Induction of oxidative stress in Prototheca zopfii by indole-3-acetic acid/HRP or 2,4-pentanedione/HRP systems and their oxidation products.

We investigated the toxic effects on Prototheca zopfii of indole-3-acetic acid (IAA) and 2,4-pentanedione (PD) combined with horseradish peroxidase (HRP) alongside the oxidation products of 3-methyl-2-oxindole (MOI) and indole-3-carbinol (I3C) from the IAA/HRP system and methylglyoxal (MGO) from the PD/HRP system. The microorganism was incubated in the absence (control) or presence of IAA, PD, IAA/HRP, PD/HRP, MOI, I3C and MGO and determined: (1) cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay; (2) growth inhibitory concentration by resazurin assay and (3) antioxidant enzymes activities of: catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). P. zopfii was more susceptible to IAA at 40 mM than PD at the same concentration, which seems to indicate that IAA was more effective at initiating cell death. These data corroborate results from the resazurin assay. Concentrations of 40 mM of IAA, IAA/HRP and PD/HRP, 20 mM of PD/HRP, 10 mM of MOI, 2 mM of I3C and 8 mM of MGO inhibited the growth of P. zopfii. With sub-inhibitory concentrations of IAA and IAA/HRP at 30 mM, MOI at 8 mM and I3C at 1 mM, the activities of CAT and GR increased, whereas no statistical difference was observed for CAT activity with IAA/HRP. Thus, PD at 30 mM and MGO at 6 mM increased the activities of CAT and GR, whereas PD/HRP system at 15 mM decreased CAT activity and PD/HRP and MGO showed no statistical difference for SOD activity. In conclusion, IAA/HRP or PD/HRP systems and their oxidation products exert cytotoxic effects on P. zopffi; however, I3C and MGO appear to exert greater microbicidal effect on P. zopfii.