Testing liver toxicity in HepG2 WT with differents methods (HCI, resazurin…)
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ABSTRACT:
ORGANISM(S): human
SUBMITTER: Laia Tolosa
PROVIDER: S-TOXR1193 | biostudies-other |
REPOSITORIES: biostudies-other
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| S-TOXR1191 | biostudies-other
Project description:(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 (TiO2), 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 (EC50 3.8 µg/cm2) than in 3D cultures (EC50 > 30 µg/cm2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC50 10.1-16.2 µg/cm2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO2-NPs showed no toxicity (EC50 > 75 µg/cm2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.
| S-EPMC7153628 | biostudies-literature
Project description:Hepatic metabolism catalyzed by the cytochrome P450 (CYP) superfamily affects liver toxicity associated with exposures to natural compounds and xenobiotic agents. Previously we generated a battery of HepG2-derived stable cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). In this study, we comprehensively characterized each cell line for its CYP expression and enzyme activity. Specifically, we measured the mRNA expression, protein expression, and metabolite formation. Using CYP3A4, 2D6, and 2C9-overexpressing cells as representatives, we examined the stability of these cells in long-term cultures for up to 10 passages. The results showed that CYPs can be stably overexpressed for up to 10 cell culture passages without losing their activities. The robustness of responses to stimuli among the cells at different passages was also investigated in CYP3A4-overexpressing cells and the response to amiodarone and dronedarone showed no difference between the cells at the passage 2 and 10. Moreover, the mRNA expression level of most CYPs was higher in CYP-overexpressing HepG2 cells than that in HepaRG cells and primary human hepatocytes. This study confirmed the stability of CYP-overexpressing HepG2 cell lines and provided useful information for a broader use of these cells in pharmacologic and toxicologic research.
| S-EPMC7931144 | biostudies-literature
Project description:The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.
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| S-TOXR1383 | biostudies-other
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| S-TOXR1384 | biostudies-other
Project description:Twenty-four hour median lethal concentration (LC50) toxicity tests were performed with five species of nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, Pristionchus pacificus, Oscheius tipulae, and Oscheius myriophila) in response to copper chloride and zinc chloride. In addition, lethality tests were also performed with seven strains of C. elegans (N2 > 1 year in culture, N2 newly acquired, N2 ancestral, ED3053, JU258, JU1171, and MY1) exposed to copper chloride. Nominal chemical concentrations were validated and analyzed according to U.S. Environmental Protection Agency method 6010 using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). This paper combines the datasets previously published separately by Heaton et al. (2020, 2022). The goal is to catalog all raw and analyzed toxicity data collected from both studies in a single consistent information source for use by the scientific community.
| S-EPMC8591337 | biostudies-literature
Project description:Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.
| S-EPMC5818600 | biostudies-other
Project description:The spice saffron (Crocus sativus) has anticancer activity in several human tissues, but the molecular mechanisms underlying its potential therapeutic effects are poorly understood. We investigated the impact of safranal, a small molecule secondary metabolite from saffron, on the HCC cell line HepG2 using untargeted metabolomics (HPLC-MS) and transcriptomics (RNAseq). Increases in glutathione disulfide and other biomarkers for oxidative damage contrasted with lower levels of the antioxidants biliverdin IX (139-fold decrease, p = 5.3 × 105), the ubiquinol precursor 3-4-dihydroxy-5-all-trans-decaprenylbenzoate (3-fold decrease, p = 1.9 × 10-5), and resolvin E1 (-3282-fold decrease, p = 45), which indicates sensitization to reactive oxygen species. We observed a significant increase in intracellular hypoxanthine (538-fold increase, p = 7.7 × 10-6) that may be primarily responsible for oxidative damage in HCC after safranal treatment. The accumulation of free fatty acids and other biomarkers, such as S-methyl-5'-thioadenosine, are consistent with safranal-induced mitochondrial de-uncoupling and explains the sharp increase in hypoxanthine we observed. Overall, the dual omics datasets describe routes to widespread protein destabilization and DNA damage from safranal-induced oxidative stress in HCC cells.
| S-EPMC9219844 | biostudies-literature