Yoon Hee Cho

Prenatal Smoke Exposure and Genomic DNA Methylation in a Multiethnic Birth Cohort

Background: Exposure to prenatal tobacco smoke (PTS) has been associated with a number of health outcomes in the offspring, including some childhood cancers. Lower levels of genomic DNA methylation have also been associated with several types of cancers. We investigated whether PTS was associated with global DNA methylation levels in the offspring. Methods: Our sample was drawn from a birth cohort of women born between 1959 and 1963 in New York City (n = 90). We measured methylation of repetitive elements (Sat2, Alu, LINE-1) from peripheral blood granulocytes. We combined prospectively collected data on PTS with adult epidemiologic data and blood samples collected in 2001 to 2007 (mean age, 43 years). We used linear regression to assess the association between PTS and repetitive element methylation. Results: Thirty-six percent of mothers smoked during pregnancy. We observed an inverse association between PTS and Sat2 methylation. This inverse association remained even after adjustment for potential mediators including child environmental tobacco smoke exposure, birth size, postnatal weight and height changes, and adult smoking status and alcohol intake (β = −0.22, 95% confidence interval = −0.40 to −0.03 for ever exposed to PTS vs. never exposed using models of log-transformed methylation levels). PTS exposure was not statistically significantly associated with LINE-1 or Alu methylation. Conclusions: PTS exposure, measured at the time of pregnancy and not retrospectively reported, was associated with a decrease in Sat2 methylation but not LINE-1 or Alu methylation. Impact: If replicated in larger studies, this study supports a persistent effect of PTS on DNA methylation levels, as measured by Sat2, in adulthood. Cancer Epidemiol Biomarkers Prev; 20(12); 2518–23. ©2011 AACR.

Early life socioeconomic factors and genomic DNA methylation in mid-life

Epigenetic modifications may be one mechanism linking early life factors, including parental socioeconomic status (SES), to adult onset disease risk. However, SES influences on DNA methylation patterns remain largely unknown. In a US birth cohort of women, we examined whether indicators of early life and adult SES were associated with white blood cell methylation of repetitive elements (Sat2, Alu and LINE-1) in adulthood. Low family income at birth was associated with higher Sat2 methylation (β = 19.7, 95% CI: 0.4, 39.0 for lowest vs. highest income quartile) and single parent family was associated with higher Alu methylation (β = 23.5, 95% CI: 2.6, 44.4), after adjusting for other early life factors. Lower adult education was associated with lower Sat2 methylation (β = -16.7, 95% CI: -29.0, -4.5). There were no associations between early life SES and LINE-1 methylation. Overall, our preliminary results suggest possible influences of SES across the life-course on genomic DNA methylation in adult women. However, these preliminary associations need to be replicated in larger prospective studies.

The effect of extremely low frequency electromagnetic fields (ELF-EMF) on the frequency of micronuclei and sister chromatid exchange in human lymphocytes induced by benzo(a)pyrene

The interaction of extremely low frequency electromagnetic fields (ELF-EMF) on the frequency of micronuclei (MN) and sister chromatid exchange (SCE) induced by benzo(a)pyrene (BP) in human lymphocytes was examined. A 60 Hz ELF-EMF of 0.8 mT field strength was applied either alone or with the tumor initiator, BP for 24 h. The frequencies of MN and SCE induced by BP increased in a dose-dependent manner. The co-exposure of cells to BP and 0.8 mT ELF-EMF for 24 h, followed by BP exposure for 48 h led to significant increases in the frequencies of MN and SCE compared to BP treatment for 72 h alone (P<0.05), but no significant difference was observed between field exposed and sham exposed control cells. The obtained results suggest that low density ELF-EMF could act as an enhancer of the initiation process of BP rather than as an initiator of mutagenic effects in human lymphocytes.

Aberrant Methylation of RASSF1A in Plasma DNA Before Breast Cancer Diagnosis in the Breast Cancer Family Registry

In addition to classic genetic mechanisms such as deletions and mutations, growth regulatory genes can be inactivated via methylation of cytosine-residues in their promoter regions. Hypermethylation of promoter CpG islands is now recognized as an important and early event in carcinogenesis. Detection of methylated DNA in serum or plasma has been suggested to be a marker for early cancer development. We examined methylation changes in RASSF1A, a growth regulatory gene in plasma DNA from blood collected before diagnosis from women with breast cancer and from controls. Samples were from two sets of subjects, 28 women with breast cancer and 10 of their unaffected siblings, and 33 women with breast cancer and 29 age- and ethnicity-matched population-based controls. Using methylation specific PCR, we found 11 of 61 (18%) cases were positive for methylation of RASSF1A in their plasma DNA collected before diagnosis. Two of 10 healthy high-risk sibling controls (20%) had plasma DNA positive for RASSF1A methylation in their plasma DNA compared with 0 of 29 (0%) population-based controls. Tumor tissue was available for 12 cases and all were positive for RASSF1A methylation. These results, if replicated, suggest that aberrant promoter hypermethylation in serum/plasma DNA may be common among high-risk women and may be present years before cancer diagnosis. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2723–5)

Alterations in DNA methylation corresponding with lung inflammation and as a biomarker for disease development after MWCNT exposure

Abstract Use of multi-walled carbon nanotubes (MWCNT) is growing which increases occupational exposures to these materials. Their toxic potential makes it important to have an in-depth understanding of the inflammation and disease that develops due to exposure. Epigenetics is one area of interest that has been quickly developing to assess disease processes due to its ability to change gene expression and thus the lung environment after exposure. In this study, promoter methylation of inflammatory genes (IFN-γ and TNF-α) was measured after MWCNT exposure using the pyrosequencing assay and found to correlate with initial cytokine production. In addition, methylation of a gene involved in tissue fibrosis (Thy-1) was also altered in a way that matched collagen deposition. In addition to using epigenetics to better understand disease processes, it has also been used as a biomarker of exposure and disease. In this study, global methylation was determined in the lung to ascertain whether MWCNT alter global methylation at the site of exposure and if those alterations coincide with disease development. Then, global methylation levels were determined in the blood to ascertain whether global methylation could be used as a biomarker of exposure in a more easily accessible tissue. Using the LuUminometric Methylation Assay (LUMA) and 5-Methylcytosine (5-mC) Quantification assay, we found that MWCNT lead to DNA hypomethylation in the lung and blood, which coincided with disease development. This study provides initial data showing that alterations in gene-specific methylation correspond with an inflammatory response to MWCNT exposure. In addition, global DNA methylation in the lung and blood coincides with MWCNT-induced disease development, suggesting its potential as a biomarker of both exposure and disease development.

Effects of Extremely Low-Frequency Electromagnetic Fields on Delayed Chromosomal Instability Induced by Bleomycin in Normal Human Fibroblast Cells

This study was carried out to examine the interaction of extremely low-frequency electromagnetic fields (ELF-EMF) on delayed chromosomal instability by bleomycin (BLM) in human fibroblast cells. A micronucleus–centromere assay using DNA probes for chromosomes 1 and 4 was performed and a 60-Hz ELF-EMF of 0.8 mT field strength was applied either alone or with BLM throughout the culture period. The frequencies of micronuclei (MN) and aneuploidy were analyzed at 28, 88, and 240 h after treatment with BLM. The coexposure of cells to BLM and ELF-EMF led to a significant increase in the frequencies of MN and aneuploidy compared to the cells treated with BLM alone. No difference was observed between field-exposed and sham-exposed control cells. The frequency of MN induced by BLM was increased at 28 h, and further analysis showed a persistent increase up to 240 h, but the new levels were not significantly different from the level at 28 h. BLM increased the frequencies of aneuploidy at 28, 88, and 240 h, and significantly higher frequency of aneuploidy was observed in the cells analyzed at 240 h compared to the cells examined at 28 h. No interaction of ELF-EMF on delayed chromosomal instability by BLM was observed. Our results suggest that ELF-EMF enhances the cytotoxicity of BLM. BLM might induce delayed chromosomal instability, but no effect of ELF-EMF was observed on the BLM-induced delayed chromosomal instability in fibroblast cells.

Sources of polycyclic aromatic hydrocarbons are associated with gene-specific promoter methylation in women with breast cancer

BACKGROUND Tobacco smoke, diet and indoor/outdoor air pollution, all major sources of polycyclic aromatic hydrocarbons (PAHs), have been associated with breast cancer. Aberrant methylation may be an early event in carcinogenesis, but whether PAHs influence the epigenome is unclear, particularly in breast tissue where methylation may be most relevant. We aimed to evaluate the role of methylation in the association between PAHs and breast cancer. METHODS In a population-based case-control study, we measured promoter methylation of 13 breast cancer-related genes in breast tumor tissue (n=765-851 cases) and global methylation in peripheral blood (1055 cases/1101 controls). PAH sources (current active smoking, residential environmental tobacco smoke (ETS), vehicular traffic, synthetic log burning, and grilled/smoked meat intake) were evaluated separately. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS When comparing methylated versus unmethylated genes, synthetic log use was associated with increased ORs for CDH1 (OR=2.26, 95%CI=1.06-4.79), HIN1 (OR=2.14, 95%CI=1.34-3.42) and RARβ (OR=1.80, 95%CI=1.16-2.78) and decreased ORs for BRCA1 (OR=0.44, 95%CI=0.30-0.66). Residential ETS was associated with decreased ORs for ESR1 (OR=0.74, 95%CI=0.56-0.99) and CCND2 methylation (OR=0.65, 95%CI=0.44-0.96). Current smoking and vehicular traffic were associated with decreased ORs for DAPK (OR=0.53, 95%CI=0.28-0.99) and increased ORs for TWIST1 methylation (OR=2.79, 95%CI=1.24-6.30), respectively. In controls, synthetic log use was inversely associated with LINE-1 (OR=0.59, 95%CI=0.41-0.86). DISCUSSION PAH sources were associated with hypo- and hypermethylation at multiple promoter regions in breast tumors and LINE-1 hypomethylation in blood of controls. Methylation may be a potential biologic mechanism for the associations between PAHs and breast cancer incidence.

Micronucleus-centromere assay and DNA repair gene polymorphism in lymphocytes of industrial radiographers

The micronucleus-centromere assay using a pan-centromeric probe was used to assess chromosomal damage in lymphocytes of 47 industrial radiographers occupationally exposed to low dose ionizing radiation and 47 controls. The influence of genotype of DNA repair genes (XRCC1(399), XRCC3(241) and XPD(751)) on micronuclei (MN) frequency was also investigated. Centromere negative micronuclei (MNC-) frequency was significantly higher in radiographers than in controls, whereas similar centromere positive micronuclei (MNC+) frequency was observed in both groups. Poisson regression analyses revealed that the MNC- frequency was significantly associated with radiation occupational exposure and with cumulative-radiation doses in radiographers, after adjusting for confounding variables such as age, smoking, alcohol intake and genotypes. Compared to homozygous wild-type subjects, MNC- frequency in radiographers with variant XRCC3 genotype was significantly higher using univariate analysis. There were no differences in MNC- or MNC+ frequencies by genotype in controls. In conclusion, scoring of MNC- is a useful cytogenetic biomonitoring method for radiographers. Polymorphisms in XRCC3 might contribute to the increased genetic damage in individuals occupationally exposed to chronic ionizing radiation.

Alterations in DNA methylation and airway hyperreactivity in response to in utero exposure to environmental tobacco smoke.

Abstract Growing evidence indicates that prenatal exposure to maternal smoking is a risk factor for the development of asthma in children. However, the effects of prenatal environmental tobacco smoke (ETS) exposure on the genome and lung immune cells are unclear. This study aims to determine whether in utero ETS exposure alters DNA methylation patterns and increases airway hyperreactivity (AHR) and inflammation. Pregnant C57BL/6 mice were exposed daily to a concentration of 1.0 mg/m3 ETS. AHR was determined in the 6-week-old offspring by measurement of airway resistance. Global and gene promoter methylation levels in lung DNA from offspring were analyzed by luminometric methylation and pyrosequencing assays, respectively. Offspring exposed to ETS showed a marked increase in the number of alveolar macrophages in the bronchoalveolar lavage fluid and level of IL-13 in the airways compared with offspring of filtered-air exposed dams (controls). ETS exposure significantly augmented AHR compared with controls. In the methylation analysis, ETS-exposed offspring had a significantly lower level of global DNA methylation than the controls. We observed a significant increase in IFN-γ, and significant decrease in IL-13 methylation levels in the ETS group compared with controls. Collectively, these data suggest that in utero ETS exposure increases the risk of pulmonary inflammation and AHR through altered DNA methylation, but additional studies are needed to fully determine the causal link between changes in methylation and cytokines levels, as well as AHR.

Promoter Hypermethylation in White Blood Cell DNA and Breast Cancer Risk

The role of gene-specific methylation in white blood cells (WBC) as a marker of breast cancer risk is currently unclear. We determined whether promoter hypermethylation in blood DNA of candidate tumor suppressor genes frequently methylated in breast tumors can be used as a surrogate biomarker for breast cancer risk. Promoter methylation of BRCA1, CDH1 and RARβ was analyzed in WBC DNA from a population-based sample of 1,021 breast cancer patients and 1,036 controls by the MethyLight assay. Gene-specific promoter methylation in the DNA of 569 tumor tissue samples was also analyzed to determine the correlation of methylation levels with blood from the same individual. Hypermethylation of BRCA1 (OR: 1.31; 95% CI: 0.98-1.75) in WBC was associated with an increased risk of breast cancer when positive methylation was defined as ≥0.1% methylated. There was lack of concordance between tumor tissue and paired WBC DNA methylation. These results provide limited support that hypermethylation of BRCA1 in WBC DNA may be useful for determination of breast cancer risk. Additional studies with larger numbers of genes are needed to fully understand the relationship between WBC methylation and breast cancer risk.