DNA In Comet Tail Research Articles

Cancer pulmonaire et avancées physiopathologiques : des applications proches ?

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1 ± 11.8) compared to whole blood (43.0 ± 12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p = 0.01) in ex-/non-smokers (41.0 ± 2.1%) compared to smokers (50.3 ± 2.7%). This phase of repair was also slower (p = 0.02) in HNSCC patients (36.8 ± 2.1%) compared to controls matched on age and smoking (46.4 ± 3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.

Long-term genotoxic effect of monocrotophos in different tissues of freshwater fish Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

Monocrotophos, commonly known as azodrin, is one of the organophosphate pesticides extensively used in agricultural practices throughout the world. However, little information is available on its long-term genotoxic effects in different tissues of fish using genotoxicity biomarkers. The objective of the present study was to detect DNA damage, induced by monocrotophos in freshwater teleost fish Channa punctatus using the comet assay. The LC 50-96 h value of technical-grade monocrotophos was estimated for the fish species in a semi-static system. On the basis of the LC 50 value, the sublethal and nonlethal concentrations were determined. The DNA damage was measured in the gill, kidney and lymphocytes as the percentage of DNA in comet tails of fish exposed to different sublethal and nonlethal concentrations of monocrotophos. In general, significant effects ( P < 0.01) from both concentration and time of exposure were observed in exposed fish. It was found that the tissues at all concentrations exhibited the highest DNA damage on day 4, after which there was a decline in percentage tail DNA. The comparison of DNA damage among tissues at different concentrations indicated that the gill cells were more sensitive to the pesticide than the kidney cells and lymphocytes. This study also explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential of various agents.

Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1 ± 11.8) compared to whole blood (43.0 ± 12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower ( p = 0.01) in ex-/non-smokers (41.0 ± 2.1%) compared to smokers (50.3 ± 2.7%). This phase of repair was also slower ( p = 0.02) in HNSCC patients (36.8 ± 2.1%) compared to controls matched on age and smoking (46.4 ± 3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.

Studies on the Genotoxicity of Endosulfan in Different Tissues of Fresh Water Fish Mystus vittatus Using the Comet Assay

Endosulfan, a widely used organochlorine pesticide, is readily bio-accumulative in fishes and can be indirectly harmful to human populations. Limited efforts have been made to study long-term genotoxic effects of endosulfan in different tissues of fish using gentoxicity biomarkers. Therefore, the current investigation was undertaken to detect single-cell DNA strand breaks induced by endosulfan in the fresh water teleost fish Mystus vittatus using the comet assay. The LC(50) value of technical grade endosulfan was first determined for the fish species in a semistatic system, and on the basis of the LC(50) value, the sublethal and nonlethal concentrations were determined. The DNA damage was measured in gill, kidney, and erythrocytes as the percentage of DNA in comet tails of fish specimens exposed to the sublethal and nonlethal concentrations of endosulfan. In general, significant effects (p < 0.01) from both concentration and time of exposure were observed in exposed fishes. It was found that all the tissues at all concentrations exhibited the highest DNA damage on day 1, after which there was a nonlinear decline in the percentage of tail DNA. The comparison of DNA damage among the tissues at different concentrations could not show the sensitivity of particular tissue to endosulfan. The current study explored the utility of the comet assay for in vivo laboratory studies using fish species to screen the genotoxic potential of chemical agents.

Sperm Preparation: DNA Damage by Comet Assay in Normo- and Teratozoospermics

The present study was carried out on semen samples of human fertile and infertile subjects, teratozoospermics (TZs) and idiopathics (IDs), with neat semen and sperm prepared by swim up or Percoll density gradient centrifugation procedures. Sperm morphology analysis revealed that only head and midpiece defects in TZs and IDs were significantly (P < 0.001) higher compared to fertile subjects. Infertile subjects indicated significantly higher (P < 0.001) sperm DNA damage compared to fertile subjects. Fertile subjects with sperm prepared from neat and Percoll density gradient centrifugation exhibited a comet tail DNA percentage of 20% and 15%, respectively. The TZs and IDs infertile subjects had higher levels of comet tail DNA of 33% and 25% and 25% and 19%, respectively. A significant (F = 24.01; P = 0.0059) decrease in mean comet head DNA percentage or sperm DNA integrity was observed in neat samples from fertile and infertile subjects by Repeated Measures ANOVA. In Percoll prepared samples from fertile, TZs, and IDs, there was a significant increase in sperm DNA integrity. Similarily, there was a decrease in abnormal sperm morphology in swim up and Percoll prepared sperm compared to neat samples. The Percoll density gradient centrifugation procedure yields sperm with an increase in sperm DNA integrity relative to swim up. Sperm DNA damage of TZs with both sperm preparation methods was significantly (P < 0.01) higher when compared to fertile and IDs. But the level of DNA damage was higher in IDs compared to fertile subjects. Compared to the other methods tested, the Percoll method yielded sperm with improved DNA integrity. In conjunction with semen analysis, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating the patients to specific assisted reproductive treatments.

Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture

Antrodia cinnamomea, a well-known tradition Chinese medicine, possesses anti-tumor, anti-oxidation activities and stimulates the immune system. The aim of this study was to investigate the protective effect of water-soluble polysaccharides from the fermented filtrate and mycelia of Antrodia cinnamomea in submerged culture (ACSC) on hydrogen peroxide-induced cytotoxicity and DNA damage in Chang liver cells. Oxidative DNA damage was evaluated by single cell gel electrophoresis (Comet assay) or by the formation of 8-hydroxy-deoxyguanosine (8-OHdG) adducts. The polysaccharides isolated by ion-exchange chromatography contained glucose, xylose, galactose, arabinose, and mannose. The results showed that incubation of Chang liver cells with isolated polysaccharides at 200 μg/mL for 5 h prior to H 2O 2 treatment (50 μM, 30 min) significantly reduced oxidative DNA damage as detected by the formation of comet tail DNA and 8-OHdG adducts by 89% and 69%, respectively. Pre-treatment Chang liver cells with polysaccharides also reduced the levels of thiobarbituric acid reactive substances (TBARS) ( p < 0.01) and intracellular reactive species (ROS) ( p < 0.01) induced by H 2O 2. Moreover, glutathione S-transferase (GST) and the GSH/GSSG ratio were significantly increased in Chang liver cells pre-incubated with the polysaccharides ( p < 0.01). These results demonstrate that polysaccharides in ASCS have antioxidant properties which may involve up-regulation of GST activity, maintenance of normal GSH/GSSG ratio, and scavenging of ROS.

Improvement of wrinkles by an all- trans-retinoic acid derivative, d-δ-tocopheryl retinoate

All- trans -retinoic acid (RA) is well known as a potent anti-aging drug. However, RA is generally difficult to use topically due to its irritancy and mutagenicity . Therefore, we synthesized a new RA derivative , d -δ-tocopheryl retinoate (TR). To evaluate the potency of TR on anti-aging effects, investigating responses of human skin cells, keratinocytes and fibroblasts. To examine the potential for TR to remove reactive oxygen species (ROS), we used an electron spin resonance (ESR) spin-trapping method and a chemical assay. To investigate the effects of TR on skin cells, we used HaCaT cells, human keratinocytes, and human fibroblasts. Further, clinical test was carried out. In ESR study, TR showed the quenching abilities against singlet oxygen . In a biological system, TR significantly suppressed the elongation of DNA comet tails in HaCaT keratinocytes after UVB irradiation. The sum of those results suggests that TR has a capability to remove oxidative stress . TR had no effect on matrix metalloprotainase-1 (MMP-1) expression by UVA-irradiated fibroblasts. TR increased collagen production by fibroblasts and hyaluronic acid (HA) production by keratinocytes. We conducted a clinical study on wrinkle improvement using eight human volunteers. Treatment with TR exhibited a significant reduction in the depth of linear wrinkles, showing the potency of TR to improve wrinkles. Anti-wrinkle efficacy of TR is suggested to involve removing oxidative stress and modulating the metabolism of the extracellular matrix .

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay

To assess genotoxic effects of sodium arsenite (NaAsO 2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO 2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO 2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (≥120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO 2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.

Effects of extremely low frequency electromagnetic fields on DNA of testicular cells and sperm chromatin structure in mice

To study the effects of 50 Hz electromagnetic fields (EMFs) on DNA of testicular cells and sperm chromatin structure in mice. Mice were exposed to 50 Hz, 0.2 mT or 6.4 mT electromagnetic fields for 4 weeks. DNA strand breakage in testicular cells was detected by single-cell gel electrophoresis assay. Sperm chromatin structure was analyzed by sperm chromatin structure assay with flow cytometry. After 50 Hz, 0.2 mT or 6.4 mT EMFs exposure, the percentage of cells with DNA migration in total testicular cells increased from the control level of 25.64% to 37.83% and 39.38% respectively. The relative length of comet tail and the percentage of DNA in comet tail respectively increased from the control levels of 13.06% +/- 12.38% and 1.52% +/- 3.25% to 17.86% +/- 14.60% and 2.32% +/- 4.26% after 0.2 mT exposure and to 17.88% +/- 13.71% and 2.35% +/- 3.87% after 6.4 mT exposure (P < 0.05). Exposure to EMFs had not induced significant changes in S.D.alphaT and XalphaT, but COMPalphaT (cells outside the main population of alpha t), the percentage of sperms with abnormal chromatin structure, increased in the two exposed groups. 50 Hz EMFs may have the potential to induce DNA strand breakage in testicular cells and sperm chromatin condensation in mice.

Toxicity testing of herbicide norflurazon on an aquatic bioindicator species – the planarian Polycelis felina (Daly.)

Norflurazon is a bleaching, preemergence herbicide. Due to its mobility and long half-life it presents a potential for groundwater contamination. The aim of our study was to investigate toxic effects of norflurazon on non-targeted aquatic bioindicator organism, the planarian Polycelis felina (Daly.). Animals were exposed to water solutions of norflurazon in concentrations 200, 20, 2 and 0.2 μM. Mortality, locomotive and morphological changes were monitored. Histological changes were studied both on treated and control animals with light microscopy. The primary DNA damage on single planarian cells was studied using the alkaline comet assay. Three comet parameters were studied: tail length, percentage of DNA in comet tail and tail moment. The results showed that norflurazon caused mortality, locomotive, morphological and histological changes in treated animals compared to corresponding controls. The most prominent histological changes were damage of the outer mucous layer, lack of rhabdites, damage to epidermis and extensive damage to parenchyma cells. The results of alkaline comet assay indicated that norflurazon in concentrations of 2 and 0.2 μM induces significant increase of primary DNA damage in planarian cells compared to the corresponding control animals. The mean values of all three measured parameters were significantly elevated on the fourth day of the treatment compared with the first and the seventh day. Based on the results of mortality and locomotive observations, we conclude that the fourth day of the treatment represents a certain threshold within planarian metabolism followed by the beginning of detoxification and recovery. However, histological preparations and comet data statistics show results indicating that high toxicity on the seventh day of the treatment gave the results of decrease of DNA damage due to the tissue/cell damage (apoptosis) and not recovery. The present study showed the ability of norflurazon to induce a wide range of different toxicological responses in freshwater planarian Polycelis felina (Daly.).

Influence of hOGG1, XRCC1 and XRCC3 genotypes on biomarkers of genotoxicity in workers exposed to cobalt or hard metal dusts

Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. We examined whether variations in genes involved in base-excision ( hOGG1, XRCC1) and double strand break ( XRCC3) DNA repair contribute to inter-individual differences in genotoxic effects induced in the lymphocytes of 21 cobalt (Co) exposed, 26 hard metal (WC–Co) exposed and 26 matched control male workers. Genotyping was performed by PCR-RFLP. DNA single strand breaks and alkali-labile sites were measured by the alkaline Comet assay. Chromosomal rearrangements resulting from chromosome loss or acentric fragments were assessed as micronucleated mononucleates (MNMC) and binucleates (MNCB) with the cytokinesis-block micronucleus test. Urinary 8-hydroxydeoxyguanosine (8-OHdG) levels were used as an indicator of systemic oxidative DNA damage. A significantly higher frequency of MNMC was observed in WC–Co exposed workers with variant hOGG1 326 genotype. Multivariate analysis performed with genotypes, age, exposure status, type of plant, smoking and their interaction terms as independent variables indicated that MNMC and Comet tail DNA (TD) were influenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MNMC frequencies. Further studies will demonstrate whether genotyping for hOGG1 and XRCC3 polymorphisms is useful for a better individual monitoring of workers.

145 STAGE-SPECIFIC EFFECT OF OXIDATIVE STRESS ON DEVELOPMENTAL COMPETENCE, ROS GENERATION AND DNA DAMAGE OF PORCINE PARTHENOGENETIC EMBRYOS

We investigated the effect of oxidative stress on stage specific developmental ability, reactive oxygen species (ROS) generation and DNA damage of parthenogenetically activated porcine embryos. Cumulus-oocyte complexes (COCs) were aspirated from follicles on the surface of ovaries. The COCs were matured in NCSU-23 containing 10% (vol/vol) porcine follicular fluid and 10 IU/mL hCG during the first 22 h followed by an extra 22 h of culture in the hormone free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for activation. Oocytes were activated by a 100-�sec pulse of 1.5 kV/cm DC with 1-mm electrodes in 0.3 m mannitol, 0.1 mm MgSO4, and 0.05 mm CaCl2. Activated oocytes were then cultured for 5 h in NCSU-23 containing 5 mg/mL BSA, 10 �g/mL EGF and 7.5 �g/mL cytochalasin B. Embryos were then cultured for 6 days in PZM-5. In Experiment 1, after parthenogenetic activation, embryos were cultured at 38.5�C under 5% O2, 5% CO2 and 90% N2 (defined as 5% O2) or 5% CO2 in air (20% O2). The oxygen concentration for embryo culture was changed from 5% to 20% on day 1, 2, 3, 4, and 5 post-activation, respectively. Embryos were also cultured throughout 6 days in 5 and 20% O2. About 100 embryos were used in each experiment. The number of embryos cleaved and developed to blastocyst stage was observed on day 2 and 6, respectively. In Experiment 2, 10 to 20 embryos cultured in 5 and 20% O2 were collected on Days 2, 4, and 6 for the detection of ROS, intracellular glutathione (GSH) levels and DNA damage. Intracellular ROS and GSH levels, were measured with fluorescent dyes (22,72-dichlorodihydrofluorescein diacetate for ROS and Cell Tracker" Blue for GSH). DNA damage of individual embryos was detected with a comet assay. DNA damage was quantified by measuring the length of the streak of DNA comet tail between the edge of the zona pellucida and the end of the visible comet tail by image analysis software. The rate of migrated DNA area per total DNA was also quantified. In Exp. 1, the rate of blastocyst formation was significantly decreased (P &lt; 0.001) when embryos were cultured for 6 days under 20% O2 (17.8 � 4%) than 5% O2 (38.5 � 5%). The rates of blastocyst formation were significantly decreased (P &lt; 0.05) when O2 concentration was changed from 5 to 20% before Day 3. After Day 4, high O2 concentration did not affect the development. In Exp. 2, relative ROS levels were significantly higher (P &lt; 0.05) on Day 2 (1.5 � 0.03) and Day 4 (1.4 � 0.06) in embryos cultured under 20% O2 than in those cultured under 5% O2 (1.0). No difference was observed in GSH level. DNA damage was significantly increased (P &lt; 0.05) in Day 2 embryos cultured under 20% O2 (161 � 54 �m) than 5% O2 (65 � 8.8 �m). These results indicate that the oxidative stress to embryo development by high O2 concentration is stage specific, that embryos are more sensitive in early stages, and that the oxidative stress has correlation with the increase of intracellular ROS and DNA damage.

Urinary levels of insecticide metabolites and DNA damage in human sperm.

Members of the general population are exposed to non-persistent insecticides at low levels. The present study explored whether environmental exposures to carbaryl and chlorpyrifos are associated with DNA damage in human sperm. Subjects (n=260) were recruited through a Massachusetts infertility clinic. Individual exposures were measured as spot urinary metabolite concentrations of chlorpyrifos [3,5,6-trichloro-2-pyridinol (TCPY)] and carbaryl [1-naphthol (1N)], adjusted using specific gravity. Sperm DNA integrity was assessed by neutral comet assay and reported as comet extent, percentage DNA in comet tail (Tail%) and tail distributed moment (TDM). A statistically significant increase in Tail% was found for an interquartile range (IQR) increase in both 1N [coefficient=4.1; 95% confidence interval (CI) 1.9-6.3] and TCPY (2.8; 0.9-4.6), while a decrease in TDM was associated with IQR changes in 1N (-2.2; -4.9 to 0.5) and TCPY (-2.5; -4.7 to -0.2). A negative correlation between Tail% and TDM was present only when stratified by comet extent, suggesting that Tail% and TDM may measure different types of DNA damage within comet extent strata. Environmental exposure to carbaryl and chlorpyrifos may be associated with increased DNA damage in human sperm, as indicated by a change in comet assay parameters.

Impact of ethanol and acetaldehyde on DNA and cell viability of cultured neurones

Ethanol consumption has long been associated with brain damage. However, the mechanism underlying this deleterious effect remains unclear. Among different hypotheses, acetaldehyde is regarded by certain authors as playing a major role in the expression of ethanol toxicity, but there are still some uncertainties about the exact nature of its implication. We therefore tried to characterize the profile of the alterations of neuronal viability and DNA integrity obtained after either a direct exposure to ethanol or to acetaldehyde. Ethanol at concentrations within the range of blood alcohol levels in intoxicated humans (< or = 100 mmol/L) induced DNA alterations without any apparent effect on cell viability. Acetaldehyde (< or = 1000 micromol/L) can also induce DNA alterations but with a different profile of the DNA cellular alterations. The comparison between the distributions of the comet tail DNA indicated that ethanol induced strong breaks (tail DNA > or = 60 a.u.) generation whereas acetaldehyde rather induced lower breaks (20 < or = tail DNA < or = 50 a.u.) formation but affecting a greater number of neurones. Acetaldehyde had thus a different genotoxic potential which may suggest a different mode of action or a different cellular target. Furthermore, when a single 100 mmol/L ethanol exposure did not lead to any loss of cell viability, the addition of an inhibitor of aldehyde dehydrogenase was followed by a significant loss in viability. In contrast, the inhibition of catalase, which suppresses acetaldehyde synthesis, led to no reduced viability in the same exposure conditions. ROS also reduced viability, but this was observed only after both cytochrome P450 stimulation and catalase inhibition. These combined results could suggest that acetaldehyde may play a significant role in the expression of ethanol toxicity in brain.

DNA damage and cytotoxicity in pancreatic β-cells expressing human CYP2E1

Epidemiological studies have identified nitrosamines as a risk factor for Type I (insulin dependent) diabetes mellitus. These compounds require bioactivation by cytochrome P450 2E1 (CYP2E1) for exertion of their toxic effects. Two mammalian insulin secreting pancreatic β-cell lines BRIN BD11h2E1 and INS-1h2E1, which express human full length CYP2E1 cDNA, were used to elucidate the role of CYP2E1-mediated nitrosamine bioactivation in pancreatic β-cell dysfunction and destruction. These cell lines were shown to metabolise dimethylnitrosamine to produce formaldehyde at rates of 3.41 ± 0.24 and 3.65 ± 0.26 nmol/min mg microsomal protein, respectively. Following incubation with various concentrations of the nitrosamines dimethylnitrosamine, N-nitrosopyrrolidine and 1-nitrospiperidine, all of which are bioactivated by CYP2E1, cytotoxicity and DNA damage were assessed using either the neutral red assay or comet assay respectively. Exposure of CYP2E1 expressing cells to nitrosamines resulted in significant dose-dependent decreases in cell viability, which were not seen in cells which did not express CYP2E1. Following culture with nitrosamine concentrations as low as 2.5 mM 1-nitrosopiperidine, cell viability was significantly lower in BRIN BD11h2E1 and INS-1h2E1 cell lines in comparison to the BRIN BD11 and INS-1 parental cell lines (72.5 ± 4.96 and 66.4 ± 3.09% in BRIN BD11h2E1 and INS-1h2E1 versus 109.0 ± 3.40 and 100.0 ± 3.25% in BRIN BD11 and INS-1 respectively, P < 0.001). The highest dose of any of the nitrosamines tested failed to significantly reduce cell viability in the cells which lacked CYP2E1. Expression of CYP2E1 did not cause any change in the basal level of DNA damage in any of the cell lines. However, 16 h exposure to various nitrosamines resulted in significant dose-dependent DNA damage in the BRIN BD11h2E1 and INS-1h2E1 cells compared to their respective non CYP2E1-expressing parental controls, e.g. DNA damage increased from 34.38 ± 1.25 to 44.01 ± 1.56% DNA in comet tail in BRIN BD11h2E1 cells incubated with 10 or 40 mM N-nitrosopyrrolidine, respectively ( P < 0.001). Similar treatment of the BRIN BD11 and INS-1 cell lines did not result in a significant increase in DNA damage (20.33 ± 1.0 and 22.4 ± 0.98% DNA in comet tail). The pancreatic β-cell is richly vascularised and expresses CYP2E1. This study suggests that expression of human CYP2E1 in pancreatic β-cells make them highly susceptible to cytotoxicity and DNA damage by nitrosamines and other agents bioactivated by CYP2E1.

Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters.

Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm-prepared sperm were processed using a modified comet assay 3-6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.

The comet assay detects adaptation to MNU-induced DNA damage in barley

We have established the comet assay for detection of DNA damage in barley. Immediately after treatment with the monofunctional alkylating agent MNU, a dose-dependent increase of DNA damage (mainly DNA breaks) was detected by the alkaline denaturation/neutral gel electrophoresis (A/N) variant of the comet assay in nuclei isolated from root tip meristems and from young leaves. A reduction of damage was observed within meristematic nuclei but not in differentiated leaf nuclei 48 h after treatment. Adaptive pretreatment with a nontoxic dose of cadmium chloride prior to challenge treatment with MNU reduced the frequency of chromatid type aberrations, micronuclei and aneuploid cells as well as the amount of DNA in comet tails of meristematic nuclei.

Levels of peripheral blood cell DNA damage in insulin dependent diabetes mellitus human subjects

Increased production of reactive oxygen species (ROS) in vivo can lead to cellular biomolecule damage. Such damage has been suggested to contribute to the pathogenesis of insulin dependent diabetes mellitus (IDDM). In this study, we used the alkaline comet assay to measure DNA damage (single-stranded DNA breaks and alkali-labile sites) in freshly isolated whole blood, lymphocytes, monocytes, and neutrophils from 23 subjects with IDDM and 32 age- and sex-matched controls. Analysis of the results showed elevated levels of DNA damage (expressed as % comet tail DNA) in the lymphocyte (4.10±0.47, 3.22±0.22), monocyte (4.28±0.47, 3.49±0.18), and whole blood (4.93±0.51, 4.51±0.23) fractions from IDDM subjects compared to controls, respectively, but the increases observed were not statistically significant. However, we found significantly elevated basal levels of DNA damage in the neutrophil fraction (8.38±0.64, 4.07±0.23; p<0.001, Mann–Whitney U test) in IDDM subjects compared to controls. Given these novel neutrophil findings, we extended the study to include a total of 50 IDDM subjects and 50 age- and sex-matched control subjects and determined basal levels of DNA damage in the neutrophils of all 100 subjects. We found significantly elevated mean levels of DNA damage (8.40±0.83, 4.34±0.27; p<0.001, Mann–Whitney U test) in the neutrophils from the IDDM subjects when compared to controls. Our results show that even with acceptable glycaemic control there is a significantly elevated level of DNA damage within diabetic neutrophils in vivo.

Characterisation of biotransformation enzyme activities and DNA integrity in isolated cells of the digestive gland of the common mussel, Mytilus edulis L.

Characterisation of biotransformation and antioxidant enzyme activities and DNA integrity was carried out on isolated cells of the major biotransformation organ (digestive gland) of Mytilus edulis as a first step in developing a cell culture system for use in toxicology. Digestive gland cells were isolated by trypsin or non-protease tissue dissociation procedures, followed by filtration and differential centrifugation. Both dissociation methods produced a mixture of smaller cell types and large digestive cells at a viability of over 90% (EOSIN Y exclusion). The specific activities (per milligramme of protein) of 10 000× g supernatants from freshly isolated digestive gland mixed-cell populations produced by either dissociation method were similar to those from whole digestive gland for the antioxidant enzymes superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), but from 3- to 10-fold lower for the biotransformation enzyme activities benzo[a]pyrene hydroxylase, glutathione- S-transferase (EC 2.5.1.18; substrate: 1-chloro-2,4-dinitrobenzene) and NADPH-dependent DT-diaphorase (EC 1.6.99.2). Cells produced by trypsin dissociation showed significant increased DNA strand breakage as measured by the single cell electrophoretic ‘comet’ assay, viz.% comet tail DNA increased from 10.3±2.3 (control) to 19.8±4.1 (0.01% w/v trypsin) to 23.7±3.2 (0.1%) (mean±S.D.). Cell yields from digestive gland were only slightly lower for non-enzymic compared to trypsin dissociation for the same time period of dissociation, indicating the former as a preferred method of cell culture preparation.