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Exposure of HepaRG Cells to Sodium Saccharin Underpins the Importance of Including Non-Hepatotoxic Compounds When Investigating Toxicological Modes of Action Using Metabolomics
Received: 10 October 2019 / Accepted: 30 October 2019 / Published: 4 November 2019
Metabolites represent the most downstream information of the cellular organisation. Hence, metabolomics experiments are extremely valuable to unravel the endogenous pathways involved in a toxicological mode of action. However, every external stimulus can introduce alterations in the cell homeostasis, thereby obscuring the involved endogenous pathways, biasing the interpretation of the results. Here we report on sodium saccharin, which is considered to be not hepatotoxic and therefore can serve as a reference compound to detect metabolic alterations that are not related to liver toxicity. Exposure of HepaRG cells to high levels of sodium saccharin (>10 mM) induced cell death, probably due to an increase in the osmotic pressure. Yet, a low number (n = 15) of significantly altered metabolites were also observed in the lipidome, including a slight decrease in phospholipids and an increase in triacylglycerols, upon daily exposure to 5 mM sodium saccharin for 72 h. The observation that a non-hepatotoxic compound can affect the metabolome underpins the importance of correct experimental design and data interpretation when investigating toxicological modes of action via metabolomics.
in vitro; HepaRG; sodium saccharin; reference toxicants
The metabolome is a dynamic level of the cellular organisation. External stimuli theoretically invoke a response of the cell, resulting in a change of the metabolome. Sodium saccharin is considered to be a non-hepatotoxic chemical, which makes it an ideal compound to select markers of exposure not necessarily related to hepatotoxicity [8
The acquisition of the metabolome of cells exposed to a hepatotoxicant in comparison to vehicle only would reveal alterations that would all be addressed to a toxic mode of action. The main consequence of this assumption is the questionable predictive value of the observed metabolic alterations, especially in small-scale experiments (one dose, one time point exposure of a single chemical exposure). This consideration stresses the importance of an exposure to a non-toxic negative control during the experiment.
Few features of the lipidome were changed significantly (n = 15) and included the presence of triacylglycerols and a lower presence of phospholipids. The absence of significant changes in the polar fraction suggested that the cell culture did not implement major adaptations in metabolism as a response to the external stimuli.
Effects related to hyperosmolarity have also been described in human cell cultures, which showed the increased presence of monosaccharides and amino acids to retain the osmotic balance [26
]. However, it is possible that these effects were not observed in this current experiment due to the deliberate choice to avoid these hyperosmolar (non-physiological) concentrations. The choice of a supra-physiological dose would imply a bias to select metabolic alterations related to modes of action not relevant in physiological conditions. This bias is especially relevant in in vitro techniques, as exposure concentrations can be increased to unrealistic, non-physiological levels. A potential prevention is the use of toxicokinetic data to confirm the plausibility of the exposure conditions.
García-Cañaveras et al. [27
] and Ramirez et al. [28
] classified different toxicants according to the mode of action, observing specific fingerprints for different end-points of toxicity. Ramirez et al. described the downregulation of carnitine, creatine, phosphocreatine, and pantothenic acid during exposure to peroxisome proliferating agents, the decrease of oleic acid, galactose and acetyl aspartate in combination with an increase of tryptophan and alanine during exposure to enzyme-inducing xenobiotics [28
]. Garcia et al. compared the fingerprint of xenobiotics inducing oxidative stress, steatosis and phospholipidosis, in which they observed alterations in glutamate levels, oxido-reductive status, lysophospholipid/phospholipid ratio and lipid accumulation [27
]. The inclusion of non-hepatotoxic compounds in their experimental design, such as citrate and ketotifen, states the importance of reference compounds for non-hepatotoxicity as metabolic changes were observed, albeit with a different fingerprint.
A qualitative comparison of the metabolic alterations for steatosis and cholestasis obtained in previous experiments are presented in Table 3
]. The clear alterations of the metabolome during hepatotoxic modes of action can be discriminated from the exposure to a non-hepatotoxic toxicant. Next to significant differences in the polar metabolome, the lipidome of a cell culture exposed to hepatotoxic compounds showed clear and strong alterations, with multiple lipid species of several classes involved in the downstream effect, whereas the effects during exposure to sodium saccharin were not substantial.
A mechanistic interpretation based on the observed metabolic alterations provides an additional value to the localisation of the specific molecular initiating event and the potential adverse outcome, as this observation is an additional argument for a toxic cascade. Although metabolic alterations were observed upon exposure to sodium saccharin, no specific affected pathway could be identified. Based on all observations, the use of a non-hepatotoxic compound instead of a vehicle-only negative control group may help reducing false positive results without jeopardising the sensitivity of toxic insults.
Conceptualization, R.M.R. and T.V.; Data curation, C.B. and K.L.; Investigation, M.C., T.V. and A.C.; Methodology, M.C., C.B. and R.M.R.; Software, C.B.; Supervision, R.M.R., K.L., T.V. and A.C.; Writing—original draft, M.C.; Writing—review & editing, K.L., T.V. and A.C.
Matthias Cuykx has been funded by the Fonds voor Wetenschappelijk Onderzoek (FWO, Belgium; fellowships 12H2216N and 11Z3318N). Further funding of this research has been provided by the Vrije Universiteit Brussel (VUB, Belgium) and the University of Antwerp (UA, Belgium).
Conflicts of Interest
The authors declare no conflict of interest.
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Averaged viability curve for the neutral red uptake (NRU)-assay upon daily exposure to sodium saccharin for a period of 72 h. Cytotoxicity is observed from 10 mM onwards, which is equal to an osmotic pressure of ±20 mOsm/L.
PCA plots of the non-polar fraction in positive mode during the 72-h exposure showing PC1 vs. PC2. There is strong overlapping of the different exposure groups in all principal components, indicating the variance is not related to exposure. Only a slight trend is visible between the negative control group and the group exposed to the higher dose.
AUCs of the random forest classifiers comparing the negative control group against the different exposures.
|Exposure||Non-Polar Positive||Non-Polar Negative||Polar Positive||Polar Negative|
R2 and Q2 values for the PLS-DA discrimination between the exposure groups and the negative control group.
|Exposure||Non-Polar Positive||Non-Polar Negative||Polar Positive||Polar Negative|
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