THE USE OF TETRAZOLIUM SALTS TO DETERMINE SITES OF DAMAGE TO THE MITOCHONDRIAL ELECTRON-TRANSPORT CHAIN IN INTACT-CELLS FOLLOWING IN-VITRO PHOTODYNAMIC THERAPY WITH PHOTOFRIN-II

A method is described utilizing the tetrazolium salts neotetrazolium c hloride (NTC), triphenyltetrazolium chloride (TTC), ,N-diphenyl-N'-4,5 -dimethylthiazol-2-yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of i ntact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transpo rt chain through Complex I, as evidenced by the fact that the inhibiti on of MTT reduction was reversible by the addition of malic acid to th e culture media. A second site of dark toxicity was found to be Comple x IV (cytochrome oxidase). After photoirradiation of the cells, Comple x I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the additi on of succinic acid, whose electrons enter the transport chain at Comp lex II. A second and more sensitive site of photoirradiation damage wa s found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoi rradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contr ast, TTC reduction was found to be inhibited almost immediately indica ting Complex IV is extremely susceptible to photoirradiation damage. C ompared to other assays of mitochondrial function requiring subcellula r fractionation, the use of tetrazolium salts is simpler to perform an d can be done using physiologically relevant conditions.