Multiomics: Functional Molecular Biomarkers of Micronutrients for Public Health Application.

Adequate micronutrient intake and status are global public health goals. Vitamin and mineral deficiencies are widespread and known to impair health and survival across the life stages. However, knowledge of molecular effects, metabolic pathways, biological responses to variation in micronutrient nutriture, and abilities to assess populations for micronutrient deficiencies and their pathology remain lacking. Rapidly evolving methodological capabilities in genomics, epigenomics, proteomics, and metabolomics offer unparalleled opportunities for the nutrition research community to link micronutrient exposure to cellular health; discover new, arguably essential micronutrients of microbial origin; and integrate methods of molecular biology, epidemiology, and intervention trials to develop novel approaches to assess and prevent micronutrient deficiencies in populations. In this review article, we offer new terminology to specify nutritional application of multiomic approaches and encourage collaboration across the basic to public health sciences to advance micronutrient deficiency prevention.

Low magnesium in conjunction with high homocysteine increases DNA damage in healthy middle aged Australians.

PURPOSE: Magnesium is one of the most common elements in the human body and plays an important role as a cofactor of enzymes required for DNA replication and repair and many other biochemical mechanisms including sensing and regulating one-carbon metabolism deficiencies. Low intake of magnesium can increase the risk of many diseases, in particular, chronic degenerative disorders. However, its role in prevention of DNA damage has not been studied fully in humans so far. Therefore, we tested the hypothesis that magnesium deficiency either on its own or in conjunction with high homocysteine (Hcy) induces DNA damage in vivo in humans. METHODS: The present study was carried out in 172 healthy middle aged subjects from South Australia. Blood levels of magnesium, Hcy, folate and vitamin B12 were measured. Cytokinesis-Block Micronucleus cytome assay was performed to measure three DNA damage biomarkers: micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) in peripheral blood lymphocytes. RESULTS: Data showed that magnesium and Hcy are significantly inversely correlated with each other (r = - 0.299, p < 0.0001). Furthermore, magnesium is positively correlated both with folate (p = 0.002) and vitamin B12 (p = 0.007). Magnesium is also significantly inversely correlated with MN (p < 0.0001) and NPB (p < 0.0001). Individuals with low magnesium and high Hcy exhibited significantly higher frequency of MN and NPBs compared to those with high magnesium and low Hcy (p < 0.0001). Furthermore, there was an interactive effect between these two factors as well in inducing MN (p = 0.01) and NPB (p = 0.048). CONCLUSIONS: The results obtained in the present study indicate for the first time that low in vivo levels of magnesium either on its own or in the presence of high Hcy increases DNA damage as evident by higher frequencies of MN and NPBs.

Reduced SIRT1 and SIRT3 and Lower Antioxidant Capacity of Seminal Plasma Is Associated with Shorter Sperm Telomere Length in Oligospermic Men.

Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.

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.

Red Blood Cell Fatty Acid Profiles Are Significantly Altered in South Australian Mild Cognitive Impairment and Alzheimer's Disease Cases Compared to Matched Controls.

Nutritional imbalances have been associated with a higher risk for cognitive impairment. This study determined the red blood cell (RBC) fatty acid profile of newly diagnosed mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients compared to age and gender-matched controls. There was a significant increase in palmitic acid (p < 0.00001) for both MCI and AD groups. Saturated fatty acids were significantly elevated in the MCI group, including stearic acid (p = 0.0001), arachidic acid (p = 0.003), behenic acid (p = 0.0002), tricosanoic acid (p = 0.007) and lignoceric acid (p = 0.001). n-6 polyunsaturated fatty acids (PUFAs) were significantly reduced in MCI, including linoleic acid (p = 0.001), γ-linolenic acid (p = 0.03), eicosatrienoic acid (p = 0.009) and arachidonic acid (p < 0.00004). The n-3 PUFAs, α-linolenic acid and docosahexaenoic acid, were both significantly reduced in MCI and AD (p = 0.0005 and p = 0.00003). A positive correlation was evident between the Mini-Mental State Examination score and nervonic acid in MCI (r = 0.54, p = 0.01) and a negative correlation with γ-linolenic acid in AD (r = -0.43, p = 0.05). Differences in fatty acid profiles may prove useful as potential biomarkers reflecting increased risk for dementia.

Low Magnesium in Conjunction with High Homocysteine and Less Sleep Accelerates Telomere Attrition in Healthy Elderly Australian.

The relationship between sleep and micronutrients, including magnesium, is implicated in its regulation. The effects of low magnesium and other micronutrients on sleep disruption and telomere loss are not well understood. The present study was carried out in 172 healthy elderly subjects from South Australia. Plasma micronutrients including magnesium were measured. Each participant provided information about their sleep hours (<7 h or ≥7 h). Lymphocyte telomere length (TL) was measured by real-time qPCR assay. Plasma magnesium level was significantly low in subjects who sleep less than 7 h (p = 0.0002). TL was significantly shorter in people who are low in magnesium and sleep less than 7 h (p = 0.01). Plasma homocysteine (Hcy) is negatively associated with magnesium (r = −0.299; p < 0.0001). There is a significant interaction effect of magnesium and Hcy on sleep duration (p = 0.04) and TL (p = 0.003). Our results suggest that inadequate magnesium levels have an adverse impact on sleep and telomere attrition rate in cognitively normal elderly people, and this may be exacerbated by low levels of vitamin B12 and folate that elevate Hcy concentration.

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.

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.

Dietary Pattern, Genomic Stability and Relative Cancer Risk in Asian Food Landscape.

The incidence of cancer globally is increasing, partly due to lifestyle factors. Despite a better understanding of cancer biology and advancement in cancer management and therapies, current strategies in cancer treatment remain costly and cause socioeconomic burden especially in Asian countries. Hence, instead of putting more efforts in searches for new cancer cures, attention has now shifted to understanding how to mitigate cancer risk by modulating lifestyle factors. It has been established that carcinogenesis is multifactorial, and the important detrimental role of oxidative stress, chronic inflammation, and genomic instability is evident. To date, there is no study linking dietary pattern and genomic stability in cancer risk in the Asian food landscape. Thus, this present review article discusses recent literature on dietary pattern and genomic stability and its relationship with cancer risk in Asia.

Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress.

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.

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.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

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.

Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time.

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.

Use of micronucleus assays for the prediction and detection of cervical cancer: a meta-analysis.

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.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

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.

Dietary sugars and related endogenous advanced glycation end-products increase chromosomal DNA damage in WIL2-NS cells, measured using cytokinesis-block micronucleus cytome assay.

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 Nε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu-BSA model. Nε-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 (R2 = 0.7704; P = 0.0031), BN with NPBs (R2 = 0.9311; P < 0.0001) and BN with NBuds (R2 = 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.

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.

Mechanistic insights into the phenomena of increasing capacity with cycle number: using pulsed-laser deposited MoO2 thin film electrodes.

Lithium ion batteries typically lose capacity or energy storage density (i.e. capacity fading) over the course of extended cycling which can be problematic for applications and appears to be exaggerated when high current rates are used. However, in some cases fluctuations in capacity with cycle number and even increases in capacity with cycle number are noted with predominantly thin film based electrodes. Here we demonstrate the synthesis and in-depth characterisation of laser deposited MoO2 thin film anodes and its unconventional mechanism. A MoO2 electrode shows an initial capacity of 79 mA h g-1 which increases to capacities of 600 mA h g-1 at 15.8 A g-1 after 90 000 cycles. A maximum capacity of 1714 mA h g-1 was achieved in an electrode cycled at 1.5 A g-1 for over 3800 cycles, the highest recorded capacity in MoOx anodes to date. The most intriguing aspects of this work is the fact that capacity is shown to fluctuate and typically increase well above the theoretical capacity of MoO2. A combination of electrochemical cycling, X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, focused ion beam milling and transmission electron microscopy at various states of cycling is used to illustrate a proposed mechanism. The mechanism illustrated is based on exfoliation of layers of MoO2 off the pulsed laser deposition (PLD) grown MoO2 electrodes during cycling that creates additional surface area and easier access for Li-ions to both adsorb to the surface and insert/react with the host material. Further features in the capacity evolution are rationalised by this mechanism and methods to control the capacity evolution are detailed. These results present a rational explanation for when an electrode undergoes a substantial increase in capacity over its extended cycling life.

Leukocyte telomere length in relation to risk of lung adenocarcinoma incidence: Findings from the Singapore Chinese Health Study.

Telomeres are crucial in the maintenance of chromosome integrity and genomic stability. Critically short telomeres can trigger programed cell death while cells with longer telomeres may have increased likelihood of replicative errors, resulting in genetic mutations and chromosomal alterations, and ultimately promoting oncogenesis. Data on telomere length and lung cancer risk from large prospective cohort studies are spare. Relative telomere length in peripheral blood leukocytes was quantified using a validated monochrome multiplex quantitative polymerase chain reaction (qPCR) method in 26,540 participants of the Singapore Chinese Health Study. After a follow-up of 12 years, 654 participants developed lung cancer including 288 adenocarcinoma, 113 squamous cell carcinoma and 253 other/unknown histological type. The Cox proportional hazard regression was used to estimate hazard ratio (HR) and 95% confidence interval (CI). HR of lung adenocarcinoma for individuals in the highest comparing the lowest 20 percentile of telomere length was 2.84 (95% CI 1.94-4.14, ptrend < 0.0001). This positive association was present in never smokers (ptrend < 0.0001), ever smokers (ptrend = 0.0010), men (ptrend = 0.0003), women (ptrend < 0.0001), and in shorter (ptrend = 0.0002) and longer (ptrend = 0.0001) duration of follow-up. There was no association between telomere length and risk of squamous cell carcinoma or other histological type of lung cancer in all or subgroups of individuals. The agreement of results from this prospective cohort study with those of previous prospective studies and Mendelian randomization studies suggest a possible etiological role of telomere length in the development of lung adenocarcinoma.

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.

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.