Benzene Uptake and Glutathione S-transferase T1 Status as Determinants of S-Phenylmercapturic Acid in Cigarette Smokers in the Multiethnic Cohort

Research from the Multiethnic Cohort (MEC) demonstrated that, for the same quantity of cigarette smoking, African Americans and Native Hawaiians have a higher lung cancer risk than Whites, while Latinos and Japanese Americans are less susceptible. We collected urine samples from 2,239 cigarette smokers from five different ethnic groups in the MEC and analyzed each sample for S-phenylmercapturic acid (SPMA), a specific biomarker of benzene uptake. African Americans had significantly higher (geometric mean [SE] 3.69 [0.2], p<0.005) SPMA/ml urine than Whites (2.67 [0.13]) while Japanese Americans had significantly lower levels than Whites (1.65 [0.07], p<0.005). SPMA levels in Native Hawaiians and Latinos were not significantly different from those of Whites. We also conducted a genome-wide association study in search of genetic risk factors related to benzene exposure. The glutathione S-transferase T1 (GSTT1) deletion explained between 14.2–31.6% (p = 5.4x10^-157) and the GSTM1 deletion explained between 0.2%-2.4% of the variance (p = 1.1x10^-9) of SPMA levels in these populations. Ethnic differences in levels of SPMA remained strong even after controlling for the effects of these two deletions. These results demonstrate the powerful effect of GSTT1 status on SPMA levels in urine and show that uptake of benzene in African American, White, and Japanese American cigarette smokers is consistent with their lung cancer risk in the MEC. While benzene is not generally considered a cause of lung cancer, its metabolite SPMA could be a biomarker for other volatile lung carcinogens in cigarette smoke.     

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene

Objective: This study aims to assess the effects of low-dose benzene on DNA damage and O⁶-methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers.

Materials and methods: We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay.

Results: The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p?<?0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage.

Discussion and conclusion: MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

Personal exposure to different levels of benzene and its relationships to the urinary metabolites S-phenylmercapturic acid and trans,trans-muconic acid

This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe-making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography-tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure.     

Changes in Poly(ADP-Ribosyl)ation Patterns in Workers Exposed to BTX

Occupational exposure to (benzene, toluene and xylene, BTX is common in the Chinese workplace. Chronic occupational exposure to benzene is associated with an increased risk of hematological malignancies such as acute myeloid leukemia (AML), but the underlying mechanisms are still unclear. This study investigates changes in poly(ADP-ribosyl)ation and DNA methylation in subjects occupationally exposed to a BTX. Blood DNA samples and exposure data were obtained from subjects with different levels of exposure, including 132 decorators, 129 painters, and 130 unexposed referents in a container-manufacturing factory in Shenzhen, China. Occupational exposure assessment included personal monitoring of airborne benzene, toluene and xylene. Hematological parameters were measured and the cytokinesis-block micronucleus (CBMN) assay was used to detect DNA damage in peripheral lymphocytes. Quantitative real-time PCR was used to detect the mRNA expression of poly(ADP-ribose) polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG), DNA methyltransferases (DNMTs) including DNMT1, DNMT3a and DNMT3b, methyl-CpG-binding domain protein 2(MBD2). PARP1 assay was used to measure PARP activity. Airborne levels of benzene, toluene and xylene in the two exposed groups were significantly higher than those of controls (P<0.001). The two exposed groups (decorators, painters) showed decreased PARP1, DNMTs and MBD2 expression relative to controls (P<0.05), and PARP activity was also decreased (P<0.05). Decreased PARP1, DNMT1, DNMT3a, DNMT3b and MBD2 mRNA expression was correlated with increased airborne BTX (Pearson''s r: −0.587, −0.314, −0.636, −0.567 and −0.592 respectively, P<0.001). No significant differences in hematological parameters and CBMN were found among the three groups. Together, these results suggest that decreased DNMTs, MBD2 and PARP1 might be involved in the global hypomethylation associated with BTX exposure, and the imbalance of PARP/PARG might participate in the down-regulation of DNMTs. This is the first human study to link altered poly(ADP-ribosyl)ation patterns, which reproduce the aberrant epigenetic patterns found in benzene-treated cells, to chronic occupational exposure to BTX.     

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

A quantitative approach to evaluate urinary benzene and S-phenylmercapturic acid as biomarkers of low benzene exposure

Context: Benzene is a ubiquitous pollutant; smoking habit, genetic polymorphisms, and analytical difficulties impact the identification of the best biomarker.

Objective: To apply a systematic quantitative approach to evaluate urinary benzene (BEN-U) and S-phenylmercapturic acid (SPMA) as biomarkers of low benzene exposures.

Methods: Seventy-one blue collar refinery workers, 97 white collar refinery workers and 108 general population subjects were included. Intrinsic characteristics, sampling and analytical issues were compared.

Results: BEN-U and SPMA were detected in 99% and 78% of samples, which correlated with benzene exposure (r?=?0.456 and r?=?0.636, respectively) and with urinary cotinine (r?=?0.630 and r?=?0.570, respectively). Intrinsic characteristics were similar for the two biomarkers: specificity (0.64 and 0.69 for BEN-U and SPMA), sensitivity (0.74 and 0.83), as well as intra- and inter-individual variability (150% and >14 for both).

Conclusion: BEN-U and SPMA show similar intrinsic characteristics; analytical issues in detecting SPMA suggest that BEN-U is more convenient for investigating low exposure levels.     

LINE-1 Hypomethylation in Blood and Tissue Samples as an Epigenetic Marker for Cancer Risk: A Systematic Review and Meta-Analysis

Objective

A systematic review and a meta-analysis were carried out in order to summarize the current published studies and to evaluate LINE-1 hypomethylation in blood and other tissues as an epigenetic marker for cancer risk.

Methods

A systematic literature search in the Medline database, using PubMed, was conducted for epidemiological studies, published before March 2014. The random-effects model was used to estimate weighted mean differences (MDs) with 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by sample type (tissue or blood samples), cancer types, and by assays used to measure global DNA methylation levels. The Cochrane software package Review Manager 5.2 was used.

Results

A total of 19 unique articles on 6107 samples (2554 from cancer patients and 3553 control samples) were included in the meta-analysis. LINE-1 methylation levels were significantly lower in cancer patients than in controls (MD: −6.40, 95% CI: −7.71, −5.09; p<0.001). The significant difference in methylation levels was confirmed in tissue samples (MD −7.55; 95% CI: −9.14, −65.95; p<0.001), but not in blood samples (MD: −0.26, 95% CI: −0.69, 0.17; p = 0.23). LINE-1 methylation levels were significantly lower in colorectal and gastric cancer patients than in controls (MD: −8.33; 95% CI: −10.56, −6.10; p<0.001 and MD: −5.75; 95% CI: −7.75, −3.74; p<0.001) whereas, no significant difference was observed for hepatocellular cancer.

Conclusions

The present meta-analysis adds new evidence to the growing literature on the role of LINE-1 hypomethylation in human cancer and demonstrates that LINE-1 methylation levels were significantly lower in cancer patients than in control samples, especially in certain cancer types. This result was confirmed in tissue samples, both fresh/frozen or FFPE specimens, but not in blood. Further studies are needed to better clarify the role of LINE-1 methylation in specific subgroups, considering both cancer and sample type, and the methods of measurement.     

Enrichment and properties of urinary pre-S-phenylmercapturic acid (pre-SPMA)

In benzene metabolism, pre-S-phenylmercapturic acid (pre-SPMA) is the precursor to S-phenylmercapturic acid (SPMA). Urinary pre-SPMA/SPMA ratios are variable. For the determination of urinary SPMA as a biomarker of exposure to benzene it is essential to completely convert pre-SPMA to SPMA. We developed a procedure for the enrichment and determination of urinary pre-SPMA by LC–MS/MS which allowed us to trace the conversion of pre-SPMA to SPMA. Complete conversion was found upon treatment of urine with HCl (37%) at pH 1.1. Previously reported treatment of urine with concentrated H₂SO₄ was found to yield SPMA levels higher than after HCl treatment. The origin of that extra SPMA amount is unknown. In conclusion, our findings suggest that pre-treatment of urine with HCl to adjust the pH to 0.5–1 is essential for complete conversion of pre-SPMA to SPMA and should be applied prior to analysis of SPMA in urine.     

Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including ^mC^mC, ^uC^uC, ^mC^uC and ^uC^mC. Of these, ^mC and ^uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of ^mC^mC and ^uC^uC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each ^mC^uC or ^uC^mC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %^mC^mC and %^uC^uC increased, while the %^mC^uC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %^mC^uC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %^mC^uC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered ^mC^uC to ^mC^mC and ^uC^uC forms, and changes ^uC^mC to ^uC^uC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.     

Polycomb group genes are targets of aberrant DNA methylation in renal cell carcinoma

The combined effects of genetic and epigenetic aberrations are well recognized as causal in tumorigenesis. Here, we defined profiles of DNA methylation in primary renal cell carcinomas (RCC) and assessed the association of these profiles with the expression of genes required for the establishment and maintenance of epigenetic marks. A bead-based methylation array platform was used to measure methylation of 1,413 CpG loci in ∼800 cancer-associated genes and three methylation classes were derived by unsupervised clustering of tumors using recursively partitioned mixture modeling (RPMM). Quantitative RT-PCR was performed on all tumor samples to determine the expression of DNMT1, DNMT3B, VEZF1 and EZH2. Additionally, methylation at LINE-1 and AluYb8 repetitive elements was measured using bisulfite pyrosequencing. Associations between methylation class and tumor stage (p = 0.05), LINE-1 (p < 0.0001) and AluYb8 (p < 0.0001) methylation, as well as EZH2 expression (p < 0.0001) were noted following univariate analyses. A multinomial logistic regression model controlling for potential confounders revealed that AluYb8 (p < 0.003) methylation and EZH2 expression (p < 0.008) were significantly associated with methylation class membership. Because EZH2 is a member of the Polycomb repressive complex 2 (PRC2), we next analyzed the distribution of Polycomb group (PcG) targets among methylation classes derived by clustering the 1,413 array CpG loci using RPMM. PcG target genes were significantly enriched (p < 0.0001) in methylation classes with greater differential methylation between RCC and non-diseased kidney tissue. This work contributes to our understanding of how repressive marks on DNA and chromatin are dysregulated in carcinogenesis, knowledge that might aid the development of therapies or preventive strategies for human malignancies.Key words: EZH2, DNA methylation, renal cell carcinoma, polycomb, microarray     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

LINE-1 methylation in granulocyte DNA and trihalomethane exposure is associated with bladder cancer risk

DNA methylation changes contribute to bladder carcinogenesis. Trihalomethanes (THM), a class of disinfection by-products, are associated with increased urothelial bladder cancer (UBC) risk. THM exposure in animal models produces DNA hypomethylation. We evaluated the relationship of LINE-1 5-methylcytosine levels (LINE-1%5mC) as outcome of long-term THM exposure among controls and as an effect modifier in the association between THM exposure and UBC risk. We used a case-control study of UBC conducted in Spain. We obtained personal lifetime residential THM levels and measured LINE-1%5mC by pyrosequencing in granulocyte DNA from blood samples in 548 incident cases and 559 hospital controls. Two LINE-1%5mC clusters (above and below 64%) were identified through unsupervised hierarchical cluster analysis. The association between THM levels and LINE-1%5mC was evaluated with β regression analyses and logistic regression was used to estimate odds ratios (OR) adjusting for covariables. LINE-1%5mC change between percentiles 75^th and 25^th of THM levels was 1.8% (95% confidence interval (CI): 0.1, 3.4%) among controls. THM levels above vs. below the median (26 μg/L) were associated with increased UBC risk, OR = 1.86 (95% CI: 1.25, 2.75), overall and among subjects with low levels of LINE-1%5mC (n = 975), OR = 2.14 (95% CI: 1.39, 3.30), but not associated with UBC risk among subjects’ high levels of LINE-1%5mC (n = 162), interaction P = 0.03. Results suggest a positive association between LINE-1%5mC and THM levels among controls, and LINE-1%5mC status may modify the association between UBC risk and THM exposure. Because reverse causation and chance cannot be ruled out, confirmation studies are warranted.     

DNA damage in lymphocytes of benzene exposed workers correlates with trans,trans-muconic acids and breath benzene levels

Benzene causes many kinds of blood disorders in workers employed in many different environments. These diseases include myelodisplastic syndrome and acute and chronic myelocytic leukemia. In the present study, five occupational work places, including six industrial process types, namely, printing, shoe-making, methylene di-aniline (MDA), nitrobenzene, carbomer, and benzene production were selected, and the levels of breath benzene, and trans,trans-muconic acids (t,t-MA) and phenol in urine were evaluated, as well as hematological changes and lymphocyte DNA damage. The concentration of benzene in breath was less than 3 ppm in the workplaces, and benzene exposure was found to be higher in work places where benzene is used, than in those where benzene is produced. At low levels of benzene exposure, urinary t,t-MA correlated strongly with benzene in air. Highest Olive tail moments were found in workers producing carbomer. Levels of breathzone benzene were found to be strongly correlated with Olive tail moment values in the lymphocytes of workers, but not with hematological data in the six workplaces types. In conclusion, the highest benzene exposures found occurred in workers at a company, which utilized benzene in the production of carbomer. In terms of low levels of exposure to benzene, urinary t,t-MA and DNA damage exhibited a strong correlation with breath benzene, but not with hematological data. We conclude that breath benzene, t,t-MA and lymphocytic DNA damage are satisfactory biomonitoring markers with respect to benzene exposure in the workplace.     

A Prospective Study of LINE-1DNA Methylation and Development of Adiposity in School-Age Children

Background

Repetitive element DNA methylation is related to prominent obesity-related chronic diseases including cancer and cardiovascular disease; yet, little is known of its relation with weight status. We examined associations of LINE-1 DNA methylation with changes in adiposity and linear growth in a longitudinal study of school-age children from Bogotá, Colombia.

Methods

We quantified methylation of LINE-1 elements from peripheral leukocytes of 553 children aged 5–12 years at baseline using pyrosequencing technology. Anthropometric characteristics were measured periodically for a median of 30 months. We estimated mean change in three age-and sex-standardized indicators of adiposity: body mass index (BMI)-for-age Z-score, waist circumference Z-score, and subscapular-to-triceps skinfold thickness ratio Z-score according to quartiles of LINE-1 methylation using mixed effects regression models. We also examined associations with height-for-age Z-score.

Results

There were non-linear, inverse relations of LINE-1 methylation with BMI-for-age Z-score and the skinfold thickness ratio Z-score. After adjustment for baseline age and socioeconomic status, boys in the lowest quartile of LINE-1 methylation experienced annual gains in BMI-for-age Z-score and skinfold thickness ratio Z-score that were 0.06 Z/year (P = 0.04) and 0.07 Z/year (P = 0.03), respectively, higher than those in the upper three quartiles. The relation of LINE-1 methylation and annual change in waist circumference followed a decreasing monotonic trend across the four quartiles (P trend = 0.02). DNA methylation was not related to any of the adiposity indicators in girls. There were no associations between LINE-1 methylation and linear growth in either sex.

Conclusions

Lower LINE-1 DNA methylation is related to development of adiposity in boys.     

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA methylation in histologically normal tissues could provide insight into early stages of carcinogenesis. In a cross-sectional study of 121 healthy women with no prior history of cancer who underwent reduction mammoplasty, we examined associations between plasma and breast folate, genetic variation in one-carbon metabolism, and percent LINE-1 methylation using multivariable regression models (adjusting for race, oral contraceptive use, and alcohol use). Results are expressed as the ratio of LINE-1 methylation relative to that of the referent group, with the corresponding 95% confidence intervals (CI). We found no significant associations between plasma or breast folate and percent LINE-1 methylation. Variation in MTHFR, MTR, and MTRR were significantly associated with percent LINE-1 methylation. Variant allele carriers of MTHFR A1289C had 4% lower LINE-1 methylation (Ratio 0.96, 95% CI 0.93–0.98), while variant allele carriers of MTR A2756G (Ratio 1.03, 95% CI 1.01–1.06) and MTRR A66G (Ratio 1.03, 95% CI 1.01–1.06) had 3% higher LINE-1 methylation, compared to those carrying the more common genotypes of these SNPs. DNA methylation of LINE-1 elements in histologically normal breast tissues is influenced by polymorphisms in genes in the one-carbon metabolism pathway. Future studies are needed to investigate the sociodemographic, environmental and additional genetic determinants of DNA methylation in breast tissues and the impact on breast cancer susceptibility.     

Quality control and statistical modeling for environmental epigenetics: A study on in utero lead exposure and DNA methylation at birth

DNA methylation data assayed using pyrosequencing techniques are increasingly being used in human cohort studies to investigate associations between epigenetic modifications at candidate genes and exposures to environmental toxicants and to examine environmentally-induced epigenetic alterations as a mechanism underlying observed toxicant-health outcome associations. For instance, in utero lead (Pb) exposure is a neurodevelopmental toxicant of global concern that has also been linked to altered growth in human epidemiological cohorts; a potential mechanism of this association is through alteration of DNA methylation (e.g., at growth-related genes). However, because the associations between toxicants and DNA methylation might be weak, using appropriate quality control and statistical methods is important to increase reliability and power of such studies. Using a simulation study, we compared potential approaches to estimate toxicant-DNA methylation associations that varied by how methylation data were analyzed (repeated measures vs. averaging all CpG sites) and by method to adjust for batch effects (batch controls vs. random effects). We demonstrate that correcting for batch effects using plate controls yields unbiased associations, and that explicitly modeling the CpG site-specific variances and correlations among CpG sites increases statistical power. Using the recommended approaches, we examined the association between DNA methylation (in LINE-1 and growth related genes IGF2, H19 and HSD11B2) and 3 biomarkers of Pb exposure (Pb concentrations in umbilical cord blood, maternal tibia, and maternal patella), among mother-infant pairs of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) cohort (n = 247). Those with 10 μg/g higher patella Pb had, on average, 0.61% higher IGF2 methylation (P = 0.05). Sex-specific trends between Pb and DNA methylation (P < 0.1) were observed among girls including a 0.23% increase in HSD11B2 methylation with 10 μg/g higher patella Pb.     

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002–03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p = 0.04) and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p = 0.02) and lower levels of fasting HDL (p = 0.009). The findings from this study confirm that DNA “global” hypomethylation (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.Key words: cardiovascular disease, HDL, LDL, LINE-1, DNA methylation, Samoa     

Effects of Formaldehyde on Lymphocyte Subsets and Cytokines in the Peripheral Blood of Exposed Workers

Formaldehyde (FA) is a well-known irritant, and it is suggested to increase the risk of immune diseases and cancer. The present study aimed to evaluate the distribution of major lymphocyte subsets and cytokine expression profiles in the peripheral blood of FA-exposed workers. A total of 118 FA-exposed workers and 79 controls were enrolled in the study. High performance liquid chromatography, flow cytometry, and cytometric bead array were used to analyze FA in air sample and formic acid in urine, blood lymphocyte subpopulations, and serum cytokines, respectively. The FA-exposed workers were divided into low and high exposure groups according to their exposure levels. The results showed that both the low and high FA-exposed groups had a significant increase of formic acid in urine when compared to the controls. Both the low and high exposure groups had a significant increase in the percentage of B cells (CD19⁺) compared to the control group (p<0.01). A significant increase in the percentage of the natural killer (NK) cells (CD56⁺) was observed in the low exposure group compared to the control (p = 0.013). Moreover, the FA-exposed workers in both exposure groups showed a significant higher level of IL-10 but lower level of IL-8 than the control (p<0.01). Subjects in the high exposure group had a higher level of IL-4 but a lower level of IFN-γ than the control (p<0.05). Finally, there is a significant correlation between the levels of IL-10, IL-4, and IL-8 and formic acid (p<0.05). The findings from the present study may explain, at least in part, the association between FA exposure and immune diseases and cancer.