Increase In DNA Strand Breaks Research Articles

Expression of the pem homeobox gene in Sertoli cells increases the frequency of adjacent germ cells with deoxyribonucleic acid strand breaks.

Pem is a member of the homeobox transcription factor family that is expressed in somatic cells in male and female reproductive tissues. In the murine testis, Pem is specifically expressed in Sertoli cells, where it is dramatically induced at the initiation of meiosis during the first wave of spermatogenesis and then later is restricted to stages IV-VIII of the seminiferous epithelial cycle. To study the function of Pem in Sertoli cells, we generated transgenic mice that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in a large increase (6-fold) in the number of preleptotene spermatocytes with double-strand DNA breaks and a modest increase (2.5-fold) in the number of elongating spermatids (steps 9 and 10) with single-strand DNA breaks, based on terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling and comet analysis. The average number of DNA strand breaks in these germ cell populations also increased. Despite the transient increase in DNA strand breaks, Pem transgenic mice exhibited no significant anomalies in spermatogenesis, fertility, or fecundity. Our results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells.

Resistance of p53 knockout cells to doxorubicin is related to reduced formation of DNA strand breaks rather than impaired apoptotic signaling

The anthracycline doxorubicin (adriamycin) is an important chemotherapeutic agent used in the treatment of solid epithelial and mesenchymal tumors as well as leukemias. A variety of mechanisms has been proposed to be involved in doxorubicin-induced cytotoxicity such as DNA intercalation, oxidative stress, DNA strand breakage by inhibition of topoisomerase II, activation of death receptors, and altered p53 expression. Concerning doxorubicin resistance and p53 status data reported are contradictory. Here, we show that mouse fibroblasts deficient in p53 (p53 −/−) are more resistant to doxorubicin than p53 wild-type (p53 wt) cells. This is in contrast to other genotoxic agents (UV-light, alkylating drugs) for which p53 −/− fibroblasts proved to be more sensitive. Resistance of p53 −/− cells to doxorubicin is related to reduced induction of apoptosis. This is not likely to be due to altered apoptotic signaling since the expression of Bax and Bcl-2 was unchanged and the induction of Fas/CD95/APO-1 receptor and caspase-8 was the same in p53 −/− and p53 wt cells on treatment with doxorubicin. However, we observed a clearly lower level of doxorubicin-induced DNA strand breaks in p53 −/− cells compared to the wt. P170 glycoprotein was equally expressed and the accumulation and elimination of the drug occurred with identical kinetics in both cell types. p53 deficient cells were cross-resistant to another topoisomerase II inhibitor etoposide, which also provoked increased DNA strand breakage in p53 wt cells. Based on the data we conclude that the p53 status significantly impacts the generation of DNA strand breaks because of drug-induced topoisomerase inhibition rather than death receptor signaling. Since human tumors are frequently mutated in p53 the findings bear clinical implications.

Endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K additively enhance arsenic-induced DNA strand breaks in human cells.

We report here that sequential digestion with endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K in Tris buffer markedly increased the sensitivity for detecting DNA damage in arsenic-treated cells. These three enzymes increased DNA strand breaks in an additive manner. By using this sequential-enzyme-digestion comet assay, we demonstrated that trivalent inorganic arsenic induced more DNA damage than monomethylarsonous acid, monomethylarsonic acid, and dimethylarsinic acid in human blood cell lines. However, trivalent inorganic arsenic was far less potent than monomethylarsonous acid in inhibiting pyruvate dehydrogenase activity. Therefore, different mechanisms are involved in inhibiting pyruvate dehydrogenase activity and inducing DNA damage. Our results also indicate while trivalent inorganic arsenic induced more endonuclease III-digestible adducts, monomethylarsonous acid and monomethylarsonic acid induced more proteinase K-digestible adducts. These results suggest there is a difference in the mechanism for inducing DNA damage between inorganic and organic methylated arsenic compounds.

Effects of hexachlorobenzene on embryonic mammalian cells

Hexachlorobenzene (HCB) is a persistent organic pollutant that has been found to be bioaccumulating in several Arctic regions of the world. The response of embryonic cells to HCB exposure has been evaluated for several different cytotoxic responses, including genotoxic effects. NIH 3T3 (mouse) and WS1 (human) embryonic cells were exposed to HCB at 1 and 10 times the average newborn cord blood concentrations of Alaska Arctic natives. Assays were then performed to determine the subsequent effects of HCB on overall cell number, viability, cell growth fraction and long-term survival, as well as specific biochemical and molecular responses. Exposure of both cell types to HCB results in cell membrane damage, a short-term decrease in cell number, increased DNA strand breaks, and a long-term decrease in colony survival. WS1 cells also undergo cell-cycle arrest and modulations in glutathione concentration after exposure to HCB. In addition, NIH 3T3 cells exhibit an increased number of transformed foci after repeated exposure to HCB. This study demonstrates that relevant environmental concentrations of HCB have significant effects on mammalian embryonic cells in culture.

Protective effect of deoxynucleotide triphosphates on DNA damage in different mammalian cells exposed to -radiation

DNA is generally considered to be the most critical cellular target when considering the lethal, carcinogenic and mutagenic effects of drugs, radiation and environmental chemicals. So the study aim to the determination the damaging effect of -radiation on DNA and the protective effect of deoxynucleotide triphosphates (dNTPs). The study includes three cell types, lymphocytes, kidney cells of African gree monkey (Vero) and hepatocellular carcinoma of human (HePG2) exposed to 1-5 Gy of -radiation and by using fluorometric analysis of DNA unwinding (FADU) method, DNA damage was measured after radiation. The cells were divided into two groups: The first received 5x10-5 dNTPs from 0-30 minutes after radiation, while the second group was not supplemented with deoxynucleotides. Clonogenic survival for vero and HePG2 cell lines was measured. The results revealed that the increase of irradiation dose precipitates an increase of DNA strand breaks. The slope curve of initial DNA damage and mean inactivation dose (D ) differ between vero and HepG2 cell line by a factor of up 3.5 and 2, respectively. dNTPs have clear ameliorating effect on DNA damage. FADU method can play an important role in the choice of a suitable treatment (radiation or drugs) and its dosage according to measurement of DNA damages in selective malignant tissues. Moreover, using dNTPs mixture can reduce the side effect of these treatment especially after experimentally on live mammals (mice).

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.

Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability.

Lymphocytes are routinely used in human biomonitoring to assess the potential toxic and cytoprotective effects of diet on both DNA damage and repair and, by implication, health. Logistically, samples may require to be cryopreserved and stored. How this affects cells used in human biomonitoring is often not considered. In this study we have evaluated the influence of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity and DNA repair capability in human peripheral blood lymphocytes. Neither isolation nor freezing increased DNA strand breakage above endogenous levels found in freshly isolated human lymphocytes. Oxidized bases (both pyrimidines and purines) and misincorporated uracil, were similar for fresh and frozen lymphocytes. Fresh and frozen lymphocytes responded almost identically to hydrogen peroxide. Quercetin-mediated cytoprotection against hydrogen peroxide-induced strand breakage was maintained in cryopreserved lymphocytes after short-term (24 h) and longer term (2 months) storage compared with freshly isolated and treated cells. Hydrogen peroxide-induced DNA strand breakage was repaired in fresh lymphocytes. Cryopreserved lymphocytes were unable to repair oxidant-induced DNA strand breaks. Frozen human lymphocytes can therefore be successfully used for most aspects of DNA damage biomonitoring, but not for repair.

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.

Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage.

Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H 2O 2/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H 2O 2/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H 2O 2 suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H 2O 2/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H 2O 2 were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro .

Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells

Arsenic is generally recognized as a nonmutagenic carcinogen because sodium arsenite induces DNA damage only at very high concentrations. In this study we demonstrate that arsenite concentrations above 0.25 μM induce DNA strand breaks in both human leukemia cells and Chinese hamster ovary cells. Therefore, DNA damage may be involved in arsenic-induced carcinogenesis. Formamidopyrimidine-DNA glycosylase and proteinase K greatly increased DNA strand breaks in arsenite-treated cells, providing evidence that a large portion of arsenite-induced DNA strand breaks come from excision of oxidative DNA adducts and DNA-protein cross-links. Because DNA strand breaks appear only temporarily during excision repair, the level of detectable DNA strand breaks will be low at any given time point. For this reason many previous studies have only detected low levels of DNA strand breaks. We also show that catalase, and inhibitors of calcium, nitric oxide synthase, superoxide dismutase, and myeloperoxidase, could modulate arsenite-induced DNA damage. We conclude that arsenite induces DNA adducts through calcium-mediated production of peroxynitrite, hypochlorous acid, and hydroxyl radicals.

DNA Adduct Formation and DNA Strand Breaks in Green-lipped Mussels ( Perna viridis) Exposed to Benzo[a]pyrene: Dose- and Time-Dependent Relationships

Green-lipped mussels, Perna viridis, were exposed to 0, 0.3, 3 and 30 μg l −1 (nominal concentrations) B[a]P under laboratory conditions over a period of 24 days. Mussels were collected on day 0, 1, 3, 6, 12, 18 and 24, and the levels of DNA adducts and DNA strand breaks in their hepatopancreas tissues monitored. Mussels exposed to 0.3 and 3 μg l −1 B[a]P showed marked increases in strand breaks after 1 day of exposure. DNA strand break levels in these mussels remained high and significantly different from the control values until day 3 for the 0.3 μg l −1 treatment group, and day 6 for the 3 μg l −1 treatment group. This was followed by a gradual reduction in strand breaks. After 12 days, the levels of both groups had returned to the same level as that of the control. No increase in DNA strand breaks was observable in mussels exposed to 30 μg l −1 B[a]P in the first 12 days of exposure, but a significant increase was observed from day 12 to day 24. Increasing B[a]P concentrations resulted in elevated DNA adduct levels after 3–6 days of exposure, but this pattern of dose-related increase disappeared after 12 days. These results indicate that a better understanding of the complex interactions between exposure levels and durations is crucially important before DNA adduct levels and DNA strand breaks in P. viridis can be used as effective biomarkers for monitoring genotoxicants in marine waters.

DNA breakage due to metronidazole treatment

The mutagenicity of metronidazole [1-(hidroxyethyl)-2-methyl-5-nitroimidazole] (MTZ) has been shown in different prokaryotic systems. However, data on human cells are still contradictory. In this study DNA damage was determined by the single cell gel electrophoresis (SCGE) assay, in lymphocytes from 10 healthy subjects treated with therapeutic doses of this drug. Samples were obtained before treatment, as well as 1 and 15 days after ending treatment. Results showed a significant increase of DNA strand breaks 1 day after ending treatment, although, an inverse correlation between the amount of DNA damage and plasma concentrations of MTZ was obtained. Thus, the observed damage may be induced by some MTZ metabolite rather than by the parent drug. Interestingly, the amount of DNA damage returned to basal levels 15 days after ending treatment, except in two individuals. This persistent damage should be further investigated.

DNA-Protein Crosslink and DNA Strand Break Formation in HL-60 Cells Treated with Trans,trans-Muconaldehyde, Hydroquinone and Their Mixtures

The toxicity of benzene, a human leukemogen and ubiquitous environmental pollutant, is mediated in part by ring-hydroxylated metabolites including hydroquinone (HQ) and ring-opened metabolites including trans,trans-muconaldehyde (muconaldehyde, MUC), and their interactions. DNA-protein crosslinks (DNAPC) and DNA strand breaks (DNASB) are toxic lesions associated with the mechanism(s) of toxicity of carcinogenic compounds. In the present studies, we examined the hypothesis that individual and interactive effects of MUC and HQ are involved in the formation of DNAPC and DNASB. We extended our previous studies on DNAPC induction by MUC in HL-60 cells to HQ and mixtures of MUC and HQ, and determined DNASB levels, including 3'OH DNASB. Treatment of HL-60 cells with 25 to 100 μM HQ followed by incubation for 4 hours resulted in 1.3- to 2.8-fold increases in DNAPC levels compared with control, as determined by a K+/sodium dodecyl sulfate (SDS) precipitation assay. At 25 and 100 μM, MUC was 1.8 and 4.9 fold more effective at inducing DNAPC than HQ. Treatment with equimolar mixtures of 25 or 50 μM MUC and HQ resulted in higher DNAPC formation relative to the DNAPC levels expected if the effects were only additive. 3'OH DNASB levels as determined by the TUNEL assay showed a significant concentration-dependent increase 1 hour after treatment with 5 to 25 μM MUC, whereas HQ treatment had no effect. Cotreatment with 25 and 50 μM MUC/HQ mixtures resulted in significant decreases in TUNEL labeling relative to treatment with MUC alone. HL-60 cells treated with 1 to 50 μM MUC or HQ exhibited concentration- and time-dependent increases in DNASB as determined by the FADU assay, which measures a variety of single- and double-strand breaks and alkali labile sites. Exposure to 10 μM MUC gave Qdnasb values (1 Qdnasb ≍100 DNASB/cell) of 7.5 ± 1.2 and 15.4 ± 1.4 at the 1- and 2-hour time points respectively, compared with 0.1 ± 3.8 and 0.0 ± 1.5 for the corresponding time controls. The Qdnasb values after treatment with 10 μM HQ were 4.4 ± 0.7 and 17.7 ± 2.1 at the 1- and 2-hour time points, respectively, compared with 0.0 ± 0.5 and 0.0 ± 1.3 for the corresponding time controls. Induction of DNASB was additive 1 hour after treatment with equimolar MUC/HQ mixtures of 5 to 50 μM. These in vitro findings are significant in that DNAPC and DNASB lesions induced by MUC and HQ as well as their interactions could contribute to benzene-induced hematotoxicity and leukemogenesis.

DNA-protein crosslink and DNA strand break formation in HL-60 cells treated with trans,trans-muconaldehyde, hydroquinone and their mixtures.

The toxicity of benzene, a human leukemogen and ubiquitous environmental pollutant, is mediated in part by ring-hydroxylated metabolites including hydroquinone (HQ) and ring-opened metabolites including trans,trans-muconaldehyde (muconaldehyde, MUC), and their interactions. DNA-protein crosslinks (DNAPC) and DNA strand breaks (DNASB) are toxic lesions associated with the mechanism(s) of toxicity of carcinogenic compounds. In the present studies, we examined the hypothesis that individual and interactive effects of MUC and HQ are involved in the formation of DNAPC and DNASB. We extended our previous studies on DNAPC induction by MUC in HL-60 cells to HQ and mixtures of MUC and HQ, and determined DNASB levels, including 3'OH DNASB. Treatment of HL-60 cells with 25 to 100 microM HQ followed by incubation for 4 hours resulted in 1.3- to 2.8-fold increases in DNAPC levels compared with control, as determined by a K+/sodium dodecyl sulfate (SDS) precipitation assay. At 25 and 100 microM, MUC was 1.8 and 4.9 fold more effective at inducing DNAPC than HQ. Treatment with equimolar mixtures of 25 or 50 microM MUC and HQ resulted in higher DNAPC formation relative to the DNAPC levels expected if the effects were only additive. 3'OH DNASB levels as determined by the TUNEL assay showed a significant concentration-dependent increase 1 hour after treatment with 5 to 25 microM MUC, whereas HQ treatment had no effect. Cotreatment with 25 and 50 microM MUC/HQ mixtures resulted in significant decreases in TUNEL labeling relative to treatment with MUC alone. HL-60 cells treated with 1 to 50 microM MUC or HQ exhibited concentration- and time-dependent increases in DNASB as determined by the FADU assay, which measures a variety of single- and double-strand breaks and alkali labile sites. Exposure to 10 microM MUC gave QDNASB values (1 QDNASB approximately equals 100 DNASB/cell) of 7.5 +/- 1.2 and 15.4 +/- 1.4 at the 1- and 2-hour time points respectively, compared with 0.1 +/- 3.8 and 0.0 +/- 1.5 for the corresponding time controls. The QDNASB values after treatment with 10 microM HQ were 4.4 +/- 0.7 and 17.7 +/- 2.1 at the 1- and 2-hour time points, respectively, compared with 0.0 +/- 0.5 and 0.0 +/- 1.3 for the corresponding time controls. Induction of DNASB was additive 1 hour after treatment with equimolar MUC/HQ mixtures of 5 to 50 microM. These in vitro findings are significant in that DNAPC and DNASB lesions induced by MUC and HQ as well as their interactions could contribute to benzene-induced hematotoxicity and leukemogenesis.

Effects of (−)-epigallocatechin gallate (EGCG) on DNA strand breaks as evaluated by single-cell gel electrophoresis (SCG) in human lymphocytes

(-)-Epigallocatechin gallate (EGCG), a catechin polyphenol component, is the main ingredient of green tea extract. Although the anti-carcinogenic and cancer inhibitory effects of EGCG have been widely reported, its genotoxicity is not clear and seldom reported. In this study, we examined the effects of EGCG on DNA strand breaks in the isolated lymphocytes and whole blood lymphocytes obtained from two smoking subjects and a nonsmoking healthy subject using a single cell gel electrophoresis (SCG) assay. The results showed that after 2 hrs of treating the isolated lymphocytes from the smokers, EGCG induced a significant, increase in DNA strand breaks at concentrations from 2.5×10(-5) M to 2.0×10(-4) M, while after 2 hrs of treating the whole blood obtained from the same smokers, EGCG suppressed the DNA strand breaks in the lymphocytes at concentrations of 1.0×10(-4) M and 2.0×10(-4) M. A similar suppressive result was also shown in the whole blood lymphocytes from the nonsmoker at nearly the same concentrations, while at concentrations of 1.0×10(-3) M or 2.0×10(-3) M, EGCG induced a significant increase in DNA strand breaks in the whole blood lymphocytes from the nonsmoker. This result suggests that EGCG is not only inhibitory against DNA strand breaks in whole blood, but also genotoxic to the isolated or whole blood lymphocytes at high concentrations. Thus, more research is needed to comprehensively assess the effects of EGCG on genetic materials.

Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro.

The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy. © 2001 Cancer Research Campaign http://www.bjcancer.com

Increased uracil misincorporation in lymphocytes from folate-deficient rats.

The development of certain human cancers has been linked with inadequate intake of folates. The effects of folate deficiency in vivo on DNA stability (strand breakage, misincorporated uracil and oxidative base damage) in lymphocytes isolated from rats fed a diet deficient in folic acid was determined. Because the metabolic pathways of folate and other methyl donors are closely coupled, the effects of methionine and choline deficiency alone or in combination with folate deficiency were determined. Feeding male Hooded Lister rats a folate-free diet for 10 weeks created a moderate folate deficiency (25–50% (approx.) decrease in plasma, red blood cell and hepatic folate concentrations (P < 0.05) and a 20% rise in plasma homocysteine (P < 0.05)). Lymphocyte DNA strand breakage was increased successively in all groups after 4 weeks and 8 weeks on the diet (50–100% (approx.) after 8 weeks). Only low folate specifically and progressively induced uracil misincorporation throughout the study (100% (approx.) after 8 weeks). Neither folate deficiency nor choline/methionine deficiency altered oxidative DNA base damage. In summary, moderate folate deficiency in vivo is associated with a decrease in DNA stability, measured as increased DNA strand breakage and misincorporated uracil. © 2000 Cancer Research Campaign http://www.bjcancer.com

Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity

Gossypol, a male antifertility and potential anticancer agent, was found to induce DNA strand breaks in rat lymphocytes. DNA breaks were measured with the single-cell gel electrophoresis (SCGE) or ‘comet’ assay. A significant increase in DNA breaks was observed after 1 h incubation at concentrations of 2 μg/ml or greater. The inclusion of 10% fetal bovine serum in the media reduced the toxicity of gossypol, and DNA breaks were only observed at a concentration of 80 μg/ml. However, the increase in DNA strand breaks, for incubations with and without serum, only occurred when cell viability was reduced to less than 70%. Examination of cell morphology and DNA fragmentation at incubations up to 5 h yielded no evidence that DNA strand breaks were occurring due to apoptosis. We conclude that gossypol is not primarily genotoxic in this cell type, and that the DNA breaks observed arose secondary to cytotoxicity.

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 μM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K +-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K −-MN). The increase in K −-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K +-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.