The cytokinesis-block micronucleus (CBMN) assay in human populations exposed to styrene: a systematic review and meta-analysis

Styrene is a building-block of several compounds used in a wide array of materials and products. The most important human exposure to this substance occurs in industrial settings, especially among reinforced-plastics industry workers. The effect of occupational exposure to styrene on cytogenetics biomarkers has been previously reviewed with positive association observed for chromosomal aberrations, and inconclusive data for the micronucleus assay. Some limitations were noted in those studies, including inadequate exposure assessment and poor epidemiological design. Furthermore, in earlier studies micronuclei frequency was measured with protocols not as reliable as cytokinesis-block micronucleus (CBMN) assay. Aim of the present systematic review and meta-analysis is to investigate genomic instability and DNA damage as measured by the CBMN assay in lymphocytes of subjects exposed to styrene. A total of 11 studies published between 2004 and 2012 were included in the meta-analysis encompassing 479 styrene-exposed workers and 510 controls. The quality of each study was estimated by a quality scoring system which ranked studies according to the consideration of major confounders, exposure characterization, and technical parameters. An overall increase of micronuclei frequencies was found in styrene-exposure workers when compared to referents (meta-MR 1.34; 95% CI 1.18–1.52), with significant increases achieved in six individual studies. The consistency of results in individual studies, the independence of this result from major confounding factors and from the quality of the study strengthens the reliability of risk estimates and supports the use of the CBMN assay in monitoring genetic risk in styrene workers.

Cyto- and Genotoxicity assessment of manufactured nano cerium dioxide in the A549 cell line

In the past decades the growing application of nanomaterials (NMs) in diverse consumer products has raised various concerns in the field of toxicology. They have been extensively used in a broad range of applications and cover most of the industrial sectors as well as the medicine and the environmental areas. The most common scenarios for human exposure to NMs are occupational, environmental and as consumers and inhalation is the most frequent route of exposure, especially in occupational settings. Cerium dioxide NMs (nano-CeO2) are widely used in a number of applications such as in cosmetics, outdoor paints, wood care products as well as fuel catalysts. For such reason, nano-CeO2 is one of the selected NMs for priority testing within the sponsorship program of the Working Party of Manufactured Nanomaterials of the OECD. In this context, the aim of this study is to assess the safety of nano-CeO2 (NM-212, Joint Research Center Repository) through the characterization of its cytotoxicity and genotoxicity in a human alveolar epithelial cell line. A dispersion of the NM in water plus 0.05% BSA was prepared and sonicated during 16 minutes, according to a standardized protocol. DLS analysis was used to characterize the quality of the NM dispersion in the culture medium. To evaluate the cytotoxicity of nano-CeO2 in the A549 cell line, the colorimetric MTT assay was performed; the capacity of cells to proliferate when exposed to CeO2 was also assessed with the Clonogenic assay. The genotoxicity of this NM was evaluated by the Comet Assay (3 and 24h of exposure) to quantify DNA breaks and the FPG-modified comet assay to assess oxidative DNA damage. The Cytokinesis-Block Micronucleus (CBMN) assay was used to further detect chromosome breaks or loss. The nano-CeO2 particles are spherical, displaying a diameter of 33 nm and 28 m2/g of surface area. The results of the MTT assay did not show any decreased in cells viability following treatment with a dose-range of nano-CeO2 during 24h. Nevertheless, the highest concentrations of this NM were able to significantly reduce the colony forming ability of A549 cells, suggesting that a prolonged exposure may be cytotoxic to these cells. Data from both genotoxicity assays revealed that nano-CeO2 was neither able to induce DNA breaks nor oxidative DNA damage. Likewise, no significant micronucleus induction was observed. Taken together, the present results indicate that this nano-CeO2 is not genotoxic in this alveolar cell line under the tested conditions, although further studies should be performed, e.g., gene mutation in somatic cells and in vivo chromosome damage (rodent micronucleus assay) to ensure its safety to human health.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities.