EFFECT OF HIGH LEVELS OF HEMIN ON THE ANTIOXIDANT DEFENSE SYSTEM OF TRYPANOSOMA CRUZI

Trypanosomatid protozoa need heme compounds for growth in vitro. For T. cruzi epimastigotes grown under different hemin concentrations in the medium, we have previously observed that 5 mg/l hemin was the concentration yielding optimum growth, whilst concentrations above 15 mg/l clearly decreased growth rate, producing undesiderable morphologic changes.

Because porphyrins are an important source of oxidative stress in biologycal systems, we have evaluated the oxidant response to the presence of high levels of hemin in the culture medium of T. cruzi. Employing concentrations between 0-30 mg/l we have measured: 1) total protein content, 2) the antioxidant defense system including the enzymes: superoxide dismutase (SOD), ascorbate peroxidase (APx), trypanothione reductase (TryR) and total thiols content.

Level of total proteins was determined for hemin concentrations 0, 10 and 30 mg/l between 3-7 days of growth. Without hemin, protein concentration progressively increased from 6.41 ± 0.18 to 8.73 ± 0.25 mg/10⁶cel, whilst for hemin 10 mg/l the values were 10-15% higher, showing a maximum level at day 5 of growth, without changing thereafter. For hemin 30 mg/l a different behaviour was obtained, the concentration of proteins increased within 3-5 days (from 9.26 ± 0.10 to 10.12 ± 0.08 mg/10⁶cel), and then progressively decrease to 7.24 ± 0.25 mg/10⁶cel. The antioxidant enzymes showed a maximum activity at hemin 5 mg/l (SOD 4.50 ± 0.05 UE/mg, APx 9.50 ± 0.04 UE/mg and TryR 3.72 ± 0.08 UE/mg). For hemin 5-30 mg/l TryR and SOD activity decreased about 30-35% whilst APx activity diminished 20%. Thiols levels showed a constant value (15.53 ± 0.89 nmoles/mg prot) for hemin 0-10 mg/l and then decreased to 9.54 ± 0.46 nmoles /mg prot for hemin 30 mg/l.

These findings provide strong evidence for the existence of a direct correlation between high extracellular hemin concentrations and cellular oxidative damage.