Thiazolidinedione toxicity to isolated hepatocytes revealed by coherent multiprobe fluorescence microscopy and correlated with multiparameter flow cytometry of peripheral leukocytes

Abstract

Thiazolidinediones (TZDs) are effective for the treatment of adult-onset insulin-resistant diabetes. Unfortunately, TZDs are associated with sporadic hepatic dysfunction that is not predictable from experimental animal studies. We investigated the response of isolated rat and human hepatocytes to various TZDs using biochemical assays, coherent multiprobe fluorescence microscopy and flow cytometric analyses. The results identified direct effects of TZD on mitochondria from live human and rodent hepatocytes. The multiprobe fluorescence assays showed disruption of mitochondrial activity as an initiating event followed by increased membrane permeability, calcium influx and nuclear condensation. Other TZD-related cellular effects were increased hepatic enzyme leakage, decreased reductive metabolism and cytoplasmic adenosine triphosphate depletion. Mitochondrial effects were similar in cryopreserved hepatocytes from diabetic or non-diabetic donors. Peripheral blood mononuclear cells (PBMCs) had baseline mitochondrial energetics and metabolism comparable with isolated hepatocytes. Mitochondrial effects in isolated hepatocytes were found in human PBMCs exposed to the TZDs. The relative potency of TZDs for causing hepatocyte and PBMC effects was troglitazone > pioglitazone > rosiglitazone. These studies clearly demonstrated that hepatic alterations in vitro are characteristic of TZDs, with only quantitative differences in subcellular organelle dysfunction. Monitoring mitochondrial function in isolated PBMCs may be beneficial in diabetics undergoing TZD therapy.

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

“…36 Since then, numerous studies have identified the crucial role of ROS in causing cellular damage, [36][37][38][39] authorizing the technique as the valid and reliable substitute for other biochemical methods to detect cellular ROS. Flow cytometric detection of ROS applied in both the previous toxicologic study on hepatocytes 31 and the present study has been shown to be efficient to reveal the functional influences of potential toxicants, such as troglitazone, which have hitherto been not detected by con- ventional premarketing screening, including any animal experiments. 40,41 In this study, the OUMS-29 cells showed a gradual, but significant, decrease in unit ⌬⌿ m after long exposure to troglitazone, although not resulting in clear cell death.…”

“…40,41 In this study, the OUMS-29 cells showed a gradual, but significant, decrease in unit ⌬⌿ m after long exposure to troglitazone, although not resulting in clear cell death. Lack of obvious cell death in troglitazone-treated hepatocytes has also been reported, 31 suggesting that the troglitazone-induced ⌬⌿ m attenuation was not a critical inducer, but a possible priming, for hepatocyte death. It was speculated that enhanced accumulation in troglitazone in the liver of poor metabolizers might attenuate ⌬⌿ m in hepatocytes, triggering subsequent events for severe hepatic damage, although candidate genetic mutations in the metabolic enzymes in the poor metabolizers, such as CYP2C8, CYP3A4, and CYP2C19, have not yet been determined.…”

“…Recently, mitochondrial dysfunction associated with troglitazone has been demonstrated in hepatocytes using flow cytometry, consistent with our findings of the ⌬⌿ m attenuation. 31 Furthermore, the sulfate conjugate of troglitazone, as well as the parent drug, has been found to be involved in hepatotoxicity, showing both mitochondrial damage and inhibition in bile formation, mainly through impairment in the bile salt excretory pump. 32,33 Hepatocellular retention of the hydrophobic toxic bile salts causes hepatocyte apoptosis via mitochondrial membrane permeability transition.…”

Animal Models of Human Disease in Drug Safety Assessment

“…While normal hepatocytes tolerated troglitazone concentrations up to 100 µM without detectable mitochondrial effects, hepatocytes from one diabetic donor exhibited decreases in the mitochondrial membrane potential upon exposure to 50 µM and higher (in a second diabetic donor this was not observed) (Haskins et al, 2001). Whether liver cells from humans with T2D indeed react differently to potentially toxic drugs remains to be determined.…”

Mitochondrial dysfunction and delayed hepatotoxicity: another lesson from troglitazone

“…Thus, such responses as alteration of bioenergetics, metabolism and mitochondrial function are PPARγ-independent [17]. The order of toxicity by which TZDs cause mitochondrial dysfunction is troglitazone>pioglitazone and rosiglitazone [14,43]. The hierarchy of clinical and cellular hepatotoxicity correlates strongly to mitochondrial impairment and not to PPARγ affinity [14,17,43].…”