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High intake of red meat and/or dairy products may increase the concentration of iron and calcium in plasma—a risk factor for prostate cancer (PC). Despite our understandings of nutrients and their effects on the genome, studies on the effects of iron and calcium on radiation sensitivity of PC patients are lacking. Therefore, we tested the hypothesis that high plasma levels of iron and calcium could increase baseline or radiation-induced DNA damage in PC patients relative to healthy controls. The present study was performed on 106 PC patients and 132 age-matched healthy individuals. CBMN assay was performed to measure mi-cronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds) in lymphocytes. Plasma concentrations of iron and calcium were measured using inductively coupled plasma atomic emission spectroscopy. MN, NPBs, and NBuds induced by radiation ex vivo were significantly higher in PC patients with high plasma iron (P  = .004, P  = .047, and P  = .0003, respectively) compared to healthy controls. Radiation-induced MN and NBuds frequency were also significantly higher in PC patients (P  = .001 and P  = .0001, respectively) with high plasma calcium levels relative to controls. Furthermore, radiation-induced frequency of NBuds was significantly higher in PC patients (P  < .0001) with high plasma levels of both iron and calcium relative to controls. Our results support the hypothesis that high iron and calcium levels in plasma increases the sensitivity to radiation-induced DNA damage and point to the need of developing nutrition-based strategies to minimize DNA damage in normal tissue of PC patients undergoing radiotherapy. [ABSTRACT FROM AUTHOR]

Red blood cell (RBC) fatty acid status is used as a biomarker of dietary intake of fats; however, there is still a paucity of evidence regarding individual fatty acids and modulation of endogenous advanced glycation end product (AGE) levels. Due to membrane polyunsaturated fatty acid (PUFA) being a well-known target for peroxidation, we hypothesized that cellular PUFAs are positively associated with circulatory Nε-carboxymethyllysine (CML) that is also influenced by glyoxal levels in healthy cohorts. To test this, we investigated the association between RBC fatty acids and circulatory AGE biomarkers in healthy individuals. The results showed a negative association between saturated fatty acids (SFA) and CML, and stepwise multivariate regression analysis indicated that stearic acid was negatively associated with CML levels (β = -0.200, p = .008) after adjusting for age, body mass index (BMI), and gender. In addition, stearic acid:palmitic acid ratio was also negatively correlated with plasma concentrations of CML (rp = -.191, p = .012) and glucose (rp = -.288, p = .0001). PUFAs showed a positive association with CML levels, particularly docosapentaenoic acid, γ-linolenic acid, arachidonic acid, and docosadienoic acid. However, these associations were not evident after the multiple regression analysis adjusted for age, BMI, and gender. A strong negative correlation (rp = -.98, p < .0001) between total PUFA and total SFA was observed. Furthermore, the SFA:PUFA ratio was inversely correlated with CML (rp = -.227, p < .003). Overall, this study indicates that different fats and their combinations may influence the formation of AGEs and that carefully controlled interventions are required to further test this hypothesis. [ABSTRACT FROM AUTHOR]

Significant alterations in sleep duration and/or quality of sleep become more pronounced as people get older. Poor sleep in elderly people is associated with adverse health outcomes and cellular aging. We examined the relationship between telomere length (TL) and sleep duration, Health Promotion Index (HPI), and tested whether the presence of Apolipoprotein-E4 (ApoE-ε4) allele affects both sleep and TL. The present study was carried out in 174 healthy participants (21% male; mean age 53.79 years) from South Australia. Lymphocyte TL was measured by real-time quantitative PCR (qPCR) and ApoE genotype was determined by TaqMan assay. HPI was calculated from a questionnaire regarding 8 lifestyle habits, including sleeping hours. Multivariate regression analysis was used to establish these associations adjusted for specified confounders. TL was found to be inversely associated with age (r = -0.199; p = .008) and body mass index (r = -0.121; p = .11), and was significantly shorter in participants who slept for less than 7 hours (p = .001) relative to those sleeping ≥7 hours. TL was positively correlated with HPI (r = 0.195; p = .009). ApoE-ε4 allele carriers who slept for less than 7 hours had shortest TL (p = .01) compared to noncarriers. Plasma soluble receptor for advanced glycation end product (sRAGE) level was significantly (p = .001) lower in individuals who sleep less than 7 hours and ApoE-ε4 carriers. Our results suggest that inadequate sleep duration or poor HPI is associated with shorter TL in cognitively normal people and that carriage of APOE-ε4 genotype may influence the extent of these effects. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis. [ABSTRACT FROM AUTHOR]

Lymphocyte telomere length (LTL) is a biomarker of aging that may be modified by dietary factors including fat. Red blood cell fatty acid status is a well-validated indicator of long-term dietary intake of fat from various sources. Recent findings from epidemiological studies of LTL in relation to fatty acids in red blood cells are not conclusive. The present study was carried out to investigate if red blood cell fatty acid status in 174 healthy older South Australians is associated with LTL. Lymphocyte telomere length was measured by real-time qPCR and fatty acid content in red blood cells was measured by gas chromatography. Our results indicate that the majority of saturated fatty acids and monounsaturated fatty acids are negatively associated with LTL, whereas polyunsaturated fatty acids are positively associated with LTL. Multiple regression analysis revealed that arachidonic acid (C20:4n-6) is significantly, independently, positively correlated with LTL (β = 0.262; p = .000). The significant association of fatty acids, particularly C20:4n-6, with telomere length warrants further research. [ABSTRACT FROM AUTHOR]

Cervical cancer (CC) is the fourth most common cancer in women; the survival rates depend strongly on its early detection. The Pap test is the most frequently used diagnostic tool, but due to its limited sensitivity/specificity, additional screening tests are needed. Therefore, we evaluated the use of micronucleus (MN) assays with cervical cells for the prediction and diagnosis of CC. MN reflects structural and numerical chromosomal aberrations. A search was performed in Pubmed, Scopus, Thomson ISI and Google Scholar. Subsequently, meta-analyses were performed for different grades of abnormal findings in smears and biopsies from patients which were diagnosed with CC. Results of 21 studies in which findings of MN experiments were compared with data from Pap tests show that higher MN frequencies were found in women with abnormal cells that are indicative for increased cancer risks. MN frequency ratios increased in the order inflammation (2.1) < ASC-US and ASC-H (3.3) < LGSIL (4.4) < HGSIL (8.4). Furthermore, results are available from 17 investigations in which MN were scored in smears from patients with neoplasia. MN rates increased with the degree of neoplasia [CIN 1 (4.6) < CIN 2 (6.5) and CIN 3 (10.8)] and were significantly higher (8.8) in CC patients. Our meta-analysis indicates that the MN assay, which is easy to perform in combination with Pap tests, may be useful for the detection/prediction of CC. However, standardization (including definition of the optimal cell numbers and stains) and further validation is necessary before the MN test can be implemented in routine screening. [ABSTRACT FROM AUTHOR]

APOE ε4 allele is an established risk factor for Alzheimer's disease and hypercholesterolemia. However, its association with metabolic and genetic risk factors related to glycation is not clear. We tested the hypothesis that, apart from high plasma cholesterol, APOE ε4 carriers may also have higher advanced glycation end products (AGEs) and total soluble extracellular domain of RAGE (sRAGE) and that these biomarkers may be modified by the common Gly82Ser (G82S) polymorphism (rs2070600) in the RAGE gene. To test this, we measured these biomarkers in 172 healthy cognitively normal individuals, of which 32 were APOE ε4 carriers and 140 noncarriers. APOE ε4 carriers showed higher levels of cholesterol (p < .001), glyoxal (p < .001), fluorescent AGEs (p < .001), Nε-carboxymethyllysine (p < .001) and sRAGE (p = .018) when compared to noncarriers. Furthermore, sRAGE was also higher in those that did not carry the A allele of the RAGE gene that codes for serine instead of glycine (p = .034). Our study indicates that APOE ε4 carriers have a greater propensity to glycation than noncarriers which may further increase their risk for diabetes and dementia. The increased sRAGE levels in APOE ε4 carriers suggests a defensive response against AGEs that may be further influenced by the RAGE G82S polymorphism. [ABSTRACT FROM AUTHOR]

Apolipoprotein-ε4 (APOE-ε4)-common variant is a major genetic risk factor for cognitive decline and Alzheimer's disease (AD). An accelerated rate of biological aging could contribute to this increased risk. Glycation of serum proteins due to excessive glucose and reactive oxygen species leads to the formation of advanced glycation end products (AGEs)-a risk factor for diabetes and AD, and decline in motor functioning in elderly adults. Aim of present study was to investigate impact of APOE-ε4 allele containing genotype and accumulation of AGEs in plasma on telomere length (TL). Results showed that TL is significantly shorter in APOE-ε4 carriers compared with non-APOE-ε4 carriers (p = .0003). Higher plasma glucose level was associated with shorter TL irrespective of APOE-ε4 allele containing genotype (r = -.26; p = .0004). With regard to AGEs, higher plasma glyoxal and fluorescent AGEs concentrations were inversely related to TL (r = -.16; p = .03; r = -.28; p = .0001), however, plasma Nε-(carboxymethyl)lysine levels didn't correlate with TL (r = -.04; p = .57). Results support the hypotheses that APOE-ε4 carriers have shorter telomeres than noncarriers and telomere erosion is increased with higher concentration of glucose, fluorescent AGEs, and glyoxal. [ABSTRACT FROM AUTHOR]

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring. [ABSTRACT FROM AUTHOR]

This study investigated the effect of dietary sugars and advanced glycation end-products (AGE) on telomere dynamics in WIL2-NS cells. Dietary sugars [glucose (Glu) and fructose (Fru); 0.1 M each] were incubated with bovine serum albumin (BSA) (10 mg/ml) at 60 ± 1°C for 6 weeks to generate AGE-BSA. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed total AGE levels as 87.74 ± 4.46 nmol/mg and 84.94 ± 4.28 nmol/mg respectively in Glu-BSA and Fru-BSA model. Cell treatment studies using WIL2-NS cells were based on either glucose, fructose (each 2.5–40 mM) or AGE-BSA (200–600 µg/ml) in a dose-dependent manner for 9 days. Telomere length (TL) was measured using qPCR. Nitric oxide (NO) production and tumour necrosis factor-α (TNF-α) levels were measured in WIL2-NS culture medium. An increasing trend for TNF-α and NO production was observed with higher concentration of glucose (R 2 = 0.358; P = 0.019; R 2 = 0.307; P = 0.027) and fructose (R 2 = 0.669; P = 0.001; R 2 = 0.339; P = 0.006). A decreasing trend for TL (R 2 = 0.828; P = 0.000), and an increasing trend for NO production (R 2 = 0.352; P = 0.031) were observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant trend on TL (R 2 = 0.135; P = 0.352) with increasing concentration, however, a significant reduction was observed at 600 µg/ml (P < 0.01) when compared to BSA treatment. No trends for TNF-α levels and a decreasing trend on NO production (R 2 = 0.5201; P = 0.019) was observed with increasing Fru-BSA treatment. In conclusion, this study demonstrates a potential relationship between dietary sugars, AGEs and telomere attrition. AGEs may also exert telomere shortening through the production of pro-inflammatory metabolites, which ultimately increase the risk of diabetes complications and age-related disease throughout lifespan. [ABSTRACT FROM AUTHOR]

This study investigated the effect of glucose and fructose, and advanced glycation end-products (AGEs) on genome damage in WIL2-NS cells, measured using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The effect of AGEs was investigated using the bovine serum albumin (AGE-BSA) model system induced either with glucose (Glu–BSA) or with fructose (Fru–BSA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed higher N ε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu–BSA model. N ε-Carboxyethyllysine (CEL; 7.87 ± 0.19 nmol/mg BSA) and methylglyoxal-derived hydroimidazolone-1 (MG-H1; 69.77 ± 3.74 nmol/mg BSA) levels were higher in the Fru–BSA model. Genotoxic effects were measured using CBMN-Cyt assay biomarkers [binucleated(BN) cells with micronuclei (MNi), BN with nucleoplasmic bridges (NPBs) and BN with nuclear buds (NBuds)] following 9 days of treatment with either glucose, fructose, Glu–BSA or Fru–BSA. Fructose treatment exerted a significant genotoxic dose–response effect including increases of BN with MNi (R 2 = 0.7704; P = 0.0031), BN with NPBs (R 2 = 0.9311; P < 0.0001) and BN with NBuds (R 2 = 0.7118; P = 0.0091) on cells, whereas the DNA damaging effects of glucose were less evident. High concentrations of AGEs (400–600 µg/ml) induced DNA damage; however, there was no effect on cytotoxicity indices (necrosis and apoptosis). In conclusion, this study demonstrates a potential link between physiologically high concentrations of reducing sugars or AGEs with increased chromosomal damage which is an important emerging aspect of the pathology that may be induced by diabetes. Ultimately, loss of genome integrity could accelerate the rate of ageing and increase the risk of age-related diseases over the long term. These findings indicate the need for further research on the effects of glycation on chromosomal instability and to establish whether this effect is replicated in humans in vivo. [ABSTRACT FROM AUTHOR]

Accumulation of DNA damage in the first 1000 days may increase risk of accelerated ageing and degenerative diseases in adult life such as cancers. The extent of DNA damage in infants and the correlation of maternal factors during pregnancy with neonate birth outcomes and DNA damage is not known in infants born in Australia. Therefore, we performed a prospective cohort study to collect data on DNA damage in lymphocytes of Australian infants (aged 0, 3 and 6 months), using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay. The study also explored correlation of CBMN-Cyt biomarkers with infant birth outcomes and maternal anthropometric and lifestyle variables. Peripheral blood lymphocytes were isolated from the infants at birth (cord blood) (n = 82), 3 months (n = 64) and 6 months (n = 53) after birth. DNA damage biomarkers measured ex vivo in binucleated lymphocytes (BNC) included: micronuclei (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUD). Apoptotic and necrotic lymphocytes were also scored and nuclear division index (NDI) was measured using the frequency of mono-, bi- and multinucleated lymphocyte. MN and NBUD were also scored in mononucleated lymphocytes (MNC). The mean (± SD) frequency of MN, NPB and NBUD in BNCs at birth was 2.0 (± 1.2), 5.8 (± 3.7) and 11.1 (± 5.7) per 1000 BNC, respectively, and tended to decrease significantly at 3 months (P < 0.01, P < 0.0001, P < 0.001, respectively) and 6 months (P < 0.05, P < 0.0001, P < 0.0001, respectively) after birth relative to cord blood when compared with the same cohort of infants (n = 48 at birth, 48 at 3 months and 39 at 6 months). None of the CBMN cytome biomarkers measured at birth was associated with maternal smoking status, alcohol and folic acid intake during pregnancy. The mean gestation age correlated positively with MN (r = 0.38, P = 0.006), NPB (r = 0.30, P = 0.03) and negatively with NDI (r = -0.29, P = 0.03). Infant birth weight associated positively with MN, NPB and NBUD in cord blood (r = 0.24, P = 0.08; r = 0.32, P = 0.02; r = 0.28, P = 0.04, respectively), birth length associated positively with NPB (r = 0.32, P = 0.02) and NBUD (r = 0.27, P = 0.04) while head circumference associated negatively with apoptotic cells (r = -0.27, P = 0.06). APGAR score at 1 and 5 min after birth associated positively with NDI at birth (r = 0.3, P = 0.05, r = 0.28, P = 0.06, respectively). Mother's weight and body mass index (BMI) recorded at the time of recruitment associated positively with NPB (r = 0.38, P = 0.006, r = 0.32, P = 0.02, respectively) and negatively with APGAR score at 5 min (r = -0.25, P = 0.07). The significant positive associations of infant birth weight and length and maternal BMI with CBMN-Cyt biomarkers suggest the possibility of a genotoxic effect of metabolic processes that promote excessive growth and high BMI. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20 nM folate medium compared with 2000 nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza- 2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by γH2AX immunofluorescence (r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level. [ABSTRACT FROM AUTHOR]

Dietary deficiencies of folate and other B vitamin cofactors involved in one-carbon metabolism, together with genetic polymorphisms in key folate-methionine metabolic pathway enzymes, are associated with increases in circulating plasma homocysteine, reduction in DNA methylation patterns, and genome instability events. All of these biomarkers have also been associated with pre-eclampsia. The aim of this review was to explore the literature and identify potential knowledge gaps in relation to the role of folate at the genomic level in either the etiology or the prevention of pre-eclampsia. A systematic search strategy was designed to identify citations in electronic databases for the following terms: folic acid supplementation AND preeclampsia, folic acid supplementation AND genome stability, folate AND genome stability AND pre-eclampsia, folic acid supplementation AND DNA methylation, and folate AND DNA methylation AND pre-eclampsia. Forty-three articles were selected according to predefined selection criteria. The studies included in the present review were not homogeneous, which made pooled analysis of the data very difficult. The present review highlights associations between folate deficiency and certain biomarkers observed in various tissues of women at risk of pre-eclampsia. Further investigation is required to understand the role of folate in either the etiology or the prevention of pre-eclampsia. [ABSTRACT FROM AUTHOR]

The buccal micronucleus cytome (BMCyt) assay is a minimally invasive approach for measuring DNA damage, cell proliferation, cell differentiation and cell death in exfoliated buccal cells. The main limitation for its use is the lack of knowledge about inter- and intra-laboratory variability in scoring micronuclei and other end points included in the cytome approach. In order to identify the main sources of variability across the BMCyt biomarkers, a scoring exercise was carried out between three experienced laboratories using the same set of slides and an identical set of detailed scoring criteria and associated images for the different end points. Single batches of slides were prepared from pooled samples of four groups of subjects characterised by different frequencies of cell types and micronuclei, namely Down syndrome patients, head and neck cancer patients undergoing radiotherapy and two age- and gender-matched control groups. A good agreement among the laboratories in the identification of normal differentiated cells and of micronuclei was obtained. A 3-fold and 20-fold increase in the frequency of micronucleated cells and micronuclei in differentiated cells of Down syndrome patients and in cancer patients, respectively, compared to matched controls, was a consistent result in the three laboratories. The scores of other cell types and nuclear anomalies, such as basal, binucleated, condensed chromatin and karyorrhectic cells showed significant disagreement between and within laboratories indicating that their evaluation using the current visual scoring protocol does not yield robust results for these parameters. The guidelines for BMCyt assay application could be improved by combining the anomalies associated with cell death (condensed chromatin and karyorrhectic cells) in a single category and by defining more stringent criteria in classifying basal cell, binucleated cells and buds. [ABSTRACT FROM AUTHOR]

Wood dust is recognised as a human carcinogen, based on the strong association of wood dust exposure and the elevated risk of malignant tumours of the nasal cavity and paranasal sinuses [sino-nasal cancer (SNC)]. The study aimed to assess genetic damage in workers exposed to wood dust using biomarkers in both buccal and nasal cells that reflect genome instability events, cellular proliferation and cell death frequencies. Nasal and buccal epithelial cells were collected from 31 parquet layers, installers, carpenters and furniture workers (exposed group) and 19 non-exposed workers located in Switzerland. Micronucleus (MN) frequencies were scored in nasal and buccal cells collected among woodworkers. Other nuclear anomalies in buccal cells were measured through the use of the buccal micronucleus cytome assay. MN frequencies in nasal and buccal cells were significantly higher in the exposed group compared to the non-exposed group; odds ratio for nasal cells 3.1 [95% confidence interval (CI) 1.8–5.1] and buccal cells 1.8 (95% CI 1.3–2.4). The exposed group had higher frequencies of cells with nuclear buds, karyorrhectic, pyknotic, karyolytic cells and a decrease in the frequency of basal, binucleated and condensed cells compared to the non-exposed group. Our study confirms that woodworkers have an elevated risk for chromosomal instability in cells of the aerodigestive tract. The MN assay in nasal cells may become a relevant biomonitoring tool in the future for early detection of SNC risk. Future studies should seek to standardise the protocol for MN frequency in nasal cells similar to that for MN in buccal cells. [ABSTRACT FROM AUTHOR]

The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or ‘Q-fly’) to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose–response of γH2AvB (0–400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies. [ABSTRACT FROM AUTHOR]

Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally susceptible to DNA damage. [ABSTRACT FROM PUBLISHER]

The ultraviolet (UV)-B spectrum in solar UV radiation is essential for stimulating the epidermal production of vitamin D but also damages DNA and causes cancer in exposed cells. We examined the role of solar UV in inducing DNA damage in blood lymphocytes and the possible modulation of this damage by serum 25-hydroxy vitamin D (25(OH)D) in 207 male and female participants from South Australia. Personal solar UV exposure was estimated from hours of outdoor exposure recalled at the time of blood collection for analysis of DNA damage in lymphocytes, using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and of serum 25(OH)D. We examined the association between solar UV exposure, serum 25(OH)D and DNA damage using multiple linear regression, with age, sex, body mass index and alcohol consumption as covariates. The frequency of cells with micronuclei (a biomarker of chromosome breakage or loss) increased with increasing sun exposure [% increase = 5.24; 95% confidence interval (CI): 0.35 to 10.37 P-value = 0.04] but cells with nucleoplasmic bridges (a biomarker of misrepair of DNA strand breaks or telomere end fusions) decreased (% increase = −8.38; 95% CI: −14.32 to −2.03 P-value = 0.01). There was also a fall in the nuclear division index (NDI) (% increase = −1.01; 95% CI: −2.00 to 0.00 P-value = 0.05), suggesting diminished mitogenic response and, possibly, immune suppression. There was no overall relationship between 25(OH)D and DNA damage. There were, however, weak modulating effects of 25(OH)D on the associations of solar UV exposure with micronucleus formation and with NDI (P-interaction = 0.03 and 0.05, respectively), where the increase in micronuclei and fall in NDI with increasing solar UV were greater at serum 25(OH)D levels <50 nmol/l. Thus, the influence of solar UV exposure in causing DNA damage or immune suppression in internal tissues may be stronger when vitamin D levels are low. [ABSTRACT FROM AUTHOR]

Excessive alcohol consumption is associated with an increased risk of a variety of cancers. The specific association between alcohol consumption and increased risk of breast cancer has been a consistent finding in numerous studies to date; however, the biological mechanism remains unknown. One possibility is that alcohol induces chromosome instability and aneuploidy events commonly seen in cancer. The cytokinesis-block micronucleus cytome assay was used to assess the ability of alcohol to induce DNA damage and aneuploidy in two human B lymphoblastoid cell lines—WIL2-NS and GM13705. The cells were treated chronically with physiologically relevant levels of alcohol (0.36 and 1.35% v/v) for a period of 6 weeks. Results demonstrate that in these cell lines, chronic treatment with alcohol induces micronuclei, nucleoplasmic bridges and nuclear buds, indicative of the various genome damaging events of chromosome loss and breakage, asymmetric chromosome rearrangement and gene amplification, respectively. Using chromogenic in situ hybridisation, we measured chromosome 17 aneuploidy in these cell lines. Results from this assay indicate that chronic treatment of alcohol induces aneuploidy (measured as chromosome 17 aneuploidy) in both cell lines. The results from this study support the hypothesis that alcohol is a probable cause of cancer initiation by inducing chromosomal instability and aneuploidy, which may be a result of multiple indirect mechanisms. [ABSTRACT FROM PUBLISHER]

These are personal reflections on the development of methods to use micronuclei as a measure of genetic damage and their use in research and in toxicology by four people who have been intimately involved with this work, a personal rather than a comprehensive history. About 6000 papers have been published using such methods in many tissues in vivo or in cultured cells of many organisms from plants to humans, but the majority of the work has been on mammalian erythrocytes and human lymphocytes, the areas in which we have worked primarily. Although this is by no means a complete history, those working in the field may be interested in some of the personal events that lie behind the development and acceptance of methods that are now standard. [ABSTRACT FROM AUTHOR]

Micronucleus (MN) frequency in cytokinesis-blocked peripheral blood lymphocytes (PBL) has become one of the best-established biomarkers for studying DNA damage occurring in vivo in humans. The application of this method in population biomonitoring studies requires a deep understanding of how lifestyle and common host variables may influence MN frequency in PBL. In this mini-review, an update is provided on results from studies reporting on the impact of age, gender, diet and lifestyle factors (e.g. exercise, alcohol, smoking and recreational drugs) on this biomarker. Evidence from these studies shows that each of these factors, either in isolation or in combination, can significantly influence MN frequency. Proper control for these factors is required to enable better measurement of the impact of other conditions, such as environmental exposure to genotoxins or a susceptible genetic background, on MN frequency in PBL. [ABSTRACT FROM AUTHOR]

Although micronuclei (MNi) have been extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their formation was not completely addressed until recently, due to limitations of traditional experimental methods. The development of live-cell imaging, combined with genetically engineered chromosome labelling techniques makes it possible to investigate the origin of a micronucleus in a single cell in a real-time and high-throughput manner. Here, we review all the available studies on the origins of MNi in live cells and discuss novel findings based on this recently emerged methodology. Some unsolved questions on MNi formation and limitations of live-cell imaging in the investigation of MNi have also been discussed. [ABSTRACT FROM AUTHOR]

The International Human Micronucleus (HUMN) Project (www.humn.org) was founded in 1997 to coordinate worldwide research efforts aimed at using micronucleus (MN) assays to study DNA damage in human populations. The central aims were to (i) collect databases on baseline MN frequencies and associated methodological, demographic, genetic and exposure variables, (ii) determine those variables that affect MN frequency, (iii) establish standardised protocols for performing assays so that data comparisons can be made more reliably across laboratories and countries and (iv) evaluate the association of MN frequency with disease outcomes both cross-sectionally and prospectively. In the first 10 years of the HUMN project, all of these objectives were achieved successfully for the MN assay using the cytokinesis-block micronucleus (CBMN) assay in human peripheral blood lymphocytes and the findings were published in a series of papers that are among the most highly cited in the field. The CBMN protocol and scoring criteria are now standardised; the effect of age, gender and smoking status have been defined, and it was shown prospectively using a database of almost 7000 subjects that an increased MN frequency in lymphocytes predicts cancer risk. More recently in 2007, the HUMN coordinating group decided to launch an equivalent project focussed on the human MN assay in buccal epithelial cells because it provides a complementary method for measuring MN in a tissue that is easily accessible and does not require tissue culture. This new international project is now known as the human MN assay in exfoliated cells (HUMNxL). At present, a database for >5000 subjects worldwide has been established for the HUMNxL project. The inter-laboratory slide-scoring exercise for the HUMNxL project is at an advanced stage of planning and the analyses of data for methodological, demographic, genetic, lifestyle and exposure variables are at a final stage of completion. Future activities will be aimed at (i) defining the genetic variables that affect MN frequencies, (ii) validation of the various automated scoring systems based on image analysis, flow cytometry and laser scanning cytometry, (iii) standardisation of protocols for scoring micronuclei (MNi) in cells from other tissues, e.g. erythrocyte and nasal cells and (iv) prospective association studies with pregnancy complications, developmental defects, childhood cancers, cardiovascular disease and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The micronucleus (MN) assay is increasingly being used to study the association between DNA damage and infertility or pregnancy complications in humans. This review provides a brief overview of the studies published to date. The results of these studies appear to support the plausibility of the following hypotheses: (i) MN in spermatids in semen may be indicative of infertility risk, (ii) MN in peripheral blood lymphocytes in males correlate positively with DNA damage in sperm, (iii) infertile couples exhibit higher frequencies of MN than fertile couples and (iv) an abnormally high frequency of MN in peripheral blood lymphocytes is associated with pregnancy complications including miscarriage, intra-uterine growth restriction and pre-eclampsia. The studies published to date consistently indicate an association of MN in peripheral blood lymphocytes with impaired reproductive capacity. However, the conclusions of these studies, although statistically significant, are limited by small sample sizes and the need for verification in other independent cohorts. In conclusion, more attention should be given to the possibility of using MN assays in peripheral blood lymphocytes and reproductive tissues as a biomarker of risk for infertility and pregnancy complications in humans. [ABSTRACT FROM AUTHOR]

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus (MN) background frequency, by which only in vivo exposures in excess of 0.2–0.3 Gy X-rays can be detected.In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation. [ABSTRACT FROM AUTHOR]

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials. [ABSTRACT FROM AUTHOR]

Successful maintenance of accurate DNA replication and repair is critical to human health. Once this homeostatic balance is impaired, genomic instability events occur, compromising the integrity of the genome, which may initiate fundamental events leading to human diseases. Biomarkers of DNA damage, such as the micronucleus (MN) index, are elevated both in developmental and degenerative diseases and have been shown to be predictive of increased cancer risk and cardiovascular disease mortality. Several micronutrients have been identified as being effective in reducing and/or protecting against DNA damage. Micronutrients act as co-factors for enzymes required in DNA repair or maintenance of methylation patterns essential for optimal gene expression. In this review, published human intervention studies are examined with respect to the efficacy of micronutrient supplementation in reducing MN frequency in both lymphocytes and buccal cells. Important knowledge gaps and future research directions are also explored. The outcomes of these studies suggest that supplementation with antioxidant vitamins and certain B vitamins may cause a substantial reduction in MN frequency. [ABSTRACT FROM AUTHOR]

The cytokinesis-block micronucleus cytome (CBMNCyt) assay is a widely used technique for measuring DNA damage in human populations. The formation of micronuclei (MN) in dividing cells can result from chromosome breakage due to unrepaired or mis-repaired DNA lesions or chromosome malsegregation due to mitotic malfunction. The sensitivity of the MN assay to polymorphisms in various genes involved in DNA repair, activation/deactivation of carcinogens/chemicals/drugs/alcohol, folate metabolism pathway and micronutrient transport has been extensively reported in the literature. MN frequency is also an important index for determining DNA repair efficiency phenotype (including mis-repair), response to environmental exposure and identifying various dietary factors required for optimal genome stability. The aim of the present study is to review the reported in vivo associations between genotype and MN frequency in humans taking into considerations the presence of interactions with nutrients levels and/or exposure to genotoxins. One hundred and eleven publications linking MN frequency in peripheral blood lymphocytes to gene polymorphism were retrieved from PubMed. After applying exclusion criteria, only 37 studies were evaluated in the present review. Polymorphisms in XRCC1 (Arg280His), ERCC2 (Lys751Gln), CYP2E1 (c1/c2) and MTR (A2756G) were consistently associated with the MN formation. These results contribute substantial evidence to the hypothesis that genotype may influence MN frequency in human cells. [ABSTRACT FROM AUTHOR]

Over a century ago, Theodor Boveri paved the way to mechanistic studies linking chromosomal abnormalities to cancer pathogenesis. Since then, theoretical and empirical evidence has been accumulated, supporting a causal role of these events in the aetiology of human cancer. A powerful tool for measurement of chromosomal abnormalities is the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The validation of the micronucleus (MN) as marker of phenotypic susceptibility to cancer has received decisive support from mutagens sensitivity studies, particularly from a recent case–control study on lung cancer, which showed increased frequency of tobacco carcinogen-induced MN, nuclear buds and especially nucleoplasmic bridges in cancer patients (odds ratios of 2.3, 10.0 and 45.5, respectively). Recently, a large international cohort study showed a significant association between MN frequency in healthy subjects and cancer risk. The study assembled data on 6718 individuals from 10 countries (62 980 person-years). Cancers incidence was significantly higher in groups with medium (RR = 1.84; 95% confidence interval: 1.28–2.66) and high MN frequency (RR = 1.53; 95% CI: 1.04–2.25). This study provided preliminary evidence that MN frequency in peripheral blood lymphocytes is predictive of cancer risk, suggesting that increased MN formation is associated with early events in carcinogenesis. These results, in combination with mechanistic evidence, prospected the use of MN frequency in cancer screening programmes. However, issues such as interindividual variability and preventive strategies in high-risk groups need to be further addressed to consolidate these achievements. [ABSTRACT FROM AUTHOR]

DNA damage is a fundamental cause of developmental and degenerative diseases. The in vitro cytokinesis-block micronucleus cytome (CBMN-Cyt) assay is an established comprehensive method for assessing cytostasis and chromosome stability in cells. Originally developed to study the acute effects of single environmental genotoxicants, creative applications and adaptations to the basic protocol have allowed its use in evaluating the impacts of dietary micronutrients and micronutrient combinations (nutriomes) on DNA damage. In this review, we examine some of these studies and the important findings they have generated with respect to nutrient/nutrient, nutrient/genotype and nutrient/genotoxicant interactions, as well as assessment of the carcinogenic (or protective) potential of whole dietary patterns. In addition, we outline current knowledge gaps and technical limitations and propose future adaptations to enhance the applicability of the CBMN-Cyt method for in vivo predictions. [ABSTRACT FROM AUTHOR]

Micronuclei (MNi) can originate either from chromosome breakage or chromosome malsegregation events and are therefore ideal biomarkers to investigate genomic instability. Studies in peripheral lymphocytes of patients with neurodegenerative diseases, mainly Alzheimer's disease (AD) and Parkinson's disease (PD), revealed an increased micronucleus (MN) frequency in both disorders but originating mainly from chromosome malsegregation events in AD and from chromosome breakage events in PD. Studies in other neurodegenerative diseases are largely missing, and some data in premature ageing disorders characterised by neurodegeneration and/or neurological complications, such as Ataxia telangiectasia, Werner's syndrome, Down's syndrome (DS) and Cockayne's syndrome, indicate that MNi increase with ageing in cultured cells. An increased frequency of aneuploidy characterises several tissues of AD patients, as well as of individuals at increased risk to develop AD, such as mothers of DS individuals and DS subjects themselves. The use of the buccal MN cytome assay in AD and DS subjects allowed finding significant changes in the MN frequency as well as other cellular modifications reflecting reduced regenerative capacity compared to age- and gender-matched controls. These changes in buccal cytome ratios may prove useful as potential future diagnostics to identify individuals of increased risk for these disorders. [ABSTRACT FROM AUTHOR]

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure. [ABSTRACT FROM AUTHOR]

The study of DNA damage in exfoliated buccal cells is a minimally invasive method for monitoring populations for exposure to genotoxic agents. The presence of micronuclei (MN) and other nuclear anomalies within these cells has been shown to be associated with genetic defects in genome maintenance, accelerated ageing, genotoxic damage and some degenerative diseases. To identify important information gaps regarding these biomarkers, a new initiative was launched within the framework of the HUman MicroNucleus (HUMN) collaborative programme, the HUMNXL project (‘XL’ designating eXfoLiated cell). An invitation to join the project was sent out together with a questionnaire to all laboratories that have published on the buccal micronucleus assay. Overall, 188 messages were delivered and 58 laboratories from 25 countries agreed to participate (43 contributing data). The questionnaire was designed to collect methodological information regarding the laboratory's performance of the assay and to assess the extent and type of epidemiological data that are routinely collected. The results provide an overview of the most commonly used methods for buccal cell collection and preparation, slide preparation, staining, scoring criteria and an evaluation of epidemiological data, including demographics, genetic background, gender, health status, occupation, exposure, lifestyle and dietary habit. According to this survey, a potential base of 15 103 subjects can be included in future pooled analyses. A number of protocol discrepancies emerged, implying that method standardization is a major priority. The results of this survey will contribute to (i) identify technical and epidemiological key variables that impact on buccal MN frequency in human populations, (ii) drive the design of future intra- and interlaboratory validation studies and (iii) determine the role of MN frequency and other biomarkers, in monitoring genomic damage and predicting cancer and other degenerative diseases. [ABSTRACT FROM PUBLISHER]

We hypothesized that the genomic response to folate deficiency might be different between breast cancer cases and healthy subjects. To test this hypothesis, we performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid (FA) deficiency on primary human lymphocytes from 19 breast cancer patients and 20 age-matched healthy females from Yunnan, China using the cytokinesis-block micronucleus assay. Lymphocytes from the volunteers were cultured in RPMI1640 medium containing 30, 120 or 240 nM FA for 9 days. The results showed that 30 nM FA was associated with increased frequencies of micronucleated binucleated cell (MNed BNC), nucleoplasmic bridges (NPB), nuclear buds (BUD), apoptosis (APO) and necrosis (NEC) relative to 120 and 240 nM FA (P < 0.001) in lymphocytes of case and control groups in vitro, however there were no significant differences between the 120 and 240 nM FA within each sampling group. The case group showed significantly higher frequencies of MNed BNC than control at 120 and 240 nM FA (P < 0.05–0.001) but not at 30 nM FA (P = 0.052). NEC was significantly higher in breast cancer group than control at all concentrations of FA (P < 0.005). FA concentration explained 60, 39, 39, 52 and 71% of the variance of MNed BNC, NPB, BUD, APO and NEC, respectively compared with breast cancer status which only explained 6 and 7% of the variance of MNed BNC and NEC(Two way ANOVA, P < 0.0001). Difference of difference analysis showed that breast cancer cases were not abnormally sensitive to the genome-damaging effect of folate deficiency. We concluded that (i) increased concentrations of FA abolished the genome-damaging effect of FA deficiency in lymphocytes of both breast cancer patients and controls to a similar extent and (ii) FA concentration is much more important than breast cancer status in determining genomic instability and cell death. [ABSTRACT FROM AUTHOR]

The evidence of a direct link between increased genome/epigenome damage and elevated risk for adverse health outcomes during the various stages of life, such as infertility, foetal development and cancer is becoming increasingly stronger. The latter is briefly reviewed against a background of evidence indicating that genome and epigenome damage biomarkers, in the absence of overt exposure of genotoxins, are themselves sensitive indicators of deficiency in micronutrients required as cofactors or as components of DNA repair enzymes, for maintenance methylation of CpG sequences and prevention of DNA oxidation and/or uracil incorporation into DNA. The latter is illustrated with cross-sectional and dietary intervention data obtained using the micronucleus assay and other efficient biomarkers for diagnosing genome and/or epigenome instability. The concept of recommended dietary allowances for genome stability and how this could be achieved is discussed. The ‘Genome Health Nutrigenomics’ concept is also introduced to define and focus attention on the specialized research area of how diet impacts on genome stability and how genotype determines nutritional requirements for genome health maintenance. The review concludes with a vision for a paradigm shift in disease prevention strategy based on the diagnosis and nutritional treatment of genome/epigenome damage on an individual basis, i.e. The Genome Health Clinic. [ABSTRACT FROM AUTHOR]

We have performed experiments using the WIL2-NS human B lymphoblastoid cell line and primary human lymphocytes to: (i) determine the importance of including measurements of nucleoplasmic bridges (NPB) in the cytokinesis-block micronucleus (CBMN) assay; (ii) provide evidence that NPB originate from dicentric chromosomes and centric ring chromosomes. In addition, we describe theoretical models that explain how dicentric chromosomes and centric ring chromosomes may result in the formation of NPB at anaphase. The results with WIL2-NS showed that it was possible to distinguish genotoxic effects induced by different oxidizing agents in terms of the NPB/micronucleus frequency ratio. The results with lymphocytes indicated a strong correlation: (i) between NPB, centric ring chromosomes and dicentric chromosomes in metaphases (r > 0.93, P < 0.0001); (ii) between micronuclei (MNi), acentric chromosome fragments and acentric ring chromosomes (r > 0.93, P < 0.0001). The dose–response curves with γ-rays were very similar for NPB, ring chromosomes and dicentric chromosomes, as were the dose–response curves for MNi, acentric rings and fragments. However, not all acentric chromosomes and dicentric chromosomes/centric rings were converted to MNi and NPB respectively, depending on the dose of radiation. Preliminary data, using FISH, suggest that NPB often represent DNA from a structural rearrangement involving only one or two homologous chromosomes. The results from this study validate the inclusion of NPB in the CBMN assay which provides a valuable measure of chromosome breakage/rearrangement that was otherwise not available in the micronucleus assay. The CBMN assay allows NPB measurement to be achieved reliably because inhibition of cytokinesis prevents the loss of NPB that would otherwise occur if cells were allowed to divide. [ABSTRACT FROM PUBLISHER]

We have tested the hypothesis that the alcoholic and phenolic components of wine are protective against the DNA-damaging and cytotoxic effects of hydrogen peroxide and γ-radiation in vitro. The components of wine tested were ethanol, glycerol, a mixture of the phenolic compounds catechin and caffeic acid and tartaric acid, all at concentrations that were 2.5 or 10.0% of the concentration in a typical Australian white wine (Riesling). These components were tested individually or combined as a mixture and compared to a white wine stripped of polyphenols, as well as a Hanks balanced salt solution control, which was the diluent for the wine components. The effect of the components was tested in lymphocytes, using the cytokinesis-block micronucleus assay, after 30 min incubation in plasma or whole blood for the hydrogen peroxide or γ-radiation challenge, respectively. The results obtained showed that ethanol, glycerol, the catechin–caffeic acid mixture, the mixture of all components and the stripped white wine significantly reduced the DNA-damaging effects of hydrogen peroxide and γ-radiation (P = 0.043–0.001, ANOVA). The strongest protective effect against DNA damage by γ-irradiation was observed for the catechin–caffeic acid mixture and the mixture of all components (30 and 32% reduction, respectively). These two treatments as well as ethanol produced the strongest protective effects against DNA damage by hydrogen peroxide (24, 25 and 18%, respectively). The protection provided by the mixture did not account for the expected additive protective effects of the individual components. Ethanol was the only component that significantly increased baseline DNA damage rate, however, this effect was negated in the mixture. In conclusion, our results suggest that the main phenolic and alcoholic components of wine can reduce the DNA-damaging effects of two important oxidants, i.e. hydrogen peroxide and ionizing radiation, in this physiologically relevant in vitro system. [ABSTRACT FROM PUBLISHER]

Folic acid (FA), the most oxidized and stable form of folate, is commonly used as a dietary supplement and in culture media. FA must be reduced and methylated to become the metabolically active form found in blood and utilized by tissues, i.e. 5-methyltetrahydrofolate (5-MeTHF). 5-MeTHF is the methyl group donor required for the conversion of homocysteine to methionine catalyzed by vitamin B12-dependent methionine synthase. It is hypothesized that 5-MeTHF may be more effective than FA in reducing spontaneous DNA damage and improving cell proliferation because, unlike FA, it can donate a methyl group for methionine synthesis, which is required for cell division via polyamine production and for maintenance methylation of DNA after its conversion to S-adenosylmethionine. We aimed to determine whether FA and 5-MeTHF differed in their capacity to prevent genetic damage and cell proliferation of human lymphocytes in vitro. Lymphocytes from eight female volunteers (40–48 years) were cultured in RPMI 1640 medium containing 12–120 nM FA or 5-MeTHF for 9 days. Mitogenesis was stimulated with phytohemagglutinin and the medium changed on days 3 and 6. Cytokinesis was inhibited by adding cytochalasin B on day 8 and cells were harvested and transferred to microscope slides on day 9. Chromosome damage, cell death and cytostasis was measured using the cytokinesis-block micronucleus assay in its comprehensive mode. The results showed that the frequency of micronucleated binucleate cells was significantly lower at 120 nM FA compared with 120 nM 5-MeTHF (P < 0.05), however, at 12 nM concentration both forms of folate were associated with increased frequency of micronuclei and nuclear buds relative to 120 nM (P < 0.05). Apoptosis tended to be significantly higher in 5-MeTHF cultures compared with FA cultures, however, necrosis and nuclear division were similar between cultures. We conclude that 5-MeTHF is not more efficient than FA in preventing human lymphocyte genomic instability in this in vitro system. Further research is needed to clarify the role of choline and methionine concentration and the importance of the reduced folate carrier and the folate receptor in determining the relative bioavailability of 5-MeTHF and FA with regard to genome stability. [ABSTRACT FROM PUBLISHER]

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12–120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive. [ABSTRACT FROM PUBLISHER]

The underlying cause(s) of methionine-dependency and its relevance to cancer remains unclear. We aimed to determine whether (i) normal human lymphocytes exhibit methionine-dependency, (ii) baseline levels of genetic damage are related to methionine-dependency and (iii) methionine-dependence can be explained, in part, by common polymorphisms in methionine synthase and methylenetetrahydrofolate reductase (MTHFR). Genetic damage was measured in lymphocytes of 52 volunteers (29–65 years) using the cytokinesis-block micronucleus assay. Methionine-dependency was assessed by culturing cells in serum-free media containing 0.1 mM l-methionine and 0 mM d,l-homocysteine (met+hcy–) or 0 mM l-methionine and 0.2 or 0.4 mM d,l-homocysteine (0.2/0.4-hcy+)(met–hcy+). Mitogenesis was stimulated with phytohaemagglutinin. Cytokinesis was inhibited by adding cytochalasin B at 44 h. Ninety-six hours after PHA, cells were transferred to microscope slides. Cell proliferation was measured by counting binucleated cell frequency and calculating nuclear division index. Volunteers were classified into tertiles of methionine-dependence according to the growth of their cells in met–hcy+ media (relative to growth in met+hcy– media). Average cell division, as a percentage of division in met+hcy– media, was approximately 5, 26 and 70% in 0.2-hcy+ media and 29, 70 and 142% in 0.4-hcy+ media for the high, mid and low tertiles of methionine-dependence, respectively. Micronucleus frequency did not vary between these tertiles (P > 0.6). In both met+hcy– and met–hcy+ media, cell division was not affected by polymorphisms in MTHFR (C677T, A1298C) or methionine synthase (A2756G). Cell division in met–hcy+ media was negatively correlated with division in met+hcy– media (P = 0.05 and 0.007 for 0.2 and 0.4-hcy+, respectively). Methionine-dependent lymphocytes had higher levels of cell proliferation in met+hcy– media than methionine-independent lymphocytes (P = 0.089 and 0.01 for 0.2 and 0.4-hcy+, respectively). However, this difference was not apparent in previous experiments when cells were grown in media containing 10% fetal calf serum. These findings show that there is a wide inter-individual variation in the degree of methionine-dependency of normal human lymphocytes in vitro. Methionine-dependency does not appear to alter the risk for chromosomal mutation as measured by the micronucleus assay. We discuss the possible relevance to cancer of increased cell division in methionine-dependent cells under methionine-replete and serum-free media conditions. [ABSTRACT FROM PUBLISHER]

Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hcy concentration is also reported to correlate positively with the micronucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hcy is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hcy are genotoxic to human lymphocytes in vitro using the cytokinesis-block micronucleus assay. Eighteen lymphocyte cultures were initiated in Met-free and serum-free RPMI 1640 medium for each of four male volunteers aged 22-23 years. At 0, 24, 44 and 72 h, cultures were spiked with L-Hcy or L-Met to achieve concentrations ranging between 50 and 400 μM. The concentration of Hcy at 96 h ranged from 19.45 ± 2.34 to 149.02 ± 28.16 μM in Hcy cultures and 0.91 ± 0.17 to 2.15 ± 0.9 μM in Met cultures spiked with 50 and 400 μM of metabolite, respectively. Forty-four hours after mitogen stimulation, cytokinesis was inhibited with cytochalasin B. After 96 h, cells were transferred to microscope slides and the frequency of micronucleated-binucleate and necrotic cells was scored. Neither Hcy (P = 0.24) nor Met (P = 0.93) had an apparent dose effect on micronucleus frequency. However, when data were pooled, micronucleus frequency was moderately higher (50.1%) in Hcy- than in Met-spiked cultures (P = 0.04; paired t-test). Hcy concentration was positively correlated with necrosis (P < 0.0005; r²= 0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hcy-spiked cultures (P= 0.01; paired t-test). Further research is required to define more clearly the genotoxic and cytotoxic potential of homocysteine and its metabolites. [ABSTRACT FROM AUTHOR]

Human biomonitoring of early genetic effects requires accurate, sensitive and, if possible, easy and not too time-consuming methodologies to assess mutations. One of the most promising methodologies at the present time is the cytokinesis block micronucleus (MN) assay (CBMN), which detects both chromosome breakage and chromosome loss in once-divided binucleated (BN) cells. Many studies have been published with this methodology, but before its extensive application is recommended, it is necessary to evaluate its strengths and limitations. Recently, Fenech et al. reviewed the advantages of the CBMN assay for biomonitoring purposes. However, up to now information present in mononucleated (MONO) cells has rarely been taken into account, although it might be complementary to that assessed in BN cells. Indeed, MONO cells should indicate damage which was present in vivo before the start of culture and BN cells may contain pre-existing micronuclei (MNi) plus lesions which are expressed as MNi during in vitro culture. To address this question, the objectives of this paper were as follows. (i) To situate the CBMN assay in a historical and mechanistic perspective. (ii) To consider whether impaired mitotic capacity in vitro may be responsible for false negative biomonitoring studies if MN in MONO cells are not taken into account in the CBMN test. The following factors were considered: division delay for repair and mitotic block, in vitro apoptosis and necrosis of damaged cells, mitotic slippage and correlation between MN expression in vitro versus in vivo. (iii) To analyse the factors which may cause a negative result in the CBMN assay in biomonitoring when exposure to specific genotoxins is evident. The specific effects of aneugens and of adaptive responses to chronic low level exposure were examined. (iv) To compare the sensitivity of MONO and BN cells in relation to the genotoxic mechanism. (v) To propose an adequate sampling scheme to study MN in both MONO and BN cells. It was concluded that a more comprehensive assessment of DNA damage may be achieved if the CBMN assay includes measures of: (i) MNi in MONO cells; (ii) MNi in BN cells; (iii) apoptotic cells; (iv) necrotic cells. It is probable that the 24 h post-phytohaemagglutinin time point may be the optimal time to assess the frequency of MNi in MONO cells, apoptotic cells and necrotic cells. It is also practical to include these measures when scoring MNi in BN cells after cytokinesis block. [ABSTRACT FROM PUBLISHER]

A mathematical model is described that predicts the effect of altered nuclear/cell division kinetics and cytotoxicity on micronucleus expression in vitro when the micronucleus assay is performed without discriminating between cells that have divided once and cells that have not divided after genotoxic insult. The model is based on the probabilities of: (i) a viable cell completing nuclear division; (ii) micronucleus expression in a cell that completes nuclear division after genotoxic insult; (iii) a cell not dividing and surviving as a mononuclear cell; (iv) a cell dying by necrosis or apoptosis. The model predicts: (i) false negative results for relatively weak chromosome damaging agents that also inhibit nuclear division, if micronuclei are scored in mononucleated cells without discriminating between divided and non-divided cells; (ii) this tendency for a false negative result when scoring micronuclei without discriminating between non-divided and once-divided mononuclear cells increases with cell lines and culture conditions that do not result in optimal rates of nuclear division (i.e. >90% of dividing cells); (iii) the absolute increment in micronucleus frequency in binucleated cells is at least 2-fold greater than that observed in mononucleated cells when nuclear division is not inhibited and this difference increases with increasing nuclear inhibition. The number of dead cells does not influence the micronucleus frequency if only viable cells are considered when determining the micronucleus frequency ratio. The results from this model suggest that the micronucleus assay when performed by scoring mononucleated cells, without restricting the score to those cells that have divided once after genotoxic insult, is prone to produce false negative results and, therefore, cannot be considered reliable or conclusive. Scoring of micronuclei in cytokinesis-blocked binucleated cells is predicted by the model to provide consistent results under all culture conditions and based on these theoretical results should be considered the preferred choice. [ABSTRACT FROM PUBLISHER]

We have developed a method that can detect the DNA-damaging and cytotoxic effects of physiological levels of reactive oxygen species (ROS) and activated human neutrophils. This was achieved using WIL2-NS cells, a human B lymphoblastoid cell line, as target cells and the cytokinesis-block micronucleus (CBMN) assay. With this method, we observed a 4- and a 30-fold increase in the frequency of micronucleated binucleated cells (MNed BNC) when cells were exposed to 10 and 30 μM hydrogen peroxide, for 1 h, respectively. A dose-dependent increase in the frequency of MNed BNC was also detected when cells were exposed to hypoxanthine (HX)/xanthine oxidase (XO), a superoxide generating system: a 50-fold increase in the frequency of MNed BNC was observed at the highest XO dose (12.5 mU/ml). In this CBMN assay, nucleoplasmic bridges (NPB) in BNC and necrotic cells were also readily detected, especially at the higher exposure doses of hydrogen peroxide or HX/XO. When WIL2-NS cells were exposed to neutrophils stimulated with phorbol 12-myristate acetate (PMA) for 1 h, the frequencies of MNed BNC in WIL2-NS cells increased in a dose-dependent manner (30-fold increase at 100 nM PMA) and with an increasing neutrophil:WIL2-NS co-culture ratio. The frequencies of MNed BNC were closely related to the production of ROS, especially hydrogen peroxide, by the neutrophils. Differentiated HL60 cells (DMSO-treated HL60) also produced ROS in response to PMA. In this case, we used a `Transwell' system to expose WIL2-NS cells to DMSO-treated HL60 cells, because direct contact with DMSO-treated HL60 cells impaired cell division in WIL2-NS target cells. Exposure to PMA-stimulated DMSO-treated HL60 cells resulted in a PMA dose-dependent increase in the frequency of MNed BNC in WIL2-NS cells. MNed BNC frequencies were positively correlated with NPB (r = 0.61–0.93) and necrosis (r = 0.55–0.86) and negatively correlated with nuclear division index (r = –0.72 to –0.91) in all of the above experiments. These results suggest that the CBMN assay using WIL2-NS cells is a sensitive assay system to examine ROS-induced chromosomal damage and necrosis by activated human neutrophils. [ABSTRACT FROM PUBLISHER]

We investigated whether high dose vitamin C influenced the viability of human lymphocytes in plasma, in the presence or absence of hydrogen peroxide (512 μM) by scoring necrotic, apoptotic and micronucleated cells using the cytokinesis-block micronucleus assay. The in vitro results showed that vitamin C (0.57–2.27 mM) on its own had no effect on the above parameters. However, in the presence of hydrogen peroxide vitamin C significantly reduced the number of dividing cells and apoptosis, and increased necrosis and micronucleated cells. A double-blind placebo controlled intervention, with a cross-over, involving 11 male subjects, aged 20–40 years, was performed to determine whether high plasma vitamin C concentration resulting from vitamin C supplementation promotes or protects against genetic damage and cell death ex vivo. Venous blood samples were collected before and after an anti-oxidant-poor diet which reduced plasma vitamin C concentrations by 15% (P < 0.05), and was followed with a 2 g vitamin C supplement, which raised plasma concentrations by 115 and 125% (0.12 mM) after 2 and 4 h, respectively (P < 0.05). Plasma collected post-vitamin C ingestion did not alter micronucleus expression or apoptosis in control or hydrogen peroxide-treated lymphocytes, but it moderately increased necrosis (P < 0.08). Analysis of combined data showed that necrotic cell frequency correlated positively with micronucleated cell frequency (r = 0.66, P < 0.0001) and negatively with apoptotic cell frequency (r = –0.81, P < 0.0001). Overall, vitamin C supplementation did not appear to cause DNA damage under normal physiological conditions nor did it protect cells against hydrogen peroxide-induced toxicity. [ABSTRACT FROM PUBLISHER]

A method is described for the inclusion of apoptotic and necrotic cells in the cell counts obtained in the cytokinesis-block micronucleus (CBMN) assay, which is conventionally used solely for the assessment of chromosome breakage, chromosome loss and frequency of dividing cells. The morphological criteria for the recognition and discrimination between necrotic, apoptotic and viable cells are described. Using this comprehensive method we have evaluated the cytotoxic and genotoxic effects of hydrogen peroxide (0–100 μM) in lymphocytes exposed in RPMI 1640 medium. The results obtained indicated significant (P < 0.05) correlations between hydrogen peroxide concentration and the frequency of micronucleated cells (r = 0.39), necrotic cells (r = 0.73), apoptotic cells (r = –0.26) and binucleated cells (r = –0.55). Almost similar results were obtained using the cytosine arabinoside modification of the CBMN assay, which enables excision-repaired sites to be converted to micronuclei. Some of the above end-points were significantly (P < 0.05) correlated with each other (necrosis and apoptosis, R = –0.39; necrosis and micronucleated cell frequency, R = 0.46; necrosis and binucleated cells, R = –0.78; apoptosis and binucleated cells, R = 0.32). It was therefore necessary to use multiple regression analysis to identify the main event induced by hydrogen peroxide, which was necrosis (β = 0.57, P = 0.0001) and not micronucleus formation (β = 0.15, P = 0.1332). Using an ELISA assay we showed that hydrogen peroxide did not induce 8-hydroxydeoxyguanosine. Our data show that the proposed comprehensive test system may provide a better procedure for classifying potential toxic chemicals and enable discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects. The integration of apoptosis and necrosis into the micronucleus assay may also be of practical use in radiosensitivity studies. [ABSTRACT FROM PUBLISHER]

We performed a biochemical and cytogenetic epidemiological study to establish if there are significant differences between vegetarians (V) and non-vegetarians (NV) in their peripheral blood lymphocyte micronucleus (MN) index, which is a measure of chromosome damage rate. The levels of plasma vitamin C (VIT-C), vitamin E (VIT-E), vitamin B (B12) and folic acid were also analysed to assess if differences in chromosome damage rates were associated with these potentially antimutagenic micronutrients. Volunteers were classified as either ‘vegetarian’ if they had abstained from eating any flesh foods for at least 3 years prior to the study or ‘non-vegetarian’ if they consumed meat or meat products at least 5 days/week for at least 3 years before participation in the study. The volunteers in the study consisted of 47 male and 79 female V and 66 male and 72 female NV, all of whom were non-smokers for at least 3 years prior to the study. The age of the volunteers varied between 20 and 89 years. There was no significant difference in the slope of the age-related increase in MN index of V and NV of either sex. However, the MN index was significantly lower in NV males in the age group 20–40 years and significantly lower for V males in the 4140 years age group. No difference between the MN index of older males was detectable and there also was no difference in the MN index of V and NV females across all age groups. V were generally found to have significantly higher plasma levels of VIT-C and folic acid, significantly lower levels of B12, and similar levels of VIT-E when compared with NV. VIT-C correlated positively with MN index in young males, but the reverse was true for B12. In young females folate and B12 appeared to correlate negatively with MN index. VIT-E had no apparent impact on MN index. These data suggest that the level of folate and B12 may be more important than VIT-C or VIT-E in minimizing chromosome damage rates in human lymphocytes. Overall, the data from this study do not support the hypothesis that V have a lower genetic damage rate than NV. [ABSTRACT FROM PUBLISHER]

We have tested the hypothesis that moderate wine drinking can protect somatic cells against the DNA-damaging effect of hydrogen peroxide which is an endogenous source of reactive oxygen metabolites. In this preliminary investigation, four male volunteers were placed on a plantpolyphenol- free (PPF) diet to ensure that the wine provided was the only main source of plant phenolic compounds. After 48 h on the PPF diet the volunteers were required to consume 300 ml of red or white wine and blood samples collected 1, 3, 8 and 24 h post-consumption while still on a PPF diet. Plasma was isolated from the blood samples and stored frozen for subsequent assays. In the subsequent assays, fresh lymphocytes from each donor were incubated in their corresponding plasma from the various intervention time-points for 30 min. The capacity of the plasma to prevent damage to DNA in lymphocytes by hydrogen peroxide was assessed using the cytokinesis-block micronucleus technique. The data from this preliminary investigation indicated that there was a strong inhibition (>70%) of hydrogen peroxide-induced micronucleated cells by the plasma samples from the blood collected 1 h after consumption of wine as compared to plasma samples from blood immediately before the consumption of wine. This protective effect was apparent for both red and white wine although statistical significance (P = 0.0068) was achieved only in the white wine intervention. A higher degree of statistical significance (P = 0.0008) was achieved when the data for samples following the consumption of red and white wine were combined. There was no difference in the hydrogen-peroxide-induced micronucleated cell frequency when comparing results immediately before starting on the PPF diet, before consumption of wine, 8 h after or 24 h after wine consumption. The hydrogen peroxide-induced micronucleated cell frequency in cells incubated with plasma from blood collected 3 h after wine consumption was intermediate to that observed for plasma after 1 and 8 h after wine intake. The protective effect of plasma against DNA damage cannot be readily explained by the red wine content of phenolic compounds because results for red wine were similar to those for white wine even though white wine had a much lower level of total polyphenols. A possible explanation could be that alcohol, glycerol and ascorbate in wine together with specific wine phenolic compounds that are also equally present in red and white wine (e.g. hydroxycinnamates) may have contributed to the observed protection of nuclear material from hydrogen peroxide-derived reactive oxygen metabolites. This explanation is supported by data from in vitro experiments showing that incubation of lymphocytes either with alcohol or wine stripped of phenolic compounds resulted in a statistically significant (P < 0.05) dose-related reduction (up to 87% reduction) in hydrogen peroxide-induced micronucleated cell frequency. [ABSTRACT FROM AUTHOR]