Manosij Ghosh

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation

ABSTRACT Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offsprings Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3–6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.

Epigenetic effects of carbon nanotubes in human monocytic cells

Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine–phosphate–guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography–mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

Cyto-genotoxic and DNA methylation changes induced by different crystal phases of TiO2-np in bronchial epithelial (16-HBE) cells

With the increase in use of TiO2-np, a better understanding of their safety is important. In the present study the effect of different crystal phases of TiO2-np (anatase, rutile and anatase: rutile mixture; 20-26nm) were studied for cyto-genotoxicity and global DNA methylation and hydroxymethylation. Cytotoxic response was observed at a concentration of 25μg/ml for the particles tested. Results of comet and micronucleus (with and without CytB) assays revealed significant genotoxic effect of these particles. Flow cytometry revealed cell cycle arrest in the S-phase. Based on the results, toxicity of the particles could be correlated with their physico-chemical properties (i.e. smaller size and hydrodynamic diameter and larger surface area), anatase form being the most toxic. From the results of the cyto-genotoxicity assays, concentrations were determined for the epigenetic study. Effect on global DNA methylation and hydroxymethylation levels were studied at cyto-genotoxic (25μg/ml), genotoxic (12.5μg/ml) and sub cyto-genotoxic (3.25μg/ml) concentrations using LC-MS/MS analysis. Though no significant changes were observed for 3h treatment schedule; significant hypomethylation were observed at 24h for anatase (significant at 3.25 and 25μg/ml), rutile (significant at 3.25 and 25μg/ml) and anatase: rutile mixture (significant at 25μg/ml) forms. The results suggest that epigenetic changes could occur at sub cyto-genotoxic concentrations. And hence for complete characterization of nanoparticle toxicity, epigenetic studies should be performed along with conventional toxicity testing methods.

Body distribution of SiO₂-Fe₃O₄ core-shell nanoparticles after intravenous injection and intratracheal instillation.

Abstract Nano-silicon dioxide (SiO2) is used nowadays in several biomedical applications such as drug delivery and cancer therapy, and is produced on an industrial scale as additive to paints and coatings, cosmetics and food. Data regarding the long-term biokinetics of SiO2 engineered nanoparticles (ENPs) is lacking. In this study, the whole-body biodistribution of SiO2 core-shell ENPs containing a paramagnetic core of Fe3O4 was investigated after a single exposure via intravenous injection or intratracheal instillation in mice. The distribution and accumulation in different organs was evaluated for a period of 84 days using several techniques, including magnetic resonance imaging, inductively coupled plasma mass spectrometry, X-ray fluorescence and X-ray absorption near edge structure spectroscopy. We demonstrated that intravenously administered SiO2 ENPs mainly accumulate in the liver, and are retained in this tissue for over 84 days. After intratracheal instillation, an almost complete particle clearance from the lung was seen after 84 days with distribution to spleen and kidney. Furthermore, we have strong evidence that the ENPs retain their original core-shell structure during the whole observation period. This work gives an insight into the whole-body biodistribution of SiO2 ENPs and will provide guidance for further toxicity studies.

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.

The effect of paternal methyl-group donor intake on offspring DNA methylation and birth weight

Most nutritional studies on the development of children focus on mother-infant interactions. Maternal nutrition is critically involved in the growth and development of the fetus, but what about the father? The aim is to investigate the effects of paternal methyl-group donor intake (methionine, folate, betaine, choline) on paternal and offspring global DNA (hydroxy)methylation, offspring IGF2 DMR DNA methylation, and birth weight. Questionnaires, 7-day estimated dietary records, whole blood samples, and anthropometric measurements from 74 fathers were obtained. A total of 51 cord blood samples were collected and birth weight was obtained. DNA methylation status was measured using liquid chromatography-tandem mass spectrometry (global DNA (hydroxy)methylation) and pyrosequencing (IGF2 DMR methylation). Paternal betaine intake was positively associated with paternal global DNA hydroxymethylation (0.028% per 100 mg betaine increase, 95% CI: 0.003, 0.053, P=0.03) and cord blood global DNA methylation (0.679% per 100 mg betaine increase, 95% CI: 0.057, 1.302, P=0.03). Paternal methionine intake was positively associated with CpG1 (0.336% per 100 mg methionine increase, 95% CI: 0.103, 0.569, P=0.006), and mean CpG (0.201% per 100 mg methionine increase, 95% CI: 0.001, 0.402, P=0.049) methylation of the IGF2 DMR in cord blood. Further, a negative association between birth weight/birth weight-for-gestational age z-score and paternal betaine/methionine intake was found. In addition, a positive association between choline and birth weight/birth weight-for-gestational age z-score was also observed. Our data indicate a potential impact of paternal methyl-group donor intake on paternal global DNA hydroxymethylation, offspring global and IGF2 DMR DNA methylation, and prenatal growth.

Exposure to Polycyclic Aromatic Hydrocarbons Leads to Non-monotonic Modulation of DNA and RNA (hydroxy)methylation in a Rat Model

Besides genetic modifications, rapidly growing evidence has linked environmental pollutants with epigenetic variations. To date, only a few studies have been performed on DNA methylation changes of polycyclic aromatic hydrocarbons (PAH), which showed contradictory results. These discrepancies might be partially explained by differences in used agents. Generally in in vitro studies, a single compound is used, while in humans environmental studies, multi-residue exposure is investigated. The present study aimed to study epigenetic alterations induced by multi-residue exposure to PAH. Female Long Evans rats were exposed to a mixture of 16 US-EPA priority PAH, 3 times per week over a 90-day period. The livers were used to assess the (hydroxy)methylation status of genomic DNA/RNA, together with reduced and oxidized forms of glutathione. The results of this study demonstrate that a multi-residue exposure to PAH affects glutathione status, DNA (hydroxy)methylation, and RNA (hydroxy)methylation, together with DNA PAH-adducts formation. In addition, a non-monotonic response relationship was demonstrated between PAH concentration, the levels of glutathione and DNA (hydroxy)methylation levels at environmental relevant doses. This hormetic response gives a novel insight concerning the toxicity of environmental pollutants such as PAH and the biological response that may be different depending on the level of exposure.

Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition

BackgroundSubtle DNA methylation alterations mediated by carbon nanotubes (CNTs) exposure might contribute to pathogenesis and disease susceptibility. It is known that both multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) interact with nucleus. Such, nuclear-CNT interaction may affect the DNA methylation effects.In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). In addition, the presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy.ResultsGenerally, a higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. Nonetheless, both CNT types were in physical contact with the nuclei. While particle type dependency was noticed for the identified genome-wide and gene-specific alterations, no global DNA methylation alteration on 5-methylcytosine (5-mC) sites was observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites).Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) at different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. On the other hand, SWCNT exposure showed hypermethylation on functionally important genes, such as SKI proto-oncogene (SKI), glutathione S-transferase pi 1 (GTSP1) and shroom family member 2 (SHROOM2) and neurofibromatosis type I (NF1), which the latter is both hypermethylated and downregulated.ConclusionAfter exposure to both types of CNTs, epigenetic alterations may contribute to toxic or repair response. Moreover, our results suggest that the observed differences in the epigenetic response depend on particle type and differential CNT-nucleus interactions.

Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells

Recent studies have identified carbon nanotube (CNT)-induced epigenetic changes as one of the key players in patho-physiological response. In the present study, we investigated whether CNT exposure is associated with epigenetic changes in human bronchial epithelial cells (16 HBE), in vitro. We focused on global DNA methylation, methylation of LINE-1 elements and promoter sequence of twelve functionally important genes (SKI, DNMT1, HDAC4, NPAT, ATM, BCL2L11, MAP3K10, PIK3R2, MYO1C, TCF3, FGFR 1 and AGRN). Additionally, we studied the influence of CNT exposure on miRNA expression. Using a LC-MS/MS method and pyrosequencing for LINE-1, we observed no significant changes in global DNA methylation (%) between the concentrations of multi-walled and single-walled CNT (MWCNT and SWCNT, respectively). Significant changes in sequence-specific methylation was observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). While changes in DNA methylation of the genes were relatively small, these changes were associated with changes in RNA expression, especially for MWCNT. However, the study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Based on our results, mainly MWCNT influence DNA methylation and expression of the studied genes and could have significant impact on several critical cellular processes.

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.