DNA Alkaline Unwinding Assay Research Articles

MANGIFERIN PROTECTS RENAL IMPAIRMENT AGAINST BENZO(A)PYRENE INDUCED TOXICITY BY REGULATING MITOCHONDRIAL AND DNA INTEGRITY

Contaminated food and water ingestion and inhalation of polycyclic aromatic hydrocarbon are the main sources of human exposure to benzo(a)pyrene leading to cause renal injury and provoking a nephropathic response. Mangiferin is a highly potent antioxidant present in mango and known for several medicinal properties. The present study was undertaken to evaluate the renoprotective effect of mangiferin against benzo(a)pyrene induced toxicity. Benzo(a)pyrene was administered at a dose of 120.0 mg/kg once alone or in combination with mangiferin at 10.0 and 20.0 mg/kg for seven days in Swiss albino mice. Exposure to benzo(a)pyrene decreased the activities of glutathione peroxidase and increased glutathione-S-transferase level in the kidneys of mice. Moreover, DNA alkaline unwinding assay exhibit a significant decrease in F-value for the toxic control group in comparison to the normal mice, which is a marker for alteration in DNA integrity. Benzo(a)pyrene treatment also revealed an increased number of micronuclei in polychromatic erythrocytes in comparison with the control group indicating chromosomal damage in erythrocytes of bone marrow. Mangiferin pretreatment significantly improved the renal mitochondrial antioxidant status and restored the renal DNA integrity, thus demonstrating the protective effect in benzo(a)pyrene-treated mice. Dietary inclusion of mangiferin could exert protective effects against renal toxicity resulting from benzo(a)pyrene exposure.Keywords: Benzo(a)pyrene, GPx, GST, Mangiferin, Micronucleus Â

Cytotoxic and genotoxic responses of human lung cells to combustion smoke particles of Miscanthus straw, softwood and beech wood chips

Inhalation of particulate matter (PM) from residential biomass combustion is epidemiologically associated with cardiovascular and pulmonary diseases. This study investigates PM0.4-1 emissions from combustion of commercial Miscanthus straw (MS), softwood chips (SWC) and beech wood chips (BWC) in a domestic-scale boiler (40 kW). The PM0.4-1 emitted during combustion of the MS, SWC and BWC were characterized by ICP-MS/OES, XRD, SEM, TEM, and DLS. Cytotoxicity and genotoxicity in human alveolar epithelial A549 and human bronchial epithelial BEAS-2B cells were assessed by the WST-1 assay and the DNA-Alkaline Unwinding Assay (DAUA). PM0.4-1 uptake/translocation in cells was investigated with a new method developed using a confocal reflection microscope.SWC and BWC had a inherently higher residual water content than MS. The PM0.4-1 emitted during combustion of SWC and BWC exhibited higher levels of Polycyclic Aromatic Hydrocarbons (PAHs), a greater variety of mineral species and a higher heavy metal content than PM0.4-1 from MS combustion. Exposure to PM0.4-1 from combustion of SWC and BWC induced cytotoxic and genotoxic effects in human alveolar and bronchial cells, whereby the strongest effect was observed for BWC and was comparable to that caused by diesel PM (SRM 2 975), In contrast, PM0.4-1 from MS combustion did not induce cellular responses in the studied lung cells. A high PAH content in PM emissions seems to be a reliable chemical marker of both combustion efficiency and particle toxicity. Residual biomass water content strongly affects particulate emissions and their toxic potential. Therefore, to minimize the harmful effects of fine PM on health, improvement of combustion efficiency (aiming to reduce the presence of incomplete combustion products bound to PM) and application of fly ash capture technology, as well as use of novel biomass fuels like Miscanthus straw is recommended.

Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay

This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2 ) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl2 . In vitro exposure of hydrogen peroxide (H2 O2 ; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment.

Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage

In spite of proficient results of several phytochemicals in preclinical settings, the conversion rate from bench to bedside is not very encouraging. Many reasons are attributed to this limited success, including inefficient systemic delivery and bioavailability under in vivo conditions. To achieve improved efficacy, polyphenolic constituents of black (theaflavin [TF]) and green (epigallocatechin-3-gallate [EGCG]) tea in poly(lactide-co-glycolide) nanoparticles (PLGA-NPs) were entrapped with entrapment efficacy of ~18% and 26%, respectively. Further, their preventive potential against 7,12-dimethylbenzanthracene (DMBA)-induced DNA damage in mouse skin using DNA alkaline unwinding assay was evaluated. Pretreatment (topically) of mouse skin with either TF or EGCG (100 μg/mouse) doses exhibits protection of 45.34% and 28.32%, respectively, against DMBA-induced DNA damage. However, pretreatment with TF-loaded PLGA-NPs protects against DNA damage 64.41% by 1/20th dose of bulk, 71.79% by 1/10th dose of bulk, and 72.46% by 1/5th dose of bulk. Similarly, 51.28% (1/20th of bulk), 57.63% (1/10th of bulk), and 63.14% (1/5th of bulk) prevention was noted using EGCG-loaded PLGA-NP doses. These results showed that tea polyphenol-loaded PLGA-NPs have ~30-fold dose-advantage than bulk TF or EGCG doses. Additionally, TF- or EGCG-loaded PLGA-NPs showed significant potential for induction of DNA repair genes (XRCC1, XRCC3, and ERCC3) and suppression of DNA damage responsive genes (p53, p21, MDM2, GADD45α, and COX-2) as compared with respective bulk TF or EGCG doses. Taken together, TF- or EGCG-loaded PLGA-NPs showed a superior ability to prevent DMBA-induced DNA damage at much lower concentrations, thus opening a new dimension in chemoprevention research.

RETRACTED: Allethrin-induced genotoxicity and oxidative stress in Swiss albino mice

Allethrin (C(19)H(26)O(3)) is non-cyano-containing pyrethroid insecticide that is used extensively for controlling flies and mosquitoes. Apart from its neurotoxic effects in non-target species, allethrin is reported to be mutagenic in bacterial systems. In this study, we observed oxidative damage-mediated genotoxicity caused by allethrin in Swiss albino mice. The genotoxic potential of allethrin was evaluated using chromosome aberrations (CAs) and a micronuclei (MN) induction assay as genetic end-points. The oral intubation of allethrin (25 and 50mg/kg b.wt.) significantly induces CAs and MN in mouse bone marrow cells. The DNA-damaging potential of allethrin was estimated in mouse liver using the DNA alkaline unwinding assay (DAUA) and by measuring the levels of 8-hydroxy-2'-deoxy-guanosine (8-OH-dG). Furthermore, a dose-dependent increase in reactive oxygen species (ROS) generation and lipid peroxidation (LPO), with a concurrent decrease in superoxide dismutase (SOD) and catalase, confirm its pro-oxidant potential. The DNA-damaging potential of allethrin was found to be mediated through the modulation of p53, p21, GADD45α and MDM-2. These results confirm the genotoxic and the pro-oxidant potential of allethrin in Swiss albino mice.

Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 μg.L−1), Pb2+ (50, 250, and 500 μg L−1) and Cd2+ (5, 25, and 50 μg L−1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 μg L−1 Pb2+ treatment group at day 3 and the 50 μg L−1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 μg L−1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.

Seasonal Liza aurata tissue-specific DNA integrity in a multi-contaminated coastal lagoon (Ria de Aveiro, Portugal)

In this study, the DNA integrity of golden grey mullet (Liza aurata) collected in differently contaminated sites of a coastal lagoon, Ria de Aveiro (Portugal), was assessed, over the period of 1 year, using the DNA alkaline unwinding assay, in four different tissues (gill, kidney, liver and blood) and compared to a reference site. The four tissues displayed different DNA integrity basal levels, clearly affected by seasonal factors. Gill and kidney were, respectively, the most and least sensitive tissues. All sites demonstrated the capacity to interfere with DNA integrity. The sites displaying the highest and lowest DNA damage capability were, respectively, Barra (subject to naval traffic) and Vagos (contaminated with polycyclic aromatic hydrocarbons). In terms of seasonal variability, autumn seems to be the more critical season (more DNA damage) unlike summer when no DNA damage was found in any tissue. Data recommend the continued monitoring of this aquatic system.

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure

BackgroundEpidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water.MethodsDNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB.ResultsCCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs.ConclusionCCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.

DNA alkaline unwinding assay for monitoring the impact of environmental genotoxins

DNA alkaline unwinding assay for monitoring the impact of environmental genotoxins

DNA alkaline unwinding assay for monitoring the impact of environmental genotoxins

DNA alkaline unwinding assay for monitoring the impact of environmental genotoxins

Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves

DNA alkaline unwinding methods were used to detect DNA strand breaks in tissues of marine bivalves following field and laboratory exposures and subsequent recoveries in the laboratory. Field deployments of mussels ( Mytilus edulis) or oysters ( Crassostrea virginica) into two highly contaminated urban estuaries (New Bedford Harbor, Massachusetts, and Elizabeth River, Virginia, respectively) resulted in significantly increased DNA strand breaks in gill tissues. DNA strand breaks did not persist in tissues from oysters that were deployed in the Elizabeth River and then allowed to recover in the laboratory for 7–10 days. Laboratory studies were conducted to characterize the sensitivity and specificity of the DNA unwinding method to detect DNA strand breaks in bivalve tissues. Increased DNA strand breaks resulted from acute exposures to sublethal concentrations of the genotoxic agent N-methyl-N'-nitro-N-nitrosoguanidine, but were not persistent for more than 48 h. Acute or chronic (continuous 7-day) exposures of mussels to sublethal concentrations of copper (not generally considered to be genotoxic) did not result in increased DNA strand breaks. These studies demonstrate that DNA alkaline unwinding methods can be used to reveal potentially genotoxic environmental exposures of bivalves. These methods can be used to evaluate the persistence of DNA damage and species-specific DNA repair capacities.

The further development of a mammalian DNA alkaline unwinding bioassay with potential application to hazard identification for contaminants from environmental samples.

Recently, we have detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells (Daniel et al., 1985). In this paper we present further development of this assay, including: (1) studies on the relationship between DNA adducts and DNA strand breaks; (2) evaluation of the role of cytotoxicity in DNA strand breaks; and (3) application of the DAUA to cell preparations from the liver of mice dosed with methylating agents. The level of DNA adducts produced in human CCRF-CEM cells by treatment with benzo(a)pyrene diol-epoxide (BPDE), N-acetoxy-2-acetyl aminofluorene (AAAF), and various methylating agents was linear with concentration over several orders of magnitude. Likewise, the level of strand breaks increased with the concentration over the same dose range. The strand breaks/adduct ratio ranged from 0.05 for the methyl adducts to 0.001 for the BPDE adducts. Using these values and the inherent sensitivity of the DAUA (circa 100 to 1000 breaks/cell), (Daniel et al., 1985), the ability of the assay to detect DNA damage induced by various classes of chemical carcinogens can be calculated. The DAUA appears to be useful for assessing the relative potency of various environmental genotoxic effects on mammalian cells. In addition, it can be conducted on cells isolated from target organs of whole animals.

Using the DNA alkaline unwinding assay to detect DNA damage in laboratory and environmentally exposed cells and tissues

The DNA alkaline unwinding assay is being evaluated for use in the detection of DNA damage in marine animals exposed to environmental pollutants. In preliminary work, DNA unwinding methods were used with in vitro cell systems to demonstrate DNA strand breaks. Cultured mammalian fibroblasts and sperm from marine fish and invertebrates ( Pseudopleuronectes, Arbacia and Mytilus) showed concentration-dependent increases in DNA strand breaks after brief exposures to alkylating agents. DNA unwinding methods were also used on DNA extracted from marine animals injected with genotoxicants and from animals exposed in situ at an estuarine site. Gills from blue mussels caged at the New Bedford Harbor Superfund Site (MA, USA) highly contaminated with many organic (e.g. PCBs) and inorganic contaminants, were also examined. A significant increase in DNA strand breaks was seen in gill tissues of animals held in the contaminated site for as little as 3 days. Although not as severe, an increase in strand breaks was also seen in animals held at the control site for 28 days. This apparent damage may have resulted from low-level contamination and/or from generally poor tissue condition. These results suggest that DNA alkaline unwinding results from controlled experiments using cells briefly exposed to toxicants can be used to identify DNA strand breaking agents. While whole-animal exposures to toxicants may affect DNA integrity, unwinding results may not specifically reflect genotoxicity.