The role of plasma membrane in bioreduction of two tetrazolium salts, MTT,and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bio reduction of these compounds have not been fully elucidated. We investigate d the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimet hylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts ap pear to represent examples of species that are reduced by different mechani sms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damag ed and remain metabolically active for at least 45 min. In contrast, CTC re mains extracellular with respect to viable cells and thus requires plasma m embrane permeable electron carrier to be reduced efficiently. However, redu ction of CTC in the presence of an electron carrier inflicts damage on plas ma membranes. The intracellular vs extracellular sites of reduction of tetr azolium salts were established on the basis of deposition of formazans. Cry stals of formazan were detected using fluorescence or backscattered light c onfocal laser microscopy. We postulate that the capacity of a tetrazolium s alt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the o utcome of tetrazolium assays designed to measure cellular production of oxy gen radicals, activity of mitochondrial, cytosolic, or outer membrane reduc tases, etc. (C)2000AcademicPress.