Ou X, Thomas G R, Chacón M R, Tang L, Selkirk M E
Wellcome Research Centre for Parasitic Infections, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Exp Parasitol. 1995 May;80(3):530-40. doi: 10.1006/expr.1995.1065.
The sensitivity of microfilariae and adult Brugia malayi to hydrogen peroxide (H2O2) was determined in vitro, and parasite viability assessed by incorporation of 2-deoxy-D-[1-3H]glucose. Both stages were surprisingly resistant to peroxide stress. Microfilariae tolerated the direct addition of H2O2 to medium in which they were incubated at concentrations up to 50 microM, whereas adult worms survived the addition of 100 microM H2O2 and showed slightly impaired viability at 150 microM H2O2. Higher concentrations were lethal in both cases. This observation of differential susceptibility was reproducible when parasites were subjected to continuous generation of H2O2 via glucose/glucose oxidase. Microfilariae remained viable over a 4-hr period when challenged with concentrations which generated 20 microM H2O2 in the absence of parasites. Adults survived higher concentrations of glucose oxidase, which generated 200 microM H2O2 over the same time period. Under these conditions the parasites effectively countered the rate of peroxide generation by metabolising the product. Protein carbonyl formation was detectable at sublethal concentrations of glucose/glucose oxidase, but malonaldehyde formation was only detectable coincident with parasite death. The rate of H2O2 consumption by parasites was determined and showed that adult worms metabolised it at a rate 23x faster than microfilariae, expressed as activity per wet weight. Assessment of enzyme activities in parasite extracts demonstrated that H2O2 metabolism was effected principally by catalase activity, which was elevated in adult worms relative to microfilariae. Cytochrome c peroxidase activity was also detected and was roughly equivalent in both stages. Glutathione peroxidase and NADH/NADPH-dependent consumption of H2O2 were absent, and the rate of nonenzymic reduction of H2O2 coupled to glutathione oxidation did not contribute significantly to metabolism. Glutathione reductase activity and total glutathione content were equivalent in adults and microfilariae. This study illustrates that Brugia malayi are much more resistant to H2O2 than other filarial species examined to date and can effectively metabolise levels in excess of those potentially generated by activated leucocytes.
