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After E. coli cells (WP2 and WP2uvrA) were treated with chemical mutagens (methyl methanesulfonate, MMS; N-methyl-N-nitrosourea, MNU; 4-nitroquinoline 1-oxide, 4NQO) in 1/15 M phosphate buffer, the mutability of the treated cells plated on a D2O-agar plate was compared with that plated on an ordinary H2O-agar plate. The mutation frequency decreased more or less on the D2O-agar plate. The D2O-substitution effects, as termed by the relative mutation frequencies (MFD2O/MFH2O), are 0.92 for MMS, 0.29 for MNU, and 0.42 for 4NQO in WP2, and 0.68 for MMS, 0.49 for MNU, and 0.16 for 4NQO in WP2uvrA. The D2O effect seemed to be partly related to the function of the uvrA gene-associated products. The pH dependence of mutability was discussed in connection with the D2O-substitution effect. 相似文献
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The mouse NQO2 cDNA and gene with flanking regions were cloned and sequenced. Analysis of the primary structure of the mouse NQO2 protein revealed the presence of glycosylation, myristylation, protein kinase C and caseine kinase II phosphorylation sites. These sites are conserved in the human NQO2 protein. The mouse NQO2 gene promoter contains several important cis-elements, including the antioxidant response element (ARE), the xenobiotic response element (XRE), and an Sp1 binding site. Northern analysis of eight mouse tissues indicated wide variations in the expression of the NQO2 and NQO1 genes. NQO2 gene expression was higher in liver and testis compared with the NQO1 gene, which was highest in the heart. NQO1 gene expression was undetectable in the testis. Mouse kidney showed significantly higher expression levels of NQO1 compared with NQO2. Brain, spleen, lung, and skeletal muscle showed undetectable levels of NQO2 and NQO1 gene expression. NQO2 activity followed a more or less similar pattern of tissue-specific expression as NQO2 RNA. Interestingly, the NQO2 activity remained unchanged in the NQO1-/-mice tissues compared with NQO1+/+ mice, with the exception of the liver. The livers from NQO1-/-mice showed a 45% increase in NQO2 activity compared with the NQO1+/+ mice. The mouse NQO2 cDNA was subcloned into the pMT2 eukaryotic expression vector which, upon transfection in monkey kidney COS1 cells, produced a significant increase in NQO2 activity. Deletion of 54 amino acids from the N-terminus of the mouse NQO2 protein resulted in the loss of NQO2 expression and activity in transfected COS1 cells. This indicates that deletion of exon(s) encoding the N-terminus of NQO2 from the endogenous gene in mouse embryonic (ES) stem cells should result in NQO2-null mice. 相似文献
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Human lymphocyte were treated in G1 with 4-nitroquinoline-N-oxide (4NQO) and methyl methanesulfonate (MMS) and then incubated in the presence or absence of cytosine arabinoside (ara-C). There was an increase in aberration frequency in those cells incubated with ara-C compared with those treated with 4NQO or MMS alone. This increase was restricted to chromosome-type aberrations. When cells were treated in G2 with 4NQO and then incubated with ara-C until fixation, there was an increase in deletions compared with cells treated with 4NQO alone. No exchange aberrations were observed following any treatment even when deletion frequencies were high, as in the case with 4NQO plus ara-C treatment. These results suggest that ara-C can inhibit the repair of DNA damage induced by 4NQO and MMS that is converted into aberrations. They also show that the terms “S-dependent” and “S-independent” used to describe the modes of action of chemical clastogens are not valid. 相似文献
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NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO). 相似文献
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J Futran J D Kemp E H Field A Vora R F Ashman 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(3):787-792
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Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents. 相似文献
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M Nakajima M Kikuchi K Saeki Y Miyata M Terada F Kishida R Yamamoto C Furihata S W Dean 《Mutation research》1999,444(2):321-336
As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration. 相似文献
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Regulation of genes encoding NAD(P)H:quinone oxidoreductases 总被引:15,自引:0,他引:15
Jaiswal AK 《Free radical biology & medicine》2000,29(3-4):254-262
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J Tremblay V Hadrava U Kruppa T Hashimoto P Hamet 《Canadian journal of physiology and pharmacology》1992,70(4):565-572
Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Three mutagen-sensitive mutants, MS-1, M10 and Q31, were isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline-1-oxide (4NQO); and Q31 cells are cross-sensitive to UV and 4NQO. MMS-, X-ray- and UV-sensitive markers in these mutants behaved recessively in hybrids between pairs of these mutants as in hybrids between L5178Y and these mutants as reported before (Shiomi et al., 1982b). Complementation analyses were carried out by forming hybrids between two MMS-sensitive mutants (MS-1 and M10) and between two 4NQO-sensitive mutants (M10 and Q31). MMS and 4NQO survivals were measured in these hybrid cells. MS-1 and M10 were found to belong to different complementation groups for MMS-sensitive phenotypes. The hybrid clones between M10 and Q31 were as sensitive to 4NQO as each of the mutants, indicating codominance of 4NQO sensitivity in these mutants. The hybrids constructed with L5178Y and three mutants were stable as to their chromosome constitution for 100 days of cultivation without selective pressure. From the segregation studies on these hybrids, it is concluded that neither the X-ray-sensitive mutation in M10 nor the UV-sensitive mutation in Q31 is located on the X chromosome. 相似文献
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We describe a cytophotometric assay for unscheduled DNA synthesis (UDS) in asynchronously growing cells. Monolayer cultures of human HEp-2 and mouse MCa-11 cells were incubated with the carcinogen methyl-methane sulfonate (MMS), as well as with hydroxyurea and (3H)thymidine. Slides were prepared, and the DNA contents and areas of nuclei were measured by absorption cytophotometry. The labeling of the nuclei, determined on the basis of their DNA content to be in G0/G1, was selectively measured after the preparation of autoradiographs. The labeling of the G0/G1 cells increased with increasing doses of MMS. We also found that the increased nuclear labeling after MMS treatment was not due to induction of replicative DNA synthesis or selective destruction of G0/G1 cells. The results of this assay compared favorably with a standard biochemical method for measuring unscheduled DNA synthesis by benzoylated naphthoylated DEAE cellulose chromatography. 相似文献