Loss of DNA repair capacity during successive subcultures of primary rat fibroblasts

Cultures of fibroblasts from newborn rats and successive subcultures of these cells were treated with 4-nitroquinoline-1-oxide to induce DNA repair. DNA from the cultures was examined by velocity sedimentation in alkaline sucrose gradients immediately after drug treatment and after a post-treatment incubation period of 3 h. Early passage cells were able to repair the damage that appeared as single strand breaks, however, by the seventh subculture this activity was not apparent. Measurements of repair synthesis showed a partial loss of this capacity with successive subculture. The results fit a model in which 4NQO causes two kinds of DNA modification, one of which is alkali labile and appears as a single-strand break. Both modifications are subject to excision repair, but each is recognized initially by a specific endonuclease. In the late passage cells, the endonuclease specific for the alkali labile modification is absent.     

Preferential DNA repair of alkali-labile sites within the active insulin gene

DNA damage and repair were studied in a DNA fragment containing the insulin gene after treatment of cells with methylnitrosourea. For these studies, two clonal isolates from the same rat insulinoma cell line which differ in that the insulin gene is transcribed in one (RINr 38) and is silent in the other (RINr B2) were utilized. Both the determination of immunologically reactive insulin released and the expression of insulin mRNA were used to verify that the gene was transcribed in the RINr 38 cells and not in the RINr B2 cells. Repair kinetics for the removal of alkali-labile sites were comparable across the entire genome in the RINr 38 and RINr B2 cells as determined using alkaline sucrose gradient sedimentation and a 32P end-labeling assay for the quantitation of N7-methylguanine. Quantitative DNA blot analysis was utilized to assess the formation and repair of alkali-labile sites within the restriction fragment containing the insulin gene. Alkali-labile sites appeared to be formed equally within the restriction fragment containing the insulin gene in both the RINr 38 and RINr B2 cells. However, at 24 h, 60% of the lesions were removed from the fragment in the RINr 38 cells, where the gene was transcribed, compared to the removal of only 20% in the RINr B2 cells, where the gene was silent. Thus, it appears that alkali-labile sites induced by exposure to methylnitrosourea are repaired more efficiently in the DNA fragment containing the insulin gene when it is actively transcribed.     

Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells

We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.     

The modifying effect of sodium ascorbate on DNA damage and repair after N-methyl-N′-nitro-N-nitrosoguanidine treatment in vivo

A biological reducing agent, sodium ascorbate, was used to modify both the damage induced by N-methyl-N′-nitro-N-nitrosoguanidine to mouse gastric mucosal cell DNA and the repair of that damage $\text{in vivo}$. Freshly-mixed carcinogen and sodium ascorbate enhanced DNA fragmentation as measured by shifts in alkaline sucrose gradient sedimentation profiles whereas incubation of the two compounds for a short period resulted in reduced DNA fragmentation. Furthermore, periodic administration of sodium ascorbate following stomach cell DNA damage with carcinogen inhibited DNA repair.     

Azaserine: further evidence for DNA damage in Escherichia coli

Azaserine causes DNA damage in stationary-phase cells. In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques. Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks. Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced. Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs. Several other studies also failed to detect DNA damage induced directly by azaserine. Increased levels of beta-galactosidase were induced in an E. coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product. In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E. coli strain.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.     

Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay

The comet assay, also called the single-cell gel electrophoresis (SCGE) assay, is a rapid and sensitive method for the detection of DNA damage (strand breaks and alkali-labile sites) in individual cells. The assay is based on the embedding of cells in agarose, their lysis in alkaline buffer and finally subjection to an electric current. In the present study, alkaline SCGE was used to evaluate the extent of primary DNA damage and DNA repair in peripheral blood lymphocytes of workers employed in pesticide production. After the period of high pesticide exposure, lymphocytes of the occupationally exposed workers manifested increased tail length and tail moment compared to the control group. After the workers spent 6 months out of the pesticide exposure zone, both endpoints were still above that of the control but significantly decreased as compared to the results of the first analysis.     

Repair,replication and survival in UV-irradiatedEscherichia coli

The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficientEscherichia coli K-12 after a flux of 20 J m⁻². In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage ia of primary importance for excision-proficient cells which can easily cope with persisting dimers.     

Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA.

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.     

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a water-soluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkali-labile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.     

Formation of single-stranded breaks in newly-synthesized Escherichia coli DNA following treatment with bleomycin

Changes in molecular weight of newly synthesized DNA was studied after bleomycin treatment of Escherichia coli cells. The treatment by this drug causes only the increase of dispersion in sedimentation profiles of daughter DNA strands in wild type cells. There are two alternative explanation of this fact. First, single-strand breakage does not occur in newly synthesized DNA, i.e. bleomycin-induced athyminic sites do not block cellular DNA polymerases. Second, it is possible to explain it by quick rejoining of given breaks by cell repair systems. The sedimentation profile of daughter DNA strands of recA mutant rules out the first possibility. Observed shift to low molecular weight fractions region strongly indicates the formation of single-strand breaks in newly synthesized DNA. Extensive daughter DNA degradation in xthA mutant supports the idea of the existence of very effective excision repair in the case of apyrimidinic sites. Thus, non-eliminated bleomycin-induced damage causes the formation of single-strand breaks in newly synthesized DNA strands. These breaks may be repaired in the course of recA-dependent post-replication repair.     

Photogenotoxicity of folic acid

Folic acid (FA), also named vitamin B9, is an essential cofactor for the synthesis of DNA bases and other biomolecules after bioactivation by dihydrofolate reductase (DHFR). FA is photoreactive and has been shown to generate DNA modifications when irradiated with UVA (360 nm) in the presence of DNA under cell-free conditions. To investigate the relevance of this reaction for cells and tissues, we irradiated three different cell lines (KB nasopharyngeal carcinoma cells, HaCaT keratinocytes, and a melanoma cell line) in the presence of FA and quantified cytotoxicity and DNA damage generation. The results indicate that FA is phototoxic and photogenotoxic by two different mechanisms. First, extracellular photodecomposition of FA gives rise to the generation of H₂O₂, which causes mostly DNA strand breaks. If this is prevented, e.g., by the presence of catalase, DNA damage generated by intracellular FA becomes evident. The damage spectrum in this case consists predominantly of oxidatively generated purine modifications sensitive to the repair glycosylase Fpg, as characteristic for type I photoreactions, and is associated with the formation of micronuclei. In KB cells, the DNA damage is strongly enhanced after pretreatment with the DHFR inhibitor methotrexate, which prevents the loss of the chromophore associated with the intracellular reduction of FA by DHFR. The results indicate that FA is photoreactive in cells and gives rise to nuclear DNA damage under irradiation.     

Application of alkaline unwinding assay for detection of mutagen-induced DNA strand breaks

The frequency of single-strand breaks in parental DNA and gaps in nascent DNA in various cells exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) was investigated by alkaline unwinding assay using two types of alkaline lysis conditions, 22°C lysis versus 0°C lysis. The DNA damage induced by MMS and MNU is considered to be characteristic of lesions produced in DNA by alkylating agents. The aim of our research project was to adjust this method to be able to detect the greatest number of DNA lesions induced by alkylating agents in parental DNA of different mammalian cells. In our experiments we used human cell lines EUE, GM637 and XP12, Chinese hamster V79 cells, and Syrian hamster embryo cells. The higher level of strand interruptions was detected under conditions of lysis of cells at 22°C. Probably the level of strand interruptions found after the lysis of cells at 22°C correlates with the increased number of disrupted alkali-labile sites of DNA. It is remarkable that the different lysis conditions did not influence the number of gaps detected in nascent DNA of alkylated cells. Comparing induction of breaks and gaps in radiolabelled strands of parental and daughter DNA under different lysis conditions, we succeeded in defining the optimum conditions for detection of alkali-labile sites of parental DNA.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks

Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium. While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain. In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.     

Genotoxicity of acrylamide in human lymphocytes

Acrylamide is used in the industry and can be a by-product in a high-temperature food processing. It is reported to interact with DNA, but the mechanism of this interaction is not fully understood. In the present study, we investigated the DNA-damaging potential of acrylamide (ACM) in normal human lymphocytes using the alkaline-, neutral- and 12.1 versions of the comet assay and pulsed-field gel electrophoresis. We also investigated effect of acrylamide on caspase-3 activity as well as its influence on the repair process of hydrogen peroxide-induced DNA damage. Acrylamide at 0.5-50 microM induced mainly alkali-labile sites. This damage was repaired during a 60-min repair incubation. Post-treatment of the damaged DNA with repair enzymes: thymine glycol DNA N-glycosylase (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II (Alk A), recognizing alkylated bases, caused an increase in the extent of DNA damage, indicating the induction of oxidative and alkylative DNA base modifications by acrylamide. Pre-treatment of the lymphocytes with N-tert-butyl-alpha-phenylnitrone (PBN), a spin trap, as well as vitamins C and E decreased the DNA-damaging effect of acrylamide, which suggest that free radicals/reactive oxygen species may be involved in this effect. Acrylamide impaired the repair of DNA damaged by hydrogen peroxide and increased the activity of caspase-3, which may indicate its potential to induce apoptosis. Our results suggest that acrylamide may exert a wide spectrum of diverse effects on DNA of normal cells, including mostly DNA base modifications and apoptosis. Acrylamide may also impair DNA repair. Free radicals may underline these effects and some dietary antioxidants can be considered as protective agents against genotoxic action of acrylamide. As normal lymphocytes contain cyp2e1 and P450, engaged in the bioactivation of ACM to glicidamide it is uncertain whether acrylamide causes all of measured effect per se or this is the result of the action of its metabolites.     

Viscoelastic behavior of mammalian DNA.

The viscoelastic behavior of rat 9L cellular DNA was studied as a function of the detergent used for lysis, the pH and duration of lysis, and gamma ray dose. For nondenaturing lysis conditions, a model of the DNA was proposed to account for the effects of these agents on the viscoelastic retardation time. It was concluded that these agents affect the hydrodynamic radius of the DNA rather than its molecular weight. For denaturing lysis conditions, molecular weights calculated from the relaxation time were consistent with those calculated from alkaline sucrose sedimentation profiles.     

Detection of DNA damage and repair by alkaline elution using human lymphocytes

The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.     

Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid

Highly purified, (32)P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of (32)P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified (32)P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.