Hepato(Geno)toxicity assessment of nanoparticles in a HepG2 liver spheroid model

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC 3.8 µg/cm 2) than in 3D cultures (EC > 30 µg/cm 2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 10.1-16.2 µg/cm 2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO-NPs showed no toxicity (EC > 75 µg/cm 2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs

Risk-benefit assessment of sunscreen: Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and Environment

publishedVersio

Risk-benefit assessment of sunscreen: Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and Environment

publishedVersio

Risk assessment of caffeine exposure from diet and personal care products. Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and Environment

publishedVersio

Risk-benefit assessment of sunscreen - Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and Environment

VKM has performed a risk-benefit assessment of sunscreen use and six UV filters. This task was undertaken on the initiative of a VKM Panel in response to the apparent paradox between the need for protective measures, such as use of sunscreens, to reduce Norway’s high incidence and mortality of skin cancer and a consumer concern for the safety of sunscreens. Concerns include safety of ingredients and sunscreens’ effect on vitamin D synthesis. Sunscreen products are legally regulated as cosmetic products in the EU, and only approved UV filters up to a maximum determined concentration are allowed in the ready-foruse preparation. VKM used a systematic approach to assess risks and benefits of sunscreen use and risks of six selected UV filters: bis-ethyl-hexyloxyphenol methoxyphenyl triazine (BEMT), butyl methoxydibenzoyl methane (BMDBM), 2-ethylhexyl salicylate (EHS), ethylhexyl triazone (EHT), octocrylene (OC), and titanium dioxide in nanoform (NP-TiO2). These UV filters are among the most frequently used in sunscreens on the Norwegian market. Sunscreen sprays and lip products were not included. Scientific publications and reports up to 2020 were retrieved to assess adverse and protective effects of sunscreen and adverse effects of UV filters. We assessed risk of bias in the studies and evidence for health outcomes with the aid of validity tools, and estimated exposure to each UV filter using probabilistic methods. The evidence showed that sunscreens were beneficial in protecting against certain skin cancers. Insufficient evidence precluded determination of the hazard associated with sunscreen use. The UV filters occurred in concentrations similar to or below the limits set in the EU cosmetics regulative. VKM considered that little to no hazard was associated with use of the six evaluated UV filters. VKM concludes that the risks related to use of the six evaluated UV filters are negligible since the real-life use of these UV filters is several-fold lower than the amounts that may cause any adverse health effect. The evidence for harmful health effects of sunscreens is insufficient to determine risk. Sunscreen use protects against certain skin cancers and is beneficial for the general Norwegian population.Risk-benefit assessment of sunscreen - Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and EnvironmentpublishedVersio

Risk-benefit assessment of sunscreen - Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and Environment

VKM has performed a risk-benefit assessment of sunscreen use and six UV filters. This task was undertaken on the initiative of a VKM Panel in response to the apparent paradox between the need for protective measures, such as use of sunscreens, to reduce Norway’s high incidence and mortality of skin cancer and a consumer concern for the safety of sunscreens. Concerns include safety of ingredients and sunscreens’ effect on vitamin D synthesis. Sunscreen products are legally regulated as cosmetic products in the EU, and only approved UV filters up to a maximum determined concentration are allowed in the ready-foruse preparation. VKM used a systematic approach to assess risks and benefits of sunscreen use and risks of six selected UV filters: bis-ethyl-hexyloxyphenol methoxyphenyl triazine (BEMT), butyl methoxydibenzoyl methane (BMDBM), 2-ethylhexyl salicylate (EHS), ethylhexyl triazone (EHT), octocrylene (OC), and titanium dioxide in nanoform (NP-TiO2). These UV filters are among the most frequently used in sunscreens on the Norwegian market. Sunscreen sprays and lip products were not included. Scientific publications and reports up to 2020 were retrieved to assess adverse and protective effects of sunscreen and adverse effects of UV filters. We assessed risk of bias in the studies and evidence for health outcomes with the aid of validity tools, and estimated exposure to each UV filter using probabilistic methods. The evidence showed that sunscreens were beneficial in protecting against certain skin cancers. Insufficient evidence precluded determination of the hazard associated with sunscreen use. The UV filters occurred in concentrations similar to or below the limits set in the EU cosmetics regulative. VKM considered that little to no hazard was associated with use of the six evaluated UV filters. VKM concludes that the risks related to use of the six evaluated UV filters are negligible since the real-life use of these UV filters is several-fold lower than the amounts that may cause any adverse health effect. The evidence for harmful health effects of sunscreens is insufficient to determine risk. Sunscreen use protects against certain skin cancers and is beneficial for the general Norwegian population.Risk-benefit assessment of sunscreen - Opinion of the Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food, and Cosmetics of the Norwegian Scientific Committee for Food and EnvironmentpublishedVersio

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.publishedVersio

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.publishedVersio

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R’s to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs