Alkaline DNA Unwinding Research Articles

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.

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells

The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (γH2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

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.

Neuroprotection by Minocycline Caused by Direct and Specific Scavenging of Peroxynitrite

Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.

EN

Genotoxicity tests are essential to identify compounds that have a potential to compromise not only the environment but also human and animal health, including compounds that increase the risk of cancer. At present, no single test is capable of detecting all types of genotoxic effects; therefore a battery of in vitro and, if positive, in vivo tests is necessary to determine the genotoxicity of a substance. However, the respective specificities of current assays have been criticized for their high percentage of false positive results. We recently published an automated version of the "Fluorimetric detection of Alkaline DNA Unwinding" (FADU) assay for measuring DNA strand breaks in human peripheral blood mononuclear cells or in cell lines. Using this new technology we show detection of DNA strand breaks in cells treated with several compounds known to induce DNA strand breaks by various mechanisms. We also tested toxic compounds that were not expected to induce DNA strand breaks; these were negative in the assay as expected. Finally, we included zinc oxide nanoparticles of high production volume to explore further fields of potential FADU applications. The main advantages of this assay are high reproducibility, easy handling, lack of operator bias, high-throughput, speed, and low cost.

High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay

DNA interstrand crosslinks (ICL) are induced both by several cytotoxic anti-cancer drugs as well as by the chemical warfare agent sulphur mustard (SM). Although measurement of ICL formation could be used in risk assessment or provide valuable predictive information on the response of malignant cells to crosslinking chemotherapeutic agents, respectively, it is currently not applied due to lack of appropriate standardized methodology. Here we describe a fast and convenient procedure for detection of ICL in human peripheral blood mononuclear cells (PBMC) as high-throughput method, termed ‘reverse FADU assay’. This assay detects ICL based on the prevention of time-dependent alkaline unwinding of double-stranded DNA in a cell lysate that starts from single or double strand breaks. We have successfully established and optimized the reverse FADU assay by using human PBMC exposed to the model compounds mitomycin C, melphalan and SM. Our fully automated assay version is faster than currently used methods and possesses similar sensitivity. It operates in a 96-well format, thus allowing parallel analysis of multiple samples. Furthermore, we describe optimized protocols for sample preparation, with sample volume minimized to 100 μl of blood, storage and shipment conditions. We conclude that the reverse FADU assay is an attractive candidate method for monitoring DNA damage induced by DNA crosslinking agents.

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H₂O₂), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (> pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H₂O₂ could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H₂O₂ could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time.

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.

1,4-Naphthoquinones as inducers of oxidative damage and stress signaling in HaCaT human keratinocytes

Selected biological effects of 1,4-naphthoquinone, menadione (2-methyl-1,4-naphthoquinone) and structurally related quinones from natural sources – the 5-hydroxy-naphthoquinones juglone, plumbagin and the 2-hydroxy-naphthoquinones lawsone and lapachol – were studied in human keratinocytes (HaCaT). 1,4-naphthoquinone and menadione as well as juglone and plumbagin were highly cytotoxic, strongly induced reactive oxygen species (ROS) formation and depleted cellular glutathione. Moreover, they induced oxidative DNA base damage and accumulation of DNA strand breaks, as demonstrated in an alkaline DNA unwinding assay. Neither lawsone nor lapachol (up to 100 μM) were active in any of these assays. Cytotoxic and oxidative action was paralleled by stimulation of stress signaling: all tested quinones except lawsone and lapachol strongly induced phosphorylation of the epidermal growth factor receptor (EGFR) and the related ErbB2 receptor tyrosine kinase. EGFR activation by plumbagin, juglone and menadione was attenuated by a superoxide dismutase mimetic, indicating that ROS-related mechanisms contribute to EGFR activation by these naphthoquinones.

A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

BackgroundFormation and repair of DNA single-strand breaks are important parameters in the assessment of DNA damage and repair occurring in live cells. The 'Fluorimetric Detection of Alkaline DNA Unwinding (FADU)' method [Birnboim HC, Jevcak JJ. Cancer Res (1981) 41:1889–1892] is a sensitive procedure to quantify DNA strand breaks, yet it is very tedious to perform.ResultsIn order (i) to render the FADU assay more convenient and robust, (ii) to increase throughput, and (iii) to reduce the number of cells needed, we have established a modified assay version that is largely automated and is based on the use of a liquid handling device. The assay is operated in a 96-well format, thus greatly increasing throughput. The number of cells required has been reduced to less than 10,000 per data point. The threshold for detection of X-ray-induced DNA strand breaks is 0.13 Gy. The total assay time required for a typical experiment to assess DNA strand break repair is 4–5 hours.ConclusionWe have established a robust and convenient method measuring of formation and repair of DNA single-strand breaks in live cells. While the sensitivity of our method is comparable to current assays, throughput is massively increased while operator time is decreased.

Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks)

An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0-1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 microg ml(-1). The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively.

Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction

Imbalance between pro- and antioxidant species (e.g. during aging) plays a crucial role for vascular function and is associated with oxidative gene regulation and modification. Vascular aging is associated with progressive deterioration of vascular homeostasis leading to reduced relaxation, hypertrophy, and a higher risk of thrombotic events. These effects can be explained by a reduction in free bioavailable nitric oxide that is inactivated by an age-dependent increase in superoxide formation. In the present study, mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD, SOD-2) and aldehyde dehydrogenase (ALDH-2) were investigated. Age-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-2(-/-), MnSOD(+/+), and MnSOD(+/-) mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolated heart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mtDNA damage. In contrast, middle-aged ALDH-2(-/-) mice showed a marked vascular dysfunction that was similar in old ALDH-2(-/-) mice suggesting that ALDH-2 exerts age-dependent vasoprotective effects. Aged MnSOD(+/-) mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.

MnSOD and ALDH‐2 deficiency potentiate aging‐associated vascular dysfunction, mitochondrial oxidative stress and mtDNA strand breaks

Vascular aging is associated with progressive deterioration of vascular homeostasis and reduced relaxation. These effects can be explained by a reduction in free bioavailable NO that is inactivated by an age‐dependent increase in superoxide formation. In the present study mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD) and aldehyde dehydrogenase (ALDH‐2) were investigated. Age‐dependent effects on vascular function were determined in aortae of these mice by isometric tension measurements. Mitochondrial ROS formation was measured by enhanced chemiluminescence in isolated heart mitochondria. ROS‐mediated mitochondrial DNA (mtDNA) damages were detected by the fluorescent‐detection alkaline DNA unwinding (FADU)‐assay. Expression of antioxidant proteins were assayed by quantitative RT‐PCR. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and mtDNA damage. In contrast, middle‐aged ALDH‐2−/− mice showed a marked vascular dysfunction that was similar in old ALDH‐2−/− mice. Aged MnSOD+/− mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. Mitochondrial radical formation significantly contributes to age‐dependent endothelial‐dysfunction.

Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5–15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of γ-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.

An automated version of the fluorescence-detected alkaline DNA-unwinding (FADU) assay to measure DNA strand breaks and DNA repair

An automated version of the fluorescence-detected alkaline DNA-unwinding (FADU) assay to measure DNA strand breaks and DNA repair

Final report on the safety assessment of octoxynol-1, octoxynol-3, octoxynol-5, octoxynol-6, octoxynol-7, octoxynol-8, octoxynol-9, octoxynol-10, octoxynol-11, octoxynol-12, octoxynol-13, octoxynol-16, octoxynol-20, octoxynol-25, octoxynol-30, octoxynol-33, octoxynol-40, octoxynol-70, octoxynol-9 carboxylic acid, octoxynol-20 carboxylic acid, potassium octoxynol-12 phosphate, sodium octoxynol-2 ethane sulfonate, sodium octoxynol-2 sulfate, sodium octoxynol-6 sulfate, and sodium octoxynol-9 sulfate.

Octoxynols are ethoxylated alkylphenols in which the size of the molecule is related to the number of moles of ethylene oxide used in synthesis. Reactions are performed at elevated temperature, under pressure, and in the presence of NaOH. It is possible that the synthesis may leave trace amounts of ethylene oxide, 1,4-dioxane, and unreacted C9 phenols. Octoxynols of various chain lengths as well as octoxynol salts and organic acids function in cosmetics either as surfactants--emulsifying agents, surfactants--cleansing agents, surfactant--solubilizing agents, or surfactants--hydrotropes in a wide variety of cosmetic products at concentrations ranging from 0.0008% to 25%, with most less than 5.0%. The octoxynols are chemically similar to nonoxynols, the safety of which were previously considered. Long-chain nonoxynols (9 and above) were considered safe as used, whereas short-chain nonoxynols (8 and below) were considered safe as used in rinse-off products and safe at concentrations less than 5% in leave-on formulations. Acute exposure of hamsters to Octoxynol-9 by bronchopulmonary lavage produced pneumonia, pulmonary edema, and intra-alveolar hemorrhage. Octoxynol-9 at doses over 1 g/kg was toxic in rats and in mice in acute oral toxicity studies. No significant effects were noted in short-term oral studies of Octoxynol-9 in rats, in subchronic oral studies of Octoxynol-40 in rats and dogs, or in chronic oral studies of Octoxynol-40 in rats. The intraperitoneal LD50 of Octoxynol-9 in rats and mice was around 100 mg/kg. In skin irritation studies, octoxynols ranged from nonirritating to moderately irritating. Octoxynols were not ocular irritants in one rabbit study, but in others there was ocular irritation. No immune system toxicity in CF-1 female mice was noted following the intraperitoneal injection of Octoxynol-9 followed by subcutaneous immunization with sheep red blood cells (SRBCs). Octoxynol-9 produced no humoral and cell-mediated immune responses, or autoimmune response in mice. In the Ames test, Octoxynol-1 was not mutagenic with and without metabolic activation nor was Octoxynol-9 clastogenic. Results for Octoxynol-9 were negative in the following assays: unscheduled DNA synthesis, hypoxanthine guanine phosphoribosyl transferase mutation assay, malignant transformation assay, DNA alkaline unwinding test, and mouse lymphoma thymidine kinase locus forward mutation assay. Ethoxylated alkylphenols are generally considered to be estrogenic in that they mimic the effects of estradiol. Dermal exposure at three dose levels of rats to Octoxynol-9 failed to induce any malformations by category (external, visceral, or skeletal) or by individual anatomical location that were different from controls at statistically significant level. An increased incidence of a vestigial thoracic rib was observed in all dose groups. Octoxynol-9 also did not induce developmental toxicity (number of viable litters, liveborn per litter, percentage survival, birth weight per pup, and weight gain per pup) in female specific pathogen-free CD-1 mice dosed daily by gavage on gestation days 6 through 13. No reproductive toxicity was seen in male albino rats which received 5% Octoxynol-40 in the diet daily for 3 months; however, in an in vitro test, Octoxynol-9 (0.24 mg/ml) totally immobilized all human spermatozoa within 20 s. Women who used Nonoxynol-9 or Octoxynol-9 as spermicides, but who did become pregnant, did not have an increase in the overall risk of fetal malformations. In a human skin irritation study, formulations containing 2.0% Octoxynol-9 were classified as moderately irritating and minimally irritating, respectively, in a 24-h single-insult, occlusive patch test. Octoxynol-9 (1.0%) was classified as a nonirritant in a clinical study of nine subjects patch tested for 4 consecutive days. The skin sensitization potential of Octoxynols-1, -3, -5, -9, and -13 was evaluated using 50 subjects. Octoxynol-1 induced sensitization in two subjects; all other results were negative. No sensitization was observed in the following studies: 8.0% Octoxynol-9 in 103 subjects, 0.5% Octoxynol-9 in 102 subjects, and 0.1% Octoxynol-9 in 206 subjects. Concerns about even trace levels of 1,4-dioxane, ethylene oxide, or unreacted C9 led to the recommendation that levels be limited. Concerns about the ocular irritancy of short-chain octoxynols led to a recommendation that they should not be used in products that will be used in the area surrounding the eyes. A limitation on the use concentration for short-chain octoxynols (8 and below) arose from consideration of the skin sensitization potential of octoxynols and the recognition that the short-chain octoxynols could be absorbed into the skin more than the long-chain octoxynols. Overall, based on the available data, it was concluded that long-chain octoxynols (9 and above) are safe as used, whereas short-chain octoxynols (8 and below) are safe as used in rinse-off products and safe at concentrations less than 5% in leave-on formulations.

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.

Role of cytochrome P4501A1 in biotransformation of a tissue specific sarcomagen N-methyldibenzo[c,g]carbazole

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen, while its synthetic methyl derivative N-methyldibenzo[c,g]carbazole (MeDBC) is tissue specific sarcomagen. It is supposed that sarcomagenic activity of DBC depends on biotransformation at ring-carbon atoms, as with PAH, whereas the heterocyclic nitrogen plays an important role in liver carcinogenicity. The objective of this study was to elucidate the role of cytochrome P4501A1 in metabolic activation of sarcomagenic derivatives of DBC and to characterize the DNA damage profiles induced by DBC and MeDBC in relation to the mode of metabolic activation. The genetically engineered V79MZh1A1 cell line with stable expression of cDNA of human cytochrome P4501A1, the parental V79MZ cell line lacking any cytochrome P450 activity and human hepatocarcinoma Hep G2 cells were used as a model cells. Dose-dependent decrease in colony forming ability (CFA) was found in the V79MZh1A1 cell line after treatment of cells with DBC and MeDBC; however, no change in CFA was induced in parental V79MZ cells. These results were in a good correlation with DNA damaging effects of these two derivatives measured by the alkaline DNA unwinding (ADU) and the modified single cell gel electrophoresis (SCGE) techniques. Differences in DNA damage profiles induced by DBC and MeDBC were found in V79MZh1A1 and Hep G2 cells. These differences were probably the result of different reactive metabolite formation depending on chemical structure of the molecule and ways of biotransformation. This study showed that the cytochrome P4501A1 took part in activation of sarcomagenic DBC derivatives. Moreover, V79 cell lines with stable expression of different cytochromes P450 in combination with DNA repair endonucleases should be a useful tool for characterization of the role of individual cytochromes in metabolic activation pathways of DBC and MeDBC.

Marine molluscs and fish as biomarkers of pollution stress in littoral regions of the Red Sea, Mediterranean Sea and North Sea

The intensive development of industry and urban structures along the seashores of the world, as well as the immense increase in marine transportation and other activities, has resulted in the deposition of thousands of new chemicals and organic compounds, endangering the existence of organisms and ecosystems. The conventional single biomarker methods used in ecological assessment studies cannot provide an adequate base for environmental health assessment, management and sustainability planning. The present study uses a set of novel biochemical, physiological, cytogenetic and morphological methods to characterize the state of health of selected molluscs and fish along the shores of the German North Sea, as well as the Israeli Mediterranean and Red Sea. The methods include measurement of activity of multixenobiotic resistance-mediated transporter (MXRtr) and the system of active transport of organic anions (SATOA) as indicators of antixenobiotic defence; glutathione S-transferase (GST) activity as an indicator of biotransformation of xenobiotics; DNA unwinding as a marker of genotoxicity; micronucleus test for clastogenicity; levels of phagocytosis for immunotoxicity; cholinesterase (ChE) activity and level of catecholamines as indicators of neurotoxicity; permeability of external epithelia to anionic hydrophilic probe, intralysosomal accumulation of cationic amphiphilic probe and activity of non-specific esterases as indicators of cell/tissue viability. Complete histopathological examination was used for diagnostics of environmental pathology. The obtained data show that the activity of the defensive pumps, MXRtr and SATOA in the studied organisms was significantly higher in the surface epithelia of molluscs from a polluted site than that of the same species from control, unpolluted stations, providing clear evidence of response to stress. Enhanced frequency of DNA lesions (alkaline and acidic DNA unwinding) and micronucleus-containing cells was significantly higher in samples from polluted sites in comparison to those from the clean sites that exhibited genotoxic and clastogenic activity of the pollutants. In all the studied molluscs a negative correlation was found between the MXRtr levels of activity and the frequency of micronucleus-containing hemocytes. The expression of this was in accordance with the level of pollution. The complete histopathological examination demonstrates significantly higher frequencies of pathological alterations in organs of animals from polluted sites. A strong negative correlation was found between the frequency of these alterations and MXRtr activity in the same specimens. In addition to these parameters, a decrease in the viability was noted in molluscs from the polluted sites, but ChE activities remained similar at most sites. The methods applied in our study unmasked numerous early cryptic responses and negative alterations of health in populations of marine biota sampled from the polluted sites. This demonstrates that genotoxic, clastogenic and pathogenic xenobiotics are present and act in the studied sites and this knowledge can provide a reliable base for consideration for sustainable development.