INHIBITION OF NA+ K+-ATPASE BY PHENOXYL RADICALS OF ETOPOSIDE (VP-16)- ROLE OF SULFHYDRYLS OXIDATION/

In the present work, we studied the effects of phenoxyl radicals, gene rated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na+/K+-ATPase by character izing interactions of VP-16 phenoxyl radicals with the enzyme's SH-gro ups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na+/K+-ATPase which was dependent on the incubati on time and concentration of tyrosinase. The inhibition of Na+/K+-ATPa se was accompanied by a decrease of DTNB (5,5'-dithiobis-(2-nitrobenzo ic acid)-titratable SH-groups. In the presence of Na+/K+-ATPase, a typ ical ESR signal of the VP-16 phenoxyl radical could be observed only f ollowing a lag period the duration of which was proportional to the co ncentration of the Na+/K+-ATPase added. Our HPLC measurements demonstr ated that Na+/K+-ATPase protected VP-16 against tyrosinase-catalyzed o xidation. Combined these results suggest that redox-cycling of VP-16/V P-16 phenoxyl radical by SH-groups of Na+/K+-ATPase occurred. Ascorbat e which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-gr oups, Reduction of VP-16 phenoxyl radicals by ascorbate was directly o bserved by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na+/K+-ATPase may be responsible for at least some of its clin ical side effects (e.g., cardiotoxicity) which can be prevented by asc orbate.