Inactivación de la tripanotiona reductasa de trypanosoma cruzi por radicales libres catiónicos de fenotiazinas

Peroxidase/H₂O₂/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H₂O₂/phenotiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H₂O₂/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 μM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100% inactivation of TR. With the MPO/H₂O₂ systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H₂O₂/PZ, / TMPZ,/TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) anal -Cl, -CF₃, -SCH₃, COCH₂CH₃ and -CN in position C2 exerted significant influence on phenotiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H₂O₂/PZ and MPO/H₂O₂/PZ systems. The HRP/H₂O₂ and MPO/H₂O₂/ phenothiazines systems generated the corresponding cationic radicals (FTZ^̇+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ^+̇; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H₂O₂/phenothiazine systems, as well as with the FTZ^̇+ role in phenothiazine cytotoxicity.