Mitochondrial toxicity is one of the pathogenic mechanisms of severe drug-induced liver injury (DILI). It is desirable to identify severe DILI risks early in development, however it is extremely difficult to recognize this risk in pre-clinical animal species. Mitochondrial dysfunction accounts for a growing proportion of preclinical drug attrition and post-market withdrawals or black box warnings by the U.S. Food and Drug Administration (FDA).
Figure 1. Mitochondria toxicity assessment with PXB-cells and Cryopreserved Human Hepatocytes. Image modified from the Graphical Abstract of the Ikeyama Y. et al. Toxicol. in Vitro 2020; 65: 104785
Under standard cell culture conditions (high glucose and low oxygen concentration), mitochondrial respiratory activity is suppressed, compared to in vivo physiological conditions. This happens because cultured cells in vitro produce ATP mainly through glycolysis, while in vivo mitochondria produce ATP mainly by oxidative phosphorylation. Thus, the mitochondrial toxicity is often overlooked in the in vitro assays.
In the recent paper by Ikeyama Y. et al. “Successful energy shift from glycolysis to mitochondrial oxidative phosphorylation in freshly isolated hepatocytes from humanized mice liver”, authors directly compare the mitochondrial function in the cryopreserved human hepatocytes (CHH) lots and in the PXB-cells® – freshly isolated hepatocytes from cDNA-uPA/SCID chimeric mouse with humanized liver (PXB-mouse®).
It is known that cryopreserved human hepatocytes have such disadvantages as unstable supply from the same donor and potential organelle damage due to cryopreservation. In contrast, PXB-cells® are used without cryopreservation and are supplied on demand from the same donor lot.
The authors goal was to identify suitable human hepatocytes for the mitochondrial toxicity assessment.
To achieve this goal, they compared CHH (from three donors) and PXB-cells® in terms of energy metabolism and mitochondrial function after sugar resource substitution from glucose to galactose. PXB-cells® in the galactose culture had a higher level of intracellular ATP than those in the glucose culture. This result was not observed in CHH lots: a total of 5 out of 6 CHH lots tested were not able to survive after sugar resource substitution with galactose. PXB-cells® showed high viability regardless the substitution of sugars.
In addition, PXB-cells® were more sensitive to rotenone (mitochondrial respiratory chain complex I inhibitor) in the galactose medium than in the glucose medium. The sensitivity of PXB-cells® to other respiratory chain inhibitors (RCI) was examined as well. The anti-diabetic drug, phenformin, and the anti-cancer drug, flutamide were used as RCIs, while metformin and bicalutamide were used as negative control. Although the toxicity enhancement was observed in all the cases with sugar source substitution, the effects of phenformin and flutamide were much more pronounced.
In conclusion, the authors suggest that the substitution of CHH with PXB-cells® would allow more reliable assessment of mitochondrial toxicity. With further studies, PXB-cells® could be used for the evaluation of other types of mitochondrial toxicities, such as reactive oxygen species (ROS)-mediated toxicity and mitochondrial permeability transition (MPT)-mediated toxicity.
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