Chemical and genotoxic characterization of bioaccessible fractions as a comprehensive in vitro tool in assessing the health risk due to dust-bound contaminant ingestion.

In the last two decades, awareness grew on the matter of the impact of environment on human health. Contaminants sorbed onto soil and settled dust can be ingested and thus represent a hazard, particularly to young children, who play on the ground and bring their hands and objects to their mouth. Metal(loid)s and polycyclic aromatic hydrocarbons (PAHs) are of concern as they are both carcinogenic to humans and ubiquitous in outdoor environments. The present study aims to assess the total and bioaccessible fractions of PAHs and metal(loid)s present in settled dust of four preschools located in industrial, urban, and suburban areas. On the one hand, children's incremental life cancer risks (ILCR) were calculated according to ingestion pathway. On the other hand, the genotoxicities of the bioaccessible dust-bonded contaminants were determined on gastric cells. PAH concentrations ranged from 50.9 to 2267.3 ng/g, and the bioaccessible fraction represented 10.7% of the total in average. Metal(loid) concentration ranged from 12,430 to 38,941 µg/g, and the mean bioaccessibility was of 40.1%. Cancer risk ranged from 2.8.105 to 8.6.105, indicating that there is a potential cancer risk for children linked to the ingestion of settled dust. The inorganic bioaccessible fraction induced little DNA (< 20%TailDNA) and chromosomal damages (30% increase in micronuclei), whereas the organic bioaccessible fraction induced higher DNA (17-63%TailDNA) and chromosomal damages (88% increase in micronuclei). Such experimental approach needs to be deepen, as a tool complementary to cancer risk calculation, since the latter only lays on a set of targeted contaminants with known toxicity values.

Toward an interdisciplinary approach to assess the adverse health effects of dust-containing polycyclic aromatic hydrocarbons (PAHs) and metal(loid)s on preschool children.

Settled dust can function as a pollutant sink for compounds, such as polycyclic aromatic hydrocarbons (PAHs) and metal(loid)s (MMs), which may lead to health issues. Thus, dust represents a hazard specifically for young children, because of their vulnerability and hand-to-mouth behavior favoring dust ingestion. The aim of the present study was to explore the influence of the season and the microenvironment on the concentrations of 15 PAHs and 17 MMs in indoor and outdoor settled dust in three preschools (suburban, urban, and industrial). Second, the potential sources and health risks among children associated with dust PAHs and MMs were assessed. Third, domestic factors (risk perception, knowledge and parental style) were described to explore protective parental behaviors toward dust hazards. The suburban preschool had the lowest concentrations of dust PAHs and MMs, while the industrial and urban preschools had higher but similar concentrations. Seasonal tendencies were not clearly observed. Indoor dusts reflected the outdoor environment, even if specific indoor sources were noted. Source analysis indicated mainly vehicular emissions, material release, and pyrogenic or industrial sources. The non-cancer health risks were non-existent, but potential cancer health risks (between 1.10-6 and 1.10-4) occurred at all sampling locations. Notably, the highest cancer risk was observed in a playground area (>1.10-4) and material release should be further addressed. Whereas we assessed higher risk indoors, parents perceived a higher risk in the open-air environment and at the preschool than at home. They also perceived a lower risk for their own children, revealing an optimism bias, which reduces parental anxiety.

Gaining insight into genotoxicity with the comet assay in inhomogenoeous exposure scenarios: The effects of tritiated steel and cement particles on human lung cells in an inhalation perspective.

The comet assay was recently applied for the first time to test the genotoxicity of micrometric stainless steel and cement particles, representative of those produced in the dismantling of nuclear power plants. A large dataset was obtained from in vitro exposure of BEAS-2B lung cells to different concentrations of hydrogenated (non-radiative control) and tritiated particles, to assess the impact of accidental inhalation. Starting from the distributions of the number of nuclei scored at different extent of DNA damage (% tail DNA values), we propose a new comet data treatment designed to consider the inhomogeneity of the action of such particles. Indeed, due to particle behavior in biological media and concentration, a large fraction of cells remains undamaged, and standard averaging of genotoxicity indicators leads to a misinterpretation of experimental results. The analysis we propose reaches the following goals: genotoxicity in human lung cells is assessed for stainless steel and cement microparticles; the role of radiative damage due to tritium is disentangled from particulate stress; the fraction of damaged cells and their average level of DNA damage are assessed separately, which is essential for carcinogenesis implications and sets the basis for a better-informed risk management for human exposure to radioactive particles.

In Vitro Genotoxicity Evaluation of PAHs in Mixtures Using Experimental Design.

Settled dusts are sinks for environmental pollutants, including Polycyclic Aromatic Hydrocarbons (PAHs) that are ubiquitous, persistent, and carcinogenic. To assess their toxicity in mixtures, Toxic Equivalent Factors (TEFs) are routinely used and based on the hypothesis of additive effects, although PAH interactions may occur and remain an open issue. This study investigated genotoxic binary interaction effects for six PAHs in mixtures using two in vitro assays and estimated Genotoxic Equivalent Factors (GEFs) to roughly predict the genotoxicity of PAH in mixtures. The Design of the Experiment approach was used with the micronucleus assay for cytostasis and micronuclei frequency and the alkaline comet assay for DNA damage. GEFs were determined for each PAH independently and in a mixture. For the cytostasis endpoint, no PAHs interaction was noted. BbF and BaP had a synergistic effect on DNA damage. All the PAH interacted between them regarding chromosomal damage. Although the calculated GEFs were similar to the TEFs, the latter may underestimate the genotoxic potential of a PAH mixture. GEFs calculated for PAH alone were lower than GEFs for PAHs in mixtures; thus, mixtures induce greater DNA/chromosomal damage than expected. This research helps to advance the challenging issue of contaminant mixtures' effects on human health.

Tritiated Steel Micro-Particles: Computational Dosimetry and Prediction of Radiation-Induced DNA Damage for In Vitro Cell Culture Exposures.

Biological effects of radioactive particles can be experimentally investigated in vitro as a function of particle concentration, specific activity and exposure time. However, a careful dosimetric analysis is needed to elucidate the role of radiation emitted by radioactive products in inducing cyto- and geno-toxicity: the quantification of radiation dose is essential to eventually inform dose-risk correlations. This is even more fundamental when radioactive particles are short-range emitters and when they have a chemical speciation that might further concur to the heterogeneity of energy deposition at the cellular and sub-cellular level. To this aim, we need to use computational models. In this work, we made use of a Monte Carlo radiation transport code to perform a computational dosimetric reconstruction for in vitro exposure of cells to tritiated steel particles of micrometric size. Particles of this kind have been identified as worth of attention in nuclear power industry and research: tritium easily permeates in steel elements of nuclear reactor machinery, and mechanical operations on these elements (e.g., sawing) during decommissioning of old facilities can result in particle dispersion, leading to human exposure via inhalation. Considering the software replica of a representative in vitro setup to study the effect of such particles, we therefore modelled the radiation field due to the presence of particles in proximity of cells. We developed a computational approach to reconstruct the dose range to individual cell nuclei in contact with a particle, as well as the fraction of "hit" cells and the average dose for the whole cell population, as a function of particle concentration in the culture medium. The dosimetric analysis also provided the basis to make predictions on tritium-induced DNA damage: we estimated the dose-dependent expected yield of DNA double strand breaks due to tritiated steel particle radiation, as an indicator of their expected biological effectiveness.

Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models.

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAirTM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAirTM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

Evaluation of PIG-A-mutated granulocytes and ex-vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans.

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.

Potentially harmful elements in house dust from Estarreja, Portugal: characterization and genotoxicity of the bioaccessible fraction.

Due to their behavioral characteristics, young children are vulnerable to the ingestion of indoor dust, often contaminated with chemicals that are potentially harmful. Exposure to potentially harmful elements (PHEs) is currently exacerbated by their widespread use in several industrial, agricultural, domestic and technological applications. PHEs cause adverse health effects on immune and nervous systems and can lead to cancer development via genotoxic mechanisms. The present study is an integrated approach that aims at assessing the genotoxicity of bioaccessible PHEs following ingestion of contaminated house dust. A multidisciplinary methodology associating chemical characterization of five house dust samples, extraction of the bioaccessible PHEs in gastric extracts by the unified BARGE method, determination of the bioaccessible fraction and in vitro genotoxicity of gastric extracts in adenocarcinoma gastric human (AGS) cells was developed. The five gastric extracts induced dose-dependent genotoxicity in AGS cells. Copper (bioaccessible concentration up to 111 mg/kg) was probably the prevalent PHE inducing primary DNA damage (up to 5.1-fold increase in tail DNA at 0.53 g/l of gastric extract). Lead (bioaccessible concentration up to 245 mg/kg) was the most prevalent PHE inducing chromosome-damaging effects (r = 0.55; p < 0.001 for micronucleated cells induction). The association of principal component analysis and Spearman's correlations was decisive to understand the chromosome-damaging properties of the bioaccessible PHEs in AGS cells. This methodology could be used on a larger-scale study to provide useful information for science-based decision-making in regulatory policies, and a better estimation of human exposure and associated health risks.

Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways.

BACKGROUND: Poorly soluble cobalt (II, III) oxide particles (Co3O4P) are believed to induce in vitro cytotoxic effects via a Trojan-horse mechanism. Once internalized into lysosomal and acidic intracellular compartments, Co3O4P slowly release a low amount of cobalt ions (Co(2+)) that impair the viability of in vitro cultures. In this study, we focused on the genotoxic potential of Co3O4P by performing a comprehensive investigation of the DNA damage exerted in BEAS-2B human bronchial epithelial cells. RESULTS: Our results demonstrate that poorly soluble Co3O4P enhanced the formation of micronuclei in binucleated cells. Moreover, by comet assay we showed that Co3O4P induced primary and oxidative DNA damage, and by scoring the formation of γ-H2Ax foci, we demonstrated that Co3O4P also generated double DNA strand breaks. CONCLUSIONS: By comparing the effects exerted by poorly soluble Co3O4P with those obtained in the presence of soluble cobalt chloride (CoCl2), we demonstrated that the genotoxic effects of Co3O4P are not simply due to the released Co(2+) but are induced by the particles themselves, as genotoxicity is observed at very low Co3O4P concentrations.

Cerium dioxide nanoparticles affect in vitro fertilization in mice.

Due to their catalytic and oxidative properties, cerium dioxide nanoparticles (CeO2NPs) are widely used as diesel additive or as promising therapy in cancerology; yet, scarce data are available on their toxicity, and none on their reproductive toxicity. We showed a significant decrease of fertilization rate, assessed on 1272 oocytes, during in vitro fertilization (IVF) carried out in culture medium containing CeO2NP at very low concentration (0.01 mg.l(-1)). We also showed significant DNA damage induced in vitro by CeO2NP on mouse spermatozoa and oocytes at 0.01 mg.l(-1) using Comet assay. Transmission Electron Microscopy did not detect any nanoparticles in the IVF samples at 0.01 mg.l(-1), but showed, at high concentration (100 mg.l(-1)), their endocytosis by the cumulus cells surrounding oocytes and their accumulation along spermatozoa plasma membranes and oocytes zona pellucida. We did not observe any nanoparticles in the cytoplasm of spermatozoa, oocytes or embryos. This study demonstrates for the first time the impact of CeO2NP on in vitro fertilization, as well as their genotoxicity on mouse spermatozoa and oocytes, at low nanoparticle concentration exposure. Decreased fertilization rates may result from: (1) CeO2NP's genotoxicity on gametes; (2) a mechanical effect, disrupting gamete interaction and (3) oxidative stress induced by CeO2NP. These results add new and important insights with regard to the reproductive toxicity of nanomaterials requesting urgent evaluation, and support several publications on metal nanoparticles reprotoxicity. Our data highlight the need for in vivo studies after low-dose exposure.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei.

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C-MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C-MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.

Biological properties of a neutralized 2.5% sodium hypochlorite solution.

OBJECTIVES: The objective of this study was to evaluate the influence of neutralizing a 2.5% NaOCl solution on its cytotoxicity, genotoxicity, and tissue-dissolving potential. STUDY DESIGN: The cytotoxicity and the genotoxicity of Dakin, a 2.5% NaOCl solution, and a neutralized 2.5% NaOCl solution were assessed according to ISO 10993 standards. The weight of palatal mucosa samples placed in neutralized 2.5% NaOCl, 2.5% NaOCl was recorded over time as well as the pH of the solutions. RESULTS: The neutralized 2.5% NaOCl solution was 10-fold more cytotoxic than the 2.5% NaOCl solution. None of the solutions was genotoxic. The 2.5% NaOCl solution had a better tissue-dissolving capacity than the neutralized 2.5% NaOCl solution. The pH of the 2.5% NaOCl solution and neutralized 2.5% NaOCl solution decreased from 12 to 9 and from 7.5 to 5.6, respectively. CONCLUSION: Neutralizing a 2.5% NaOCl solution increased its cytotoxicity, did not induce any genotoxic effect, and reduced its tissue-dissolving ability.

[Changes in chromosome number, genetic instability, and occupational exposures]. / Anomalies chromosomiques de nombre, instabilité génétique et expositions professionnelles.

Aneugenic compounds cause chromosome missegregation during cell division and induce aneuploidy in cells that do not die. Aneuploidy is a key step in the progression from a normal cell into a cancerous cell, and it could represent an early event in the carcinogenic process. Missegregation of chromosome during anaphase often originates from centrosome abnormality, which plays a key role in the formation of the mitotic spindle during cell division. Micronuclei (MN) are thought to be biomarkers of chromosome damage due to genetic instability or exposure to environmental mutagens or carcinogens (occupational exposure for example). The MN assay in combination with fluorescent in situ hybridization discriminates between MN containing acentric chromosome fragments (chromosome breakage) and MN containing whole chromosomes (chromosome loss), consecutively to clastogenic and aneugenic events, respectively. Centromere-positive micronuclei are due to alteration in mitotic apparatus proteins. Two pathways could be involved : chromosome migration impairment would lead to MN containing a single chromosome whereas centrosome amplification would induce MN containing several chromosomes. For biomonitoring purposes, numerous confounding factors (host factors, lifestyle, genetic polymorphism) influence the MN biomarker. Thus, the separated analysis of MN containing a single or several centromeres could be useful, as centrosome abnormalities seem to be linked with an increase in genetic instability.

Assessment of occupational exposure to welding fumes by inductively coupled plasma-mass spectroscopy and by the alkaline Comet assay.

Welding fumes are classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer. In the current study, blood and urine concentrations of aluminum (Al), cadmium (Cd), cobalt (Co), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn) were monitored by inductively coupled plasma-mass spectrometry (ICP-MS) in 30 welders and in 22 controls. In addition, DNA damage was examined in the lymphocytes of these subjects by the alkaline Comet assay. Two biological samples were taken from the welders at the beginning (BW) and at the end (EW) of a work week. In controls, collection of samples was limited to BW. Blood concentrations of Cd, Co, Cr, Ni, and Pb were higher in the welders than in the control group while higher concentrations of Al, Cd, Co, Cr, Ni, and Pb were detected in welder urines. There was no significant difference in the metal concentrations for the BW and EW welder samples. Increased levels of DNA damage were found in lymphocytes from welders as compared to the controls, and 20/30 welders had higher levels of DNA lesions in the EW than in the BW samples. Age had a significant effect on DNA damage in the control group. Spearman's rank correlation analysis indicated that there were positive correlations between blood concentrations of Al, Co, Ni, and Pb and the levels of DNA damage. A negative correlation was found between DNA damage and Mn in blood, while there was a positive correlation between urinary Mn concentration and DNA damage. These data indicate that occupational exposure to welding fumes increases DNA damage in lymphocytes.

Okadaic acid: chromosomal non-disjunction analysis in human lymphocytes and study of aneugenic pathway in CHO-K1 cells.

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.

Patterns of gene expressions induced by arsenic trioxide in cultured human fibroblasts.

Arsenic exposure is associated with several human diseases and particularly, with neoplasia. Although the mechanism of arsenic toxicity is not fully understood, several recent works pointed out the involvement of oxidative stress in arsenic-induced DNA damage that, in living cells, correlates with changes in gene expressions. In cultured human fibroblasts exposed for 24 h to micromolar arsenic concentrations, we studied, using real-time RT-PCR, the expression profile of a limited number of genes: genes coding for a stress protein (HSP70), transcription factors (cJUN, cFOS, ETR103, ETR101 and TTP) and cell cycle or DNA repair proteins (P21, GADD153). We observed that the expression profile of genes followed individual different patterns that can be summed up in early-transient gene expression by contrast to delayed gene expression.

[Interest of studying the in vitro genotoxicity of an antineoplastic drug on healthy human cells: paclitaxel example]. / Intérêt de l'étude de la génotoxicité in vitro d'une molécule de chimiothérapie sur cellules humaines : exemple du paclitaxel.

Paclitaxel is often used as an adjuvant antineoplastic agent. However, very little data is available in literature on its potential genotoxicity on healthy human cells. Our objective is to study the potential in vitro genotoxicity of paclitaxel, by using the cytokinesis-blocked micronucleus assay in combination with fluorescent in situ hybridization of nonspecific centromeric probes on human T-lymphocytes. Paclitaxel was found to significantly increase the micronucleated lymphocyte rates with a concentration-dependant manner. This increase is observed as early as with the weakest concentration tested (2,5 nM). Over 85% of those micronuclei contained one or more whole chromosomes, indicating that paclitaxel is a strong aneugenic drug. Paclitaxel induces the formation of aneuploid daughter cells, as a consequence of abnormalities in the distribution of chromosomes during the cell division. However, aneuploidy is probably one of the first events in the oncogenesis process, and it often seems to be linked to a dysfunction of the centrosome. An interaction between paclitaxel and centrosome could explain a considerable amount of the centromere-positive micronuclei due to multipolar mitosis. Paclitaxel, whose influence is being questioned in clinical practice in the occurrence of secondary acute myeloid leukemia, is therefore an in vitro aneugenic drug, which could be carcinogenic.