*3.6. Modifications in Redox Signaling Induced by Depletion of Fe in Culture Medium*

The mutual regulation of hexokinases and redox signaling comprises important events in parasites that cause severe diseases, e.g., *T. cruzi* [58]. This is the reason why we decided to investigate two enzymes involved in *T. cruzi* redox signaling and see the influence of Fe depletion: (i) superoxide dismutase (TcSOD), which has Fe as a cofactor in *T. cruzi* [59], and (ii) ascorbate peroxidase (TcAPX) [60], an enzyme that is absent in the human host [61], which metabolizes H2O2 to H2O. We measured TcSOD activity by following two strategies. First, by evaluating the formation of nitroblue tetrazolium formazan (NBT formazan) after oxidation of xanthine to uric acid and generation of O2 •−, a reaction in which the higher

superoxide dismutase activity is, the lower NBT formazan formation will be. Second, measuring the H2O2 formed through different processes, including the premature transfer of electrons to O2 in the *T. cruzi* mitochondrion. Table 5 shows that the SOD activity associated with the O2 •− formed during the oxidation of xanthine increased by approximately 200% in parasites grown in IDM regardless of supplementation with Fe. Moreover, when the total H2O2 production was assayed, a significant decrease of about 40% was found in IDM parasites, which was suppressed in the IDM + Fe group. Finally, the expression profile of TcSOD was the mirror of the activity, whereas that of TcAPX matched the activity, i.e., decreased in the parasites grown in IDM, with recovered levels in the IDM + Fe group.

**Table 5.** Effect of exogenous Fe on redox signaling \*.


\* SOD activity (*n* = 4) and production of H2O2 (*n* = 3) were assessed in living epimastigotes maintained in RM, IDM, or IDM + Fe. The quantification of the TcSOD (*n* = 5) and of the TcAPX transcripts (*n* = 5) in *T. cruzi* epimastigotes was done using 100 ng cDNA from epimastigotes grown in each culture medium. We used one-way ANOVA with Tukey's test to assess differences between mean values; Different lower-case letters as superscripts indicate statistical differences among the mean values in the same line (*p* < 0.05).
