Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6 VI. The correlation between UV-induced DNA lesions and chromosomal aberrations,and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied.The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-β-d-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G₁ cells treated under similar conditions.Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G₁ give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations.Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single- and double-strand breaks. Substantial increase of the parameter "% of DNA in tail" was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double-strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains.     

Extreme cytotoxicity and susceptibility to hprt mutagenesis in Ku-deficient xrs-6 cells treated with bleomycin in plateau phase

In an attempt to determine the possible role of Ku-dependent end joining in mutagenesis resulting from DNA double-strand breaks, mutations induced by bleomycin at the hprt locus in plateau phase normal CHO-K1 and Ku-deficient xrs-6 cells were examined. Plateau phase xrs-6 cells were 500-fold more sensitive to chronic bleomycin treatment than were CHO-K1 cells. XRCC4-deficient XR-1 cells were approximately 100-fold and DNA-PKcs-deficient XR-C1 and V-3 cells 15- to 30-fold more sensitive than CHO-K1 cells. These hypersensitivities are much greater than those previously reported for acute treatments with bleomycin or ionizing radiation. While the induced mutation frequencies at comparable levels of survival were slightly lower in xrs-6 cells, mutations were induced by bleomycin at much lower concentrations in xrs-6 than in CHO-K1 cells. For both cell lines bleomycin treatment resulted in a marked increase in the incidence of complete hprt deletions, while point mutations in hprt cDNA were rare. The results suggest that bleomycin-induced double-strand breaks tend to generate very large deletions in both cell lines and that this effect occurs at much lower levels of double-strand breaks in Ku-deficient than in normal cells.     

Evidence to support the existence of efficient DNA double-strand break rejoining in a radiosensitive mutant of V79-4 following irradiation with 250 kVp X-rays or neutrons

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irsl. This was done in order to investigate further the likelihood that irsl carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks.The mutant displayed an equal increase in sensitivity to the lethal of neutrons, as compared to X-rays. Both irsl and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irsl also showed an exponential survival after either X-rays or neutrons.The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10–40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irsl seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irsl cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10–40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line.The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irsl again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irsl is a misrepair mutant.     

Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.     

Rejoining of DNA strand breaks induced by propylene oxide and epichlorohydrin in human diploid fibroblasts

The repair kinetics of DNA single- and double-strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half-life was estimated to be ∼15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half-life of ∼15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5–10 mMh and 0.5–1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half-life of ∼2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half-life for DSBs rejoining was estimated to be ∼2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low-to-moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO. Environ. Mol. Mutagen. 32:223–228, 1998 © 1998 Wiley-Liss, Inc.     

Differential effects of luminol,nickel, and arsenite on the rejoining of ultraviolet light and alkylation-induced DNA breaks

When Chinese hamster ovary cells were treated with ultraviolet (UV) light or methyl methanesulfonate (MMS), a large number of DNA strand breaks could be detected by alkaline elution. These strand breaks gradually disappeared if the treated cells were allowed to recover in a drug-free medium. The presence of nickel or arsenite during the recovery incubation retarded the disappearance of UV-induced strand breaks, whereas the disappearance of MMS-induced strand breaks was retarded by the presence of arsenite or of luminol, a new inhibitor for poly(ADP-ribose) synthetase. Luminol, however, had no apparent effect on the repair of UV-induced DNA strand breaks, and nickel had no effect on the repair of MMS-induced DNA strand breaks. When UV- or MMS-treated cells were incubated in cytosine arabinofuranoside (AraC) plus hydroxyurea (HU), a large amount of low molecular weight DNA was detected by alkaline sucrose sedimentation. The molecular weight of these DNAs increased if the cells were further incubated in a drug-free medium. This rejoining of breaks in cells pretreated with UV plus AraC and HU was inhibited by nickel and by arsenite, but not by luminol. The rejoining of breaks in cells pretreated with MMS plus AraC and HU was inhibited by luminol and by arsenite, but not by nickel. These results suggest that different enzymes may be used in DNA resynthesis and/or ligation during the repairing of UV- and MMS-induced DNA strand breaks, and that nickel, luminol, and arsenite may have differential inhibitory effects on these enzymes. © 1994 Wiley-Liss, Inc.     

Repair kinetics in CHO cells of X-ray induced DNA damage and chromatid aberrations during a cell cycle extended by transient hypothermia

Transient hypothermia was employed to extend the G2 phase of CHO K1 cells in order to facilitate study of the repair of X-ray induced chromatid and DNA damage. Thus G2 + 1/2M at 37 degrees C of 2.9 h was lengthened to 5.7 h at 33 degrees C and 7.3 h at 29 degrees C. While chromatid break kinetics remained essentially unaltered at 33 degrees C, at 29 degrees C there was an initial shoulder followed by a decrease in breaks similar to that at 37 and 33 degrees C. Although fewer exchanges were observed at 33 and 29 degrees C than at 37 degrees C, a similar kinetic involving a sharp initial rise followed by a plateau was observed at 33 and 29 degrees C, and, as far as could be judged, also at 37 degrees C. The failure of G2 prolongation to influence the rate of break disappearance was taken as evidence in support of the view that the disappearance of chromatid breaks represented a repair process rather than the decline of chromosomal radiosensitivity throughout this phase, though the possibility of a reduced sensitivity close to the G2/M border remained open. This hypothesis was supported by the mainly flat kinetics of exchanges. The data were taken as further evidence that chromatid rejoining and misjoining (exchanges) are essentially different processes. The rates of repair of DNA double-strand breaks as measured by neutral filter elution were similar at 37 and 33 degrees C, while there was evidence of inhibition at 29 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radioation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

Protection provided by exogenous DNA ligase in G0 human lymphocytes treated with restriction enzyme MspI or bleomycin as shown by the comet assay

DNA double-strand breaks (DSB) may arise either spontaneously during cellular processes or as a result of exposure to DNA-damaging agents such as ionizing radiation, or radiomimetic agents such as restriction endonucleases or bleomycin. It is widely accepted that nonrepaired or misrepaired DSB are the main lesions leading to the production of chromosomal aberrations, mutagenesis, oncogenic transformation, and cell killing. Studies focusing on this relationship, as well as the possible modulation of DNA repair mechanisms, are currently of major interest. A wide variety of test systems are available to study DNA damage. In the last few years, single-cell gel electrophoresis, commonly known as “comet assay,” has been considered a rapid, sensitive, and visual method for quantifying DNA strand breaks and alkali-labile damage in individual cells. In this study, making use of the comet assay, we tried to find out if under conditions that maintain chromatin structure the DNA ligase from T4 phage is able to facilitate the rejoining of strand breaks with different end structures, induced by the restriction endonuclease MspI or bleomycin in living human lymphocytes in a nonproliferating state. T4 DNA ligase, as well as the restriction endonuclease or bleomycin, were introduced together by electroporation into human lymphocytes. Our results support the idea that it is possible to modulate the DSB-rejoining of different DNA strand-breaking agents by exogenous T4 DNA ligase. Environ. Mol. Mutagen. 32: 336–343, 1998 © 1998 Wiley-Liss, Inc.     

Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1×10⁻² breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15–20%) after 24 h repair incubation in the tumor cell line, 5–10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Thymidine in the micromolar range promotes rejoining of UVC-induced DNA strand breaks and prevents azidothymidine from inhibiting the rejoining in quiescent human lymphocytes

The effect and inter-individual variation in the effect of exogenously added deoxynucleosides (2×10⁻⁶ M) on rejoining of UVC-induced DNA strand breaks was examined in quiescent human lymphocytes from 25 healthy persons. Thymidine at concentrations below 2×10⁻⁶ M, effectively and with statistically extreme significance, increased rejoining of UVC-induced DNA strand breaks in the lymphocytes of every one of the 25 persons tested (p<0.0001, Wilcoxon's signed ranks test). The mean stimulation after 20 h of postirradiation repair was 48% (range 18–78%) with an inter-individual variation of 30% (coefficient of variation, CV). Deoxyguanosine stimulated rejoining in 16, but inhibited in three of 19 test persons (mean stimulation 28%, range −31 to 71%). The stimulating effect of deoxyguanosine was also extremely significant (p<0.0004). Deoxycytidine and deoxyadenosine stimulated rejoining in some persons and inhibited it in others, and without statistical significance (p values above 0.5). The stimulating effect of thymidine was significantly inhibited by deoxycytidine (p<0.05, n=12) whereas deoxyguanosine neither promoted or inhibited the stimulation by thymidine (p=1, n=12). Rejoining of DNA strand breaks induced by methyl methanesulfonate did not appear significantly stimulated or inhibited by any of the four deoxynucleosides. Finally, the inhibiting effect of azidothymidine (AZT) on rejoining of UVC-induced DNA strand breaks was nullified by the addition of thymidine. In three donors examined, 10⁻⁴ M AZT inhibited the rejoining by about 40–50%. The presence of less than 10⁻⁵ M thymidine reduced the level of UVC-induced DNA strand breaks to below the level in control lymphocytes allowed to repair without AZT. These results indicate that among the four deoxynucleoside triphosphates, dTTP has a crucial role on the repair of UVC-induced DNA damage in quiescent lymphocytes. The results also indicate that an expansion of the dTTP pool may counteract the inhibiting effect of AZT on DNA repair in quiescent lymphocytes.     

Adriamycin-induced DNA strand breaks in HeLa and in P388 leukaemia cells detected using in situ nick translation

DNA strand breaks produced by adriamycin (ADR) were measured in HeLa cells and ADR-sensitive and -resistant P388 leukaemia cells, using the in situ nick translation method. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labelled dTTP, and were visualized by autoradiographic observation of the grains. The DNA strand breaks in the HeLa cells increased in a dose-dependent manner, compared with findings in the untreated control cells, i.e., 15.2 fold at 20 micrograms/ml of ADR for 1 h. This level correlated with DNA single-strand breaks detected by the alkaline elution method. DNA breaks were also noted in the ADR-sensitive P388 cells, but in the ADR-resistant cells the level of DNA strand breaks was low. The enhanced cytotoxicity is apparently the consequence of the enhanced potential of ADR to cause breaks in the DNA strands. Our findings show that the survival response of the cells decreases and the level of DNA strand breaks increases following exposure to ADR. ADR resistance may be mediated by a reduction in the level of DNA strand breaks.     

Effects of adenine arabinoside and coformycin on the kinetics of G2 chromatid aberrations in X-irradiated human lymphocytes.

The kinetics of chromatid aberrations have been studied in human lymphocytes exposed to X-rays in the G2 phase of the cell cycle and incubated with or without the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), known to inhibit the repair of DNA double-strand breaks. In the absence of ara A an exponential decrease in frequencies of chromatid breaks occurred which we interpret as repair. Few breaks were observed if samples were harvested immediately following irradiation. The frequency of chromatid breaks at 1 h after X-irradiation (442 per 100 cells/Gy) was similar to that previously observed in Chinese hamster ovary (CHO) K1 cells. However, the exponential decrease of chromatid breaks between X-irradiation and sampling occurred with a t1/2 of 0.87 h, a faster rate than we have previously observed in CHO K1 cells and was not inhibited by 200 microM ara A alone, in contrast with our previous findings in a human fibroblast line. However, in the presence of the ADA inhibitor coformycin, inhibition of break repair was already observed at an ara A concentration of 100 microM indicating that the apparent unresponsiveness to ara A of lymphocyte chromatid break rejoining results from the deamination of this nucleoside analogue. This deamination effect was confirmed by measurements of DNA synthesis which showed stable inhibition of synthesis by ara A only when coformycin was present. Frequencies of chromatid exchanges in irradiated cells remained constant except at the sampling time directly after irradiation, consistent with the view that chromosomal radiosensitivity remained constant throughout the G2 phase, except for the period immediately prior to mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of metabolic inhibitors to compare the excision repair of pyrimidine dimers and nondimer DNA damages in human skin fibroblasts exposed to 254-nm and sunlamp-produced greater than 310-nm ultraviolet radiation

Normal human skin fibroblasts were exposed to either 0-5 J/m2 of 254-nm ultraviolet (UV) radiation or 0-50 kJ/m2 of the Mylar-filtered UV (greater than 310 nm) produced by a fluorescent sunlamp. These cells were then incubated for 0-20 min in medium containing 10 mM hydroxyurea (HU) and 0.1 mM 1-beta-D-arabinofuranosyl cytosine (ara C), and the yield of DNA strand breaks was measured by means of the alkaline elution technique. For cells irradiated with 254-nm UV, which results primarily in the formation of cyclobutane pyrimidine dimers, a rapid increase in DNA strand breaks was detected following incubation with these metabolic inhibitors. In contrast, only a low level of strand breaks formed in cells incubated with HU and ara C after irradiation with approximately equitoxic fluences of sunlamp UV greater than 310 nm, which mainly causes the induction of nondimer DNA lesions. Hence, these results are consistent with the conclusion that the pathways involved in the repair of nondimer DNA damages induced by UV wavelengths greater than 310 nm differ from the repair of pyrimidine dimers.     

The Apicomplexan AP2 family: integral factors regulating Plasmodium development

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks.     

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.     

The effect of mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) on DNA damage, repair and aging of human diploid fibroblasts

We used mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) to examine aging at the cellular level in relation to DNA damage and repair. With the filter elution technique we monitored spontaneous single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA during in vitro aging at 34 degrees C, 37 degrees C and 39 degrees C of normal human diploid fibroblasts (HDF). DNA repair was assessed after ionizing and non-ionizing (ultraviolet) radiation of HDF at different population doubling levels (PDLs): the former was assayed by filter elution and the latter by unscheduled DNA synthesis. Survival was assessed by trypan blue dye exclusion and colony formation. Cells at 37 degrees C achieve a higher cumulative PDL (67 +/- 6) than cells at 39 degrees C (60 +/- 5) or at 34 degrees C (55 +/- 6). The level of spontaneous SSBs and DSBs, and radiosensitivity of DNA to either 6 Gy or 100 Gy gamma rays, do not change with in vitro age at any of the three temperatures. Repair of SSBs (induced by 6 Gy) and DSBs (induced by 100 Gy) does not change with in vitro age: rejoining is 86-104% complete by 60 min repair and generally does not differ across temperatures. Response to non-ionizing radiation (254 nm, 75, 150, 300 ergs/mm2) does not change with in vitro age at 37 degrees C or 39 degrees C, whereas excision repair increases with age at 34 degrees C even though cell survival does not. The results do not support the rate of living theory of aging (Pearl, R., The Rate of Living, University of London Press, London, 1928) as applied to temperature effects on HDF aging in vitro (as measured by proliferative lifespan) and on their response to radiation-induced DNA damage.