Comet Assay Research Papers - Academia.edu (original) (raw)

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- Australia, Oxidative Stress, Health, DNA damage

Phenothrin, a synthetic pyrethroid compound, is widely used to control agricultural and household insects, as well as to eliminate human louse infestation. Toxicity studies on the direct DNA-damaging effect of phenothrin are lacking. We therefore investigated whether phenothrin exposure can lead to increased DNA damage in vitro in human peripheral blood lymphocytes and in human hepatocytes. Genotoxicity was evaluated by means of the comet assay modified with formamidopyrimidine DNA-glycosylase post-treatment for the detection of oxidative base-damage in DNA. We also assessed the cytotoxic potential of this compound by use of combined fluorescence viability staining. Our results show that phenothrin induces statistically significant, dose-dependent DNA damage in the absence of marked cytotoxicity at concentrations higher than 20 µM and 50 µM in human blood peripheral lymphocytes and hepatocytes, respectively. Oxidative DNA damage could also be detected in the two cell types, although this did not reach statistical significance. These findings provide evidence of the DNA-damaging potential of phenothrin and call for additional studies to reveal the genotoxic properties of this pyrethroid. The observations also point at the importance of using caution when considering the use of phenothrin.

- by Gabor Racz
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- Oxidative Stress, DNA damage, Environmental Biotechnology, Insecticides

The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is nongenotoxic under the present test conditions.

- by Mohammed Noushad
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- Malaysia, DNA damage, Mice, Salmonella Typhimurium

Nitric oxide is a metastable radical, reacts with oxygen to produce toxic nitrogen oxides (N2O3, ONOO−) which damage DNA. Occupational exposure to nitric oxide leads to increased frequency of chromosomal aberrations in humans. In the present study the DNA damage among the jewellery workers occupationally exposed to nitric oxide was analyzed using buccal cell comet assay. The result of this study shows increased levels of DNA damage among jewellery workers. The habit of cigarette smoking among the jewellery workers has a synergistic effect on inducing DNA damage.

- by Jayakumar Rajarajeswaran
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- DNA damage, Nitric oxide, Comet Assay, Occupational Exposure

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 8C, 25 8C, and 4 8C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20 AE 14.77% and did not differ significantly from the results of a neutral comet assay (22.0 AE 19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 8C than when incubated at 25 8C or 4 8C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing. #

- by Fabián Crespo
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- Fluorescence Microscopy, Cryopreservation, Endangered Species, Theriogenology

The antioxidant properties of the fruit of the Rosa roxburghii (RR) plant have been associated with several putative health promoting effects. The possible cytotoxic, mutagenic/antimutagenic and genotoxic effects of RR fruit extract were investigated. The effect on antioxidant status and protection against induced oxidative stress were also investigated using primary rat hepatocytes. A RR fruit extract containing 45 g/L total ascorbic acid and 65 g/L total polyphenols was used in this study. Dilutions up to 0.08% (v/v) increased significantly the antioxidant status in primary rat hepatocytes. The glutathione redox state was decreased with RR treatment but was increased in Chang liver cells and MT-2 lymphoblast. No cyto- or genotoxicity were observed at levels of up to 5% (v/v) of the fruit extract. In addition, a significant protection against t-BHP induced oxidative stress was observed in primary rat hepatocytes. The Ames test revealed no mutagenic activity using the Salmonella typhimurium strains TA98, TA100 and TA102. A significant antimutagenic effect of the extract was observed against the metabolic activated mutagens 2-acetylaminofluorene and aflatoxin B1 and to a lesser extent against methyl methanesulfonate. It is concluded that these results support the associated health promoting potential of Rosa Roxburghii fruit and in particular against oxidative stress. Copyright © 2007 John Wiley & Sons, Ltd.

- by Jeanine L Marnewick
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- Oxidative Stress, DNA damage, Biological Sciences, Antioxidants

Background: In the recent years, the use of natural antioxidants as photochemoprotective agents against skin damages produced by ultraviolet radiation gained considerable attention. Our goal was to show that the hydroethanolic extract obtained from red grape seeds, Burgund Mare (BM) variety could have a protective effect on keratinocytes exposed to UVB radiation. Materials and methods: HaCaT keratinocytes were treated with BM extract 30 min. before UVB exposure. The effect was evaluated by assessing cell viability with MTT; the generation of lipid peroxides with malondialdehide (MDA) assay; DNA damage using comet assay; the quantification of DNA photolesions by ELISA and apoptosis by immunocytochemistry with AnnexinV. Results: After irradiation with UVB, HaCaT cells pretreated with BM showed: increased cell viability compared to those exposed to UVB only; significantly lower lipid peroxides level; the lesion scores and DNA photolesions were significantly lower and a significant reduction of the cells undergoing apoptosis. Conclusions: These results recommend the use of the BM extract as photochemoprotective agent as such or in combination with sunscreens and/or other natural products with similar or complementary properties.

- by Ioana Brie
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- DNA damage, Apoptosis, Antioxidants, Cell line

The generation of reactive oxygen species and other radicals, catalyzed by iron ions at the fiber surface, is thought to play an important role in asbestos-induced cytotoxicity and genotoxicity, but a direct confirmation of this statement needs the availability of asbestos samples differing only for their iron content, without the interference of other physicochemical features. Synthetic stoichiometric chrysotile nanofibers, devoid of iron or any other contaminant, did not exert genotoxic and cytotoxic effects nor elicited oxidative stress in a murine alveolar macrophage cell line; on the contrary, the same nanofibers, loaded with 0.57% and 0.94% (w/w) iron, induced DNA strand breaks, lipoperoxidation, inhibition of redox metabolism and alterations of cell integrity, similarly to natural chrysotile. On the other hand, the incubation with ferric nitrilotriacetate, a cell-permeating iron complex, even if it caused an intracellular overloading of iron very similar to that elicited by iron-loaded synthetic chrysotile and by natural chrysotile, did not exert any of these effects. This suggests that chrysotile is not toxic by acting simply as a carrier of iron into the cell, but rather that the redox activity of iron is potentiated when organized at the fibers surface into specific crystallographic sites having coordination states able to activate free radical generation. Synthetic chrysotile fibers may be proposed as a standard reference sample and model solids for experimental studies on asbestos fibers aiming to clarify the mechanisms of its toxicity and to synthesize new fibers devoid of pathogenic effects.

- by Canzio Romano
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- Inorganic Chemistry, Organic Chemistry, Free Radicals, Scanning Electron Microscopy

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32 P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides Abbreviations: ara-C, cytosine arabinoside; B[a]P, benzo[a]pyrene; BNF, ␤-naphthoflavone; C3H10T1/2, C3H10T1/2CL8; trans-B[a]P-4,5-diol, (±) trans-4,5-dihydro-4,5-dihydroxyB[a]P; BPDE, anti-trans-B[a]P-7,8-diol-9,10-epoxide; MMS, methyl methanesulfonate; PAH, polycyclic aromatic hydrocarbon; ROS, reactive oxygen species ଝ The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. (S. Nesnow). 1383-5718/02/$ -see front matter. Published by Elsevier Science B.V. PII: S 1 3 8 3 -5 7 1 8 ( 0 2 ) 0 0 2 1 8 -8

- by Maria Pimentel
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- Genetic Toxicology, DNA damage, Environmental Biotechnology, System Design

Single stranded DNA breakage induced by lead nitrate in mice has been studied in vivo using alkaline single cell gel electrophoresis (comet assay). Mice were administered orally 0.7, 1.4, 2.8, 5.6, 11.2, 22.4, 44.8 and 89.6 mg/kg body weight of lead nitrate and the assay was performed on whole blood at 24, 48, 72 h, 1st and 2nd week. Significant increase in mean tail-length of DNA was observed at all time intervals after treatment with lead nitrate when compared to controls. The mean tail-length did not show a dose-related increase and the elevation in the mean tail-length was of a fluctuating type. Increase in mean tail-lengths clearly gives evidence that lead nitrate causes DNA damage effectively. The study indicates that the alkaline comet assay is a sensitive and rapid method to detect DNA damage caused by heavy metals.

- by Kaiser Jamil
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- Toxicology, DNA damage, DNA repair, DNA

Ž. Aflatoxin B AFB is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on 1 1 Ž. Cancer IARC. The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB in fish using the Comet assay. 1 Two different species of fish were selected as models due to previously established sensitivity to AFB : rainbow trout 1 Ž. Ž. sensitive and channel catfish resistant. Fish were i.p. injected with 0.5 mg AFB r1 ml DMSOr1 kg body weight. The 1 Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney Ž. Ž. tissue tested displayed significant p-0.05 and extensive DNA damage shown by increased tail moment after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB and for evaluating organ specific effects of these agents in 1 different species.

- by Refaat Elfayoumi
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- Genetic Toxicology, DNA damage, Environmental Biotechnology, FISH

In the last decade, molecular epidemiological studies have provided new perspectives on studying environmental risks in pediatric populations, based on the growing understanding that children may be more susceptible to toxicants than adults. Protecting children's health is a social priority, and specific research programs have been initiated with this purpose in the United States and Europe. These programs address the development of (i) less invasive methods for biological specimens collection, (ii) specific tools for interpretation and validation of biomarkers, (iii) methods for translating biomarker results into intervention strategies and for integrating them with environmental monitoring and health data, (iv) optimal ways to obtain consent and provide information to children and/or their parents participating in the studies and (v) techniques for the effective communication with policy makers and the public. Critical issues in children's environmental research discussed in this paper include specific needs of study design, exposure assessment, sample collection and ethics. Special consideration is given to the autonomy of the child in giving consent, the details and nature of the information provided, and the need to warrant controlled access to sensitive information. The use of incentives such as gifts and payment to ensure the participation of school-aged children is specifically discussed. Examples of field studies that are focused on the effects of pesticides, air pollution and formaldehyde are used to illustrate advantages and limitations of biomarker studies in children. #

- by Lisbeth Franco
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- Genetics, Epidemiology, Child Welfare, Environmental Health

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the fre-quency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents. Environ. Mol. Mutagen. 38:311-315, 2001.

- by Sharbel Weidner Maluf
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- Radiation, DNA damage, Biological Sciences, DNA
- by Giovana Gamaro
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- Chemistry, Clinical Biochemistry, Oxidative Stress, DNA damage

In order to mimic the biological effects of an oil spill in Arctic waters, we examined several types of biomarkers (genes, enzymes, metabolites, and DNA damage) in polar cod Boreogadus saida experimentally exposed to the water soluble fractions of crude oil. During 4 weeks of exposure, induction of the studied biomarkers exceeded baseline levels. The mRNA expression of the cytochrome P4501A1 (cyp1a1) gene was the most promising biomarker, with glutathione S-transferase (gst) as a suitable complement. The delayed ethoxyresorufin O-deethylase (EROD) and GST activities and their persistence following 2 weeks of depuration may allow detection of previous exposures in field samples. The composition of PAH metabolites in the bile indicated the bioavailability of different PAH size-classes. Although mRNA expressions of antioxidant defense genes were induced at start of the exposure, with the strongest responses from catalase and cytosolic superoxide dismutase, they were poor for oil monitoring purposes due to their very short response times. Significant DNA damage demonstrated genotoxicity even at low PAH concentrations (<15 g L −1 ) and was correlated with benzo(a)pyrene and pyrene metabolites in the bile.

- by Renée Bechmann
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- Water, Petroleum, Aquatic Toxicology, DNA damage

Thiosemicarbazones are versatile organic compounds that present considerable pharmaceutical interest because of a wide range of properties. In our laboratory we synthesised some new metal-complexes with thiosemicarbazones derived from natural aldehydes which showed peculiar biological activities. In particular, a nickel complex [Ni(S-tcitr) 2 ] (S-tcitr = S-citronellalthiosemicarbazonate) was observed to induce an antiproliferative effect on U937, a human histiocytic lymphoma cell line, at low concentrations (IC 50 = 14.4 lM). Therefore, we decided to study the interactions of this molecule with various cellular components and to characterise the induced apoptotic pathway. Results showed that [Ni(S-tcitr) 2 ] causes programmed cell death via down-regulation of Bcl-2, alteration of mitochondrial membrane potential and caspase-3 activity, regardless of p53 function. The metal complex is not active on G 0 cells (i.e. fresh leukocytes) but is able to induce perturbation of the cell cycle on stimulated lymphocytes and U937 cells, in which a G 2 /M block was detected. It reaches the nucleus where it induces, at low concentrations (2.5-5.0 lM), DNA damage, which could be partially ascribed to oxidative stress. [Ni(S-tcitr) 2 ] is moreover able to strongly reduce the telomerase activity. Although the biological target of this metal complex is still unknown, the reported data suggest that [Ni(S-tcitr) 2 ] could be a good model for the synthesis of new metal thiosemicarbazones with specific biological activity.

- by Annamaria Buschini
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- Chemistry, Inorganic Chemistry, Flow Cytometry, Cell Cycle

Hexavalent chromium is the major terrestrial pollutant released from electroplating industries, tanneries, thermal power plants and sewage. Cr(VI) is reported genotoxicant in vertebrates. Sub-lethal level of Cr(VI) can cause DNA damage. Earthworms are ecologically important soil invertebrates, used for ecotoxicological studies. In present study coelomocytes of Dichogaster curgensis were used for assessing Cr(VI) genotoxicity using single cell gel electrophoresis [(SCGE) i.e. comet assay] following in vitro and in vivo exposures. Cr(VI) produces reactive oxygen species (ROS) in the cellular system, which can be a major cause of DNA damage. Dose dependent DNA damage was observed during in vitro exposure, while in vivo exposure showed increasing DNA damage till 30 ppm in terms of arbitrary units and unexpected decrease in arbitrary units at 70 and 100 ppm of Cr(VI). It is speculated that the decrease in arbitrary units at higher concentrations might be due to lower DNA mobility, caused by formation of different types of cross-links.

- by Sophie Simarmeintières
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- Environmental Toxicology, DNA damage, Thermal Power Plant, Comet Assay
- by Dharmendra Maurya
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- Radiation Protection, South India, Current Science, Ganoderma Lucidum

The effect of melatonin, an indole hormone of the pineal gland, on the initiation of N-nitroso-N-methylurea (NMU)-induced carcinogenesis in rats and mutagenesis in vitro has been investigated. Two-month-old female LIO rats (groups 1 and 2) were exposed to a single injection of NMU (50 mg/kg of body weight, i.v.). Rats from group 2 were given melatonin orally (20 mg/l) from 18:00 to 09:00 h over 3 days (2 days before and 1 day after NMU injection). Animals from group 1 (control) were administered the solvent (ethanol/water, 1:1000). Rats were followed up to natural death or were sacri®ced when moribund. Tumors developed both in rats treated with NMU alone (50.0%) and in rats exposed to NMU plus melatonin (34.8%). The percentage of malignant tumor-bearing rats in group 2 (21.7%) was lower (P , 0:02) than that in the other group (41.7%). Melatonin also decreased the multiplicity of malignant tumors 1.3-fold and reduced the incidence of malignancies in some organs. Two in vitro tests were used for mutagenesis studies: the Ames test (strains TA 100 and TA 102 of Salmonella typhimurium) and the Single Cell Gel Electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells. Melatonin itself revealed no genotoxic effect in either of the tests. No protective action of melatonin (at doses of up to 2 mmol/ plate) towards NMU was found in the Ames test. In contrast, in the SCGE assay a slight, but statistically signi®cant (P , 0:001), dose-related anticlastogenic effect of melatonin (10 210 ±10 27 M) was observed. Thus, our data indicate that melatonin may act as an anti-initiating hormone in NMU-induced carcinogenesis and possess anticlastogenic activity towards NMU in CHOK1 cells. q

- by Sergei Musatov
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- DNA damage, Melatonin, Salmonella Typhimurium, Statistical Significance

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B 1 (AFB 1 ) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B 1 at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB 1 control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (Pb 0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B 1 control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB 1 control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB 1 -induced molecular alterations in hepatic cells during carcinogenesis.

- by Pradip Behare
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- Genetics, Probiotics, DNA damage, Gene expression

Ethnopharmacological relevance: Baccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as "carqueja", have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism. Aim of the study: To evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions. Materials and methods: Aqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300 g/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10 mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2 mg/kg or 42 mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102. Results: The anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3-300 g/kg) with AFBt reduced the paw edema (3 h) at lower levels (29.2-37.3%; P < 0.01), than those observed for AEBt (44.7-54.2%; P < 0.001), EFBt (49.3-58.2%; P < 0.001) or indomethacin (64.6%, P < 0.001, 10 mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC 50 182.6 g/ml), EFBt (IC 50 78.1 g/ml) and AFBt (IC 50 86.2 g/ml), with lower effects on HTC hepatic cell (IC 50 308.8 g/ml, 396.5 g/ml and 167.9 g/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC 50 372.5 g/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC 50 2.2 g/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC 50 of 0.42 g/ml; the IC 50 for doxorubicin for HEK and HTC cells was 0.28 g/ml and 14.4 g/ml, respectively, at 96 h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42 mg/kg for 15 days altered the kidney relative weight, but not at 4.2 mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively. Conclusions: Baccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells.

- by Israel Felzenszwalb
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- Water, Folk Medicine, Complementary and Alternative Medicine, Plant Biology

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line an...

- by Jeanne Perrin
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- Risk assessment, Cryopreservation, DNA damage, Mice

Aims: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. Methods and Results: CHLN was assayed at concentrations of 0AE5 and 2AE5 lg ml )1 , and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. Conclusions: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. Significance and Impact of the Study: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.

- by Mário Mantovani
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- Multidisciplinary, Applied Economics Letters, Comet Assay, Cell nucleus

Zinc (Zn) is an essential cofactor required by numerous enzymes that are essential for cell metabolism and the maintenance of DNA integrity. We investigated the effect of Zn deficiency or excess on genomic instability events and determined the optimal concentration of two Zn compounds that minimize DNA-damage events. The effects of Zn sulphate (ZnSO(4)) and Zn carnosine (ZnC) on cell proliferation were investigated in the WIL2-NS human lymphoblastoid cell line. DNA damage was determined by the use of both the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Zn-deficient medium (0μM) was produced using Chelex treatment, and the two Zn compounds (i.e. ZnSO(4) and ZnC) were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0μM. Results from an MTT assay showed that cell growth and viability were decreased in Zn-depleted cells (0μM) as well as at 32μM and 100μM for both Zn compounds (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001). DNA strand-breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). The CBMN-Cyt assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001). Elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were induced in Zn-depleted cells (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001), whereas genome damage was reduced in supplemented cultures for both Zn compounds at 4μM and 16μM, possibly suggesting that these concentrations may be optimal for genome stability. The potential protective effect of ZnSO(4) and ZnC was also investigated following exposure to 1.0Gy γ-radiation. Culture in medium containing these compounds at 4-32μM prior to irradiation displayed significantly reduced frequencies of MNi, NPBs and NBuds compared with cells maintained in 0μM medium (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001). Expression of γ-H2AX and 8-oxoguanine glycosylase measured by western blotting was increased in Zn-depleted cells. These results suggest that Zn plays important role in genomic stability and that the optimal Zn concentration-range for prevention of DNA damage and cytotoxicity in vitro lies between 4 and 16μM.

- by Peter Zalewski and +1
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- Genetic Toxicology, DNA damage, Environmental Biotechnology, Cell line

Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured. Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics. Strand breaks correlated with body mass index in the diabetic group. A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations. When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal. DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia.

- by Maria Dusinska
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- Chemistry, Free Radicals, Statistical Analysis, DNA damage

Red beetroot, a common ingredient of diet, is a rich source of a specific class of antioxidants, betalains. Our previous studies have shown the protective role of beetroot juice against carcinogen induced oxidative stress in rats. The aim of this study was to examine the effect of long term feeding (28 days) with beetroot juice on phase I and phase II enzymes, DNA damage and liver injury induced by hepatocarcinogenic N-nitrosodiethylamine (NDEA). Long term feeding with beetroot juice decreased the activities of enzymatic markers of cytochrome P450, CYP1A1/1A2 and CYP2E1. NDEA treatment also reduced the activities of these enzymes, but increased the activity of CYP2B. Moreover, combined treatment with beetroot juice and NDEA enhanced significantly CYP2B only. Modulation of P450 enzyme activities was accompanied by changes in the relevant proteins levels. Increased level and activity of NQO1 was the most significant change among phase II enzymes. Beetroot juice reduced the DNA damage increased as the result of NDEA treatment, as well as the biomarkers of liver injury.

- by Wanda Baer-dubowska
- •
- DNA damage, Western blotting, ALP, Proteins

It is well established that ultraviolet (UV) radiation from sunlight damages skin cells' DNA. Wavelengths in the UVB range are absorbed by DNA and can induce mutagenic lesions such as pyrimidine dimers. On the other hand, genotoxic effects of solar UVA are mainly mediated by the activation of endogenous photosensitizers resulting in the generation of a local oxidative stress. Exogenous chemicals, such as drugs like psoralens or fluoroquinolones, sometimes amplify UV-induced harmful effects. DNA damage can lead to mutations and genetic instability. This is one of the reasons why sunlight overexposure increases the risk of skin cancer. But DNA photolesions can also be involved in other skin-specific responses to UV radiation: erythema, immunosuppression, and melanogenesis are examples reported in the literature. The aim of this short review is to summarize the general knowledge in the field of UV-induced DNA damage. Besides the biological consequences of DNA photolesions, this article also deals with technologies used for their detection and shows how helpful such approaches can be to assess photoprotection provided by sunscreens. ( J Am Acad Dermatol 2008;58:S139-48.)

- by Jean-Roch MEUNIER
- •
- Biology, Cell Cycle, Dermatology, Oxidative Stress

DNA integrity was investigated in the lymphocytes of 50 bus drivers, 20 garagemen and 50 controls using the comet assay with excision repair enzymes. In parallel, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 15-F(2t)-isoprostane levels in the urine and protein carbonyl levels in the plasma were assessed as markers of oxidative damage to DNA, lipids and proteins. Exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) and volatile compounds was measured by personal samplers for 48 and 24h, respectively, before the collection of biological specimens. Both exposed groups exhibited a higher levels of DNA instability and oxidative damage to biological macromolecules than the controls. The incidence of oxidized lesions in lymphocyte DNA, but not the urinary levels of 8-oxodG, correlated with exposure to benzene and triglycerides increased this damage. Oxidative damage to lipids and proteins was associated with exposure to cPAHs and the lipid peroxidation levels positively correl...

- by Radim Šrám
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- Air pollution, Oxidative Stress, DNA damage, DNA

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (,0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

- by Goran Gajski
- •
- Inorganic Chemistry, Organic Chemistry, Petroleum, Tobacco

3-Methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.

- by Eva Miadoková
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- DNA damage, DNA repair, DNA, Cell line

Terminalia catappa L. is a popular folk medicine for preventing hepatoma and treating hepatitis in Taiwan. In this paper, we examined the protective effects of T. catappa leaf water extract (TCE) and its major tannin component, punicalagin, on bleomycin-induced genotoxicity in cultured Chinese hamster ovary cells. Pre-treatment with TCE or punicalagin prevented bleomycin-induced hgprt gene mutations and DNA strand breaks. TCE and punicalagin suppressed the generation of bleomycin-induced intracellular free radicals, identi®ed as superoxides and hydrogen peroxides. The effectiveness of TCE and punicalagin against bleomycin-induced genotoxicity could be, at least in part, due to their antioxidative potentials. q

- by Jih-heng Li
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- Folk Medicine, Free Radical, Reactive Oxygen Species, Anions

GSTM1, T1 and P1 are important enzymes of glutathione S-transferases (GSTs), involved in the metabolism of many endogenous and exogenous compounds. Individual genetic variation in these metabolizing enzymes may influence the metabolism of their substrates. The present study was designed to determine the genotoxic effects using DNA damage and its association with GSTM1, GSTT1, and GSTP1 (Ile105Val) genetic polymorphisms in workers occupationally exposed to organophosphate pesticides (OPs). We examined 230 subjects including 115 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using individual PCR or PCR–RFLP. Significantly higher DNA tail moment (TM) was observed in workers as compared to control subjects (14.41 ± 2.25 vs. 6.36 ± 1.41 tail % DNA, p < 0.001). The results revealed significantly higher DNA TM in workers with GSTM1 null genotype than those with GSTM1 positive (15.18 vs. 14.15 tail % DNA, p = 0.03). A significantly higher DNA TM was also observed in workers with homozygous Ile–Ile GSTP1 genotype than heterozygous (Ile–Val) and mutant (Val–Val) GSTP1 genotype (p = 0.02). In conclusion, the results show that null deletion of GSTM1 and homozygote wild GSTP1 genotype could be related to inter-individual differences in DNA damage arises from the gene–environment interactions in workers occupationally exposed to OPs.• DNA damage and its association with GSTM1, GSTT1 and GSTP1 genotypes carried out. • Workers with exposure to organophosphate pesticides found increased DNA damage. • DNA damage observed to be associated with GSTM1 null and GSTP1 Ile/Ile genotypes.

- by Shyam GROVER
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- Genetic Toxicology, DNA damage, Environmental Biotechnology, Mutation

Ocean acidification (OA) may alter the behaviour of sediment-bound metals, modifying their bioavailability and thus toxicity. We provide the first experimental test of this hypothesis with the amphipod Corophium volutator. Amphipods were exposed to two test sediments, one with relatively high metals concentrations (Σ metals 239 mg kg À1 ) and a reference sediment with lower contamination (Σ metals 82 mg kg À1 ) under conditions that mimic current and projected conditions of OA (390-1140 latm pCO 2 ). Survival and DNA damage was measured in the amphipods, whereas the flux of labile metals was measured in the sediment and water column (WC) using Diffusive Gradients in Thin-films. The contaminated sediments became more acutely toxic to C. volutator under elevated pCO 2 (1140 latm). There was also a 2.7-fold increase in DNA damage in amphipods exposed to the contaminated sediment at 750 latm pCO 2 , as well as increased DNA damage in organisms exposed to the reference sediment, but only at 1140 latm pCO 2 . The projected pCO 2 concentrations increased the flux of nickel and zinc to labile states in the WC and pore water. However, the increase in metal flux at elevated pCO 2 was equal between the reference and contaminated sediments or, occasionally, greater from reference sediments. Hence, the toxicological interaction between OA and contaminants could not be explained by effects of pH on metal speciation. We propose that the additive physiological effects of OA and contaminants will be more important than changes in metal speciation in determining the responses of benthos to contaminated sediments under OA. Our data demonstrate clear potential for near-future OA to increase the susceptibility of benthic ecosystems to contaminants. Environmental policy should consider contaminants within the context of changing environmental conditions. Specifically, sediment metals guidelines may need to be reevaluated to afford appropriate environmental protection under future conditions of OA.

- by David Roberts and +1
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- Water quality, DNA damage, Global Change Biology, Quality Control

Photosome Ò is constituted of photolyases included in liposomes. Photolyase is a bacterial enzyme that can repair ultraviolet B (UVB)-induced cyclobutane pyrimidine dimers (CPD) in eukaryotic cells. A modified version of the alkaline comet assay has been set up to evaluate the repair activity of this enzyme after a single dose of UVB (312 nm, 0.06 J/cm 2 ) in human keratinocytes. The formation of single strand breaks (SSB) induced by the UVA photoactivation of the enzyme (1.2 J/cm 2 ) was inhibited by the pretreatment of the cells with 4 mM L L-ergothioneine (ERT) during 30 min at 37°C. To increase the sensitivity of the comet assay, an additional lysis was used with a buffer containing sodium dodecyl sulfate (0.5%) and proteinase K (0.1 mg/ml) for 60 min at 37°C. Unrepaired CPD by photolyase were revealed by a second enzymatic treatment with T4 endonuclease V, a CPD specific glycosylase. UVB irradiation increased the SSB level in keratinocytes and additional T4NV treatment enhanced this SSB level by 1.5-2.0-fold confirming that CPD were the major base modifications generated by UVB irradiation. UVA-photoactivated Photosome Ò repaired CPD lesions and decreased the SSB levels by 2.6-3.3-fold. Photosome Ò could be an additional component of sunscreens to reduce the development of skin cancer.

- by Michel Méo
- •
- Keratinocytes, Skin Cancer, Enzyme, Comet Assay

Since chlorhexidine is effective against microorganisms, it is widely recommended in dentistry. However, studies have provided evidence that chlorhexidine is toxic for a variety of cell types. In order to identify potential genotoxins in different cell types, the purpose of this study was to investigate whether chlorhexidine digluconate is able to cause, in terms of DNA damage, alterations in leukocytes, liver, kidney and urinary bladder by the single cell gel (comet) assay. Ten male Wistar rats were divided into two groups: a negative control and the experimental group treated with 3 ml of 0.12% chlorhexidine digluconate by gavage once a day for 8 days. Statistically significant increases of DNA damage was observed in leukocytes and kidney cells of the chlorhexidine digluconate treated group as depicted by the mean tail moment. Taken together, the data indicate that leukocytes and kidney cells are potential targets for primary DNA damage following oral exposure to chlorhexidine digluconate as detected by single cell gel (comet) assay.

- by Daniel Ribeiro
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- DNA damage, DNA, Statistical Significance, Disinfectants
- by Joseph Neulen
- •
- Recycling, Environmental Monitoring, DNA damage, Germany

Geum quellyon Sweet, a perennial herb of the Rosaceae family, has been used in the traditional medicine of the Mapuche Amerindians of Chile to treat tooth neuralgia, gastric inflammation, prostatitis and to regulate menstruation, and for its diuretic and aphrodisiac properties. Although many benefits have been claimed for this plant, few scientific studies are available in the literature. In this study, we investigated the antioxidant activity of a methanolic extract of Geum quellyon roots. We also examined the anticancer action of this plant on Caco-2 (colon adenocarcinoma cells), DU-145 (androgen-insensitive prostate cancer cells) and KB (oral squamous carcinoma cells) human tumor cell lines. Our data showed that Geum quellyon extract, containing tannins, exhibits interesting antioxidant properties, expressed by its capacity to scavenge 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and superoxide anion (O 2

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protaminedeficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.

DNA integrity was analyzed in the lymphocytes of 65 non-smoking city policemen during January and September 2004 using the comet assay combined with excision repair enzymes. Information about inhalation exposure was obtained by (1) stationary monitoring of PM2.5 and carcinogenic polycyclic aromatic hydrocarbons (cPAHs) during the sampling periods and (2) personal exposure monitoring of cPAHs 48 h before blood sampling. The data were completed by a lifestyle questionnaire. Regardless of the season of the year, policemen working outdoors (exposed group) exhibited higher levels of DNA damage than those working indoors (controls). Within the exposed group, the levels of both unspecified and oxidative DNA damage detected in January significantly exceeded those found in September. The controls did not show analogous inter-seasonal variability. The winter levels of oxidative DNA damage positively correlated with exposure to cPAHs, probably reflecting increased oxidative stress as a result of high concentrations of PM2.5. In comparison with the wild type genotype, the carriers of at least one mutated allele, CYP1A1*2C (Ile/Val), MTHFR 2656 or MS 2656, and the EPHX1-medium phenotype appeared to be more susceptible specifically to the induction of oxidative DNA damage, while the p53 MspI mutation predisposed the carrier to a higher incidence of both breaks and oxidative lesions in DNA. In contrast, GSTM1-null and vitamin C tended rather to protect DNA integrity.

- by Radim Šrám
- •
- Police, Environmental Monitoring, Air pollution, Oxidative Stress

Malaysian locally processed raw food products are widely used as main ingredients in local cooking. Previous studies showed that these food products have a positive correlation with the incidence of cancer. The cytotoxicity effect was evaluated using MTT assay (3-(4,5-dimetil-2-thiazolil)-2,5-diphenyl-2H-tetrazolium bromide) against Chang liver cells at 2000 lg/ml following 72 h incubation. Findings showed all methanol extracts caused a tremendous drop in the percentage of cell viability at 2000 lg/ml (shrimp paste -41.69 ± 3.36%, salted fish -37.2 ± 1.06%, dried shrimp -40.32 ± 1.8%, p < 0.05). To detect DNA damage in a single cell, alkaline Comet Assay was used. None of the extracts caused DNA damage to the Chang liver cells at 62.5 lg/ml following 24 h incubation, as compared to the positive control, hydrogen peroxide (tail moment -9.50 ± 1.50; tail intensity -30.50 ± 2.50). Proximate analysis which was used for the evaluation of macronutrients in food showed that shrimp paste did not comply with the protein requirement (<25%) as in Food Act 1983. Salt was found in every sample with the highest percentage being detected in shrimp paste which exceeded 20%. Following heavy metal analysis (arsenic, cadmium, lead and mercury), arsenic was found in every sample with dried shrimps showing the highest value as compared to the other samples (6.16 mg/kg). In conclusion, several food extracts showed cytotoxic effect but did not cause DNA damage against Chang liver cells. Salt was found as the main additive and arsenic was present in every sample, which could be the probable cause of the toxicity effects observed.

- by Nor Rajab
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- Arsenic, Malaysia, Food, DNA damage

Cytokinesis-block micronucleus (CB-MN) assay and single-cell gel electrophoresis (SCGE) were employed to analyze leukocytes from 14 Fanconi anemia (FA) patients, 30 Down syndrome (DS) patients, and 30 control individuals, to examine the sensitivity of these techniques to detect genomic instability in these 2 diseases. The DS patients presented increased DNA damage as measured by SCGE in relation to controls. The frequencies of micronuclei and dicentric bridges were similar to those of controls. Micronucleus frequency, dicentric bridge frequencies, and DNA damage were higher in FA patients than in controls. The high frequency of micronuclei observed in FA patients seems to be due to clastogenic events, because an increase in the frequency of dicentric bridges was also observed. Micronuclei are expressed mutations and need cell division to appear. The damage detected by SCGE is repairable, and does not require cell division. Under alkaline conditions, SCGE assesses double-and single-strand breaks and alkali-labile sites. The 2 methods are efficient for monitoring mutagenic events in exposed populations or in individuals with genetic instability. While the damage measured by micronucleus analysis is accumulated over a long period of time, DNA damage measured by SCGE reflects recent, unrepaired events.

- by Sharbel Weidner Maluf
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- Adolescent, DNA damage, Down Syndrome, Cell Division

Natural rubber latex (NRL) has several features that make it an excellent biomaterial to promote the growth and repair of tissues, skin and bones. Most of the research with NRL membranes uses a mixture of different clones and chemical preservatives in the collection process. In this study, we compared five clones that produce NRL, seeking to identify their differences in biocompatibility. The clones studied were RRIM 600, PB 235, GT1, PR 255 and IAN 873 commonly found in plantations in Brazil. We did also study the effect of ammonia used during latex collection. NRL membranes were prepared aseptically and sterilized. In the in vitro tests, the membranes remained in direct contact with mouse fibroblasts cells for three periods, 24, 48 and 72 h. In the in vivo tests, the membranes were implanted subcutaneously in rabbits. The results indicated the biocompatibility of the membranes obtained from all clones. Membranes from the clones RRIM 600 and IAN 873 induced greater cell proliferation, suggesting greater bioactivity. It was found that the membranes made from latex that was in contact with ammonia during collection, showed cytotoxic and genotoxic effects in cultures, as well as necrosis, and increased inflammatory cells in the rabbit&amp;amp;amp;amp;amp;amp;amp;#39;s tissues close to the implant.

- by Lionel Camus
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- Chemistry, Ecotoxicology, Aquatic Toxicology, DNA damage

This study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC50 values of SN38 were observed at 5 and 10 μM of DNA-PK inhibitors. Moreover, at 1–2 μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC50 of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

- by Jean-Yves Masson and +1
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- Flow Cytometry, Breast Cancer, Cell Cycle, DNA damage

Plants of the genus Hibiscus thrives produce a diversity of molecules with bioactive properties. In a previous study of Hibiscus tiliaceus L. methanolic extract (HME) using bacteria and yeast, as test media, it has been shown that HME strongly inhibited the mutagenic action of H 2 O 2 or tert-butyl-hydroperoxide (t-BHP). Here, our interest is to evaluate the genotoxicity and the antigenotoxic/antimutagenic properties of HME using oxidative challenge with H 2 O 2 and t-BHP in V79 cells. We determined cytotoxicity using clonal survival assay; evaluated DNA damage using the comet assay and the micronucleus test in binucleated cells besides of the lipid peroxidation degree and the reduced glutathione content. We examined the ability of HME in quenching hydroxyl radical by means of a HPLC-based method utilizing the hypoxanthine/xanthine oxidase assay. At concentrations ranging from 0.001 to 0.1 mg/mL, HME was not cytotoxic, genotoxic or mutagenic. Treatment with non-cytotoxic concentrations of HME increased cell survival after H 2 O 2 and t-BHP exposure and prevented DNA damage. The pre-treatment with HME also was able to decrease the mutagenic effect of these genotoxins, evaluated using the micronucleus test. HME prevented the increase in lipid peroxidation and decrease in GSH content in response to the oxidative challenge. Therefore, the ability in preventing against H 2 O 2 -and t-BHP-induced GSH depletion and lipid peroxidation was probably a major contribution to the cytoprotective effects. Moreover, HME acts as a hydroxyl radical scavenger. In summary, HME did not have a harmful or inhibitory effect on the growth of V79 cells and presented antioxidant activity, consequently, both antigenotoxic and antimutagenic effects against oxidative DNA damage.

- by Dinara Jaqueline Moura
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- DNA damage, Cell line, Micronuclei, Glutathione

The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests:

- by Nicola Hewitt
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- Europe, Skin, Cosmetics, Comet Assay

Ethnopharmacological relevance: Essential oils of palmarosa and citronella have been extensively used in ancient Indian and South-east Asian traditional medicines. Aim of the study: These essential oils have been reported to exhibit antimicrobial, anti parasitic effects against bacteria, yeasts, filamentous fungi, and viruses. In the present study the oils were tested for their potential antigenotoxic and antioxidant properties in human lymphocyte cells. Materials and methods: The antigenotoxic effect on human lymphocyte cells (measurement of cell viability, DNA damage) was studied using trypan blue dye exclusion test, plasmid pBR322 DNA strand scission, and comet assay. Anti-oxidant activity was evaluated by DPPH + free radical scavenging and lipid peroxidation assay. Results: The essential oils showed a good antigenotoxic activity against methyl methanesulphonate (MMS) and hydrogen peroxide. In addition, a significant dose dependent antioxidant activity was observed. Conclusion: Our data provide evidence that support the usage of palmarosa and citronella essential oils in traditional herbal preparations. They can constitute a natural source of a new and safe antioxidant.

We investigated levels of DNA damage in blood cells of barn swallows (Hirundo rustica) inhabiting the Chernobyl region to evaluate whether chronic exposure to low-level radioactive contamination continues to induce genetic damage in free-living populations of animals. Blood samples were obtained from barn swallows collected at sites with different background levels of radiation, including a relatively uncontaminated area. The extent of DNA damage was evaluated using the alkaline (pH = 12.1) comet assay, a robust and sensitive electrophoresis-based technique widely employed in research ranging from biomonitoring to clinical studies. We found that levels of DNA damage, as indexed by the extent of DNA migration, were increased in barn swallows living in areas surrounding Chernobyl when compared to swallows sampled at low-level sites. The results we obtained are consistent with previous findings on this same species, which showed that swallows breeding in areas heavily contaminated with radionuclides have increased mutation rates, higher oxidative stress and incidence of morphological aberrations and tumors. Overall, these results indicate that chronic exposure to radioactive contaminants, even 20 years after the accident at the Chernobyl nuclear power plant, continues to induce DNA damage in cells of free-living animals.

- by Michael Wyatt and +1
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- Genetics, Geography, Biomarkers, Oxidative Stress

The ethanolic extract of propolis, especially the Brazilian green type, is widely and mainly used for therapeutic purposes despite the lack of knowledge about its effects and its cellular mode of action. This type of propolis, derived from Baccharis dracunculifolia (alecrim-do-campo), has been extensively commercialized and the consumers use it to enhance health. This work aimed to assess the genotoxic/mutagenic and antigenotoxic/antimutagenic potential of the ethanolic extracts of Brazilian green propolis and of B. dracunculifolia, on mammalian cells. It was not observed genotoxic and mutagenic effect by both extracts. After evaluate the exposure of the cells to each extract with a recognized mutagen, simultaneously, the results showed a significant reduction on DNA damage. The experiment carried out with a pre-incubation period was more effective than without incubation test, showing that the tested extracts were able to inactivate the mutagen before it could react with the DNA.

- by Matheus M Roberto
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- Cell Culture, Mammalian Cell Culture, Propolis, Mutagenesis