Ali Mustafa Tabish

Epigenetic factors in cancer risk: effect of chemical carcinogens on global DNA methylation pattern in human TK6 cells.

In the current study, we assessed the global DNA methylation changes in human lymphoblastoid (TK6) cells in vitro in response to 5 direct and 10 indirect-acting genotoxic agents. TK6 cells were exposed to the selected agents for 24 h in the presence and/or absence of S9 metabolic mix. Liquid chromatography-mass spectrometry was used for quantitative profiling of 5-methyl-2′-deoxycytidine. The effect of exposure on 5-methyl-2′-deoxycytidine between control and exposed cultures was assessed by applying the marginal model with correlated residuals on % global DNA methylation data. We reported the induction of global DNA hypomethylation in TK6 cells in response to S9 metabolic mix, under the current experimental settings. Benzene, hydroquinone, styrene, carbon tetrachloride and trichloroethylene induced global DNA hypomethylation in TK6 cells. Furthermore, we showed that dose did not have an effect on global DNA methylation in TK6 cells. In conclusion we report changes in global DNA methylation as an early event in response to agents traditionally considered as genotoxic.

Global Methylation and Hydroxymethylation in DNA from Blood and Saliva in Healthy Volunteers

Aims. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify and compare simultaneously global methylation and hydroxymethylation in human DNA of different tissues. Materials and Methods. Blood and saliva DNA from fourteen volunteers was processed for epigenetic endpoints using LC-MS/MS and PCR-pyrosequencing technology. Results. Global DNA methylation was significantly lower in saliva (mean 4.61% ±  0.80%), compared to blood samples (5.70% ± 0.22%). In contrast, saliva (0.036% ± 0.011%) revealed significantly higher hydroxymethylation compared to blood samples (mean 0.027% ± 0.004%). Whereas we did not find significant correlations for both epigenetic measures between the tissues, a significant association was observed between global methylation and global hydroxymethylation in saliva DNA. Neither LINE-1 nor Alu elements of blood and saliva correlated, nor were they correlated with the DNA hydroxymethylation of blood or saliva, respectively. Conclusion. Global DNA methylation and hydroxymethylation of cytosine can be quantified simultaneously by LC-MS/MS. Saliva DNA cannot be considered as a surrogate for blood DNA to study epigenetic endpoints.

Changes in DNA Methylation in Mouse Lungs after a Single Intra-Tracheal Administration of Nanomaterials

Aims This study aimed to investigate the effects of nanomaterial (NM) exposure on DNA methylation. Methods and Results Intra-tracheal administration of NM: gold nanoparticles (AuNPs) of 5-, 60- and 250-nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 h to BALB/c mice. Study showed deregulations in immune pathways in NM-induced toxicity in vivo. NM administration had the following DNA methylation effects: AuNP 60 nm induced CpG hypermethylation in Atm, Cdk and Gsr genes and hypomethylation in Gpx; Gsr and Trp53 showed changes in methylation between low- and high-dose AuNP, 60 and 250 nm respectively, and AuNP had size effects on methylation for Trp53. Conclusion Epigenetics may be implicated in NM-induced disease pathways.

Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.

Epigenetic changes in lymphocytes of solvent-exposed individuals

AIM We investigated global DNA methylation alterations in lymphocytes of solvent workers and chronic toxic encephalopathy (CTE) patients and explored potential gene-environment interactions for GST. POPULATION & METHODS: A cross-sectional study was set up in 41 referents, 128 solvent workers and 23 CTE patients. RESULTS We found a global DNA hypermethylation in the solvent-exposed population compared with the referents (p = 0.001, r = -0.544). Global DNA methylation was negatively associated with exposure. Furthermore, GSTP1 genotypic polymorphism was found to be significantly associated (p = 0.033) with global DNA hypomethylation, which indicates a potential role for gene-environment interaction in the etiology of solvent-induced neurobehavioral disorders. CONCLUSION This study indicates that solvent-induced DNA methylation alterations have an impact on neurotoxicity and development of CTE.

Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing ® of Repetitive Elements

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

Changes in DNA methylation induced by multi-walled carbon nanotube exposure in the workplace

Abstract This study was designed to assess the epigenetic alterations in blood cells, induced by occupational exposure to multi-wall carbon nanotubes (MWCNT). The study population comprised of MWCNT-exposed workers (n=24) and unexposed controls (n=43) from the same workplace. We measured global DNA methylation/hydroxymethylation levels on the 5th cytosine residues using a validated liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. Sequence-specific methylation of LINE1 retrotransposable element 1 (L1RE1) elements, and promoter regions of functionally important genes associated with epigenetic regulation [DNA methyltransferase-1 (DNMT1) and histone deacetylase 4 (HDAC4)], DNA damage/repair and cell cycle pathways [nuclear protein, coactivator of histone transcription/ATM serine/threonine kinase (NPAT/ATM)], and a potential transforming growth factor beta (TGF-β) repressor [SKI proto-oncogene (SKI)] were studied using bisulfite pyrosequencing. Analysis of global DNA methylation levels and hydroxymethylation did not reveal significant difference between the MWCNT-exposed and control groups. No significant changes in Cytosine-phosphate-Guanine (CpG) site methylation were observed for the LINE1 (L1RE1) elements. Further analysis of gene-specific DNA methylation showed a significant change in methylation for DNMT1, ATM, SKI, and HDAC4 promoter CpGs in MWCNT-exposed workers. Since DNA methylation plays an important role in silencing/regulation of the genes, and many of these genes have been associated with occupational and smoking-induced diseases and cancer (risk), aberrant methylation of these genes might have a potential effect in MWCNT-exposed workers.

429 Signature of epigenetic alterations induced by carbon nanotube- in vitro, in vivo and in workers

Introduction Growing indication of toxicity and production of carbon nanotubes (CNTs), have resulted in concern about adverse effect of occupational exposure. Research have suggested carcinogenic potential of some forms of CNTs (MWCNT-7 Mitsui) and asbestos-like pathogenesis. Studying epigenetic alterations (e.g. DNA methylation) could provide important additional evidence to determine CNT toxicity and disease progression. Methods To understand epigenetic effects of CNT (SWCNT and MWCNT), we designed a translational study incorporating in vitro and in vivo experiments. The changes were compared to results of asbestos exposure study. Changes in DNA methylation were studied at global (LC/MS-MS), genome wide (illumina 450 K), sequence specific levels (bisulfite pyrosequenceing). Changes in gene expression were studied using RNA-Seq. Finally, signatures obtained from these studies were validated in 23 workers exposed to MWCNT. Result In vitro, CNTs and asbestos induced gene specific DNA methylation changes. Asbestos exposure induced alterations in genes associated with Rho mediated signal transduction, HOX genes, WNT genes. Methylation and transcriptomic profiles of CNT exposed cells revealed alterations in DNA damage repair, tp53, cell cycle, protein phosphorylation pathways. Additionally, CNTs induced sequence specific changes in promoter region of several key genes including DNMT1, HDAC4, ATM, MAP3K10, PIK3R2 and MYO1C. Some of the genes, specifically ATM was also differentially methylated by SWCNTs and MWCNTs in the in vivo study. Based on these result, we studied some of these markers in MWCNT exposed workers, where we observed significant changes in sequence specific methylation for DNMT1, ATM, SKI and HDAC4 promoter CpGs. Conclusion Epigenetic cell responses provides important insights in potential health risks and underlying mechanisms. Hence, many of these genes have been associated with occupational asbestos and smoking induced diseases and cancer. Further research needs to confirm whether methylation alterations in this set of genes can be used in monitoring changes associated CNT exposure and effect.

395 Solvent-induced DNA methylation changes: A translational study

Objectives Workers exposed to solvents are at risk for developing cancer and neurobehavioral diseases. Evidence is growing on the role of epigenetic alterations involved in the development of both diseases. In this project, we set up a translational study to investigate the impact of solvents on DNA methylation alterations and neurobehavioral changes. Methods First, we assessed global DNA methylation changes in human lymphoblastoid (TK6) cells in vitro in response to 10 solvents. Next, a cross sectional study was set-up to validate these results in 128 solvent workers. Liquid chromatography-mass spectrometry was used to quantify global DNA methylation profile in TK6 cells and in lymphocytes of the workers. Each participant underwent a series of tests based on the Neurobehavioral Evaluation System. Results Benzene, hydroquinone, styrene, carbon tetrachloride and trichloroethylene induced global DNA hypomethylation in TK6 cells. DNA methylation in solvent-workers was, after correction for age, negatively associated with total exposure time (r = -0.198, p = 0,025) and the cumulative exposure index (r = -0.244, p = 0,006). Age and smoking were associated with a global DNA hypomethylation, while use of alcohol was associated with hypermethylation. Interestingly, both DNA methylation and exposure were significant predictors for neurobehavioral effects in the multivariate regression models. Conclusions We report changes in global DNA methylation as an early event in response to solvents. Global unmethylated DNA is known to dysregulate transcription, which has an impact on the gene expression and the function of cells, e.g. loss of control of cell division. These results are suggestive for the possible involvement of epigenetic mechanisms in neurodegenerative diseases and cancer. Lymphocytes are not necessarily the target tissue, but might be a good surrogate because of their accessibility and the high correlation with methylation profiles in somatic tissues.