β -OXIDATION OF [9,10(n)- 3 H]PALMITATE BY HUMAN LEUKOCYTES: A SIMPLE IN SITU ASSAY TO ASSESS MITOCHONDRIAL TOXICITY IN THE PRESENCE OF TOXINS

We utilized the β-oxidation of [9,10(n)- 3 H]palmitate in human leukocytes as a simple assay to assess mitochondrial toxicity. Advantages of the use of tritiated fatty acids in lieu of 14 C-labeled fatty acids include less interassay variation, higher specific activities obtained at a lower cost,and the relatively small number of cells required. In our assay, only 50 μg cellular protein ( 5×104 leukocytes) are required per β-oxidation reaction as opposed to 1×10 6 leukocytes required in the beta-oxidation reaction with 14 C-labeled fatty acids. The higher specific activities obtained substantially reduced assay volumes, and 96 well microtiter plates could be used to conduct the assays. Rotenone (a mitochondrial complex I inhibitor) and antimycin (a mitochondrial complex III inhibitor) acutely inhibited the β-oxidation of [9,10(n)- 3 H]palmitate, thereby validating our assay. This assay offers an in situ or even a pseudo in vivo rendering of mitochondrial inhibition while potential effects of the cell membrane or cytosol or both on the toxicology of the compounds are simultaneously taken into account. We exploited these properties of our assay to investigate the insitu mitochondrial inhibitory properties of the Parkinsonian-inducing drug MPTP and its neurotoxic pyridinium metabolite, MPP+.