Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Abstract

Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazonetreated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (⌬⌿ m ) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2 ,7 -dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electrondense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the ⌬⌿ m per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity. (HEPATOLOGY 2003;37:136-147.) T roglitazone, a ligand for peroxisome proliferatoractivated receptor ␥ (PPAR␥), used to be administered as an antidiabetic drug until it was discovered to be associated with hepatotoxicity and withdrawn from the market after reports of several dozens of deaths or cases of severe hepatic failure requiring liver transplantation. 1,2 In contrast to troglitazone, 2 other glitazones, pioglitazone and rosiglitazone, are still on the market; however, several cautionary reports have suggested that the latter 2 drugs show hepatotoxicity as a class effect of glitazones. [2][3][4][5][6][7][8][9] Although the troglitazone-associated hepatotoxicity has been considered idiosyncratic direct hepatocyte toxicity, 10 the precise mechanism has remained unclear. In our recent study, mitochondrial damage was ultrastructurally demonstrated in troglitazone-treated human hepatoma cells, 11 allowing us to first focus on alterations in the mitochondrial structure and the function in the troglitazone-treated human hepatocytes. Troglitazone is known

Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells

“…**p<0.01 and ***p<0.001 vs control. Co Control, Tro troglitazone reports have shown that TZDs affect mitochondrial function and lower ATP production in human hepatocytes and H-2K b muscle cells [11,12,40,41]. Here, we extend these results by demonstrating that troglitazone induces acute mitochondrial membrane depolarisation, activates AMPK and accelerates glucose flux in muscle cells.…”

“…Unfortunately, troglitazone had rare but serious side effects causing subacute irreversible liver injury and failure [9] and was recently removed from the market [10]. In studies geared to uncover the cause of the hepatotoxicity using human or rat hepatocyte cell lines, several mitochondrial abnormalities were observed including the collapse of the mitochondrial membrane potential [11,12]. In those studies, the effect of troglitazone on mitochondrial membrane potential was rapid (within an hour) and occurred at concentrations approximating those considered therapeutically active.…”

Mitochondrial dysfunction and delayed hepatotoxicity: another lesson from troglitazone

“…This inferred conclusion will be based on an analysis of this case series and is also powerfully supported by cellular in-vitro studies and animal experiments [14][15][16][17]. The opposite premise, taken by some, which claims that this animal and cellular data are not relevant to the human experience, is far harder to defend.…”

“…These mitochondrial changes were inhibited by N-acetyl cysteine [15]. Furthermore, enhanced susceptibility to cell injury in conditions of preexisting oxidative stress/mitochondrial impairment has been demonstrated by Ong and Boelsterli in a mouse model of silent mitochondrial abnormality [44].…”

Effects of rosiglitazone on the liver histology and mitochondrial function in ob/ob mice

“…In fact, inhibition of complex I with rotenone or phytanic acid strongly enhances O 2 Ϫ generation. 38 A number of authors have reported that troglitazone produces mitochondrial changes in a hepatocyte cell line 39 and in HepG2 cells. 40 Scatena et al 41 and Brunmair et al 42 have demonstrated that PPAR-ligands decrease respiratory control, induce uncoupling of the oxidative phosphorylation, and reduce the activity of complex I in cell culture and tissue homogenates.…”