Objectives and achievements of the HUMN project on its 26th anniversary.

Micronuclei (MN) are a nuclear abnormality that occurs when chromosome fragments or whole chromosomes are not properly segregated during mitosis and consequently are excluded from the main nuclei and wrapped within nuclear membrane to form small nuclei. This maldistribution of genetic material leads to abnormal cellular genomes which may increase risk of developmental defects, cancers, and accelerated aging. Despite the potential importance of MN as biomarkers of genotoxicity, very little was known about the optimal way to measure MN in humans, the normal ranges of values of MN in healthy humans and the prospective association of MN with developmental and degenerative diseases prior to the 1980's. In the early 1980's two important methods to measure MN in humans were developed namely, the cytokinesis-block MN (CBMN) assay using peripheral blood lymphocytes and the Buccal MN assay that measures MN in epithelial cells from the oral mucosa. These discoveries greatly increased interest to use MN assays in human studies. In 1997 the Human Micronucleus (HUMN) project was founded to initiate an international collaboration to (i) harmonise and standardise the techniques used to perform the lymphocyte CBMN assay and the Buccal MN assay; (ii) establish and collate databases of MN frequency in human populations world-wide which also captured demographic, lifestyle and environmental genotoxin exposure data and (iii) use these data to identify the most important variables affecting MN frequency and to also determine whether MN predict disease risk. In this paper we briefly describe the achievements of the HUMN project during the period from the date of its foundation on 9th September 1997 until its 26th Anniversary in 2023, which included more than 200 publications and 23 workshops world-wide.

Effect of iron and calcium on radiation sensitivity in prostate cancer patients relative to controls.

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.

The Relationship between Telomere Length and Nucleoplasmic Bridges and Severity of Disease in Prostate Cancer Patients.

Telomeres are repetitive nucleotide (TTAGGG) sequences that stabilize the chromosome ends and play an important role in the prevention of cancer initiation and progression. Nucleoplasmic bridges (NPBs) are formed when chromatids remain joined together during mitotic anaphase either due to mis-repair of DNA breaks or due to chromatid end fusion as a result of telomere loss or telomere dysfunction. We tested the hypotheses that (i) telomere length (TL) is shorter in prostate cancer (PC) patients relative to healthy age-matched individuals, (ii) TL differs in different stages of PC and (iii) shorter TL is significantly correlated with NPBs formation in PC cases. TL was measured in whole blood by well-established quantitative PCR method and the frequency of NPBs was measured in lymphocytes using cytokinesis-block micronucleus cytome (CBMNcyt) assay. Our results indicate that TL is shorter and NPBs are increased in PC patients relative to age-matched healthy controls. Furthermore, TL was significantly shorter (p = 0.03) in patients with a Gleason score more than 7 and there was also a significant trend of decreasing TL across all three stages (p trend = 0.01; Gleason score <7, 7 and >7). Furthermore, TL was significantly inversely correlated with NPB frequency in PC patients (r = -0.316; p = 0.001) but not in controls (r = 0.163; p = 0.06) and their relationships became stronger with higher Gleason scores. More studies are required that can confirm our observations and explore mechanistic differences in the role of telomeres in NPB formation in PC cases relative to non-cancer cases.

Effect of Selenium and Lycopene on Radiation Sensitivity in Prostate Cancer Patients Relative to Controls.

Almost half of prostate cancer (PC) patients receive radiation therapy as primary curative treatment. In spite of advances in our understanding of both nutrition and the genomics of prostate cancer, studies on the effects of nutrients on the radiation sensitivity of PC patients are lacking. We tested the hypothesis that low plasma levels of selenium and lycopene have detrimental effects on ionising radiation-induced DNA damage in prostate cancer patients relative to healthy individuals. The present study was performed in 106 PC patients and 132 age-matched controls. We found that the radiation-induced micronucleus (MN) and nuclear buds (NBuds) frequencies were significantly higher in PC patients with low selenium (p = 0.008 and p = 0.0006 respectively) or low lycopene (p = 0.007 and p = 0.0006 respectively) levels compared to the controls. The frequency of NBuds was significantly higher (p < 0.0001) in PC patients who had low levels of both selenium and lycopene compared to (i) controls with low levels of both selenium and lycopene and (ii) PC patients with high levels of both selenium and lycopene (p = 0.0001). Our results support the hypothesis that low selenium and lycopene levels increase the sensitivity to radiation-induced DNA damage and suggest that nutrition-based treatment strategies are important to minimise the DNA-damaging effects in PC patients receiving radiotherapy.

Methylglyoxal Impairs Sister Chromatid Separation in Lymphocytes.

The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.

Plasma Micronutrient Profile of Prostate Cancer Cases Is Altered Relative to Healthy Controls-Results of a Pilot Study in South Australia.

Emerging evidence suggests possible roles of micronutrients in cancer prevention. The study was designed to test the hypothesis that the concentration profile of plasma micronutrients (i.e., the nutriome) in prostate cancer patients is different from that of healthy controls. Plasma samples from 116 Caucasian men diagnosed with late onset of prostate cancer and 132 matched controls from the South Australian population were collected and analysed for their concentration of micronutrients. Plasma concentrations of lutein, lycopene, α-carotene and ß-carotene were found to be significantly lower in prostate cancer patients (p = 0.03, 0.008, 0.002 and 0.002, respectively). Plasma levels of elements such as iron, copper, calcium and sulphur were significantly higher (p < 0.0001, <0.0001, <0.0001 and p = 0.0003, respectively) while that of selenium was significantly lower (p = 0.002) in prostate cancer patients. Higher prostate cancer risk is significantly associated with plasma levels below the median of lycopene (OR: 2.24), α-carotene (OR: 2.13), ß-carotene (OR: 1.97) and high levels above the median of iron (OR: 2.31), calcium (OR: 4.35) and sulphur (OR: 2.39). The results of this study suggest that the plasma nutriome could be a useful diagnostic of prostate cancer risk.

"Micronuclei and Disease" special issue: Aims, scope, and synthesis of outcomes.

The purpose of the "Micronuclei and Disease" special issue (SI) is to: (i) Determine the level of evidence for association of micronuclei (MN), a biomarker of numerical and structural chromosomal aberrations, with risk of specific diseases in humans; (ii) Define plausible mechanisms that explain association of MN with each disease; (iii) Identify knowledge gaps and research needed to translate MN assays into clinical practice. The "MN and Disease" SI includes 14 papers. The first is a review of mechanisms of MN formation and their consequences in humans. 11 papers are systematic reviews and/or meta-analyses of the association of MN with reproduction, child health, inflammation, auto-immune disease, glycation, metabolic diseases, chronic kidney disease, cardiovascular disease, eleven common cancers, ageing and frailty. The penultimate paper focuses on effect of interventions on MN frequency in the elderly. A road map for translation of MN data into clinical practice is the topic of the final paper. The majority of reviewed studies were case-control studies in which the ratio of mean MN frequency in disease cases relative to controls, i.e. the mean ratio (MR), was calculated. The mean of these MR values, estimated by meta-analyses, for lymphocyte and buccal cell MN in non-cancer diseases were 2.3 and 3.6 respectively, and for cancers they were 1.7 and 2.6 respectively. The highest MR values were observed in studies of cancer cases in which MN were measured in the same tissue as the tumour (MR = 4.9-10.8). This special issue is an important milestone in the evidence supporting MN as a reliable genomic biomarker of developmental and degenerative disease risk. These advances, together with results from prospective cohort studies, are helping to identify diseases in which MN assays can be practically employed in the clinical setting to better identify high risk patients and to prioritise them for preventive therapy.

Lymphocyte micronuclei frequencies in skin, haematological, prostate, colorectal and esophageal cancer cases: A systematic review and meta-analysis.

Micronucleus (MN) assay has been widely used as a biomarker of DNA damage, chromosomal instability, cancer risk and accelerated aging in many epidemiological studies. In this narrative review and meta-analysis we assessed the association between lymphocyte micronuclei (MNi) and cancers of the skin, blood, digestive tract, and prostate. The review identified nineteen studies with 717 disease subjects and 782 controls. Significant increases in MRi for MNi were observed in the following groups: subjects with blood cancer (MRi = 3.98; 95 % CI: 1.98-7.99; p = 0.000) and colorectal cancer (excluding IBD) (MRi = 2.69; 95 % CI: 1.82-3.98, p < 0.000). The results of this review suggest that lymphocyte MNi are a biomarker of DNA damage and chromosomal instability in people with haematological or colorectal cancers. However, the MRi for lymphocyte MNi in subjects with cancers of skin, prostate, esophagus was not significantly increased. More case-control and prospective studies are warranted to further verify the observed trends and to better understand the role of lymphocyte MNi as a biomarker of cancer risk in blood, skin, digestive tract and prostate.

Association between glycation biomarkers, hyperglycemia, and micronucleus frequency: A meta -analysis.

Micronucleus assay has been used as a biomarker of DNA damage, chromosomal instability, cancer risk and accelerated aging. In this review, a meta-analysis was performed to assess the association between micronuclei (MNi) and diseases with increased advanced glycation end products (AGEs) and HbA1c. The review identified eight studies with 632 subjects with disease and 547 controls. The Mean Ratio (MRi) for AGE levels (MRi = 2.92, 95 %CI: 2.06-4.13, P < 0.00001) and HbA1c levels (MRi = 1.32, 95 %CI: 1.12-1.56, P = 0.001) were significantly higher in the disease group compared to healthy controls. The meta-analysis indicated that the overall estimates of MRi for MNi was 1.83 (95 %CI: 1.38-2.42, p < 0.0001) in subjects with disease compared to controls. Significant increases in MRi for MNi were also observed in the following sub-groups: subjects with disease for elevated AGEs (MRi = 1.62, 95 %CI: 1.12-2.35, P = 0.01), elevated HbA1c (MRi = 2.13, 95 %CI: 1.33-3.39, P = 0.002), lymphocytes MNi (MRi = 1.74, 95 %CI: 1.29-2.33, P = 0.0003), exfoliated buccal cells MNi (MRi = 2.86, 95 %CI: 1.19-6.87, P = 0.02), type 2 diabetes mellitus (T2DM) (MRi = 1.99, 95 %CI: 1.17-3.39, P = 0.01), chronic renal disease (MRi = 1.68, 95 %CI: 1.18-2.38, P = 0.004) and other disease groups (MRi = 2.52, 95 %CI: 1.28-4.96, P = 0.008). The results of this review suggest that MNi could be used as a biomarker of DNA damage and chromosomal instability in degenerative disease where increased AGEs and HbA1c are implicated. The lack of heterogeneity for MN frequency when considered either for all studies or subgroup strengthened the MRi of the meta-analysis. However, the lack of significant association between MRi for MNi and MRi for AGEs or HbA1c indicates that the case-control studies investigated may be confounded by other variables. Thus, larger studies with long term AGE exposure is warranted to further understand the role of MN formation in the initiation and progression of diseases caused by excessive glycation.

Advanced glycation end-products accelerate telomere attrition and increase pro-inflammatory mediators in human WIL2-NS cells.

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 (R2 = 0.358; P = 0.019; R2 = 0.307; P = 0.027) and fructose (R2 = 0.669; P = 0.001; R2 = 0.339; P = 0.006). A decreasing trend for TL (R2 = 0.828; P = 0.000), and an increasing trend for NO production (R2 = 0.352; P = 0.031) were observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant trend on TL (R2 = 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 (R2 = 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.

Cytokinesis Block Micronucleus Cytome (CBMN Cyt) Assay Biomarkers and Their Association With Radiation Sensitivity Phenotype in Prostate Cancer Cases and DNA Repair Gene hOGG1 (C1245G) Polymorphism.

Prostate cancer (PC) is commonly diagnosed cancer in men but only a few risk factors, such as family history, ethnicity, and age have been established. Chromosomal instability is another possible risk factor but this has not been adequately explained previously. In this study, we tested the hypotheses that peripheral blood lymphocytes (PBL) of PC patients have (1) an abnormally high level of chromosomal instability; (2) that they are hypersensitive to ionizing radiation-induced DNA damage; and (3) that these phenotypes are affected by hOGG1 (C1245G) polymorphism. These experiments were performed using the cytokinesis-block micronucleus Cytome (CBMN cyt) assay in PC cases and controls. We found that spontaneous or radiation-induced (3G) micronucleus (MN) frequency is not significantly different between both groups. However, spontaneous frequency of nucleoplasmic bridges (NPBs) and radiation-induced nuclear buds (NBuds) were significantly higher in patients vs. controls (P < 0.0001; P = 0.0005, respectively). In addition, apoptosis and nuclear division index (NDI) was significantly higher in patients compared to controls after radiation treatment (P = 0.006; P = 0.0002, respectively). Furthermore carriage of at least one G allele of hOGG1 (C1245G) polymorphism was associated with a significantly increased odds ratio (OR) to have a base-line MN, NPB, or NBud frequency greater than medium level compared to homozygotes for C allele (OR:1.94, 1.77, 2.36, respectively, P = 0.02; 0.04, and 0.004, respectively). Our results support the hypotheses that those who develop PC have significantly higher level of genomic instability which is further increased in those who carry G allele of the hOGG1 (C1245G) polymorphism. Environ. Mol. Mutagen. 59:813-821, 2018. © 2018 Wiley Periodicals, Inc.

Occupational Exposure to Pesticides in Tobacco Fields: The Integrated Evaluation of Nutritional Intake and Susceptibility on Genomic and Epigenetic Instability.

Pesticides used at tobacco fields are associated with genomic instability, which is proposed to be sensitive to nutritional intake and may also induce epigenetic changes. We evaluated the effect of dietary intake and genetic susceptibility polymorphisms in MTHFR (rs1801133) and TERT (rs2736100) genes on genomic and epigenetic instability in tobacco farmers. Farmers, when compared to a nonexposed group, showed increased levels of different parameters of DNA damage (micronuclei, nucleoplasmic bridges, and nuclear buds), evaluated by cytokinesis-block micronucleus cytome assay. Telomere length (TL) measured by quantitative PCR was shorter in exposed individuals. Global DNA methylation was significantly decreased in tobacco farmers. The exposed group had lower dietary intake of fiber, but an increase in cholesterol; vitamins such as B6, B12, and C; ß-carotene; and α-retinol. Several trace and ultratrace elements were found higher in farmers than in nonfarmers. The MTHFR CT/TT genotype influenced nucleoplasmic bridges, nuclear buds, and TL in the exposed group, whereas TERT GT/TT only affected micronucleus frequency. We observed a positive correlation of TL and lipids and an inverse correlation of TL and fibers. The present data suggest an important role of dietary intake and subjects' genetic susceptibility to xenobiotics-induced damages and epigenetic alterations in tobacco farmers occupationally exposed to mixtures of pesticides.

Role of PON1, SOD2, OGG1, XRCC1, and XRCC4 polymorphisms on modulation of DNA damage in workers occupationally exposed to pesticides.

Tobacco farming has been proving to induce poor health outcomes in agricultural workers, genomic instability being the triggering one. This study evaluated influence of PON1 (paraoxonase 1), SOD2 (superoxide dismutase), OGG1 (8-oxoguanine glycosylase), XRCC1 (X-ray repair cross-complementing protein 1), and XRCC4 (X-ray repair cross-complementing protein 4) genes polymorphisms on DNA damage in 121 subjects occupationally exposed to pesticides mixtures and nicotine at tobacco fields and 121 non-exposed individuals. Inorganic elements (Cl, P, S and Zn) and cotinine levels were found increased in farmers, confirming exposure. Results show higher frequencies of buccal micronucleus (MN), nuclear buds (NBUD), binucleated cells (BN) and damage index (comet assay), reduced telomere length (TL), and increased parameters of oxidative stress in farmers compared to non-exposed individuals. PON1 Gln/Gln genotype was associated with increased MN frequency. SOD2 Val/Val showed association with increased frequency of MN and NBUD and decreased antioxidant activity. The XRCC1 Arg/Arg showed protective effect for MN, BN and TL, which was also positively influenced by OGG1 -/Cys. MN was decreased in XRCC4 -/Ile farmers. These genotypes also showed a risk for antioxidant activity. Our study proposes that PON1 and SOD2 variants play a role in xenobiotic-metabolizing system in farmers, while base excision repair (BER) pathway could be the repair mechanism involved in genomic instability suffered by tobacco farmers.

Chronic occupational exposure endured by tobacco farmers from Brazil and association with DNA damage.

Tobacco farming is an important economic income in Brazil, although it has been challenged as regard the occupational exposure to both pesticides and nicotine endured by farmers. Chronic occupational exposure to complex mixtures can lead to health hazardous. We examined genomic instability and epigenetic changes in tobacco farmers occupationally exposed to pesticide mixtures and nicotine at tobacco fields. DNA damage was assessed by alkaline comet assay in blood cells. Genomic DNA was isolated, and telomere length was measured using quantitative polymerase chain reaction assay. We measured 5-methyl-2'-deoxycytidine, a marker of global DNA methylation, and p16 promoter methylation. The oxidative profile was evaluated by trolox equivalent antioxidant capacity and lipid peroxidation (thiobarbituric acid reactive substances) in serum. Exposure parameters, plasma cotinine and inorganic element levels, were also measured. DNA damage was significantly elevated for farmers in relation to unexposed group (P < 0.001; Mann-Whitney test) and positively associated with years of exposure. Inverse relationship between DNA damage and total equivalent antioxidant activity was demonstrated for exposed and unexposed groups. Exposed group showed significantly shorter telomeres (P < 0.001; unpaired t-test) and DNA hypomethylation (P < 0.001; unpaired t-test), as well as p16 hypermethylation (P = 0.003; Mann-Whitney test). Lipid peroxidation was increased for exposed group in relation to unexposed one (P = 0.02; Mann-Whitney test) and presented a positive correlation with global DNA methylation (P = 0.0264). Farmers have increased plasma cotinine levels (P < 0.001) and inorganic elements (phosphorus, sulphur and chlorine) in relation to unexposed group. Elevated oxidative stress levels due to chronic occupational pesticide mixtures and nicotine exposure in tobacco farmers were associated with higher DNA damage, shorter telomeres and altered DNA methylation. Telomere-accelerated attrition due to exposure may be potential intermediate step before a disease state.

Pharmacotherapeutic potential of phytochemicals: Implications in cancer chemoprevention and future perspectives.

Cancer is a leading cause of disease burden throughout the world. Many cancers develop as a result of exposure to both lifestyle and environmental factors that are potentially modifiable. In the last few years, much of the scientific attention has drawn to the discovery of new and effective chemopreventive agents from natural sources. A multitude of phytoconstituents have been explored for their potential to prevent the occurrence of carcinogenesis both in vitro and in vivo by means of diverse cellular and molecular approaches. Key focus of this review is to highlight some significant and new information about different molecular aspects of chemopreventive ability of plant based phytochemicals in terms of their inhibitory potential on cancer growth. In addition, information regarding certain limiting factors such as whole animal physiology, tumour microenvironment and bioavailability of active components of phytoconstituents used in pre/clinical trials are further explored. This review would further assist the scientific community involved in designing efficacious chemopreventive approaches using these phytochemicals in treating cancer.

Shorter telomere length in people with schizophrenia: A preliminary study from Australia.

Schizophrenia is a complex mental illness affecting the normal functioning of the brain, interfering with the ability to think, feel and act. It can be conceptualised as a syndrome of accelerated ageing, with early onset of cardiovascular disease and high rates of premature mortality. Telomere attrition increases with oxidative stress and is considered a biomarker of ageing. Previous studies have assessed abnormalities in telomere length in schizophrenia, but the results are inconsistent. The present study used a case-control design to assess whether people with schizophrenia have shortened telomeres, indicative of accelerated ageing. Subjects were all male, aged 25-35years, living in the same urban region of Adelaide, South Australia. Telomere length was measured using a quantitative real-time polymerase chain reaction (PCR) method. We found significantly shorter telomeres in people with schizophrenia relative to healthy controls. This is the first study to show telomere attrition among people with schizophrenia in Australia. Shorter telomere length may indicate the common pathways that schizophrenia shares with other neuropsychiatric and neurodevelopmental disorders associated with increased cellular senescence. Further well-controlled larger studies in people with schizophrenia are required to fully understand (i) the role of variables that have the potential to modulate telomere length such as use of antipsychotic drugs, medical conditions, parental age, smoking, alcohol abuse and use of illicit drugs; (ii) effective treatments to slow telomere erosion and (iii) mechanisms responsible for accelerating and reducing telomere damage.

Predictors of radiation-induced gastrointestinal morbidity: A prospective, longitudinal study following radiotherapy for carcinoma of the prostate.

Background Chronic gastrointestinal (GI) morbidity occurs in ≥50% of patients after external beam radiotherapy (EBRT) for carcinoma of prostate (CaP). This prospective, longitudinal study examines which baseline measurements of: 1) homocysteine and micronutrients in plasma; 2) chromosome damage/misrepair biomarkers; and 3) anal and rectal dose volume metrics predict GI morbidity after EBRT. Patients and methods In total, 106 patients with CaP had evaluations of GI symptoms (modified LENT-SOMA questionnaires) before EBRT and at one month, one, two and three years after its completion. Other variables measured before EBRT were: 1) plasma concentrations of homocysteine and micronutrients including caroteinoids and selenium; 2) chromosome damage/DNA misrepair (micronuclei/nucleoplasmic bridge) indices; and 3) mean anal and rectal wall doses and volumes of anal and rectal walls receiving ≥40 Gy and ≥60 Gy. Univariate and multivariate analyzes examined the relationships among: 1) plasma levels of homocysteine and micronutrients; 2) indices of chromosome damage/DNA misrepair; and 3) mean anal and rectal wall doses and volumes of anal and rectal walls receiving ≥40 Gy and ≥60 Gy and total GI symptom scores from one month to three years after EBRT. Results Increased frequency and urgency of defecation, rectal mucous discharge and bleeding after EBRT resulted in sustained rises in total GI symptom scores above baseline at three years. On univariate analysis, total GI symptom scores were significantly associated with: 1) plasma selenium and α tocopherol; 2) micronuclei indices of DNA damage; 3) mean anal and rectal wall doses; and 4) volumes of anal and rectal wall receiving ≥40 Gy and ≥60 Gy (p = 0.08-<0.001). On multivariate analysis, only volume of anal wall receiving ≥40 Gy was significant for increased GI symptoms after EBRT (p < 0.001). Conclusion The volume of anal wall receiving ≥40 Gy predicts chronic GI morbidity after EBRT for CaP.

Genotoxicity and cytotoxicity of chromium, copper, manganese and lead, and their mixture in WIL2-NS human B lymphoblastoid cells is enhanced by folate depletion.

Heavy metal exposure or dietary deficiency is associated with increased genetic damage, cancer and age-related diseases. Folate (vitamin B9) required for DNA repair and synthesis may increase cellular susceptibility to metal induced genotoxicity. This study investigated the interactive effects of folic acid deficiency and sufficiency on genome instability and cytotoxicity induced by chromium (VI), copper (II), manganese (II), lead (IV), and their mixture (CCMP) in WIL2-NS human B lymphoblastoid cells. WIL2-NS cells were cultured in folic acid deficient (20 nM) and replete (2000 nM) RPMI 1640 medium treated with different concentrations (0.00-1000 µM) of the metals and CCMP for 48 h. Chromosomal damage and cytotoxicity were measured using the Cytokinesis-block Micronucleus Cytome assay. CCMP, Cr, Pb, Cu and Mn induced concentration dependent, increases in cells with chromosome damage (micronuclei, nucleoplasmic bridges, nuclear buds) and necrotic cells and decreased nuclear division index. The metals exhibited different cytotoxic and genotoxic potentials (CCMP>Cr>Pb>Cu>Mn) in both folate deficient and sufficient cells, with the cytogenotoxic effects being greater in folate deficient cells. Significant interaction between the metals and folic acid suggests that folic acid deficiency exacerbated cell proliferation inhibition and genome instability induced by metals. Folate deficiency, increasing metal concentration, and their interactions explained 3-11%, 74-92% and 4-12% of the variance of DNA damage biomarkers. In conclusion, exposure to the tested metals (0.01-1000 µM) increased chromosomal DNA damage in WIL2-NS cells and this was exacerbated by folate deficiency.

GSTP1 methylation and polymorphism increase the risk of breast cancer and the effects of diet and lifestyle in breast cancer patients.

Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. Polymorphism at exon 5 of the GST π family decreases the catalytic activity and affects the detoxification ability of the enzyme, GSTP1. GSTP1 promoter hypermethylation and loss of expression are frequently observed in various types of carcinoma. We hypothesized that somatic epigenetic modification in homozygous mutants increases the degree to which breast cancer risk is affected by lifestyle factors and dietary habits. The present study used tumor biopsies and blood samples from 215 breast cancer patients and 215 blood samples from healthy donors. GSTP1 polymorphism was studied using PCR-restriction fragment length polymorphism, methylation using methylation-specific PCR and loss of expression using immunohistochemistry and western blotting. No significant increase was observed in the breast cancer risk of individuals with the mutant (Val) allele [odds ratio (OR), 1.48; 95% confidence interval (CI), 0.97-2.26 for heterozygotes; OR, 1.42; 95% CI, 0.86-2.42 homozygous mutants]. GSTP1 promoter hypermethylation was detected in one-third of tumor biopsies (74/215) and was found to be associated with a loss of expression. Genotype and tumor methylation associations were not observed. Estrogen (ER) and progesterone (PR) receptor-positive tumors had a higher methylation frequency. GSTP1 polymorphism was not associated with increased promoter hypermethylation. The results suggest that GSTP1 methylation is a major event in breast carcinogenesis and may act as a tumor-specific biomarker.

Genetic polymorphisms of genes involved in DNA repair and metabolism influence micronucleus frequencies in human peripheral blood lymphocytes.

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.