Radu-Corneliu Duca

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.

Release of monomers from composite dust

OBJECTIVESnDental personnel are more at risk to develop asthmatic disease, but the exact reason is so far unknown. During abrasive procedures, dental personnel are exposed to nano-sized dust particles released from dental composite. The aim of this study was to investigate whether respirable composite dust may also release monomers.nnnMETHODSnRespirable (<5μm) composite dust was collected and the release of methacrylate monomers and Bisphenol A (BPA) in water and ethanol was evaluated by liquid chromatography/mass spectroscopy (LC-MS/MS). The dust was ultra-morphologically and chemically analyzed by transmission electron microscopy and energy-dispersive X-ray spectroscopy (TEM-EDS).nnnRESULTSnLC-MS/MS analysis revealed that, irrespective of the type of composite, the respirable fraction of composite dust may release relatively high concentrations of unpolymerized methacrylate monomers, both in water and ethanol. Higher release was observed in ethanol. The endocrine disruptor BPA also emanated from the composite dust particles. TEM showed that most particles were nano-sized, although particle size ranged between 6nm and 5μm with a mode value between 12 and 39nm. Most particles consisted of several filler particles in resin matrix, although single nano-filler particles could also be observed. Elemental analysis by TEM-EDS proved that the particles collected on the filters originated from the dental composites.nnnCONCLUSIONnTheoretically, composite dust may function as a vehicle to transport monomers deeply into the respiratory system. The results of this study may shed another light on the increasing incidence of respiratory disease among dental personnel, and more care should be taken to prevent inhalation of composite dust.nnnCLINICAL SIGNIFICANCEnSpecial care should be taken to prevent inhalation of composite dust, as the dust particles may release methacrylate monomers.

Temporal variability of global DNA methylation and hydroxymethylation in buccal cells of healthy adults: Association with air pollution

BACKGROUNDnEpigenetic changes, such as DNA methylation, are observed in response to environmental exposure and in the development of several chronic diseases. Consequently, DNA methylation alterations might serve as indicators of early effects. In this context, the aim of this study was to assess the temporal variability of global DNA methylation and hydroxymethylation levels in buccal cells from healthy adult volunteers.nnnMETHODSnGlobal DNA methylation (%5mdC) and hydroxymethylation (%5hmdC) levels in human buccal cells, collected from 26 healthy adults at different time points, were quantified by UPLC-MS/MS. Associations between %5mdC and %5hmdC, respectively, and short-term exposure (1-7days) to air pollutants PM2.5 and PM10 were tested with mixed-effects models including various covariates.nnnRESULTS/DISCUSSIONnDynamic short-term changes in DNA methylation and hydroxymethylation levels in buccal cells were observed, which were inversely associated with exposure to PM2.5 and PM10. An IQR increase in PM2.5 over a 7-day moving average period was significantly associated with a decrease of -1.47% (-1.74%, -1.20%) and -0.043% (-0.054%, -0.032%) in %5mdC and %5hmdC, respectively. Likewise, for PM10, a decrease of -1.42% (-1.70, -1.13) and -0.040% (-0.051%, -0.028%) was observed.nnnCONCLUSIONnGlobal DNA methylation and hydroxymethylatation varied over a time period of three weeks. The observed temporal variability was associated with exposure to ambient PM2.5 and PM10 levels. This should be taken into account when interpreting epigenetic alterations in buccal cells.

Prenatal exposure to mercury: Associations with global DNA methylation and hydroxymethylation in cord blood and in childhood

BACKGROUNDnMercury is a global pollutant, and prenatal exposure is associated with adverse health effects. To date, no studies have evaluated the association between prenatal mercury exposure and DNA hydroxymethylation, an epigenetic modification important for tissue differentiation and embryonic development.nnnOBJECTIVESnWe sought to evaluate the association between prenatal mercury exposure and offspring global DNA methylation and hydroxymethylation at birth and test for persistence of the association in childhood.nnnMETHODSnWithin Project Viva, a U.S. prebirth cohort, we examined associations of maternal second trimester red blood cell mercury (RBC-Hg) concentrations with global 5-hydroxymethylcytosine (%-5hmC) and 5-methylcytosine (%-5mC) DNA content in blood collected at birth (n=306), early childhood (n=68; 2.9 to 4.9 y), and midchildhood (n=260; 6.7 to 10.5 y).nnnRESULTSnMedian prenatal RBC-Hg concentration was 3.23μg/g [interquartileu2009rangeu2009(IQR)=3.29]. At birth, median cord blood %-5mC, %-5hmC, and their ratio were 4.95%, 0.22%, and 24.37, respectively. The mean adjusted difference [95% confidence interval (CI)] of blood %-5hmC for a doubling in prenatal RBC-Hg concentration was -0.013% (-0.029, 0.002), -0.031% (-0.056, -0.006), and 0.005% (-0.007, 0.018) at birth, early, and midchildhood, respectively. The corresponding relative adjusted change in the genomic ratio of %-5mC to %-5hmC for a doubling in prenatal RBC-Hg concentration was 4.70% (0.04, 9.58), 22.42% (7.73, 39.11), and 0.73% (-4.18, 5.88) at birth, early, and midchildhood, respectively. No associations were present between prenatal maternal RBC-Hg and %-5mC at any time point.nnnCONCLUSIONSnPrenatal mercury exposure was associated with lower %-5hmC genomic content and a corresponding increase in the ratio of %-5mC to %-5hmC in cord blood. This association was persistent in early but not midchildhood blood. Our results demonstrate the potential malleability of epigenetic modifications associated with mercury exposure in utero. https://doi.org/10.1289/EHP1467.

A Method to Quantitatively Assess Dermal Exposure to Volatile Organic Compounds

Assessing dermal exposure of workers to noxious chemicals becomes increasingly important in industrial settings. Among various chemicals, volatile organic compounds (VOCs) are widely used in industrialized countries, but still there are no validated methodologies able to accurately quantify skin exposure. In this study, we developed a sensitive methodology based on activated charcoal cloth (ACC) to quantitatively assess skin exposure to 181 VOCs. The majority of the VOCs (156) showed a constant desorption efficiency (DE) of ~100% over the studied concentration range. Seven VOCs showed a concentration dependency for the DEs, which we described by a Dubinin-Raduskevich desorption isotherm. For 18 compounds, the DEs were situated below 80% but showed to be constant over the concentration range. All tested VOCs showed a good storage stability on ACC, especially at -80°C storage. Only for n-pentane there was a decrease of ~40% when it was stored for a month. In a controlled environment test, ACC has shown to reflect well the increasing concentrations of VOCs in the air with a high linearity (R2 ≥ 0.812, except for gamma-butyrolactone where R2 = 0.570). In this study, we show that ACC is a suitable sampling material for quantitatively assessing dermal exposure to 181 VOCs in terms of sensitivity and DE. This method will allow more studies that are detailed on dermal exposure, which will lead to a better assessment of skin exposure in occupational settings.

Integrated evaluation of solvent exposure in an occupational setting: air, dermal and bio-monitoring

The assessment of dermal exposure to volatile organic compounds (VOCs) is becoming increasingly important in industrial settings. The study aimed to evaluate the overall exposure (inhalation and dermal) of workers to VOCs, and to assess the suitability of activated charcoal cloth (ACC) patches for the evaluation of the contribution of dermal exposure (vs. inhalation exposure) to the whole body burden, as reflected by human biomonitoring. Inhalation exposure to toluene, acetone and styrene (passive 3u2009M organic vapour monitors, OVMs) and dermal exposure (ACC patches on the index finger, thumb and neck) were measured simultaneously in 37 subjects performing different tasks in a factory of thermoplastic panels. Systemic exposure was assessed in urine by quantification of mandelic acid (MA) and phenyl glyoxylic acid (PGA), as biomarkers for styrene, as well as acetone and hippuric acid (HA) as biomarkers for acetone and toluene, respectively. High styrene (range 30.66-302u2009mg/m3) and acetone (range 11-644u2009mg/m3) concentrations were found in the air of the workplace, while toluene was less abundantly present (range 0.05-2.6u2009mg/m3). On the ACC patches, considerable amounts of these VOCs were found. For employees manually handling styrene, dermal exposure on the index finger and thumb were substantially higher compared to the neck ACC patch. A good correlation between air and urinary levels of acetone exposure was found. MA and PGA levels in urine, markers for styrene exposure, were correlated with both air and dermal exposure. These data suggest that there is a substantial benefit from assessing dermal exposure in the work place in addition to the more conventional air monitoring and urinary biomonitoring.

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.

Could fibrinogen and hsCRP be useful for assessing personal risk in workers exposed to a mixture of ultrafine particles and organic solvents

Abstract Purpose: Our study focuses on elucidating if two common inflammatory biomarkers, easily performed in any laboratory - high-sensitivity C-reactive protein (hsCRP), as well as fibrinogen - could be used to assess the personal health risk of workers exposed to a complex occupational exposure to ultrafine particles (UFP) and a mixture of organic solvents. Methods: To assess the inflammatory response on the body, laboratory determinations were performed by testing the serum levels of hsCRP and fibrinogen, in exposed and unexposed groups. Results: There are no statistically significant differences for hsCRPs (p-0.25), medians were similar in groups. The mean values of fibrinogen in the three groups were: in the workers group (1st group): 346.2 mg/dl, in the office staff group (2nd group): 328.7 mg/dl, and in the control group (3rd group): 284.8 mg/dl, with significant differences between 1st group vs 3rd group and between 2nd group vs 3rd group (p-0.002). UFP levels differ between the groups, as follows: 1st group were exposed to the highest levels, ranging from 48349 to 3404000 part/cm3; 2nd group, ranging from 17371 to 40595 part/cm3; and 3rd group, ranging from 213 to 16255 part/cm3. Conclusions: Our study demonstrates that fibrinogen is a useful inflammatory biomarker for exposure to a mixture of UFP and organic solvents. On the other hand, hsCRP is not a useful inflammatory biomarker in occupational exposure to UFP and organic solvents. Further studies are needed to demonstrate the extent to which fibrinogen is more or less influenced by organic solvents or UFP alone.

915 Maternal occupation is associated with maternal global dna (hydroxy) methylation in the second trimester of pregnancy

Introduction Environmental factors, such as nutrition and occupational exposure can influence epigenetic marks like DNA methylation, which play a role in the development of chronic diseases. Methods Data of the MAternal Nutrition and Offspring’s Epigenome (MANOE) study was used to assess the effect of maternal occupation on maternal and infant DNA (hydroxy)methylation levels. Mothers were categorised in job categories according to the International Standard Classification of Occupations (ISCO). Maternal global DNA (hydroxy)methylation levels during each trimester of pregnancy and at delivery (n=122) was measured in whole blood via LC-MS/MS. Data were analysed with a one-Way ANOVA. Results We found statistically significant differences in maternal global DNA methylation (p=0.008) and global DNA hydroxymethylation (p=0.004) at 20 weeks of pregnancy. Post hoc tests revealed that global DNA methylation and global DNA hydroxymethylation level was significantly lower when the mother had an intellectual/scientific/artistic profession (6.36% and 0.13%) as opposed to being a manager (7.77%, p=0.007%u2009and 0.22%, p=0.002) or administrative staff (7.71%, p=0.003%u2009and 0.2%, p=0.005). No significant differences between different working groups were found for global DNA (hydroxy)methylation in the first and third trimester of pregnancy and at delivery. Conclusion The mother’s occupation was associated with maternal global DNA (hydroxy)methylation levels only in the second trimester of pregnancy. The change in maternal global DNA (hydroxy)methylation in the second trimester of pregnancy could be due to hormonal changes during pregnancy, a shift in the one-carbon metabolism in the middle of pregnancy, but based on these results we also have to take into account maternal occupational exposure.

855 Dermal exposure to diisocyanates: development and validation of an analytical method for accurately assessment of very low levels of exposure

Introduction Dermal exposure to sensitizers such as diisocyanate have been described to promote the development of asthma in later stages when respiratory occupational exposure occurs. Therefore, we developed a reliable, sensitive and validated methodology based on dermal patches to assess skin exposure to diisocyanates. Methods An UPLC-Unispray-MS/MS method was established and validated in order to reach very low levels of detection. Custom-made dermal patches were developed in order to allow optimal sampling of diisocyanates. Their sampling capability was evaluated in a controlled environment test-chamber were patches were exposed to increasing concentrations of diisocyanates. Result The UPLC-MS/MS method using a Unispray ionisation source, based on supercritical fluids ionisation and Coanda effect, allowed reaching very low levels of detection (LoD=1u2009pg/mL) for all the targeted compounds (i.e. 4,4-MDI, 2,4-MDI, 2,6-TDI, 2,4-TDI, 1,6-HDI, and IPDI). Due to the high sensitivity of the analytical method, very low levels of diisocyanates (i.e. 25u2009pg/patch) are detected on the custom-made dermal patches. Furthermore, the patches allowed the sampling of a broad range of concentration levels (from 5 pg/cm2 to 5 ng/cm2), which have been correlated with the air levels from the controlled environment chamber-test. Discussion We succeeded to develop a method to assess dermal exposure to diisocyanates. Field studies are now necessary to further evaluate the suitability of the custom-made patches, as well as to relate low levels of exposure and potential health outcomes.