Eveline Putzeys

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.

Simultaneous analysis of bisphenol A based compounds and other monomers leaching from resin‐based dental materials by UHPLC–MS/MS

Resin-based dental materials have raised debates concerning their safety and biocompatibility, resulting in a growing necessity of profound knowledge on the quantity of released compounds into the oral cavity. In this context, the aim of this study was to develop a comprehensive and reliable procedure based on liquid chromatography with mass spectrometry for the simultaneous analysis of various leached compounds (including bisphenol A based compounds) in samples from in vitro experiments. Different experiments were performed to determine the optimal analytical parameters, comprising mass spectrometry parameters, chromatographic separation conditions, and sample preparation. Four internal standards were used as follows: deuterated diethyl phthalate and bisphenol A (commercially available), and deuterated analogues of triethylene glycol dimethacrylate and urethane dimethacrylate (custom-made). The optimized method was validated for linearity of the calibration curves and the associated correlation coefficient, lower limit of quantification, higher limit of quantification, and intra- and interassay accuracy and precision. Additionally, the developed liquid chromatography with tandem mass spectrometry method was applied to the analysis of leaching compounds from four resin-based dental materials. The results indicated that this method is suitable for the analysis of different target compounds leaching from dental materials. This method might serve as a valuable basis for quick and accurate quantification of leached compounds from resin-based dental materials in biological samples.

Freshly-mixed and setting calcium-silicate cements stimulate human dental pulp cells

OBJECTIVES To evaluate the effect of the eluates from 3 freshly-mixed and setting hydraulic calcium-silicate cements (hCSCs) on human dental pulp cells (HDPCs) and to examine the effect of a newly developed hCSC containing phosphopullulan (PPL) on HDPCs. METHODS Human dental pulp cells, previously characterized as mesenchymal stem cells, were used. To collect the eluates, disks occupying the whole surface of a 12-well plate were prepared using an experimental hCSC containing phosphopullulan (GC), Nex-Cem MTA (GC), Biodentine (Septodont) or a zinc-oxide (ZnO) eugenol cement (material-related negative control). Immediately after preparing the disks (non-set), 3ml of Dulbeccos Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) were added. The medium was left in contact with the disks for 24h before being collected. Four different dilutions were prepared (100%, 50%, 25% and 10%) and cell-cytotoxicity, cell-proliferation, cell-migration and odontogenic differentiation were tested. The cell-cytotoxicity and cell-proliferation assays were performed by XTT-colorimetric assay at different time points. The cell-migration ability was tested with the wound-healing assay and the odontogenic differentiation capacity of hCSCs on HDPCs was tested with RT-PCR. RESULTS Considering all experimental data together, the eluates from 3 freshly-mixed and setting hCSCs appeared not cytotoxic toward HDPCs. Moreover, all three cements stimulated proliferation, migration and odontogenic differentiation of HDPCs. SIGNIFICANCE The use of freshly-mixed and setting hCSCs is an appropriate approach to test the effect of the materials on human dental pulp cells. The experimental material containing PPL is non-cytotoxic and positively stimulates HDPCs.

A novel high sensitivity UPLC-MS/MS method for the evaluation of bisphenol A leaching from dental materials

There is a growing necessity to acquire more profound knowledge on the quantity of eluates from resin-based dental materials, especially with regard to bisphenol A (BPA). The aim of the present study was to develop a highly sensitive method to characterize the short-term release of BPA in saliva with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), using an extraction step and additional derivatization of BPA with pyridine-3-sulfonyl chloride. Light-cured resin-based composites were incubated at 37 °C in 1 mL artificial saliva, which was refreshed daily for one week. The final protocol allows accurate quantification of very low levels of BPA in samples of artificial saliva (i.e. 1.10 pmol BPA/mL or 250 pg/mL). The daily BPA-release from dental composites, ranging from 1.10 to 7.46 pmol BPA/mL, was characterized over a period of 7 days. The highest total amount of BPA was released from Solitaire 2 (24.72 ± 2.86 pmol), followed by G-ænial Posterior (15.51 ± 0.88 pmol) and Filtek Supreme XTE (12.00 ± 1.31 pmol). In contrast, only trace amounts of BPA were released from Ceram.x Universal. This UPLC-MS/MS method might be used for clinical research focusing on the evaluation of the clinical relevance of BPA release from dental materials.

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.

In-vitro transdentinal diffusion of monomers from adhesives

OBJECTIVES Biocompatibility of adhesives is important since adhesives may be applied on dentin near the pulp. Accurate knowledge of the quantity of monomers reaching the pulp is important to determine potential side effects. The aim of this study was to assess the transdentinal diffusion of residual monomers from dental adhesive systems using an in-vitro pulp chamber model. METHODS Dentin disks with a thickness of 300 μm were produced from human third molars. These disks were fixed between two open glass tubes, representing an in-vitro pulp chamber. The etch-and-rinse adhesive OptiBond FL and the self-etch adhesive Clearfil SE Bond were applied to the dentin side of the disks, while on in the pulpal side, the glass tube was filled with 600 μl water. The transdentinal diffusion of different monomers was quantified using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS The monomers HEMA, CQ, BisGMA, GPDM, 10-MDP and UDMA eluted from the dental materials and were able to diffuse through the dentin disks to a certain extent. Compounds with a lower molecular weight (uncured group: HEMA 7850 nmol and CQ 78.2 nmol) were more likely to elute and diffuse compared to monomers with a higher molecular weight (uncured group: BisGMA 0.42 nmol). When the adhesives were left uncured, diffusion was up to 10 times higher compared to the cured conditions. CONCLUSIONS This in-vitro research resulted in the quantification of various monomers able to diffuse through dentin and therefore contributes to a more detailed understanding about the potential exposure of the dental pulp to monomers from dental adhesives. CLINICAL SIGNIFICANCE Biocompatibility of adhesives is important since adhesives may be applied on dentin near the pulp, where tubular density and diameter are greatest.