Effect of 3-aminobenzamide on the process of ultraviolet-induced DNA excision repair

The effect of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribosyl)ation, on UV-induced DNA excision repair was investigated. HeLa cells were treated with DNA replication inhibitors, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (araCyt), before and after ultraviolet light (UV) irradiation, to accumulate DNA single-strand breaks. The activity of poly(ADP-ribosyl)ation measured in the permeable cell system of HeLa cells was enhanced in a UV dose-dependent manner after the combined treatment with HU and araCyt in vivo. However, DNA repair synthesis in vitro was not affected by addition of 1 mM 3-aminobenzamide or nicotinamide, while incorporation of [3H]NAD in the same system was completely inhibited. Furthermore, neither the magnitude of UV-induced DNA single-strand breaks accumulated by the combined treatment of HU and araCyt nor the rate of their rejoining after release from the HU and araCyt block were influenced even in the presence of 10 mM 3-aminobenzamide. As the cytotoxicity of UV irradiation was significantly potentiated by 5 mM 3-aminobenzamide, these results suggest that poly(ADP-ribosyl)ation is involved in a process other than DNA excision repair induced by UV irradiation.     

The effect of deoxyadenosine plus deoxycoformycin on replicative and repair synthesis of DNA in human lymphoblasts and isolated nuclei

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.     

DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane.

The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.     

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.Abbreviations HU hydroxyurea - dA deoxyadenosine - TSC pyridine-2-carboxaldehyde thiosemicarbazone - IMPY pyrazoloimidazole - THBA 3,4,5-trihydroxy benzohydroxamic acid - DHBA 3,4-dihydroxy benzohydroxamic acid - UDS unscheduled DNA synthesis - dT thymidine - dNTP deoxynucleoside triphosphate     

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.     

Novobiocin; an inhibitor of the repair of UV-induced but not X-ray-induced damage in mammalian cells.

In addition to inhibiting replicative DNA synthesis in HeLa cells, novobiocin has a severe effect on the cellular response to UV irradiation, reducing the number of breaks made in pre-existing DNA by the excision repair process. The inhibition of UV repair by novobiocin is reflected in enhanced UV-killing of these cells. Rejoining of DNA after X irradiation is not impaired by novobiocin. The recognition and removal of UV damage may require unwinding of the DNA by gyrase, which--in bacteria--is the target for novobiocin.     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

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-beta-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.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Repair of cytokine-induced DNA damage in cultured rat islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.     

DNA damage in HeLa S3 cells by an antitumor drug ledakrin and other antitumor 1-nitro-9-aminoacridines

Ledakrin and seven other antitumor and cytotoxic derivatives of 1-nitro-9-aminoacridine were shown to induce DNA-single strand breaks in HeLa S3 cells as found by alkaline sucrose gradient centrifugation. The induced DNA damage is of non-random character. Some of Ledakrin-induced DNA breaks are probably generated by endonucleolytic cleavage in the course of repair processes as indicated by experiments with Novobiocin, an antibiotic preventing the incision step of DNA repair. Other Ledakrin-induced DNA breaks observed on alkaline sucrose gradients may arise from alkali-labile sites in DNA. Most of such sites seem to be converted to breaks after brief exposure to alkali. The extent of DNA damage by 1-nitro-9-aminoacridines was found to be correlated with cytotoxic activities of these compounds against HeLa S3 cells. Furthermore, Ledakrin and other derivatives seem to induce DNA-repair synthesis in HeLa S3 cells as judged by the stimulation of hydroxyurea (HU)-resistant incorporation of [³H] thymidine into DNA. The agents studied differ in their concentrations required to produce a considerable stimulation of DNA repair, whereas the maximal level of this effect is similar for all the derivatives assayed. The former values are correlated with cytotoxic activites of these compounds and seem to reflect the overall extent of DNA damage by 1-nitro-9-aminoacridines. Stimulation of DNA-repair synthesis is gradually shut off during prolonged incubation of the cells with Ledakrin or during postincubation of the cells in a drug-free medium. Such postincubation results also in the gradual accumulation of DNA-single strand breaks as observed by alkaline sucrose centrifugation. Hence, HeLa S3 cells are incapable of efficiently removing DNA damage by 1-nitro-9-aminoacridines, though the drug's action activates temporarily some repair mechanisms.The reported results suggest that overall DNA damage may contribute to the cytotoxic effects of 1-nitro-9-aminoacridines besides previously found ability of these agents to form interstrand DNA cross-links.     

Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydroxyurea

The effects of hydroxyurea on Escherichia coli B/5 physiology (increases in cell mass, number of viable cells, and deoxyribonucleic acid [DNA], RNA, and protein concentrations) were studied in an attempt to find a concentration that completely inhibits DNA synthesis and increase in number of viable cells but has little or no effect on other metabolic processes. These conditions were the most closely approached at an hydroxyurea concentration of 0.026 to 0.033 m. A concentration of 0.026 or 0.033 m was used in subsequent experiments to study the site(s) of inhibition of DNA synthesis in E. coli B/5 by hydroxyurea. Hydroxyurea at a concentration of 10(-2)m was found to inhibit ribonucleoside diphosphate reductase activity completely in crude extracts of E. coli. The synthesis of deoxyribonucleotides was greatly reduced when E. coli cells were grown in the presence of 0.033 m hydroxyurea. Studies on the acid-soluble DNA precursor pools showed that hydroxyurea causes a decrease in the concentration of deoxyribonucleoside diphosphates and deoxyribonucleoside triphosphates and an increase in the total concentration of ribonucleotides. Sucrose density gradient sedimentation of DNA from cells treated with 0.026 m hydroxyurea for 30 min indicated that at this concentration hydroxyurea induces no detectable single- or double-strand breaks. In addition, both replicative and repair syntheses of DNA were found to occur normally in toluene-treated cells in the presence of relatively high concentrations of hydroxyurea. Pulse-chase studies showed that deoxyribonucleotides synthesized prior to the addition of hydroxyurea to cells are utilized normally for DNA synthesis in the presence of hydroxyurea. On the basis of these observations, we have concluded that the primary, if not the only, site of inhibition of DNA synthesis in E. coli B/5 by low concentrations of hydroxyurea is the inhibition of the enzyme ribonucleoside diphosphate reductase.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Effects of N-methyl-N-Nitrosourethane on DNA synthesis in the guinea pig pancreas

The effects of N-methyl-N-nitrosourethane (NMUT) on pancreatic DNA synthesis were investigated at sequential intervals following gavage of Hartley guinea pigs with a single dose of 30 mg/kg. There was a highly significant stimulation of DNA synthesis, as evidenced by increased incorporation of [³H] methyl-thymidine ([³H] TdR), throughout the whole pancreas and particularly in the duodenal segment, at 4 h following NMUT administration, thereafter, DNA synthesis declined sharply up to 24 h, and then recovered gradually to control levels from 24–96 h. DNA synthesis stimulated by NMUT was suppressed by hydroxyurea (HU), and hence is likely to represent replicative, rather than repair, synthesis.     

Effects of DNA replication inhibitors on UV excision repair in synchronised human cells.

When HeLa cells are irradiated with UV and treated with the DNA synthesis inhibitors hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), DNA strand breaks accumulate at sites where excision repair of DNA damage has been inhibited after the incision step. This break accumulation occurs in mitotic, G1 and S phase cells. But UV-induced repair synthesis of DNA, as measured by [3H]thymidine incorporation into unreplicated DNA, is not inhibited by HU and ara C in G1 or S phase cells, even though replicative synthesis is virtually abolished. Repair and replication must therefore utilise different DNA precursor pools, or different DNA synthetic systems; and the action of Hu and ara C in causing strand break accumulation may occur at the ligation step of excision repair.     

Repair of Cytokine-induced DNA Damage in Cultured Rat Islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1β (IL-1β), interferon γ (IFN γ) and tumour necrosis factor α (TNF α) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4?h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4?h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4?h in the presence of hydroxyurea (HU) and 1-β-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18?h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

Inhibitors of repair DNA synthesis.

We have measured repair DNA synthesis in UV-irradiated normal human fibroblasts, grown to a defined state of quiescence in order to avoid the problem of discriminating repair from replicative DNA synthesis. We have assessed the effects of various DNA synthesis inhibitors on repair. Inhibition of repair synthesis by hydroxyurea, 1-beta-D-arabinofuranosylcytosine and aphidicolin is associated with the ability to accumulate DNA breaks due to enzymic incision at DNA damage sites; the inhibition by novobiocin is in accord with its known ability to block incision.