PCR to define the affected and related genes. RESULTS: As examined previously in human ectodermal dysplasia, ENU-induced ED mice showed the specific morphologic deformities in tooth, hair, and craniofacial growth. Tooth development in the ENU-induced ED mice ceased at early cap stage. In addition, skeletal staining showed retardation in craniofacial development. Finally, the affected gene, which would be involved in the mechanism of ED, was located between the marker D3Mit14 and D3Mit319 on chromosome 3. CONCLUSIONS: The affected gene in ENU-induced ED mice showed several defects in ectodermal organogenesis and these results indicate that this gene plays an important role in mouse embryogenesis.

Pulmonary Toxicity and Recovery from Inhalation of Manual Metal Arc Stainless Steel Welding Fumes in Rats.

The objectives of this study were to examine the lung injury and inflammation caused by manual metal arc stainless steel (MMA-SS) welding fume inhalation and to evaluate the recovery process. Sprague-Dawley rats were exposed to MMA-SS welding fumes for 2 h per day in a whole-body exposure chamber, with a total suspended particulate (TSP) concentration of 51.4 ± 2.8 mg/m3 (low dose) or 84.6 ± 2.9 mg/m3 (high dose) for 30 days. The animals were sacrificed after 30 days of exposure as well as after a 30-day recovery period. To assess the inflammatory or injury responses, cellular and biochemical parameters as well as cytokines were assayed in the bronchoalveolar lavage fluid (BALF). MMA-SS welding fume exposure led to a significant elevation in the number of alveolar macrophages (AM) and polymorphonuclear cells (PMN). Additionary, the values of ß-nacetyl glucosaminidase (ß-NAG) and lactate dehydrogenase (LDH) in the BALF were increased in the exposed group when compared to controls. After 30 days of recovery from exposure, a significant reduction in inflammatory parameters of BALF was observed between the exposed and recovered groups. Slight, but significant elevations were noted in the number of AM and PMN in the recovered groups, and AM that had been ingested fume particles still remain in the lungs. In conclusion, these results indicated that welding fumes induced inflammatory responses and cytotoxicity in the lungs of exposed rats. Fume particles were not fully cleared from lungs even after a 30-day recovery period.

Comparison of high MRI T1 signals with manganese concentration in brains of cynomolgus monkeys after 8 months of stainless steel welding-fume exposure.

Several pharmacokinetic studies on inhalation exposure to manganese (Mn) have already demonstrated that Mn readily accumulates in the olfactory and brain regions. However, a shortening of the magnetic resonance imaging (MRI) T1 relaxation time or high T1 signal intensity in specific sites of the brain, including the globus pallidus and subcortical frontal white matter, as indicative of tissue manganese accumulation has not yet been clearly established for certain durations of known doses of welding-fume exposure in experimental animals. Accordingly, to investigate the movement of manganese after welding-fume exposure, six cynomolgus monkeys were acclimated and assigned to three dose groups: unexposed, low dose (31 mg/m(3) total suspended particulate [TSP], 0.9 mg/m(3) of Mn), and high dose (62 mg/m(3) TSP, 1.95 mg/m(3) of Mn) of total suspended particulate. The primates were exposed to manual metal arc stainless steel (MMA-SS) welding fumes for 2 h per day in an inhalation chamber system equipped with an automatic fume generator. Magnetic resonance imaging (MRI) studies were conducted before the initiation of exposure and thereafter every month. The tissue Mn concentrations were then measured after a plateau was reached regarding the shortening of the MRI T1 relaxation time. A dose-dependent increase in the Mn concentration was found in the lungs, while noticeable increases in the Mn concentrations were found in certain tissues, such as the liver, kidneys, and testes. Slight increases in the Mn concentrations were found in the caudate, putamen, frontal lobe, and substantia nigra, while a dose-dependent noticeable increase was only found in the globus pallidus. Therefore, the present results indicated that a shortening of the MRI T1 relaxation time corresponded well with the Mn concentration in the globus pallidus after prolonged welding-fume exposure.

Distinct pattern of allelic loss and inactivation of cadherin 1 and 5 genes in mammary carcinomas arising in p53(+/-) mice.

p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA x MSM/Ms) F(1)- p53(+/-) and (BALB/cHeA x 129/SvEv) F(1)- p53(+/-) female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F(1) mice. The MCs in the p53(+/- )mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA x MSM/Ms) F(1)- p53 (+/-) mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F(1) mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

Changes in blood manganese concentration and MRI t1 relaxation time during 180 days of stainless steel welding-fume exposure in cynomolgus monkeys.

Welders are at risk of being exposed to high concentrations of welding fumes and developing pneumoconiosis or other welding-fume exposure-related diseases. Among such diseases, manganism resulting from welding-fume exposure remains a controversial issue, as although the movement of manganese into specific brain regions has been established, the similar movement of manganese presented with other metals, such as welding fumes, has not been clearly demonstrated as being similar to that of manganese alone. Meanwhile, the competition between Mn and iron for iron transporters, such as transferrin and DMT-1, to the brain has also been implicated in the welding-fume exposure. Thus, the increased signal intensities in the basal ganglia, including the globus pallidus and subcortical frontal white matter, based on T1-weighted magnetic resonances in welders, require further examination as regards the correspondence with an increased manganese concentration. Accordingly, to investigate the movement of manganese after welding-fume exposure, 6 cynomolgus monkeys were acclimated for 1 mo and assigned to 3 dose groups: unexposed, low dose of (total suspended particulate [TSP] 31 mg/m3, 0.9 mg/m3 of Mn), and high dose of total suspended particulate (62 mg/m3 TSP, 1.95 mg/m3 of Mn). The primates were exposed to manual metal-arc stainless steel (MMA-SS) welding fumes for 2 h/day in an inhalation chamber system equipped with an automatic fume generator for 6 mo. Magnetic resonance imaging (MRI) studies of the basal ganglia were conducted before the initiation of exposure and thereafter every month. During the exposure, the blood chemistry was monitored every 2 wk and the concentrations of metal components in the blood were measured every 2 wk and compared with ambient manganese concentrations. The manganese concentrations in the blood did not show any significant increase until after 2 mo of exposure, and then reached a plateau after 90 days of exposure, showing that an exposure period of at least 60 days was required to build up the blood Mn concentration. Furthermore, as the blood Mn concentration continued to build, a continued decrease in the MRI T1 relaxation time in the basal ganglia was also detected. These data suggested that prolonged inhalation of welding fumes induces a high MRI T1 signal intensity with an elevation of the blood manganese level. The presence of a certain amount of iron or other metals, such as Cr and Ni, in the inhaled welding fumes via inhalation was not found to have a significant effect on the uptake of Mn into the brain or the induction of a high MRI T1 signal intensity.

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice.

INTRODUCTION: Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation. METHODS: BALB/cHeA-p53+/- mice were crossed with MSM/Ms-Atm+/- mice, and females of the F1 progeny ([BALB/cHeA x MSM/Ms]F1) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks. RESULTS: We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53+/- mice of the BALB/cHeA x MSM/Ms background. The singly heterozygous p53+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma. CONCLUSION: Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.

Identification of potential biomarkers of genotoxicity and carcinogenicity in L5178Y mouse lymphoma cells by cDNA microarray analysis.

In the present study, cDNA microarray analyses were performed with mouse cDNA chips in order to evaluate similarities and differences in the gene expression profiles for compounds differing in their genotoxic and carcinogenic potential. Eight test substances were evaluated, two each from four classes of compounds: genotoxic carcinogens (1,2-dibromoethane and glycidol), genotoxic noncarcinogens (8-hydroxyquinoline and emodin), nongenotoxic carcinogens (methyl carbamate and o-nitrotoluene), and nongenotoxic noncarcinogens (D-mannitol and 1,2-dichlorobenzene). Quadruplicate hybridization experiments were performed in order to identify a set of genes with significant expression changes for these four classes of substances. Twelve genes were consistently altered more than twofold by the genotoxic noncarcinogens while four genes were consistently regulated by the nongenotoxic carcinogens. One gene (Trp63) was identified whose expression was upregulated by all four genotoxic substances regardless of the presence or absence of carcinogenicity; this finding, however, was not confirmed by quantitative real-time RT-PCR. RT-PCR did confirm the change in expression of 9 of 15 genes (60%) identified by microarray analysis. Interestingly, the downregulated genes were least likely to be validated by real-time RT-PCR. Those genes showing more than a twofold change in expression level in response to at least one substance were further analyzed with hierarchical clustering after category assignment of each gene according to its main cellular function. Clustering revealed differences in the gene expression profiles between the genotoxic and nongenotoxic substances for genes involved in cell cycle control, the stress response, and the immune response. However, no clustering specific to all four carcinogenic substances was observed in any of the functional categories. Taken together, these results suggest that gene expression profiling in mouse lymphoma cells can provide valuable information for the evaluation of potential genotoxicity but may have limitations in predicting carcinogenicity.

Mapping of putative ether-anesthesia resistance gene using C57BL/6J and MSM/Ms mouse strains.

PURPOSE: We attempted to identify the locations of major mouse genes responsible for sensitivity to diethylether (ether) anesthesia, using microsatellite linkage analyses including Quantitative Trait Locus (QTL) analysis. METHODS: To determine the locations of ether anesthesia resistance genes on chromosomes, an ether anesthesia-resistant mouse strain, C57BL/6J (C57BL), and an ether anesthesia-sensitive mouse strain, MSM/Ms (MSM), were used. The sensitivity of mice to ether anesthesia was determined from the latency time required to lose the righting reflex during exposure to 4% ether vapor in air. The (C57BL x MSM) F(1) mice were found to be resistant to ether, showing that the resistant phenotype is genetically dominant. Twelve resistant and 12 sensitive mice were then selected from the 196 backcrossed F(2) mice (F(1) x MSM) at 11-16 weeks of age. Genomic DNA samples were extracted from the tails for mapping ether anesthesia-related genes using microsatellite linkage analyses. RESULTS: One major putative gene related to resistance to ether anesthesia was restricted in the region 23 to 37 cM from the centromere in chromosome 7 by primary and secondary linkage analyses. The QTL analysis narrowed the position of the gene to 29.0 cM, with a maximum logarithm of odds (LOD) score of 3.03, and it was termed Etan1 ( ether-anesthesia 1). CONCLUSION: Microsatellite linkage analyses, including QTL analysis, determined the location of the ether-resistance gene, Etan1, within a narrow range. Our findings should be helpful for further experiments, such as cloning of the gene governing the sensitivity to ether anesthesia in mice.

Fine localization of Nefl and Nef3 and its exclusion as candidate gene for lens rupture 2(lr2).

Cataract causing lr2 gene is found in the CXSD mouse, which is a recombinant inbred strain of BALB/c and STS mice. For the process of positional cloning of lr2, several candidate genes were selected in the middle region of chromosome 14, but most of them were excluded by combination of recombination and homozygosity mapping. Components of neurofilament proteins, neurofilament light polypeptide (Nefl) and neurofilament3 medium (Nef3), were linked to D14Mit87 which was not separated from the lr2 locus in the homozygosity mapping. When the expression levels of Nefl and Nef3 in eyes were compared in CXSD and BALB/c mice, there were no differences in expression levels. The cDNA sequences of the two genes from CXSD, BALB/c and STS mice were subsequently compared. Several nucleotide differences in cDNA sequences were detected between the mice strains but the majority of the changes were silent mutations that did not alter the amino acids. The sole amino acid difference, E567K in the glutamate rich region of Nfm, between BALB/c and CXSD was found to be a simple genetic polymorphism because the same substitution existed in STS, a non-cataract mouse strain. Therefore we excluded Nefl and Nef3 from the candidate genes for lr2 based on expression and mutation analyses.

Allelic loss analysis of lymphomas induced in Fas-heterozygous deficient mice.

Mutations of Fas (CD95/Apo-1) gene have been reported in various malignancies and therefore the Fas gene has been considered to be a tumor suppressor gene. To examine an involvement of Fas gene as a tumor suppressor gene in radiation lymphomagenesis, we examined the loss of heterozygosity (LOH) in lymphomas from (MSM/Ms x MRL-MpJ/Fas (lpr)) F(1) and (BALB/cHeA x MRL-MpJ/Fas (lpr)) F(1) hybrid mice. Lymphoma development by X-irradiation was efficiently observed in both F(1) hybrids. Frequent LOH was found on chromosomes 12 and 4 in the tumors from both F(1) mice, but no allelic loss on chromosome 19 containing Fas locus was found, and no wild-type allele of the Fas gene was lost in 51 lymphomas. Therefore, the putative tumor-suppressor gene regions responsible for lymphomagenesis might not considerably differ due to the Fas gene status.

cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results.

The potential application of toxicogenomics to predictive toxicology has been discussed widely, but the utility of the approach remains largely unproven. Using cDNA microarrays, we compared the gene expression profiles produced in mouse lymphoma cells by three genotoxic compounds, hydroxyurea (a carcinogen), p-anisidine (a noncarcinogen), and paclitaxel (carcinogenicity unknown). To minimize the effect of biological variability and technological limitations, quadruplicate observations were made for each compound and a subset of genes yielding reproducible induction/repression was selected for comparison. A method was applied to attach normalized expression data to genes with a low false-discovery rate (<0.1) to yield more confidence regarding differential expression. This analysis identified genotoxicity-specific gene expression. Seven genes were consistently upregulated and 12 downregulated more than 2-fold by the three genotoxic compounds. Using additional genes, the expression pattern induced by the genotoxic noncarcinogen, p-anisidine, was readily distinguished from that associated with the genotoxic carcinogen, hydroxyurea. Comparison of paclitaxel-induced expression data to data for p-anisidine and hydroxyurea suggested that paclitaxel's profile is more similar to the genotoxic noncarcinogen. With further supporting evidence it may be possible to perform large-scale monitoring of gene expression during drug and chemical development that can provide an early warning of potential toxicological responses.