Role of respiratory uncoupling in drug-induced mitochondrial permeability transition.

Mitochondrial injury contributes to severe drug-induced liver injury. Particularly, mitochondrial permeability transition (MPT) is thought to be relevant to cytolytic hepatitis. However, the mechanism of drug-induced MPT is unclear and prediction of MPT is not adequately evaluated in the preclinical stage. In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling. Herein, we investigated whether other drugs that induce MPT share similar properties as troglitazone, using isolated mitochondria from rat liver. Of the 22 test drugs examined, six drugs, including troglitazone, induced MPT and showed an uncoupling effect. Additionally, receiver operating characteristic analysis was conducted to predict the MPT potential from the respiratory control ratio, an indicator of uncoupling intensity. Results showed that 2.5 was the best threshold that exhibited high sensitivity (1.00) and high specificity (0.81), indicating that uncoupling was correlated with MPT potential. Activation of calcium-independent phospholipase A2 appeared to be involved in uncoupling-induced MPT. Furthermore, a strong relationship between MPT intensity and the uncoupling effect among similar compounds was confirmed. These results may help in predicting MPT potential using cultured cells and modifying the chemical structures of the drugs to reduce MPT risk.

SPG7 targets the m-AAA protease complex to process MCU for uniporter assembly, Ca2+ influx, and regulation of mitochondrial permeability transition pore opening.

The mitochondrial matrix ATPase associated with diverse cellular activities (m-AAA) protease spastic paraplegia 7 (SPG7) has been recently implicated as either a negative or positive regulatory component of the mitochondrial permeability transition pore (mPTP) by two research groups. To address this controversy, we investigated possible mechanisms that explain the discrepancies between these two studies. We found that loss of the SPG7 gene increased resistance to Ca2+-induced mPTP opening. However, this occurs independently of cyclophilin D (cyclosporine A insensitive) rather it is through decreased mitochondrial Ca2+ concentrations and subsequent adaptations mediated by impaired formation of functional mitochondrial Ca2+ uniporter complexes. We found that SPG7 directs the m-AAA complex to favor association with the mitochondrial Ca2+ uniporter (MCU) and MCU processing regulates higher order MCU-complex formation. The results suggest that SPG7 does not constitute a core component of the mPTP but can modulate mPTP through regulation of the basal mitochondrial Ca2+ concentration.

Sex Differences in Ischemia/Reperfusion Injury: The Role of Mitochondrial Permeability Transition.

Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity of these diseases. Ca2+ dysregulation in response to ischemia/reperfusion injury (I/RI) is a well-recognized pathogenic mechanism leading to the death of affected cells. Excess intracellular Ca2+ causes mitochondrial matrix Ca2+ overload that can result in mitochondrial permeability transition (MPT), which can have severe consequences for mitochondrial function and trigger cell death. Recent findings indicate that estrogens and their related receptors are involved in the regulation of MPT, suggesting that sex differences in I/RI could be linked to estrogen-dependent modulation of mitochondrial Ca2+. Here, we review the evidence supporting sex differences in I/RI and the role of estrogen and estrogen receptors in producing these differences, the involvement of mitochondrial Ca2+ overload in disease pathogenesis, and the estrogen-dependent modulation of MPT that may contribute to sex differences.

Impact of Pineapple on Mitochondrial Permeability Transition and Drug Metabolizing Genes in Caco-2 Cells.

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i> L.) has antioxidant and other pharmacological properties. This study examined how pineapple modified mitochondrial permeability transition and expression of drug-metabolizing enzymes, i.e., CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and the drug transporter OATP1B1 in human colorectal adenocarcinoma (Caco-2) cells. <b>Materials and Methods:</b> Caco-2 cells (2.5×10⁵ cells well¹ in 24-well plates) were incubated with pineapple (125 to 1,000 µg mL¹) for 48 hrs in a phenol red-free medium. Mitochondrial permeability transition, resazurin cell viability and AST and ALT levels were investigated. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple significantly reduced depolarized mitochondria, slightly decreased cell viability and did not change AST and ALT levels. Pineapple did not modify the mRNA expressions of CYP1A2, CYP2C9 and CYP3A4 but markedly induced UGT1A6 expression. The highest tested concentration of pineapple (1,000 µg mL¹) significantly suppressed NAT2 and OATP1B1 expression. <b>Conclusion:</b> Although pineapple slightly decreased cell viability to ~80% of control, the morphology and functions of the cells were unaffected. Pineapple showed a beneficial effect to reduce depolarized mitochondria, which consequently decreased reactive oxygen species production. Pineapple did not modify the expression of CYPs, whilst it altered the expression of phase 2 metabolizing genes UGT1A6 and NAT2 and the transporter OATP1B1. Therefore, the consumption of large amounts of pineapple is of concern for the risk of drug interaction via alteration of UGT1A6, NAT2 and OATP1B1 expression.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.