Synergistic DNA Damage Research Articles

DNA damage and oxidative stress in human B lymphoblastoid cells after combined exposure to hexavalent chromium and nickel compounds

In the present study, human B lymphoblastoid cells were exposed to potassium dichromate and/or nickel chloride for 24h or 48h. The cell viability and DNA damage induced by these compounds was measured with the CCK-8 assay and Comet assay, respectively. In addition, the generation of reactive oxygen species (ROS) and the levels of malondialdehyde (MDA) were measured using commercially available kits. Our results indicated that potassium dichromate could decrease cell viability and induce DNA damage in human B lymphoblastoid cells in a time - and concentration - dependent manner, but the toxicity of nickel chloride was not so obvious at concentrations used in our study. The results of ROS showed that both two compounds could only induce weak elevation of ROS level, but MDA levels were significantly enhanced. Antagonistic effects of cytotoxicity were mainly found between Cr (VI) and Ni (II), and synergistic effects of DNA damage and oxidative stress were partially found between these two compounds. Moreover, there were good correlations between the results of comet assay and the results of oxidative stress assays. It is suggested that synergistic DNA damage induced by simultaneously exposure of hexavalent chromium and nickel compounds is possibly related to oxidative stress.

Synergistic Cytotoxic Stress and DNA Damage in Clover (Trifolium repens) Exposed to Heavy Metal Soil from Automobile Refining Shops in Kashmir-Himalaya

Coexposure to heavy metals occurs in many occupational settings, such as automobile refining shops, pigment, and batteries production. Heavy metals around automobile refining shops were tested for their ability to induce synergistic cytogenetic effects in Trifolium repens L. by using the chromosomal aberrasions (CAs), micronucleus (MN) and comet assay. A significant increase in micronucleus (MN), chromosomal abrations (CAs), percentage of nuclei with comet tails (NCTs), the relative comet tail length (CTL), comet tail DNA (CT, DNA), and tail moment (TM) were observed with increased concentration of three heavy metals, like Cd, Pb, Hg. The present result indicate that exposure of T. repens to soils contaminated by heavy metals around automobile refining shops shows clastogenicity, cytotoxicity, and DNA damage at higher concentrations.

Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1Delta is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3Delta mutant and are essential for survival of rev3Delta cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1Delta or rrm3Delta helicase mutations, whereas mph1Delta is hypostatic to sgs1Delta. Previously reported slow growth of mph1Delta srs2Delta cells is accompanied by G(2)/M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.

Synergistic effects of S-adenosylhomocysteine and homocysteine on DNA damage in a murine microglial cell line

Background Homocysteine (Hcy) and S-adenosylhomocysteine (SAH) are 2 major metabolites of methionine. However, little is known about their interactions in human diseases. Methods We determined the interaction of Hcy with SAH on DNA damage (measured as comet formation) and DNA hypomethylation (assayed as 5-methyldeoxycytidine, 5-mdc) in BV-2 cells (immortalized murine microglia). Results Hcy at 100 μmol/l and SAH at 4 μmol/l alone caused little DNA strand breaks, whereas 100 μmol/l Hcy in combination with 0.5 to 4 μmol/l SAH led to marked DNA damage and uracil misincorporation. The combination of 100 μmol/l Hcy with 4 μmol/l SAH (SAH + Hcy) significantly increased intracellular H 2O 2, and the DNA damage induced by SAH + Hcy was strongly inhibited by addition of superoxide dismutase, catalase or desferrioxamine, suggesting the involvement of reactive oxygen species. DNA damage induced by SAH + Hcy may also involve DNA hypomethylation (i.e., decreased %5-mdc) because of the high correlation between them. The effects induced by SAH + Hcy were specific to SAH but not to Hcy because they were markedly decreased by replacing SAH with adenosine (4.0 μmol/l) but was not affected by replacing Hcy with cysteine (100 μmol/l). Conclusion SAH in combination with Hcy can cause synergistic DNA damage in BV-2 cells. It remains to be seen whether some of the Hcy-related diseases may be caused by a collaborative action of Hcy with SAH.

Synergistic DNA damage and lipid peroxidation in cultured human white blood cells exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and ultraviolet A

4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen in cigarette smoke, while ultraviolet (UV) irradiation from sunlight is a major factor for causing skin aging and skin cancer. However, little is known about the effects of the interaction between NNK and UV light on the induction of DNA damage and oxidative stress. In this study, we incubated human white blood cells (WBCs) with NNK, followed by irradiating the cells with ultraviolet A (UVA) (320-380 nm), and we measured DNA strand breaks (by the Comet or single-cell gel electrophoresis assay), lipid peroxidation (as thiobarbituric acid-reactive substances, TABRS), and the levels of intracellular reactive oxygen species (ROS). We found that preincubation with 1.0 mM NNK, followed by UVA irradiation (7.6 kJ/m2) synergistically increased DNA damage, lipid peroxidation, and the level of intracellular ROS in WBCs, while NNK or UVA alone had little or no effect. Electron spin resonance spectroscopic analyses showed that NNK plus UVA enhanced the UVA-induced generation of singlet oxygen but not hydroxyl radicals. In addition to ROS, bioactivation of NNK by cytochromes P450 (CYP) to form reactive NNK intermediates may also be involved in the synergistic damage to WBCs by NNK plus UVA. This is evidenced by the synergistic increase in N7-methylguanine (7-mGua), a major DNA adduct produced by NNK. Overall, the present study demonstrates that exposure of WBCs to both NNK and UVA synergistically increases DNA damage and lipid peroxidation and that such effects involve enhanced generation of ROS, especially singlet oxygen, and activation of NNK to 7-mGua by CYP. The results imply that NNK is a phototoxic agent.

Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro

The aim of this investigation was to study the synergistic DNA damage effects in human lymphocytes induced by 1.8 GHz radiofrequency field radiation (RFR, SAR of 3 W/kg) with four chemical mutagens, i.e. mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiomimetic agent), methyl methanesulfonate (MMS, alkylating agent), and 4-nitroquinoline-1-oxide (4NQO, UV-mimetic agent). The DNA damage of lymphocytes exposed to RFR and/or with chemical mutagens was detected at two incubation time (0 or 21 h) after treatment with comet assay in vitro. Three combinative exposure ways were used. Cells were exposed to RFR and chemical mutagens for 2 and 3 h, respectively. Tail length (TL) and tail moment (TM) were utilized as DNA damage indexes. The results showed no difference of DNA damage indexes between RFR group and control group at 0 and 21 h incubation after exposure ( P > 0.05). There were significant difference of DNA damage indexes between MMC group and RFR + MMC co-exposure group at 0 and 21 h incubation after treatment ( P < 0.01). Also the significant difference of DNA damage indexes between 4NQO group and RFR + 4NQO co-exposure group at 0 and 21 h incubation after treatment was observed ( P < 0.05 or P < 0.01). The DNA damage in RFR + BLM co-exposure groups and RFR + MMS co-exposure groups was not significantly increased, as compared with corresponding BLM and MMS groups ( P > 0.05). The experimental results indicated 1.8 GHz RFR (SAR, 3 W/kg) for 2 h did not induce the human lymphocyte DNA damage effects in vitro, but could enhance the human lymphocyte DNA damage effects induced by MMC and 4NQO. The synergistic DNA damage effects of 1.8 GHz RFR with BLM or MMS were not obvious.

Synergistic DNA damage by oxidative stress (induced by H 2O 2) and nongenotoxic environmental chemicals in human fibroblasts

Genotoxic combination effects of oxidative stress (induced by H 2O 2) and eight nongenotoxic environmental chemicals (4-chloroaniline, 2,3,4,6-tetrachlorophenol, lindane, 2,4-dichloroacetic acid (2,4-D), m-xylene, glyphosate, nitrilotriacetic acid and n-hexanol) were determined in human fibroblasts. Genotoxicity was measured quantitatively by the single cell gel electrophoresis assay. The nongenotoxic chemicals were used in non cytotoxic concentrations. H 2O 2 was used in concentrations producing low (50 μM) and no cytotoxicity (40 μM). All environmental chemicals acted in a synergistic way with H 2O 2 except DMSO which effectively inhibited H 2O 2-induced DNA damage. The most effective enhancers were 4-chloroaniline, 2,3,4,6-tetrachlorophenol, m-xylene, and n-hexanol. Synergistic effects of hexanol/H 2O 2 were still evident at a concentration of 0.09 noec (no observed effect concentration). In contrast to synergistic DNA damage in the cell antagonism was found measuring DNA breakage in isolated PM2 DNA. From the results we concluded that synergisms between H 2O 2 and nongenotoxic chemicals may be a general phenomenon which is not observed on the level of isolated DNA.

Enhancement of cytogenetic damage by inhibitors of poly(ADP-ribose)polymerase in human lymphocytes exposed to antineoplastics in vivo and in vitro.

The effect of benzamide (B) and 3-aminobenzamide (3-AB) on sister chromatid exchanges (SCEs) and cell kinetics induced in vitro by melphalan (MELPH) or thiotepa (THIO) was studied in normal human lymphocytes. The combined treatments with either MELPH or THIO plus B or 3-AB showed the potentiating ability on SCE rates and the ability to induce cell division delays of the latter chemicals. In a combined in vivo and in vitro study, lymphocytes taken from six cancer patients who had been given cytoxan by injection 2 hr before and then treated with theophylline (THEOPH) or B or 3-AB in vitro were found to have synergistically increased exchange rates and cell division delays. The frequency of SCEs in the patients own lymphocytes with and without exposure to inhibitor of Poly(ADP-ribose) polymerase (P(ADPR)polymerase) was determined before the cytostatic therapy and was used as a control for later comparison in each individual case. These results further substantiate the use of this approach for detecting the induction of cytogenetic damage concerning controlled mutagen human exposure in combined in vivo and in vitro studies. Chemically induced cytotoxicity manifested as an alteration (division delay) in cell kinetics and as synergistic DNA damage by cytostatics and inhibitors of P(ADPR)polymerase may be of use in the treatment of human cancer.