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.
Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM stages II and III. Prognostic biomarkers ...for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. miRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR-based relative quantification of miRNA expression is lacking.
A selection of frozen tumor specimens from two independent patient cohorts with TNM stage II-III microsatellite stable primary adenocarcinomas was used for laser capture microdissection. Next-generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N = 50). Differential expression analysis, comparing in metastasized and nonmetastasized tumors, identified prognostic miRNAs. Validation was performed on colon tumors from the German cohort (N = 43) using quantitative PCR (qPCR).
miR25-3p and miR339-5p were identified and validated as independent prognostic markers and used to construct a multivariate nomogram for metastasis risk prediction. The nomogram showed good probability prediction in validation. In addition, we recommend combination of miR16-5p and miR26a-5p as standard for normalization in qPCR of colon cancer tissue-derived miRNA expression.
In this international study, we identified and validated a miRNA classifier in primary cancers, and propose a nomogram capable of predicting metastasis risk in microsatellite stable TNM stage II-III colon cancer.
In conjunction with TNM staging, by means of a nomogram, this miRNA classifier may allow for personalized treatment decisions based on individual tumor characteristics.
Breast 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.
Immunohistochemical 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.
Expression 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).
When 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.
Welding fumes induce lung toxicity and are carcinogenic to humans but the molecular mechanisms have yet to be clarified. The aim of this study was to evaluate the toxicity of stainless and mild steel ...particles generated via gas-metal arc welding using primary human small airway epithelial cells (hSAEC) and ToxTracker reporter murine stem cells, which track activation of six cancer-related pathways. Metal content (Fe, Mn, Ni, Cr) of the particles was relatively homogenous across particle size. The particles were not cytotoxic in reporter stem cells but stainless steel particles activated the Nrf2-dependent oxidative stress pathway. In hSAEC, both particle types induced time- and dose-dependent cytotoxicity, and stainless steel particles also increased generation of reactive oxygen species. The cellular metal content was higher for hSAEC compared to the reporter stem cells exposed to the same nominal dose. This was, in part, related to differences in particle agglomeration/sedimentation in the different cell media. Overall, our study showed differences in cytotoxicity and activation of cancer-related pathways between stainless and mild steel welding particles. Moreover, our data emphasizes the need for careful assessment of the cellular dose when comparing studies using different in vitro models.
Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker ...is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress.
ToxTracker is an in vitro mammalian stem cell‐based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical ...exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose–response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold‐change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined‐covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose–response modeling of multivariate ToxTracker data can be used for potency ranking and mode‐of‐action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment.
Cobalt metal and cobalt sulfate are carcinogenic in rodents following inhalation exposure. The pre-carcinogenic effects associated with exposure to these cobalt substances include oxidative stress ...and genotoxicity. Some, but not all, cobalt substances induce in vitro clastogenicity or an increase in micronuclei. As a result, these substances are classified genotoxic carcinogens, having major impacts on their risk assessment, e.g. assumption of a non-thresholded dose response. Here, we investigated the potential of nine cobalt substances to cause genotoxicity and oxidative stress using the ToxTracker assay, with an extension to measure biomarkers of hypoxia. None of the nine tested substances activated the DNA damage markers in ToxTracker, and five substances activated the oxidative stress response reporters. The same five substances also activated the expression of several hypoxia target genes. Consistent with the lower tier of testing found in the preceding paper of this series, these compounds can be grouped based on their ability to release bioavailable cobalt ion and to trigger subsequent key events.
•Cobalt (Co) substances are potentially toxic and carcinogenic following inhalation.•Prediction of outcome by tiered testing of initiating and key events is described.•Two groups of Co substances with distinct effects and toxicities were identified.•The ToxTracker assay was used to investigate genotoxicity of cobalt substances.•Reactive Co substances caused oxidative stress and activated hypoxia target genes.
Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of ...their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues.
•Nucleoside analogues can be mutagenic and carcinogenic because of their MoA.•We tested well-known nucleoside analogues using our genotoxicity assay ToxTracker.•> 50% of the nucleoside analogues were found to induce genotoxicity in ToxTracker.•The data are largely in line with in vivo micronucleus and/or carcinogenicity data.•Recently developed nucleoside analogues generally have better toxicity profiles.
Abstract
Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to ...cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.
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 -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.
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