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Toxicity studies, among them hepatotoxicity, are key throughout preclinical stages of drug development to minimise undesired toxic effects that might eventually appear in the course of the clinical use of the new drug. Understanding the mechanism of injury of hepatotoxins is essential to efficiently anticipate their potential risk of toxicity in humans. The use of in vitro models and particularly cultured hepatocytes represents an easy and robust alternative to animal drug hepatotoxicity testing for predicting human risk. Here, we envisage an innovative strategy to identify potential hepatotoxic drugs, quantify the magnitude of the alterations caused, and uncover the mechanisms of toxicity. This strategy is based on the comparative analysis of metabolome changes induced by hepatotoxic and non-hepatotoxic compounds on HepG2 cells, assessed by untargeted mass spectrometry. As a training set, we used 25 hepatotoxic and 4 non-hepatotoxic compounds and incubated HepG2 cells for 24 h at a low and a high concentration (IC10 and IC50) to identify mechanism-related and cytotoxicity related metabolomic biomarkers and to elaborate prediction models accounting for global hepatotoxicity and mechanisms-related toxicity. Thereafter, a second set of 69 chemicals with known predominant mechanisms of toxicity and 18 non-hepatotoxic compounds were analysed at 1, 10, 100 and 1000 µM concentrations from which and based on the magnitude of the alterations caused as compared with non-toxic compounds, we defined a “toxicity index” for each compound. In addition, we extracted from the metabolome data the characteristic signatures for each mechanism of hepatotoxicity. The integration of all this information allowed us to identify specific metabolic patterns and, based on the occurrence of that specific metabolome changes, the models predicted the likeliness of a compound to behave as hepatotoxic and to act through a given toxicity mechanism (i.e., oxidative stress, mitochondrial disruption, apoptosis and steatosis) for each compound and concentration.

The assessment of liver steatosis is crucial in both hepatology and liver transplantation (LT) surgery. Steatosis can negatively impact the success of the LT. Steatosis is a factor for excluding...

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity., The authors wish to acknowledge the support of the European Union’s Horizon 2020 research and innovation 493 program under Grant Agreement No 681002 (EU-ToxRisk). We are indebted to CIBEREHD (ISCIII), for supporting our research program on hepatotoxicity of xenobiotics. L.T. was supported by the Miguel 494 Servet I Program financed by the Institute of Health Carlos III (Plan Estatal de I+D+i 2013-2016) 495 and co-financed by the European Regional Development Fund "A way to achieve Europe" (FEDER) 496 through grant CP16/00097. TMS acknowledges her personal predoctoral grant (FI18/00260, 497 ISCIII, Spain).