Smoking initiation is followed by the early acquisition of epigenetic change in cervical epithelium: a longitudinal study

background: To prove a causal link between an epigenetic change and an environmental or behavioural risk factor for a given disease, it is first necessary to show that the onset of exposure precedes the first detection of that epigenetic change in subjects who are still free of disease. methods: Towards this end, a cohort of women aged 15–19 years, recruited soon after they first had sexual intercourse, were used to provide sequential observations on the relationship between cigarette smoking and the detection in cervical cytological samples of methylated forms of CDKN2A (p16) using nested methylation-specific polymerase chain reaction. results: Among women who remained cytologically normal and who tested negative for human papillomavirus DNA in cervical smears during follow-up, those who first started to smoke during follow-up had an increased risk of acquiring CDKN2A methylation compared with never-smokers (odds ratio=3.67; 95% confidence interval 1.09–12.33; P=0.04). conclusion: Smoking initiation is associated with the appearance of methylated forms of CDKN2A

Sister chromatid exchanges and micronuclei in peripheral lymphocytes of shoe factory workers exposed to solvents.

We examined sister chromatid exchanges (SCEs) and micronuclei (MN; cytokinesis-block method) in cultured peripheral lymphocytes from 52 female workers of two shoe factories and from 36 unexposed age- and sex-matched referents. The factory workers showed an elevated level of urinary hippuric acid, a biomarker of toluene exposure, and workplace air contained high concentrations of various organic solvents such as toluene, gasoline, acetone, and (in one of the plants only) ethylacetate and methylenediphenyl diisocyanate. The shoe factory workers showed a statistically significant higher frequency of micronucleated binucleate lymphocytes in comparison with the referents. This finding agreed with three preliminary MN determinations (each comprising 27-32 shoe workers and 16-20 controls) performed in one of the plants 2-5 years earlier. The shoe factory workers also had a lower average level of blood hemoglobin than the referents. In contrast, no difference was found between the groups in SCE analysis. Smokers showed significantly higher mean frequencies of SCEs per cell and high frequency cells (HFC) than nonsmokers. Aging was associated with increased MN rates and reduced cell proliferation. Polymorphism of the glutathione S-transferase M1 gene (GSTM1) did not affect the individual level of SCEs; but in smoking shoe workers an effect of the occupational exposure on the frequency of micronucleated cells could be seen only in GSTM1 null subjects. The low prevalence of the glutathione S-transferase T1 (GSTT1) null genotype precluded the evaluation of the influence of GSTT1 polymorphism. Our results show that the shoe factory workers have experienced genotoxic exposure, which is manifest as an increase in the frequency of MN, but not of SCEs, in peripheral lymphocytes. The exposures responsible for the MN induction could not be identified with certainty, but exposure to benzene in gasoline and methylenediphenyl diisocyanate may explain some of the findings

Systematic evaluation and validation of reference and library selection methods for deconvolution of cord blood DNA methylation data

Background: Umbilical cord blood (UCB) is commonly used in epigenome-wide association studies of prenatal exposures. Accounting for cell type composition is critical in such studies as it reduces confounding due to the cell specificity of DNA methylation (DNAm). In the absence of cell sorting information, statistical methods can be applied to deconvolve heterogeneous cell mixtures. Among these methods, reference-based approaches leverage age-appropriate cell-specific DNAm profiles to estimate cellular composition. In UCB, four reference datasets comprising DNAm signatures profiled in purified cell populations have been published using the Illumina 450 K and EPIC arrays. These datasets are biologically and technically different, and currently, there is no consensus on how to best apply them. Here, we systematically evaluate and compare these datasets and provide recommendations for reference-based UCB deconvolution. Results: We first evaluated the four reference datasets to ascertain both the purity of the samples and the potential cell cross-contamination. We filtered samples and combined datasets to obtain a joint UCB reference. We selected deconvolution libraries using two different approaches: automatic selection using the top differentially methylated probes from the function pickCompProbes in minfi and a standardized library selected using the IDOL (Identifying Optimal Libraries) iterative algorithm. We compared the performance of each reference separately and in combination, using the two approaches for reference library selection, and validated the results in an independent cohort (Generation R Study, n = 191) with matched Fluorescence-Activated Cell Sorting measured cell counts. Strict filtering and combination of the references significantly improved the accuracy and efficiency of cell type estimates. Ultimately, the IDOL library outperformed the library from the automatic selection method implemented in pickCompProbes. Conclusion: These results have important implications for epigenetic studies in UCB as implementing this method will optimally reduce confounding due to cellular heterogeneity. This work provides guidelines for future referencebased UCB deconvolution and establishes a framework for combining reference datasets in other tissues

Utility of arsenic-treated bird skins for DNA extraction

Background: Natural history museums receive a rapidly growing number of requests for tissue samples from preserved specimens for DNA-based studies. Traditionally, dried vertebrate specimens were treated with arsenic because of its toxicity and insect-repellent effect. Arsenic has negative effects on in vivo DNA repair enzymes and consequently may inhibit PCR performance. In bird collections, foot pad samples are often requested since the feet were not regularly treated with arsenic and because they are assumed to provide substantial amounts of DNA. However, the actual influence of arsenic on DNA analyses has never been tested. Findings: PCR success of both foot pad and body skin samples was significantly lower in arsenic-treated samples. In general, foot pads performed better than body skin samples. Moreover, PCR success depends on collection date in which younger samples yielded better results. While the addition of arsenic solution to the PCR mixture had a clear negative effect on PCR performance after the threshold of 5.4 μg/μl, such high doses of arsenic are highly unlikely to occur in dried zoological specimens. Conclusions: While lower PCR success in older samples might be due to age effects and/or DNA damage through arsenic treatment, our results show no inhibiting effect on DNA polymerase. We assume that DNA degradation proceeds more rapidly in thin tissue layers with low cell numbers that are susceptible to external abiotic influences. In contrast, in thicker parts of a specimen, such as foot pads, the outermost horny skin may act as an additional barrier. Since foot pads often performed better than body skin samples, the intention to preserve morphologically important structures of a specimen still conflicts with the aim to obtain optimal PCR success. Thus, body skin samples from recently collected specimens should be considered as alternative sources of DNA

Male tobacco smoke load and non-lung cancer mortality associations in Massachusetts

<p>Abstract</p> <p>Background</p> <p>Different methods exist to estimate smoking attributable cancer mortality rates (Peto and Ezzati methods, as examples). However, the smoking attributable estimates using these methods cannot be generalized to all population sub-groups. A simpler method has recently been developed that can be adapted and applied to different population sub-groups. This study assessed cumulative tobacco smoke damage (smoke load)/non-lung cancer mortality associations across time from 1979 to 2003 among all Massachusetts males and ages 30–74 years, using this novel methodology.</p> <p>Methods</p> <p>Annual lung cancer death rates were used as smoke load bio-indices, and age-adjusted lung/all other (non-lung) cancer death rates were analyzed with linear regression approach. Non-lung cancer death rates include all cancer deaths excluding lung. Smoking-attributable-fractions (SAFs) for the latest period (year 2003) were estimated as: 1-(estimated unexposed cancer death rate/observed rate).</p> <p>Results</p> <p>Male lung and non-lung cancer death rates have declined steadily since 1992. Lung and non-lung cancer death rates were tightly and steeply associated across years. The slopes of the associations analyzed were 1.69 (95% confidence interval (CI) 1.35–2.04, r = 0.90), and 1.36 (CI 1.14–1.58, r = 0.94) without detected autocorrelation (Durbin-Watson statistic = 1.8). The lung/non-lung cancer death rate associations suggest that all-sites cancer death rate SAFs in year 2003 were 73% (Sensitivity Range [SR] 61–82%) for all ages and 74% (SR 61–82%) for ages 30–74 years.</p> <p>Conclusion</p> <p>The strong lung/non-lung cancer death rate associations suggest that tobacco smoke load may be responsible for most prematurely fatal cancers at both lung and non-lung sites. The present method estimates are greater than the earlier estimates. Therefore, tobacco control may reduce cancer death rates more than previously noted.</p

A Study of the Influence of Sex on Genome Wide Methylation

Sex differences in methylation status have been observed in specific gene-disease studies and healthy methylation variation studies, but little work has been done to study the impact of sex on methylation at the genome wide locus-to-locus level or to determine methods for accounting for sex in genomic association studies. In this study we investigate the genomic sex effect on saliva DNA methylation of 197 subjects (54 females) using 20,493 CpG sites. Three methods, two-sample T-test, principle component analysis and independent component analysis, all successfully identify sex influences. The results show that sex not only influences the methylation of genes in the X chromosome but also in autosomes. 580 autosomal sites show strong differences between males and females. They are found to be highly involved in eight functional groups, including DNA transcription, RNA splicing, membrane, etc. Equally important is that we identify some methylation sites associated with not only sex, but also other phenotypes (age, smoking and drinking level, and cancer). Verification was done through an independent blood cell DNA methylation data (1298 CpG sites from a cancer panel array). The same genomic site-specific influence pattern and potential confounding effects with cancer were observed. The overlapping rate of identified sex affected genes between saliva and blood cell is 81% for X chromosome, and 8% for autosomes. Therefore, correction for sex is necessary. We propose a simple correction method based on independent component analysis, which is a data driven method and accommodates sample differences. Comparison before and after the correction suggests that the method is able to effectively remove the potentially confounding effects of sex, and leave other phenotypes untouched. As such, our method is able to disentangle the sex influence on a genome wide level, and paves the way to achieve more accurate association analyses in genome wide methylation studies

Long Term Transcriptional Reactivation of Epigenetically Silenced Genes in Colorectal Cancer Cells Requires DNA Hypomethylation and Histone Acetylation

Epigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2′-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone. Aim: We sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis. Results: Increased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation. Conclusions: These observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes

Characterization of the Contradictory Chromatin Signatures at the 3′ Exons of Zinc Finger Genes

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription

Resveratrol inhibits benzo[a]pyrene–DNA adduct formation in human bronchial epithelial cells

Resveratrol ( trans-3,4’,5-trihydroxystilbene), a phytoalexin present in various plants and foods, has in several in vitro and in vivo studies demonstrated cancer chemopreventive and chemotherapeutic potential. We investigated the in vitro effect of resveratrol on benzo[ a] pyrene ( B[ a] P)-induced DNA adducts in human bronchial epithelial cells. This was compared to the effect of resveratrol on the expression of the cytochrome P450 (CYP) genes CYP1A1 and CYP1B1 and the formation of B[ a] P metabolites. Exposure of BEAS-2B and BEP2D cells to B[ a] P and increasing concentrations of resveratrol resulted in a dose- and time-dependent inhibition of DNA adduct formation quantified by P-32-postlabelling. Supporting this result, resveratrol was shown to inhibit CYP1A1 and CYP1B1 gene expression, as measured by real-time reverse transcriptase - polymerase chain reaction. Also, a significant correlation was found between the number of DNA adducts and the mRNA levels of these genes. Using HPLC analysis, a concomitant decrease in the formation of B[ a]P-derived metabolic products was detected. In conclusion, these data lend support to a chemopreventive role of resveratrol in polycyclic aromatic hydrocarbon-induced carcinogenesis