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The action of digitonin on rat liver mitochondria. The effects on enzyme content.

ABSTRACT: 1. The effects of repetitive treatment of rat liver mitochondria with digitonin were examined. The first treatment results in the removal of the outer membrane. Almost all the NADH-cytochrome c reductase (rotenone-insensitive) is lost whereas the major portions of the soluble and bound enzymes are retained. One exception appears to be the cytochromes, which undergo somewhat larger losses. The resulting inner-membrane complex carries out oxidative phosphorylation and P(i)-ATP exchange. 2. The properties of the inner-membrane complex are affected by the osmoticity of the medium. When it is suspended in water little protein is lost but there is a marked loss of phosphorylation. If after the suspension in water the particulate fraction is reisolated by centrifugation and treated with digitonin, or if the aqueous suspension is treated directly with digitonin and the particulate fraction then reisolated, the phosphorylation is largely restored. 3. Additional treatment of the inner mitochondrial complex with digitonin results in the formation of a particulate fraction that contains approx. 8% of the initial mitochondrial protein, no outer membrane, no soluble mitochondrial enzymes and is still capable of coupled oxidative phosphorylation and P(i)-ATP exchange. These effects cannot be reproduced by treatment with water. 4. The rat liver mitochondria and all of the resulting preparations obtained after digitonin treatment may be stored for long periods in dimethyl sulphoxide with little change of activity.

SUBMITTER: Hoppel C

PROVIDER: S-EPMC1198672 | biostudies-other | 1968 Apr

REPOSITORIES: biostudies-other

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Project description:The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

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The action of digitonin on rat liver mitochondria. The effects on enzyme content.

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