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

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

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