Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene.

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.     

Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites.

The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli. Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites. To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid. The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside. From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties. In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5. When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion. Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule. The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E. coli. The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E. coli.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA+). The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E. coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant. In the chromosome of E. coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. coli cells to the lethal effect of gamma-rays and UV light.     

Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro. II. Induction of oxidative DNA damage and mutations in the mouse lymphoma assay

We recently showed that treatment of V79 cells with hyperbaric oxygen (HBO) efficiently induced DNA effects in the comet assay and chromosomal damage in the micronucleus test (MNT), but did not lead to gene mutations at the hprt locus. Using the comet assay in conjunction with bacterial formamidopyrimidine DNA glycosylase (FPG protein), we now provide indirect evidence that the same treatment leads to the induction of 8-oxoguanine, a premutagenic oxidative DNA base modification in V79 and mouse lymphoma (L5178Y) cells. We also demonstrate that HBO efficiently induces mutations in the mouse lymphoma assay (MLA). Exposure of L5178Y cells to HBO (98% O(2); 3bar) for 2h caused a clear mutagenic effect in the MLA, which was further enhanced after a 3h exposure. As this mutagenic effect was solely due to the strong increase of small colony (SC) mutants, we suggest that HBO causes mutations by induction of chromosomal alterations. Molecular characterization of induced SC mutants by loss of heterozygosity (LOH) analysis showed an extensive loss of functional tk sequences similar to the pattern found in spontaneous SC mutants. This finding confirmed that the majority of HBO-induced mutants is actually produced by a clastogenic mechanism. The induction of point mutations as a consequence of induced oxidative DNA base damage seems to be of minor importance.     

NO-dependent pathway of the modifying of the cellular sensitivity to gamma- and neutron irradiation

The modifying effect of L-NAME, the NO-synthase inhibitor and D-NAME, the inactive enantiomer was investigated in human carcinoma cells (HeLa) and Chinese hamster fibroblasts (V-79) exposed to different doses of gamma-rays and 0.85 MeV neutrons. We estimated the level of the chromosome aberrations manifested as the bridges and fragments in anaphases. Radioprotective effect of L-NAME showed the inverse dependence on the exposure dose and at low doses (1 Gy) it was higher in the V-79 cells, than in the HeLa cells. However, at high doses (3, 4, 6 Gy) the efficiency of L-NAME for these cellular lines was almost equal (DFR = 2). The modifying effect of L-NAME was almost equal for gamma-irradiation and neutrons, although the exposure of V-79 cells to neutrons induced more the asymmetric chromosome aberrations (RBE = 4). The D-NAME had no effect on the level of the radiation-induced chromosome aberrations, although D-NAME treatment of cells increased the chromatin condensation, as well as L-NAME. The counteractive condensation does not play the major role in the radioprotective effect of L-NAME. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the inducible NO-synthase.     

Effect of synthetic polycation polyallylamine on adhesion and viability of CHL V-79 RJK Chinese hamster fibroblasts with various heat resistance

The effects of synthetic polycation polyallylamine (PAA) on the adhesion of CHL V-79 RJK fibroblasts and CHL V-79 RJK40 cells resistant to 40°C and the attachment of these cells to polycation immobilized on a polystyrene surface have been studied. We also investigated PAA cytotoxicity. It was shown that cell adhesion on polystyrene plastic coated with PAA depended on the PAA dose and did not depend on the heat resistance of the cells. The effect of PAA on cell adhesion to uncoated polystyrene surface after cell exposure to PAA depended not only on the polycation concentration but also on cell heat resistance. Pretreatment of cells with nontoxic concentrations of PAA inhibited CHL V-79 RJK cell adhesion and did not change adhesive properties of thermotolerant cells. PAA is toxic for CHL V-79 RJK and CHL V-79 RJK40 cells only at concentration of 100 μg/ml (MTT assay). PAA-induced acute toxicity was accompanied by necrotic-like cell death. Possible mechanisms of the PAA effect on the behavior of cells with different metabolic characteristics defined by heat resistance are discussed.     

The influence of sodium nitrite on the osmoresistance of Chinese hamster cells

We have previously demonstrated that incubation of V-79 cells in the medium containing the nitric oxide donor, NaNO2, increases cell resistance to damaging effect of gamma-rays, UV radiation and hyperthermia. In the present study, we investigated the effects of the nitric oxide donor on the sensitivity of V-79 cells to changes in osmomolarity of the medium by adding different amounts of sodium chloride or water. We found that pretreatment of the cells with NaNO2 resulted in a significant increase in the number of growing cells in 48 h after the treatment. The osmomolarity-dependent morphological changes in cultured cells were also substantially diminished following NaNO2 treatment. This effect could be observed under both hyper- and hypoosmosis, and was dependent on concentration of sodium chloride in hypertonic medium (being maximal under 0.17 M NaCl) and on the amount of water in hypotonic medium (being maximal under 1.1 times the dilution with water). In the experiments with increased osmomolarity, we found that the observed increase in the number of growing cells following NaNO2 treatment was accompanied with a significant increase of the mitotic index. These findings indicate that nitric oxide increases cell resistance to the damaging effects of osmotic shock in the way which is similar to the protective effect of these molecules against radiation and hyperthermia. Similarities in the effects of NaNO2 under different conditions leading to cell damage suggest that nitric oxide might serve as the universal factor participating in recovery of damaged cells and mediating increased cellular resistance to the damaging conditions.     

Mutagenic action of cadmium on the sex cells of male mice

Possible mutagenic effect of cadmium chloride was studied by determining the frequency of dominant lethal mutations induced in germ cells of male mice. Water solution of CdCl2 was injected intraperitoneally to male mice at doses of 1.0, 2.0 and 4.0 mg/kg. The results obtained did not reveal any mutagenic effect of this compound. The dose of 4.0 mg/kg CdCl2 resulted in the death of spermatocytes and spermatogonia and the sterility of male mice. Cadmium chloride at a dose of 2.0 mg/kg did not affect the frequency of dominant lethal mutation induced by gamma-rays 60Co at a dose of 450 r in germ cells of male mice.     

Experimental study of the antimutagenic properties of 5-methylresorcinol

A study was made of the effect of 5-methylresorcinol (5-MR) on mutagenic activity of benzo(a)pyrene (BP) and of the action of gamma-radiation in in-vitro and in-vivo systems. The induction of direct gene mutations in Chinese hamster cells V-79 and micronuclei in mouse bone marrow reticulocytes was efficiently suppressed by 5-MR treatment. The antimutagenic activity of 5-MR can be explained by inhibition of free-radical processes.     

Accelerated Repair and Reduced Mutagenicity of DNA Damage Induced by Cigarette Smoke in Human Bronchial Cells Transfected with E.coli Formamidopyrimidine DNA Glycosylase

Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na⁺K⁺-ATPase locus (oua^r) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells’ capacity to repair damaged DNA.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

The effect of synthetic polycation polyallylamine on adhesion and viability of CHL V-79 RJK Chinese hamster fibroblasts with different heat resistance

The effects of synthetic polycation polyallylamine (PAA) on adhesion of CHL V-79 RJK fibroblasts and CHL V-79 RJK40 cells resistant to 40 degrees C, and attachment to these cells to polycation immobilized on polystyrene surface were studied. We have also investigated the cytotoxicity of PAA. It was shown that cell adherence to polystyrene plastic coated with PAA was enhanced or decreased in dependence of the PAA concentration used for surface coating and did not depend on heat resistance of investigated cell lines. The effect of PAA on cell adhesion to uncoated polystyrene surface after cell exposure with PAA depended not only on the polycation concentration, but also on the extent of heat resistance of investigated cell lines. Pretreatment of CHL V-79 RJK cells with PAA at the nontoxic concentrations led to inhibition of cell adhesion, and no change in adhesive properties of thermoresistant cells was found under the same conditions. PAA was toxic for CHL V-79 RJK and CHL V-79 RJK40 cells only at concentrations of 100 microg/ml (MTT assay). PAA-induced acute toxicity was accompanied by necrotic-like cell damage. Possible mechanisms of the PAA effect on the behaviour of cells with different structural and metabolic characteristics that are due to the temperature of cell cultivation are discussed.     

Quantitative mutagenesis at the adenosine kinase locus in Chinese hamster ovary cells. Development and characteristics of the selection system

Stable mutants exhibiting high degree of resistance (100-1000-fold) to various nucleoside analogs viz, toyocamycin, tubercidin, 6-methyl mercaptopurine riboside (6-MeMPR) and pyrazofurin, are obtained at similar frequency (congruent to 1 X 10(-4] in CHO cells. The mutants resistant to any of the above analogs exhibit similar degree of cross-resistance to the other three nucleoside analogs, and all of the mutants examined contained no measurable activity of the purine salvage pathway enzyme adenosine kinase (AK) which converts these analogs to their phosphorylated derivatives. These results indicate that very similar mutants are selected using any of these analogs. The recovery of AK- mutants in CHO cells is not affected by cell density (up to at least 5 X 10(5) cells per 100-mm diameter dish) and after treatment with mutagen(s) maximum mutagenic effect is observed after 7-8 days, which then remains unchanged for the next several days. Treatment of CHO cells with a number of mutagenic agents e.g. ethyl methanesulfonate, ICR170, ultraviolet light, and benzo[a]pyrene, led to a nearly linear concentration-dependent increase in the frequency of the AK- mutants in cultures. The mutagenic response of the AK locus to these agents compared favorably with that of the HGPRT locus (6-thioguanine resistance) within the same experiments. These results show that the selection system for AK- mutants provides an additional valuable genetic marker for quantitative mutagenesis studies in CHO cells.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine.

The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation can be postponed or diminished by a post-irradiation treatment with 1.0 to 1.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibit depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis have no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiates X-ray-induced cell killing, this reduction in survival is due primarily to effects on cells in S-phase.     

Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells.

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.     

The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene

Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E. coli strain (JM105). The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient. Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the formation of mutations.     

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement

We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.