The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials.

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design-including treatment conditions, cell number, cell culture, format and volume of medium on the plate-is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

How does pycnogenol® influence oxidative damage to DNA and its repair ability in elderly people?

Our purpose in this randomized, double blind, placebo controlled study was to find out the possible effect of a polyphenolic pine bark extract, Pycnogenol® (Pyc) on the level of 8-oxo-7,8-dihydroguanine (8-oxoG) as representative of oxidative damage to DNA and on the DNA repair ability of elderly people. According to our results, three months of Pyc administration had no effect on the level of oxidative damage to DNA or on repair ability, but we found a relationship between the level of 8-oxoG and repair ability of DNA in this group. To conclude, even if the positive effect of Pyc was not confirmed in the case of elderly people it is important to highlight the necessity of further investigations about the mechanisms of Pyc acting on different age groups.

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Effects of age and dietary restriction on oxidative DNA damage, antioxidant protection and DNA repair in rats.

BACKGROUND: Experimentally imposed dietary restriction is known to extend the lifespan of rodents, perhaps by slowing the accumulation of oxidative damage that is thought to be one of the causes of aging. AIM OF THE STUDY: We examined the effects of restricted total food intake, and protein and calorie restriction, on DNA oxidation and related biomarkers in rats. METHODS: From 1 to 17 months, rats in group 1 received normal diet ad libitum. Group 2 received 70% of the quantity consumed by the first group. Group 3 had the same quantity as group 2, but with a reduction in protein (from 18% to 10% of the diet by weight), and group 4 were further restricted with a 30% decrease in calories. Lymphocytes were isolated from blood samples taken every two months. DNA breaks, oxidised pyrimidines, resistance to H2O2-induced damage, and strand break repair were measured with the comet assay. Organs were isolated from rats killed at 17 months, with 1 month-old rats for comparison; DNA oxidation and antioxidant enzyme activities were measured. RESULTS: DNA breaks in lymphocytes increased from 1 to 3 months but thereafter declined with age, except in ad libitum fed rats. Oxidised pyrimidines did not change significantly. Resistance to H2O2-induced damage was least at 3 months, and increased with age. Repair of DNA strand breaks was efficient at all ages. Diet had little effect on these endpoints. Diet had no influence on 8-oxo-7.8-dihydroguanine levels in DNA from liver, testis and brain of 17 month old rats. Combining data from all four groups, the levels in brain and liver were significantly higher at 17 months compared with 1 month. Antioxidant enzyme activities tended to increase between 1 and 17 months; effects of diet were not so consistent. CONCLUSIONS: While DNA damage shows a modest increase with age in some organs, antioxidant status and DNA strand break repair do not decline with age. Restricted diets (including protein and calorie restriction) have no effect on any of these markers of genetic stability.

Vitamins C and E: acute interactive effects on biomarkers of antioxidant defence and oxidative stress.

Oxidative stress is implicated in the aetiology of many diseases; however, most supplementation trials with antioxidant micronutrients have not shown expected beneficial effects. This randomized, double-blinded, placebo-controlled study evaluated acute effects (at 90, 180min and 24h [fasting] post-ingestion) of single doses of Vitamins C (500mg) and E (400IU), alone and in combination, on biomarkers of plasma antioxidant status, lipid peroxidation and lymphocyte DNA damage in 12 healthy, consenting volunteers. Plasma ascorbic acid increased significantly (P < 0.01) within 2h of ingestion of Vitamin C, and alpha-tocopherol was significantly (P < 0.01) higher at 24h post-ingestion Vitamin E. The pattern of response was not significantly different whether Vitamin C (or Vitamin E) was taken alone or in combination, indicating no augmentation of response to one by co-ingestion of the other vitamin. No significant changes were seen in plasma FRAP in the group overall (although increases (P < 0.05) were seen at 90 and 180min post-ingestion in women after Vitamin C ingestion) or in MDA across treatments, and no evidence of increased DNA damage, or of DNA protection, was seen at any time point after Vitamin C and/or E ingestion. In conclusion, the data from this first controlled study of acute effects of single doses of Vitamin C and/or E show no evidence of either a protective or deleterious effect on DNA damage, resistance of DNA to oxidant challenge, or lipid peroxidation. No evidence of a synergistic or cooperative interaction between Vitamins C and E was seen, but further study is needed to determine possible interactive effects in a staggered supplementation cycle, and study of subjects under increased oxidative stress or with marginal antioxidant status would be useful. It would be of interest also to study the effects of these vitamins ingested with, or in, whole food, to determine if they are directly protective at doses above the minimum required to prevent deficiency, if combinations with other food components are needed for effective protection, or if Vitamins C and E are largely surrogate biomarkers of a 'healthy' diet, but are not the key protective agents.

Dietary supplement use in old age: associations with childhood IQ, current cognition and health.

AIMS: Dietary supplement (DS) use is actively promoted among old people but there is little evidence in favour of DS use or information about the demographic, health and cognitive characteristics of DS users. METHOD: We examined 176 healthy, old people without dementia all born in 1921 and living independently in the community. IQ scores aged about 11 years were available for all subjects. DS users were more often female, had a lower BMI and were taking fewer prescribed medications than non-users. RESULTS: Usual dietary intake, as measured by food frequency questionnaire, did not differ between DS users and DS non-users. DS users were seen to have higher Vitamin C (p<0.05), alpha-carotene (p<0.05) and lower gamma-tocopherol (p<0.001) and homocysteine (p<0.01). DS users did not differ from DS non-users in years of education, indices of occupational code, current socio-economic category or parameters of cardiovascular or respiratory functions. DS users had higher (p<0.05) childhood IQ scores but did not differ in current Mini-Mental State Examination (MMSE) score or performance on Raven's Progressive Matrices (RPM) either before or after adjustment for childhood IQ. CONCLUSIONS: DS users may enjoy somewhat better general health than non-users but the source of this difference is unknown. Possible health benefits of DS use merit further study.

Nutritional supplementation with antioxidants decreases chromosomal damage in humans.

In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), beta-carotene (6 mg/day) and selenium (50 microg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage.

Plasma vitamin C, cholesterol and homocysteine are associated with grey matter volume determined by MRI in non-demented old people.

We studied 82 non-demented old people and, using MRI, derived measures of grey and white matter and intracranial volumes. Controlling for sex and intracranial volume, we related grey and white matter volumes to plasma concentrations of vitamins C, B(12), folate, homocysteine, cholesterol, triglycerides, high density and low density (LDL) lipoproteins, and to red blood cell folate and glycated haemoglobin concentrations (HbA1(c)). We found that lower grey matter volume was associated with lower plasma vitamin C and higher homocysteine, cholesterol and LDL. Lower blood cell folate was also associated with lower grey matter volume but HbA1(c) was not. These data are consistent with the putative benefits of dietary vitamin C and folate intake and the role of cholesterol in age related neurodegeneration.

Possible genotoxic activity of extracts of Bryonia alba roots on human lymphocytes and transformed cells.

Bryonia alba roots (BAR) are widely used as an adaptogenic and restorative drug with immunomodulatory and stress-protective properties that increase the non-specific resistance of an organism toward harmful stimuli. Potential genotoxic activity of aqueous and methanol extracts of BAR was studied on human normal (lymphocytes) and transformed (HeLa and Caco-2) cells using single cell gel electrophoresis (the comet assay). The results obtained did not show any evidence of genotoxic effects of BAR.

DNA damage and antioxidants; fluctuations through the year in a central European population group.

Dietary antioxidant levels in the blood depend on intake of fruits and vegetables and therefore might be expected to show seasonal variation. A group of healthy male subjects in Bratislava, Slovakia gave blood samples each month for 1 year. Vitamin C, alpha- and gamma-tocopherol and several carotenoids were measured in plasma, and concentrations of essential metals zinc, copper and selenium in serum. Oxidative DNA damage was assessed in lymphocytes using the comet assay. Seasonal variations in antioxidant levels did not follow a common pattern. beta-Cryptoxanthin was highest in the spring. Lycopene peaked in late summer. Lutein/zeaxanthin was higher in summer than in winter. The concentration of zinc in serum was higher in winter than in summer. DNA damage was lower in summer than in winter. Selenium as well as several antioxidants correlated negatively with indices of DNA damage, while zinc levels showed a positive correlation with DNA damage. These results provide some support for a link between consumption of antioxidants and protection against DNA oxidation.

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure.

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.

Peroxisome proliferator-activated receptor-gamma ligands inhibit nuclear but not cytosolic extracellular signal-regulated kinase/mitogen-activated protein kinase-regulated steps in vascular smooth muscle cell migration.

Vascular smooth muscle cell (VSMC) migration involves adhesion, locomotion, and invasion regulated by various signaling molecules, among which the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) play a critical role. We have shown that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands troglitazone and rosiglitazone inhibit VSMC migration downstream of ERK MAPK. The purpose of the current study was to more specifically determine which step(s) in VSMC migration are targeted by inhibition of the ERK MAPK pathway or activation of PPAR-gamma. VSMC adhesion was not affected by the ERK MAPK pathway inhibitor PD98059 or PPAR-gamma ligands. Phosphorylation and activation of myosin light chain kinase (MLCK) play important roles in cell locomotion. Platelet-derived growth factor (PDGF)-induced MLCK phosphorylation (1.7-fold) was completely blocked by PD98059 at 30 microM (p < 0.05), but not by troglitazone or rosiglitazone. PDGF-directed migration (5.8-fold) was inhibited by PD98059 (-88% at 30 microM) and the MLCK inhibitor ML9 (0.1-1 microM, -84% at 1 microM) (all p < 0.05). The transcription factor Ets-1 mediates matrix metalloproteinase induction required for tissue invasion by VSMC. PDGF (20 ng/ml) stimulated an Ets-1 protein expression (14-fold at 60 min) in VSMC, which was inhibited by PD98059 (-72% at 30 microM), troglitazone (-69% at 20 microM), and rosiglitazone (-54% at 10 microM) (all p < 0.05). Immunohistochemistry of rat aortae 2 h after balloon injury showed a dramatic upregulation of Ets-1, which was markedly inhibited in animals that had received troglitazone treatment. In contrast, phosphorylated ERK MAPK was not affected by troglitazone. These data are consistent with PPAR-gamma ligands exerting their anti-migratory effects downstream of ERK MAPK activation by blocking nuclear events, such as Ets-1 expression, required for cell invasion in response to arterial injury.

Anthocyanin-rich extract decreases indices of lipid peroxidation and DNA damage in vitamin E-depleted rats.

Anthocyanins are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. The phenolic structure of anthocyanins conveys marked antioxidant activity in model systems via donation of electrons or hydrogen atoms from hydroxyl moieties to free radicals. Dietary intakes of anthocyanins may exceed 200 mg/day, however, little is known about their antioxidant potency in vivo. Consequently, the aim of this study was to establish whether anthocyanins could act as putative antioxidant micronutrients. Rats were maintained on vitamin E-deficient diets for 12 weeks in order to enhance susceptibility to oxidative damage and then repleted with rations containing a highly purified anthocyanin-rich extract at a concentration of 1 g/kg diet. The extract consisted of the 3-glucopyranoside forms of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Consumption of the anthocyanin-repleted diet significantly improved (p <.01) plasma antioxidant capacity and decreased (p <.001) the vitamin E deficiency-enhanced hydroperoxides and 8-Oxo-deoxyguanosine concentrations in liver. These compounds are indices of lipid peroxidation and DNA damage, respectively. Dietary consumption of anthocyanin-rich foods may contribute to overall antioxidant status, particularly in areas of habitually low vitamin E intake.

Kiwifruit protects against oxidative DNA damage in human cells and in vitro.

Antioxidant micronutrients may account for the beneficial effects of fruits on human health. A direct demonstration that consumption of fruit decreases oxidative DNA damage in human cells would support this hypothesis. Kiwifruit was taken as an example of a food with putative antioxidant properties, and its effectiveness at decreasing oxidative DNA damage was assessed in ex vivo as well as in vitro tests. The comet assay (single-cell gel electrophoresis) was used to measure DNA damage in lymphocytes collected during a human supplementation trial with a single 0.5-liter drink of kiwifruit juice (with water as a control). The comet assay was also modified to assess the antioxidant effect of kiwifruit in vitro by measuring the ability of an extract to interfere with oxidative damage to DNA induced by H2O2. Ex vivo, consumption of kiwifruit led to an increased resistance of DNA to oxidative damage induced by H2O2 in isolated lymphocytes, in comparison with lymphocytes collected after a control drink of water. No effect was seen on endogenous DNA damage. In vitro, a simple extract of kiwifruit, buffered to pH 7, was more effective than a solution of vitamin C (of equivalent concentration) at protecting DNA from damage, whereas at the highest concentrations tested, neither kiwi extract nor vitamin C had a protective effect. We have demonstrated significant antioxidant activity of kiwifruit ex vivo and in vitro, not attributable entirely to the vitamin C content of the fruit. Our dual approach is appropriate for testing other fruit and vegetable products for potential antioxidant effects.

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators.

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.

DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals.

2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC(50)) and cytotoxicity (LC(50)) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage--both strand breaks and oxidised purines--was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.

An efficient procedure for genotyping single nucleotide polymorphisms.

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.