Alkaline Elution Technique Research Articles

Involvement of the Artemis Protein in the Relative Biological Efficiency Observed With the 76-MeV Proton Beam Used at the Institut Curie Proton Therapy Center in Orsay

Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.

Influences of histone deacetylase inhibitors and resveratrol on DNA repair and chromatin compaction

Accessibility of DNA is a prerequisite for both DNA damage and repair. Therefore, the chromatin structure is expected to have major impact on both processes, with opposite consequences for the stability of the genome. To analyse the influence of chromatin compaction on the generation and repair of various types of DNA modifications, we modulated the global chromatin structure of AS52 Chinese hamster ovary cells and HeLa cells by treatment with either histone deacetylase inhibitors or resveratrol and measured the repair kinetics of (i) pyrimidine dimers induced by ultraviolet B, (ii) oxidised purines generated by photosensitisation and (iii) single-strand breaks induced by H2O2, using an alkaline elution technique. The decrease of chromatin compaction (detected as reduced DNA accessibility to DNase I) after treatment with trichostatin A or butyrate slightly increased the damage generation but had no significant effect on the global repair rates. In contrast, incubation of AS52 cells with resveratrol at concentrations that caused significant chromatin compaction and that had only moderate influence on cell proliferation gave rise to a strong decrease of the repair rates of all three types of DNA modifications. Similar, but less pronounced effects were observed in HeLa cells. The effects of resveratrol on the repair rates were not antagonised by the sirtuin inhibitor EX-527 or by an increase of the intracellular thiol levels.

Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 106–108 normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2–3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC–MS/MS) is the most accurate and versatile approach whereas HPLC–electrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC–MS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.

Abstract 3519: The polyphenolic ellagitanin vescalagin is a specific inhibitor of the alpha isoform of human DNA topoisomerase II

Abstract Polyphenolic ellagitanins are natural structurally complex molecules that are often associated with the therapeutic activity of plant extracts used in traditional medicine. Studies have reported their cancer-preventing activity in animal models at concentrations found in diets. In vitro, some derivatives display cytotoxic activities at micromolar concentrations and induce apoptosis by a mechanism which remains unclear. Potential targets have been proposed, including topoisomerases (top) I and II, which are involved in the regulation of DNA topology during replication, transcription and chromosome segregation. The two human isoforms of top2: top2 alpha (top2a) and top2 beta (top2b) are the target of anticancer agents such as doxorubicin or etoposide. It is known that antitumoral activity of etoposide is primarily due the inhibition of the alpha isoform whereas inhibition of the beta isoform is primarily responsible for the development of secondary malignancies, pointing to the need for more selective top2a inhibitors. Here, we report the differential activity on top2a and top2b of new polyphenolic ellagitanins: vescalagin, methylvescalagin, acutissimin B (acuB) and epiacutissimin B (epiacuB). Using kinetoplast DNA as a substrate, we showed that top2a-mediated decatenation was strongly inhibited by all 4 derivatives at 1 µM concentration, whereas no significant inhibition of top2b could be seen. When used at 10 µM, acuB and epiacuB inhibited both isoforms and were even more active on top2b (>80% inhibition), whereas vescalagin and methylvescalagin specifically targeted top2a with a reduced effect on top2b (<20% inhibition). Vescalagin could also specifically poison top2a in treated CEM cells, since it could induce an increase in covalent top2a-DNA but not top2b-DNA cleavage complexes as detected by the immuno complex of enzyme (ICE) assay. We showed that top2a mutated CEM-VM1 etoposide-resistant cells were also resistant to vescalagin, but were still sensitive to ICRF193 suggesting that vecsalagin is a top2a poison. Using alkaline elution technique we also showed that treatment of CEM cells with 50 µM vescalagin (2h) did not induce detectable double-strand breaks conversely to etoposide. This was confirmed by the absence of gH2AX detection. Surprisingly, vescalagin could induce a significant amount of single-strand breaks. Whether generation of these single-strand breaks is mediated by top2a poisoning and is the cause of cell growth inhibition remains to be investigated. Together our preliminary data identified vescalagin as a new potent and specific top2a poison that could be used as a lead to generate alternative top2 inhibitors with reduced chances to develop secondary malignancies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3519.

Investigations of the contribution of neutrophils to the genotoxicity of respirable quartz particles

Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs).The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells.We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role.Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells

Irradiation of mammalian cells with solar light is associated with the generation of reactive oxygen species (ROS) and oxidative stress, which is mediated in part by endogenous photosensitizers absorbing in the visible range of the solar spectrum. Accordingly, oxidative DNA base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are the predominant types of DNA damage in cells irradiated at wavelengths >400 nm. We have analysed the repair of oxidative purine modifications in human skin fibroblasts and melanoma cells using an alkaline elution technique, both under normal conditions and after depletion of glutathione. Similar repair rates were observed in fibroblasts and melanoma cells from three different patients ( t 1/2 approximately 4 h). In both cell types, glutathione depletion (increased oxidative stress) caused a pronounced repair retardation even under non-toxic irradiation conditions. Furthermore, the cleavage activity at 8-oxoG residues measured in protein extracts of the cells dropped transiently after irradiation. An addition of dithiothreitol restored normal repair rates. Interestingly, the repair rates of cyclobutane pyrimidine dimers ( t 1/2 approximately 18 h), AP sites ( t 1/2 approximately 1 h) and single-strand breaks ( t 1/2 <30 min) were not affected by the light-induced oxidative stress. We conclude that the base excision repair of oxidative purine modifications is surprisingly vulnerable to oxidative stress, while the nucleotide excision repair of pyrimidine dimers is not.

Evaluation of the probe dihydrocalcein acetoxymethylester as an indicator of reactive oxygen species formation and comparison with oxidative DNA base modification determined by modified alkaline elution technique

Reactive oxygen species (ROS) play a predominant role in various diseases and the development of fast and easy methods for the quantification of intracellular ROS represents an important goal. Therefore, the aim of the present study was the evaluation of the fluorogenic probe dihydrocalcein acetoxymethylester (AM) for the detection of intracellular ROS. A flow cytometric method was developed using MCF-7 cells and the kinetics of ester hydrolysis and the cellular distribution and stability of calcein were characterized using calcein AM. Then, MCF-7 cells were challenged with model agents for the generation of singlet oxygen (illumination with visible light), peroxyl and hydroxyl radicals ( tert-butylhydroperoxide, tBHP), superoxide anion radicals (potassium dioxide), and the intracellular formation of superoxide anion radicals by redox cycling (menadione) and the formation of calcein was compared with the induction of oxidative DNA base modifications assessed by modified alkaline elution technique. Every model agent significantly induced formamidopyrimidine-DNA glycosylase-sensitive sites (i.e. oxidative DNA base modifications) and most also induced DNA strand breaks. In contrast, exclusively tBHP and illumination with visible light induced the intracellular formation of calcein. In conclusion, though intracellular oxidation of dihydrocalcein represents a fast screening method, it detects a limited spectrum of ROS.

Protective effects of vitamin A and vitamin E succinate against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced body wasting, hepatomegaly, thymic atrophy, production of reactive oxygen species and DNA damage in C57BL/6J mice.

The protective effect of vitamin A and vitamin E succinate against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced acute toxicity and measures of oxidative stress was studied. Ten mice were treated with either vitamin A (50 mg/kg every other day for eight days) or vitamin E succiante (150 mg/kg/day followed by a dose of 40 mg/kg/day for five additional days). Half of each of the above groups of animals received TCDD on day 4. Five mice received corn oil or TCDD alone. After five days of TCDD treatment, antioxidant combination treatment with vitamin A and TCDD or vitamin E succinate and TCDD resulted in a significant reduction in indicators of acute toxicity including the decrease in total body and thymus weight as compared to TCDD alone (P<0.05). The combination treatment produced also a significant reduction in the increase in liver weight as compared to TCDD only (P<0.05). Following one day of treatment with 50 microg TCDD/kg, vitamin A and vitamin E succinate produced a significant decrease in the production of superoxide anion by peritoneal lavage cells (P<0.05) and in DNA-single strand breaks in the same cells (P<0.05) as assessed by the reduction of cytochrome c and the alkaline elution technique, respectively. A significant decrease in DNA-single strand breaks in peritoneal lavage cells was observed following 5 days treatment with 50 microg TCDD/kg (P<0.05). The results indicate a potential role for oxidative stress in the acute toxicity of TCDD and a protective effect for vitamin A and vitamin E succinate in the overall toxicity of TCDD including measures of oxidative stress.

472 Alkaline elution technique and DNA fluorometric quantification: modifications, evaluation and statistics

472 Alkaline elution technique and DNA fluorometric quantification: modifications, evaluation and statistics

Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement 1,2

Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either γ or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxo-7,8-dihydro-2′-deoxyadenosine, 5-formyl-2′-deoxyuridine, 5-(hydroxymethyl-2′-deoxyuridine, 5-hydroxy-2′-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA.

Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action

The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G 2M phase. The sensitivity to ET-743 of G 1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G 2M. ET-743 concentrations up to four times higher than the IC 50 value caused no detectable DNA breaks or DNA–protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (−/−) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7–8 times less active in ERCC3/XPB and ERCC1-deficient cells than control cells. The findings that G 1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.

39] DNA damage induced by ultraviolet and visible light and its wavelength dependence

DNA damage induced by solar radiation in mammalian cells consists largely of two types of modification: pyrimidine dimers and oxidative modifications. Pyrimidine dimmers that can be subdivided into cyclobutane pyrimidine dimmers, (CPDs) and (6-4) photoproducts are the characteristic and most abundant modifications after direct excitation of DNA, although they can also be formed indirectly by energy transfer from other excited molecules such as carbonyl compounds. Oxidative DNA damage, which includes various pyrimidine and purine modifications, sites of base loss (AP sites), and strand breaks, is generated in only low yield after direct excitation of DNA (except at very short wavelengths, e.g., 193 nm 3 ). It is, however, the most frequent type of DNA damage generated by so-called “indirect mechanisms,” which emanate from the excitation of cellular chromophores other than DNA. These chromophores (endogenous photosensitizers) can react directly with DNA (type I reaction) or give rise to the formation of reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ) or superoxide (O 2 - ). This chapter describes the application of this approach to determine the wavelength dependencies (action spectra) of the generation of both cyclobutane pyrimidine dimers and oxidative DNA modifications in the range between 290 and 500 nm in cultured mammalian cells by means of the alkaline elution technique. In addition, DNA damage profiles (ratios of various types of DNA modification) induced by various wavelength ranges and by natural sunlight are described. Scope and limitations of the technique are discussed.

Accumulation of DNA damage in pre- and posthepatectomized liver of aged rats

Although the majority of the literature supports the concept that an accumulation of DNA damage or a modification in the DNA structure of postmitotic cells occurs with increasing age, there are also several reports that show no DNA changes in these cells with increasing age. In the study reported here, two components of the DNA damage hypothesis of aging were tested. Young (4–6 months) and old (18–20 month) unirradiated or irradiated, pre- and posthepatectomized male Fisher 344 rats were killed, and the posterior lateral lobe of the liver removed. Single cell/nuclei suspensions were made, and the DNA damage accumulated with age or remaining at various times after irradiation was measured using the alkaline elution technique. The results demonstrate that, 1) DNA damage accumulates in rat liver cells with age, and 2) liver cells repair their radiation-induced DNA damage slower in posthepatectomized, but not prehepatectomized aged rats.

DNA protein cross-links produced by NSC 652287, a novel thiophene derivative active against human renal cancer cells.

2, 5-bis(5-Hydroxymethyl-2-thienyl)furan (NSC 652287), is a representative of a series of thiophene derivatives that exhibit potent and selective antitumor activity against several tumor cell lines in the National Cancer Institute Anticancer Drug Screen. NSC 652287 has noticeable activity for the renal cell lines and produces cures in certain corresponding xenografts. The cellular mechanisms of action of NSC 652287 were therefore investigated in this study in greater detail. The most sensitive renal carcinoma cell line, A498, exhibited cell cycle arrest in G(0)-G(1) and G(2)-M at 10 nM NSC 652287, with increased p53 and p21(WAF1) protein. At higher concentrations, NSC 652287 still induced p53 elevation but with p21(WAF1) reduction and massive apoptosis. These results collectively suggested that NSC 652287 induced DNA damage. Using alkaline elution techniques, we found that NSC 652287 induced both DNA-protein and DNA-DNA cross-links with no detectable DNA single-strand breaks. These DNA-protein cross-links (DPC) persisted for at least 12 h after drug removal and their frequency was correlated with cytotoxicity in the renal cell lines studied. The most sensitive cells (A498) produced the highest DPC followed by the cell line with intermediate sensitivity (TK-10). DPC were minimal in the two resistant cell lines, ACHN and UO-31. Nonetheless, a similar degree of DPC occurred at doses imparting equitoxic effects. These results indicate that DNA is a primary target for the novel and potent anticancer thiophene derivative, NSC 652287. NSC 652287 did not cross-link purified DNA or mammalian topoisomerase I suggesting the importance of active metabolite(s) for the cross-linking activity.

Radiation-Induced DNA Damage in Tumors and Normal Tissues: V. Influence of pH and Nutrient Depletion on the Formation of DNA-Protein Crosslinks in Irradiated Partially and Fully Hypoxic Tumor Cells

It has been demonstrated that the yield of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) appears to depend predominantly on the oxygen concentration in the microenvironment around the DNA of mammalian cells (Radiat. Res. 142, 163-168, 1995). Consequently, these DNA lesions have the potential to be used to detect hypoxic cells or estimate the hypoxic fraction of solid tumors. Although it has been demonstrated that physiological factors (e.g. pH, temperature, nutrient depletion, etc.) have no influence on the induction and repair of both DNA strand breaks and DPCs in irradiated oxygenated cells (Radiat. Res. 140, 321-326, 1994), there are no data to suggest how these physiological factors influence the induction and repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic cells. In the present study, the influence of pH (6.6-7.3) and nutrient depletion on the formation and/or repair of radiation-induced DNA strand breaks and DPCs in partially hypoxic (O2 concentration in the gas phase of 2.0-8.0%) or fully hypoxic (O2 concentration in the gas phase of 0.3-0.4%) populations of exponential (day 3) and plateau-phase (day 5) 9L rat brain tumor cells in culture was determined. Each population of cells was irradiated at 37 degrees C with 15 Gy and trypsinized at 4 degrees C after one half-time of strand break repair at 37 degrees C, and the DNA damage was measured using our alkaline elution technique, with or without proteinase K (PK) in the lysis solution. An analysis of the DNA-protein crosslink factor as a function of the oxygen concentration and a statistical comparison of the single-strand scission factor measured at eluted fraction 7 (SSSF7), with or without PK in the lysis solution, indicate that pH and nutrient depletion do not significantly influence the formation and/or repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic 9L cells. The data also demonstrate that the DNA lesions produced in irradiated partially hypoxic cells will cause the hypoxic fraction or fractional hypoxic volume to be overestimated by this DNA damage assay, not underestimated. However, this potential overestimation of the hypoxic fraction or fractional hypoxic volume should not limit the usefulness of this DNA damage assay in most laboratory studies, because it will take the contribution of a large cohort of partially hypoxic cells to overcome the dominance of the signal from a relatively small cohort of fully hypoxic cells.

The sensitivity to DNA single strand breakage in mitochondria, but not in nuclei, of Chinese hamster V79 and variant cells correlates with their cellular sensitivity to hydrogen peroxide

To investigate whether a relation exists between the level of DNA damage by and cytotoxicity of hydrogen peroxide, we measured the initial level of H 2O 2-induced nuclear and mitochondrial DNA single strand breaks in Chinese hamster V79 and H 2O 2-resistant variant cells (Hpr-4) with an alkaline elution technique and a quantitative Southern blot technique, respectively. The frequency of DNA single strand breaks in mitochondrial DNA induced by H 2O 2 was more than one hundred times that of nuclear DNA in the parent V79 cells. While a similar frequency of nuclear DNA single strand breaks was generated in V79 and Hpr-4 cells at an equidose of H 2O 2, a lower number of mitochondrial DNA single strand breaks were generated in Hpr-4 cells than in V79 cells by H 2O 2 in the range of 100 μM to 5 mM. The sensitivity to mitochondrial DNA single strand break-induction correlated with the cellular sensitivity to H 2O 2 in Chinese hamster V79 and variant cells.

Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 μM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg −1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.

DNA Strand Break Induction and Enhanced Cytotoxicity of Propyl Gallate in the Presence of Copper(II)

The antioxidant propyl gallate (PG) induced single strand breaks in PM2 DNA at concentrations higher than 0.25 μM when it was combined with copper concentrations at 5 μM and above. In combination with 100 μM CuCl 2, extensive double strand breakage was also observed. Neither PG alone nor CuCl 2 showed any strand breaking properties. DNA strand breakage was inhibited by addition of catalase or the Cu(I) chelator neocuproine, indicating the involvement of H 2O 2 and a Cu(II)/Cu(I) redox cycle in the DNA damage. DNA damage of PG/Cu(II) was also observed in human fibroblasts. Using the alkaline elution technique concentrations of 0.15–0.5 mM PG induced DNA strand breaks in combination with 2.5 mM CuCl 2, while the single substances did not show any effect. At these concentrations cell viability measured by the MTT assay was not reduced by more than 10%; however, cell growth was inhibited by PG in combination with Cu(II). This growth inhibition was apparently due to the DNA damage incurred by PG/Cu(II). The synergistic interaction between PG and Cu(II) is probably caused by a redox reaction between both compounds, whereby reactive species such as ROS are formed, which are responsible for the observed genotoxic and cytotoxic effects. Our results demonstrate that the antioxidative and cytoprotective properties of propyl gallate may change to prooxidative, cytotoxic and genotoxic properties in the presence of Cu(II).