Drug-induced mitochondrial toxicity and its clinical manifestations
Toxicological studies show that drugs can alter mitochondrial functions, which potentially results in a deleterious range of reactions from the induction of micro- and macrovesicular steatosis to lactic acidosis [0]. These clinical manifestations result from drugs interfering with three main mitochondrial functions: aerobic respiration, beta-oxidation, and mitochondrial DNA homeostasis.
Drug-induced alteration of mitochondrial beta oxidation
Beta oxidation (β-Oxidation) is the main biochemical process by which the cell produces ATP from fatty acids. Therefore, an impairment of beta-oxidation promotes the accumulation of unconsumed fatty acid as small droplets of fat within the cytoplasm. Amiodarone is used as an anti-arrhythmic drug but disturbs mitochondrial beta-oxidation, therefore causing micro- and macro-vesicular steatosis [1]. This deleterious effect can be measured in 3D InSight™ Human Liver Microtissues as amiodarone causes a concentration-dependent increase in cellular triglyceride levels, as shown by the results of InSphero’s “Steatosis induction assay” [Figure 1].


Figure 1: Testing Drug-induced Mitochondrial Toxicity - Steatosis induction assay with Amiodarone in 3D Insight™ Human Liver Microtissues. Liver spheroids cultivated in InSphero’s BSA-free 3D Insight™ hLiMM Lean medium were exposed for 7 days to increasing concentrations of amiodarone. The triglyceride content of the model was assessed on day 7 using Promega’s Triglyceride-Glo™ assay. Statistical testing: Dunn's multiple comparisons tests, Legend: **: p-value < 0.01
Drug-induced alteration of mitochondrial respiration
Mitochondrial aerobic respiration is a biological process during which the mitochondrion uses pyruvate and oxygen to produce ATP. When Mitochondrial aerobic respiration is impaired, cellular pyruvate is instead converted to lactate for excretion (Figure 2). Phenformin is a withdrawn anti-diabetic drug that disturbs mitochondrial respiration, therefore causing lactic acidosis. This toxic effect can be measured in 3D InSight™ Liver Microtissues as phenformin causes a concentration-dependent increase in lactate secretion into the cell culture media, as shown by the results of InSphero’s “Mitochondrial function assay” [Figure 3].


Figure 2: Testing Drug-induced Mitochondrial Toxicity - Effect of mitochondrial function impairment on lactate metabolism. Top Panel: Cytosolic pyruvate diffuses into the mitochondria, in which it is converted to acetyl-CoA before entering the citric acid (TCA) cycle. Bottom panel: When mitochondrial function is impaired, the TCA cycle slows or stops and the excess of cytosolic pyruvate is converted to lactate by the lactate dehydrogenase enzyme.


Figure 3: Testing Drug-induced Mitochondrial Toxicity - Mitochondrial function assay with Phenformin 3D Insight™ Human Liver Microtissues. Liver spheroids cultivated in InSphero’s BSA-free 3D Insight™ hLiMM TOX medium were exposed for 7 days to increasing phenformin concentrations. The lactate concentration in the cell culture supernatant was measured on day 7 using Promega’s Lactate-Glo™ assay. Statistical testing: Dunn's multiple comparisons tests, Legend: *: p-value < 0.05 , **: p-value < 0.01
Why perform drug-induced mitochondrial toxicity testing in 3D InSight™ Liver Microtissues?
The effect of drugs on mitochondrial functions is currently tested with various in vitro methods that use transformed, immortalized liver cell lines such as HepG2 or HepaRG. However, there is a growing consensus in the scientific community that micro-physiological test systems using primary liver cells recapitulate the effect of drugs in vivo better than their less physiological counterparts using immortalized cell lines. 3D InSight™ Liver Microtissues are co-cultures of primary hepatocytes and non-parenchymal liver cells that model essential liver functions better than in vitro systems using immortalized cell lines (e.g. phase I & II metabolism).
References
[0] Vuda M, Kamath A. Drug-induced mitochondrial dysfunction: Mechanisms and adverse clinical consequences. Mitochondrion. 2016 Nov;31:63-74. doi: 10.1016/j.mito.2016.10.005. Epub 2016 Oct 19. PMID: 27771494
[1] Fromenty B, Fisch C, Labbe G, Degott C, Deschamps D, Berson A, Letteron P, Pessayre D. Amiodarone inhibits the mitochondrial beta-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther. 1990 Dec;255(3):1371-6. PMID: 2124623.
[2] Fimognari FL, Pastorelli R, Incalzi RA. Phenformin-induced lactic acidosis in an older diabetic patient: a recurrent drama (phenformin and lactic acidosis). Diabetes Care. 2006 Apr;29(4):950-1. doi: 10.2337/diacare.29.04.06.dc06-0012. PMID: 16567854.