Remco Derr

High nuclear expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in tumor cells correlate with decreased survival and increased relapse in breast cancer patients

BackgroundBreast cancer is a heterogeneous disease with a highly variable clinical outcome in which both genetic and epigenetic changes have critical roles. We investigated tumor expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in relation with patient survival and tumor relapse in a retrospective cohort of 460 breast cancer patients. Additionally, we correlated expression levels with tumor differentiation and tumor cell proliferation.MethodsImmunohistochemical staining for LSD1, HDAC2 and SIRT1 was performed on tissue microarrays of tumor and corresponding normal formalin-fixed paraffin-embedded tissues from breast cancer patients. Median nuclear expression levels in tumor tissues were used to divide the patients into low and high expression categories. In combined expression analyses, patients were divided into four subgroups: 1, all enzymes below-median; 2, one enzyme above-median; 3, two enzymes above-median; 4, all three enzymes above-median. The Cox proportional hazard model was used for univariate and multivariate survival analyses. The Pearson Chi-square method was used to assess correlation of combined expression levels with tumor cell proliferation and tumor differentiation.ResultsExpression of LSD1 and SIRT1, but not of HDAC2, was significantly increased in tumor tissues compared to their normal counterparts (both p < 0.001). Multivariate survival analyses identified SIRT1 as independent prognostic factor for relapse-free survival (RFS) with a hazard ratio (HR) of 1.34 (95% CI = 1.04-1.74, p = 0.02). For overall survival (OS), no significant differences were found when the individual enzymes were analyzed. Analyses of combined expression levels of the three histone-modifying enzymes correlated with OS (p = 0.03) and RFS (p = 0.006) with a HR of respectively 1.49 (95% CI = 1.07-2.08) and 1.68 (95% CI = 1.16-2.44) in multivariate analyses and were also related to tumor differentiation (p < 0.001) and tumor cell proliferation (p = 0.002).ConclusionsWhen the combined expression levels were analyzed, high expression of LSD1, HDAC2 and SIRT1 showed shorter patient survival time and shorter time to tumor relapse and correlated with poor tumor differentiation and a high level of tumor cell proliferation. Expression of these histone-modifying enzymes might therefore be involved in breast cancer pathogenesis.

The extended ToxTracker assay discriminates between induction of DNA damage, oxidative stress and protein misfolding.

Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

Genotoxic and mutagenic properties of Ni and NiO nanoparticles investigated by comet assay, γ-H2AX staining, Hprt mutation assay and ToxTracker reporter cell lines: Genotoxicity of Ni and NiO Nanoparticles

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl2. We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ‐H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl2. By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni‐containing materials. Oxidative stress was also detected in the HBEC cells following NP‐exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211–222, 2018.

The Vitotox and ToxTracker assays: A two-test combination for quick and reliable assessment of genotoxic hazards

To ensure safety for humans, it is essential to characterize the genotoxic potential of new chemical entities, such as pharmaceutical and cosmetic substances. In a first tier, a battery of in vitro tests is recommended by international regulatory agencies. However, these tests suffer from inadequate specificity: compounds may be wrongly categorized as genotoxic, resulting in unnecessary, time-consuming, and expensive in vivo follow-up testing. In the last decade, novel assays (notably, reporter-based assays) have been developed in an attempt to overcome these drawbacks. Here, we have investigated the performance of two in vitro reporter-based assays, Vitotox and ToxTracker. A set of reference compounds was selected to span a variety of mechanisms of genotoxic action and applicability domains (e.g., pharmaceutical and cosmetic ingredients). Combining the performance of the two assays, we achieved 93% sensitivity and 79% specificity for prediction of gentoxicity for this set of compounds. Both assays permit quick high-throughput analysis of drug candidates, while requiring only small quantities of the test substances. Our study shows that these two assays, when combined, can be a reliable method for assessment of genotoxicity hazard.

Mechanistic insight into reactivity and (geno)toxicity of well-characterized nanoparticles of cobalt metal and oxides

Abstract An increasing use of cobalt (Co)-based nanoparticles (NPs) in different applications and exposures at occupational settings triggers the need for toxicity assessment. Improved understanding regarding the physiochemical characteristics of Co metal NPs and different oxides in combination with assessment of toxicity and mechanisms may facilitate decisions for grouping during risk assessment. The aim of this study was to gain mechanistic insights in the correlation between NP reactivity and toxicity of three different Co-based NPs (Co, CoO, and Co3O4) by using various tools for characterization, traditional toxicity assays, as well as six reporter cell lines (ToxTracker) for rapid detection of signaling pathways of relevance for carcinogenicity. The results showed cellular uptake of all NPs in lung cells and induction of DNA strand breaks and oxidative damage (comet assay) by Co and CoO NPs. In-depth studies on the ROS generation showed high reactivity of Co, lower for CoO, and no reactivity of Co3O4 NPs. The reactivity depended on the corrosion and transformation/dissolution properties of the particles and the media highlighting the role of the surface oxide and metal speciation as also confirmed by in silico modeling. By using ToxTracker, Co NPs were shown to be highly cytotoxic and induced reporters related to oxidative stress (Nrf2 signaling) and DNA strand breaks. Similar effects were observed for CoO NPs but at higher concentrations, whereas the Co3O4 NPs were inactive at all concentrations tested. In conclusion, our study suggests that Co and CoO NPs, but not Co3O4, may be grouped together for risk assessment.