Volume 114, Issue 4, Pages 764-774 (April 1998) Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes  Alain Berson, Virginie De Beco, Philippe Lettéron, Marie Anne Robin, Claire Moreau, Johny El Kahwaji, Nicole Verthier, Gérard Feldmann, Bernard Fromenty, Dominique Pessayre  Gastroenterology  Volume 114, Issue 4, Pages 764-774 (April 1998) DOI: 10.1016/S0016-5085(98)70590-6 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of DEAEH on the mitochondrial membrane potential assessed by the fluorescence of safranine. A decrease in safranine fluorescence indicates an increase in the membrane potential. Rat liver mitochondria (0.7 mg protein/mL) were incubated with 10 μmol/L safranine, followed by the addition of 10 mmol/L succinate. Oxidation of this substrate led to the extrusion of protons from the matrix and increased the membrane potential. The uncoupler, 40 or 160 μmol/L 2,4-dinitrophenol (DNP), was used as a reference compound that causes the reentry of H+ into the matrix and decreases the membrane potential. DEAEH (50, 100, or 200 μmol/L) caused a moderate, dose-dependent decrease in the membrane potential. Tetraphenylborate (TPB) alone (3 μmol/L) had little effect. When both tetraphenylborate and 100 μmol/L DEAEH were added, there was a marked decrease in the membrane potential. Successive recordings obtained with various concentrations of DNP or DEAEH and with or without tetraphenylborate have been superimposed on the figure. FU, fluorescence unit. Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibition kinetics of MCAD by DEAEH. Matrix proteins (100 μg/mL) were incubated at 37°C with 12.5, 25, 50, 100, 200, or 400 μmol/L octanoyl-CoA (C8-CoA) and with or without 0, 25, 50, or 100 μmol/L DEAEH. The reduction of 200 μmol/L ferricenium hexafluorophosphate was monitored at 290 nm. Results are means ± SEM for 4–6 determinations. Lines are linear regression lines. (A) Double reciprocal plot; (B) Dixon's plot. Inhibition kinetics were consistent with competitive inhibition of MCAD by DEAEH. The Michaelis constant (Km) for C8-CoA in the uninhibited reaction was 5.9 μmol/L. With C8-CoA concentrations ranging from 25 to 400 μmol/L, the dissociation constant of the enzyme-inhibitor complex (Ki) for DEAEH was 11 μmol/L. Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of DEAEH on the β-oxidation of [U-14C]palmitic acid by human lymphocytes. Lymphocytes (106 cells/mL) were preincubated at 37°C for 5 minutes with (in μmol/L) 200 ATP, 40 L-carnitine, and 40 CoA, with or without 10–150 DEAEH. After addition of 40 μmol/L [U-14C]palmitic acid, the reaction was carried out at 37°C for 90 minutes. Data are expressed as means ± SEM for 6 determinations. *Significant difference from control (P < 0.05). Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A and B) Ultrastructure of hepatocytes after 3 days of culture with 50 μmol/L DEAEH. Hepatocytes exhibited microvesicular steatosis (S), elongated or giant mitochondria (M), and lysosomal phospholipidosis (L). Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 (A and B) Ultrastructure of hepatocytes after 3 days of culture with 50 μmol/L DEAEH. Hepatocytes exhibited microvesicular steatosis (S), elongated or giant mitochondria (M), and lysosomal phospholipidosis (L). Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Enhanced mitochondrial formation of 2',7'-dichlorofluorescein with DEAEH, amiodarone, and perhexiline. Mitochondria (1 mg protein/mL) were preincubated at 20°C for 5 minutes with 5 mmol/L each of glutamate and malate, 15 μmol/L 2',7'-dichlorodihydrofluorescein acetate, and 200 μmol/L each DEAEH, amiodarone, or perhexiline. ADP (0.6 mmol/L) was added, and the linear increase in the fluorescence of 2',7'-dichlorofluorescein was recorded for 5 minutes. Results are means ± SEM for 5 determinations. *Different from control (P < 0.01). Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Possible interference of DEAEH with mitochondrial function and lipid metabolism. The transfer of electrons along the respiratory chain is associated with the extrusion of protons from the mitochondrial matrix into the intermembranous space, creating a large membrane potential across the inner membrane. The proton gradient is then utilized to synthesize ATP. Unprotonated DEAEH may cross the outer membrane (step 1) and be protonated in the acidic intermembranous space (step 2). The protonated DEAEH may be “pushed” by the membrane potential into the matrix (step 3) and be partially depronated (step 4). The accumulation of DEAEH at high concentrations within mitochondria inhibits both β-oxidation enzymes (causing steatosis) and the respiratory chain. The latter effect decreases ATP and also increases the mitochondrial formation of reactive oxygen species that may oxidize fat deposits, causing extensive lipid peroxidation. Gastroenterology 1998 114, 764-774DOI: (10.1016/S0016-5085(98)70590-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions