Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.

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Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.

Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

Mitochondrial dysfunction is a common mechanism of drug-induced toxicity. Early identification of new chemical entities (NCEs) that perturb mitochondrial function is of significant importance to avoid attrition in later stages of drug development. One of the most informative ways of assessing mitochondrial dysfunction is by measuring mitochondrial oxygen consumption. However, the conventional polarographic method of measuring oxygen consumption is not amenable to high sample throughput or automation. We present an alternative, low-bulk, high-throughput approach to the analysis of isolated-mitochondrial oxygen consumption using luminescent oxygen-sensitive probes. These probes are dispensable and are analyzed in standard microtitre plates on a fluorescence plate reader. Respiratory substrate and adenosine diphosphate (ADP) dependencies of mitochondrial oxygen consumption were assessed using the fluorescence-based method, and results compared favourably to conventional polarographic analysis. To assess assay performance, the method was then applied to the analysis of a panel of classical modulators of oxidative phosphorylation. The effect of uncoupler concentration was analyzed in detail to identify factors which would be important in applying this method to large scale NCE screening and mechanistic investigations. Results demonstrate that the 96-well format can accommodate up to approximately 200 compounds/day at a single concentration or alternatively IC(50) values can be generated for approximately 25 compounds. Throughput may be increased by moving to a 384-well plate format.

This work has developed a rabbit model of PD0325901-induced RVO that may be used to characterize the cellular and molecular mechanisms of this effect in humans.

Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities and negative side-effect profiles. Early identification of mitochondrial liabilities for new chemical entities is therefore crucial for avoiding late-stage attrition during drug development. Limitations of traditional methods for assessing mitochondrial dysfunction have discouraged routine evaluation of mitochondrial liabilities. To circumvent this bottleneck, a high-throughput screen has been developed that measures oxygen consumption; one of the most informative parameters for the assessment of mitochondrial status. This technique has revealed that some, but not all, members of many major drug classes have mitochondrial liabilities. This dichotomy encourages optimism that efficacy can be disassociated from mitochondrial toxicity, resulting in safer drugs in the future.