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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.

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The in vitro metabolism of SDZ HDL 376, a thiocarbamide developed for the treatment of atherosclerosis, was investigated in rat, dog, monkey, and human liver microsomes, as well as in rat and human liver slices. [14C]SDZ HDL 376 was extensively metabolized in all the species except human. In rat liver microsomes an S-oxide was the major metabolite. In human and monkey microsomes, carbon hydroxylation was favored. The NADPH-dependent oxidation of SDZ HDL 376 resulted in covalent binding to microsomal protein. Addition of GSH to the incubations decreased protein binding in a concentration-dependent manner and resulted in a novel SDZ HDL 376-GSH adduct. Adduct formation required NADPH and was mediated predominantly by cytochrome P450. Inhibition of cytochrome P450 by 1-aminobenzotriazole resulted in a 95% decrease in adduct formation, while heat inactivation of flavin-containing monooxygenases resulted in a 10% decrease. Unlike other thiocarbamides which form disulfide adducts with GSH, the SDZ HDL 376 adduct contained a thioether linkage as characterized by LC/MS/MS and reference to a synthetic standard. Reactions performed with [35S]GSH resulted in a [35S]SDZ HDL 376-GSH adduct, demonstrating the sulfur was derived from GSH. Adduct formation was faster in rat microsomal reactions compared to human microsomes. Other structurally unrelated thiocarbamides (phenylthiourea, methimazole, 2-mercaptobenzimidazole, 2-mercaptoquinazoline, and 2-propyl-6-thiouracil) did not form similar adducts in rat liver microsomes supplemented with GSH. Therefore, the GSH adduct of SDZ HDL 376 is unique for this type of thiocarbamide. These results suggest that the bioactivation and detoxification of SDZ HDL 376 differ significantly from other thiocarbamides. Furthermore, the in vitro formation of S-oxides and GSH adducts in rat hepatic tissue, and ring hydroxylation and glucuronidation in human hepatic tissue, suggests rats may be more susceptible to the toxicity of SDZ HDL 376 compared to humans.

Cyclin-dependent kinases (CDKs) have been pursued for more than a decade for the treatment of cancer. CDK inhibitors are expected to slow the rate of cell division and potentially increase the apoptotic fraction of rapidly dividing cells. Although CDK activity is often increased in tumors, normal dividing tissues are also susceptible to the cytostatic and cytotoxic effects of CDK inhibitor action. Therefore the typical toxicity profile associated with cytotoxic anti-cancer therapy, bone marrow suppression and gastrointestinal toxicity, is expected with CDK inhibitors. Bone marrow toxicity and the ensuing delayed peripheral leukocyte suppression often limit the therapeutic application of cytotoxic anticancer drugs. Here we characterize an unusual bone marrow-independent acute toxicity toward leukocytes from broad spectrum CDK inhibitors in monkeys and rodents. The potential combination of both acute and delayed immunosuppression would likely further restrict the application of these particular compounds. Since the cells targeted were non-proliferating, it was assumed that the toxicity was not driven by the intended pharmacological mechanism thereby facilitating the development of a testing strategy to identify compounds with a reduced potential for acute leukocyte toxicity. This testing strategy resulted in a CDK inhibitor void of bone marrow-independent leukocyte toxicity that is currently undergoing clinical testing.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.

This investigation examined microRNA-208a (miR-208a) as a potential biomarker of isoproterenol (ISO)-induced cardiac injury in superoxide dismutase-2 (Sod2(+/-) ) and the wild-type mice, and the potential sensitivity of Sod2(+/-) mice to ISO-induced toxicity. A single intraperitoneal injection of ISO was administered to age-matched wild-type and Sod2(+/-) mice at 0, 80, or 160 mg/kg. Plasma miR-208a, cardiac troponin I (cTnI), and ISO systemic exposure were measured at various time points postdose. Hearts were collected for histopathology examination and for tissue expression of miR-208a and myosin heavy chain 7. ISO administration caused increases in cTnI and miR-208a plasma levels that correlated with myocardial damage; however, the magnitude of increase differed according to the types of mice. At similar ISO systemic exposure, the magnitude of cTnI was greater in wild-type mice compared to Sod2(+/) (-) mice; however, the magnitude of miR-208a was greater in Sod2(+/-) mice than that of the wild-type mice. Myocardial degeneration occurred at ≥3 hr in the wild-type and ≥6 hr in Sod2(+/) (-) mice. At ≥24 hr after ISO administration, miR-208a appeared superior to cTnI in indicating myocardial injury in both wild-type and Sod2(+/-) mice. Sod2(+/-) mice were not more sensitive than wild-type mice to ISO-induced toxicity.