The genetics and specificity of the constitutive excision repair system of Bacillus subtilis

An isogenic set of DNA repair-proficient and -deficient strains of B. subtilis, cured of all prophages, were constructed and analyzed for their sensitivities to selected mutagens. The results demonstrated that the lethal damage caused by ultraviolet (UV) radiation and by 4-nitroquinoline-1-oxide (4NQO) were repaired by the bacterial excision and/or recombination repair systems. In contrast, the lethal damages caused by ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) were removed from the DNA by the recombination repair system of the bacteria, and not by the excision repair system. Significantly, the bacteria required both a functional recombination repair system and a functional excision repair system in order to remove the DNA damage caused by the bifunctional alkylating agent mitomycin C (MC).     

Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.     

Influence of UV irradiation, 4-nitroquinoline-u-oxide, methylmethanesulfonate, and bleomycin on the acitivity of an alkaline deoxyribonuclease from human lymphocytes.

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their Separation by microdisc electrophoresis was used to determine the influence of UV irradiation, 4-nitroquinoline-1-oxide, methylmethanesulfonate, and Bleomycin on the activity of an alkaline deoxyribonuclease, specific for denatured and UV-irradiated DNA, from human lymphocytes. The activity of this endonuclease increased in UV-irradiated cells and in cells incubated with 4-nitroquinoline-1-oxide and Bleomycin. The increment was not altered by hydroxyurea inhibiting DNA synthesis, or coffeine preventing recombinational repair. It is suggested that the nuclease is involved in repair replication of mispaired DNA regions.     

Mechanisms of DNA repair disorders in human cells. Genome instability in lymphocytes of patients with myopathy related to DNA repair disorders

Increased level of cytogenetic damages was observed in lymphocytes of 10 patients with muscular dystrophy (Duchenne, Erba, Landuzi--Degerin). Analysis of DNA repair synthesis revealed inhibition of this process in lymphocytes of 10 patients observed. On the other hand, decreased reactivation of vaccinia virus and increased level of virus mutagenesis induced by 4-nitroquinoline-1-oxide and bleomycin was noted in the experiments with lymphocytes of 3 patients observed.     

Mitomycin C, 4-nitroquinoline-1-oxide and ethyl methanesulfonate induced long-lived lesions in DNA which result in SCEs during successive cell cycles in human lymphocytes

The present study was carried out in order to analyze how persistent the lesions in DNA are which elicit sister-chromatid exchanges (SCEs), induced by three different chemical agents, mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) and ethyl methanesulfonate (EMS), in proliferating human lymphocytes. Cells were exposed to the mutagens for 1 h just before starting bromodeoxyuridine substitution and SCEs were examined in third-cycle metaphases showing three-way-differential staining, by means of our previously standardized method. The results show that, in spite of the fact that these three compounds have different modes of action, the lesions induced by all of them seem to be capable of persisting in DNA and eliciting SCEs for at least three successive cell cycles.     

Simultaneous demonstration of UV-type and ionizing radiation-type DNA repair by the nucleoid sedimentation technique

The nucleoid sedimentation technique is one of the most sensitive methods for measuring DNA excision repair. With this technique, we have shown that both UV- and ionizing radiation-type repair (the latter induced by bleomycin) can be discriminated in HeLa and normal diploid cells using 1-beta-D-arabinofuranosylcytosine. The latter compound inhibits UV-type repair synthesis, and thus causes DNA breaks due to enzymic incision to persist, but has no effect on rejoining DNA after ionizing radiation-type damage. It was then possible to prove that 4-nitroquinoline-1-oxide induces both types of lesions which are repaired simultaneously. This effect could be demonstrated in HeLa and normal human diploid cells in a single experimental set-up.     

Disorders of repair processes in the lymphocytes of schizophrenia patients, cultured in vitro

Repair disorders of DNA damage induced by gamma-radiation and 4-nitroquinoline-1-oxide treatment in cultivated lymphocytes of patients with schizophrenia. 13 criteria were used for estimation of repair activity (reactivation of viral host cells) repair synthesis, reparation of DNA breaks, formation of spontaneous and induced sister chromatid exchanges.     

Enhancing effects of cinoxate and methyl sinapate on the frequencies of sister-chromatid exchanges and chromosome aberrations in cultured mammalian cells

Sister-chromatid exchanges (SCEs) induced by mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or UV-light in cultured Chinese hamster ovary cells (CHO K-1 cells) were enhanced by cinoxate (2-ethoxyethyl p-methoxycinnamate) or methyl sinapate (methyl 3,5-dimethoxy 4-hydroxycinnamate). Both substances are cinnamate derivatives and cinoxate is commonly used as a cosmetic UV absorber. Methyl sinapate also increased the frequency of cells with chromosome aberrations in the CHO K-1 cells treated with MMC, 4NQO or UV. These increasing effects of methyl sinapate were critical in the G1 phase of the cell cycle and the decline of the frequencies of UV-induced SCEs and chromosome aberrations during liquid holding was not seen in the presence of methyl sinapate. Both compounds were, however, ineffective in cells treated with X-rays. In cells from a normal human embryo and from a xeroderma pigmentosum (XP) patient, MMC-induced SCEs were also increased by the post-treatment with methyl sinapate. The SCE frequencies in UV-irradiated normal human cells were elevated by methyl sinapate, but no SCE-enhancing effects were observed in UV-irradiated XP cells. Our results suggest that the test substances inhibit DNA excision repair and that the increase in the amount of unrepaired DNA damage might cause the enhancement of induced SCEs and chromosome aberrations.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Baseline and mutagen-induced sister-chromatid exchanges in cultures of human whole blood and purified fresh or frozen lymphocytes

Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (Time 1) and 6 months (Time 6) of cryopreservation and then challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreservation of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.     

Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

The results presented in this report demonstrate that an 18–20 hour exposure/³H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin Bi and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18–20 hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18–20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10^–3M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 × 10^–4M when the 18–20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 × 10^–5M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10^–5M when the 18–20 hour exposure/labeling period was used.Abbreviations 2AAF 2-acetylaminofluorene - AB₁ aflatoxin B₁ - BENZ benzidine - DEB diepoxybutane - DEN diethylnitrosamine - DMN dimethylnitrosamine - EMS ethyl methanesulfonate - MITC mitomycin C - MMS methyl methanesulfonate - NG mean net nuclear grain counts - NM nitrogen mustard - 4NQO 4-nitroquinoline-N-oxide     

Mechanism of increased susceptibility to 4-nitroquinoline-1-oxide in cultured skin fibroblasts from patients with familial polyposis coli

About 50% of the strains of cultured fibroblasts from patients with familial polyposis coli (FPC) exhibited increased susceptibility to cytotoxicity of 4-nitroquinoline-1-oxide (4NQO) compared with cells from normal individuals. The FPC cells that showed hyper-sensitivity to 4NQO were also hyper-sensitive to mitomycin C (MMC), but susceptibilities of these cells to UV radiation, methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were within the normal range. The extent of single-strand scission of DNA in the 4NQO-sensitive FPC cells was greater than in normal cells, and the amount of [14C]4NQO bound to DNA in the FPC cells was twice as high as in normal cells. The rate of release of [14C]4NQO from DNA by the post-culture was the same as in both FPC and normal cells. The 4NQO-sensitive FPC cells exhibited increased 4NQO-reductase activity; the level of this activity was consistent with the extent of the decrease in colony formation by 4NQO. These results suggest that the enhanced ability to activate 4NQO might be an important factor in the mechanism of susceptibility of FPC cells to 4NQO rather than the reduced ability to repair DNA.     

Pollen DNA repair after treatment with the mutagens 4-nitroquinoline-1-oxide, ultraviolet and near-ultraviolet irradiation, and boron dependence of rapair

Summary Irradiation of dry, mature pollen from Petunia hybrida with near-ultraviolet light from an erythemal-sunlamp gave rise to a repair-like, unscheduled DNA synthesis during the early stages of in vitro germination. Like that brought about by farultraviolet light from a germicidal lamp, this DNA synthesis is enhanced by hydroxyurea added to the germination medium, and reduced by photoreactivating light given after ultraviolet irradiation and before germination begins. It is concluded that pollen, often receiving considerable exposure to sunlight, has, in addition to the protection afforded by the ultraviolet filtering effect of yellow pigments, also the capacity to repair ultraviolet produced changes in DNA, by both photoreactivation and dark repair processes.Because mature Petunia pollen is arrested at the G₂ stage of the cell cycle, germinating pollen provides us with a highly synchronous plant tissue with a very low background of DNA replicative synthesis suitable for sensitive measurement of DNA repair synthesis. Thus we have shown that 4-nitroquinoline-1-oxide, at concentrations greater than 0.001 mM, gives rise to an unscheduled DNA synthesis which is enhanced by hydroxyurea. Like that induced by ultraviolet radiation, the chemical mutagen brings about DNA repair only during the early stages of pollen germination, and further it has been possible to show that repair ceases at about the time that generative cell division and pollen tube elongation begins.Boron addition enhances both ultraviolet and 4-nitroquinoline-1-oxide induced repair synthesis. By delaying the chemical mutagen initiation of repair until after germination has begun, we have been able to show that boron is most beneficial during the first hour of germination. It is postulated that this is achieved through an as yet unknown effect of boron on the supply of precursors before pollen cell metabolism is fully committed to pollen tube synthesis later in the germination period.     

Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli

Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination. EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations. We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E. coli B/r WP2 cells. Each DNA stain alone showed no mutagenicity to the strain. Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I. SYBR Green I had a much greater effect. No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents. Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4',6-diamidino-2-phenylindole (DAPI). UV- and MX-induced mutations in E. coli WP2s (uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange. Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment.     

Validation of single cell gel assay in human leukocytes with 18 reference compounds

To validate the alkaline single cell gel (SCG) assay as a tool for the detection of DNA damage in human leukocytes, we investigated the in vitro activity of 18 chemicals. Thirteen of these chemicals (pyrene (PY), benzo(a)pyrene (BaP), cyclophosphamide (CP), 4-nitroquinoline-1-oxide (4NQO), bleomycin (BLM), methylmercury chloride (MMC), mitomycin C (MTC), hydrogen peroxide (HP), diepoxybutane (DEB), glutaraldehyde (GA), formaldehyde (FA), griseofulvin (GF), sodium azide (NA)) are genotoxic in at least one cell system, while five compounds (ascorbic acid (AA), glucose (GL), D-mannitol (MAN), O-vanillin (VAN), chlorophyllin (CHL)) are classified as non-genotoxic. In this in vitro SCG assay, PY, BaP and CP were positive with exogeneous metabolic activation (rat S9 mix) while 4NQO, BLM, MMC, MTC, hydrogen peroxide, and diepoxbutane were positive in the absence of metabolic activation. CHL and VAN were unexpectedly found to induce a dose-dependent increase in DNA migration. AA, GL, and MAN were negative in a non-toxic range of doses. GF gave equivocal results, while FA and GA increased DNA migration at low doses and decreased DNA migration at higher doses. This behaviour is consistent with the known DNA damaging and crosslinking properties of these compounds. These data support the sensitivity and specificity of this assay for identifying genotoxic agents.     

Evidence for a Micrococcus luteus gene homologous to uvrB of Escherichia coli

Summary Restriction fragments of Micrococcus luteus DNA that contained the gene defined by the mutation of an excision repair-deficient mutant, UV^sN1, were cloned from both the parental and mutant strains with the Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to ultraviolet, mitomycin C, and 4-nitroquinoline-1-oxide by one-step transformation. Determination of the nucleotide sequences revealed an open reading frame potentially coding for a protein of 709 amino acid residues, within which the mutation was identified as a CGTA transition causing a change from serine to phenylalanine. The putative product of the open reading frame showed an extensive amino acid sequence homology to the E. coli UvrB protein comprising 673 residues; the homologous region extended over the greater parts of both polypeptides, in which 55% and 17% of the 659 pairs of aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. This indicates that the gene defined by the UV^sN1 mutation represents a homolog of the E. coli uvrB gene, implying the presence in M. luteus of an enzyme complex homologous to the E. coli UvrABC excinuclease.Abbreviations Ap ampicillin - AP apurinic-apyrimidinic - MC mitomycin - C: 4NQO 4-nitroquinoline-1-oxide - r resistant - s sensitive - UV ultraviolet Dedicated to the memory of Shunzo Okubo (1930–1978) who played the pivotal role in our earlier studies on the M. luteus repair system     

Variations on the standard protocol design of the hepatocyte DNA repair assay

Several variations on the standard primary rat hepatocyte DNA/repair assay were evaluated for their ability to enhance the sensitivity of this genotoxicity test system. The use of hamster hepatocytes proved to be a much more sensitive system than rat hepatocytes for detecting the DNA repair inducing ability of the nitrosamines, dimethylnitrosamine and diethylnitrosamine, and the aromatic amines, 2-acetylaminofuorene, 9-aminoacridine, 1-naphthylamine and benzidine. In addition, hamster hepatocytes were a more sensitive indicator of the genotoxicity of the azo dyes, o-aminoazotoluene, Congo Red and Evans Blue. However, the azo reduction product of the azo dyes Congo Red, Trypan Blue and Evans Blue, benzidine and o-tolidine, respectively, were active in both rat and hamster hepatocytes at concentrations that were 10–100 fold lower than the parent dyes. This suggests that little or no azo reduction of the dyes occurred in the in vitro assay systems. The in vivo-in vitro variation of the rat hepatocytes DNA/repair assay exhibited a positive DNA repair response with the azo dye solvent Yellow S, which was negative in the standard in vitro assay. The in vivo-in vitro hepatocyte DNA repair assay was also more sensitive for detecting the genotoxic activity of Evans Blue, which was positive in the in vivo-in vitro assay and equivocal in the standard in vitro assay. Also, Solvent Yellow 14 was negative in the in vitro assay, but induced an equivocal DNA repair response in the in vivo-in vitro assay system. A treatment/³H-thymidine labeling period of approximately 18 hours, compared to 4 hours, was demonstrated to be superior for detecting the DNA repair elicited by the mutagens 4-nitroquinoline-1-oxide, mitomycin C, dimethylnitrosamine and methyl methanesulfonate in the in vitro rat hepatocyte assay. There was little or no difference observed between the 4 hour and 18 hour treatment/ labeling incubation periods for the detection of DNA repair induced by 2-acetylaminofluorene, aflatoxin B₁, and benzidine. The data suggest that these several variations on the standard rat hepatocyte DNA/ repair assay should be considered when evaluating the genotoxicity of chemicals for safety purposes.Abbreviations 2-AAF 2-acetylaminofluorene - o-AT o-aminoazotoluene - DMN dimethylnitrosamine - DMSO dimethylsulfoxide - FMN flavin mononucleotide - MMS methyl methanesulfonate - 4-NQO 4-nitroquinoline-1-oxide - PRI Pharmakon Research International - RTI Research Triangle Institute     

Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells

Xeroderma pigmentosum (XP) cells are dificient in the repair of damage induced by ultraviolet irradiation. Excision-repair-deficient XP cell strains have been classified into 7 distinct complementation groups, according to results of studies on cell fusion and UV irradiation. XP cells are not only abnormally sensitive to UV, but also to a variety of chemical carcinogens, including 4-nitroquinoline-1-oxide (4NQO). Complementation analysis with XP strains from 4 different complementation groups with respect to the repair of 4NQO-induced DNA damage revealed that the classification of the strains into complementation groups with respect to 4NQO-induced repair coincides with the classification based on the repair of UV damage.     

Mechanisms of disruption of DNA in human cells. Mutagenesis of a virus in xeroderma pigmentosum cells as an indicator of a defect in DNA repair

Lymphocytes of two patients with Xeroderma pigmentosum, their mothers and cell lines isolated from the third patient were examined. The reaction of vaccinia virus treated with mutagens, the index of virus-induced mutagenesis and DNA repair synthesis were the criteria of estimation of cell repair activity. Significant inhibition of DNA repair synthesis was detected in cells of all the patients observed (primary and established lines) in the experiments with UV-irradiation and 4-nitroquinoline-4-oxide. These indexes correlated with decreased virus reactivation and increased level of virus-induced mutagenesis. Inhibition of DNA repair synthesis was also revealed in lymphocytes of both mothers. These data claim a possibility of discovering heterozygotes, the carriers of mutant genes. The data on virus-induced mutagenesis serve as a simple test for detection of DNA repair disorders.