Chemiluminescence of superoxide generated byCandida albicans: differential effects of the superoxide generator paraquat on a wild-type strain and a respiratory mutant

Abstract

We previously reported that a respiration-competent parent strain (K) of Candida albicans was more susceptible to the intracellular superoxide radical (O2-) generator paraquat (PQ) than was a respiration-deficient mutant (KRD-19), although both showed a similar sensitivity to extracellularly generated O2-. To clarify the cause of the differential PQ lethality, we developed a chemiluminescence method for measuring O2- generated by C. albicans cells by using the probe methyl-Cypridina-luciferin analogue (MCLA), and examined the effects of PQ on O2- generation in both parent and mutant strains. Endogenous O2- generation without stimulation by PQ was unexpectedly low in both strains. PQ-induced O2- generation in the parent strain was maximal in logarithmic phase cells and lowered in stationary phase cells. In contrast, O2- generation in the mutant remained low throughout the growth phase, even when stimulated by PQ. The extent of PQ-induced O2- generation in the parent strain depended on the carbon source added to the assay mixture; in decreasing order, glucose, glycerol, no carbon source. The inhibitor of the cytochrome respiratory chain, antimycin A, suppressed almost completely the PQ-induced O2- generation in the parent strain. It has been established that PQ is converted to its radical form (PQ+) by receiving a single electron in cells. PQ+ then reduces molecular oxygen to O2- by redox cycling. Thus, the high tolerance to PQ of the respiration-deficient mutant can be explained by minimal PQ+/O2- production due to the limited supply of electrons from the impaired respiratory system.

Mitochondria Are a Major Source of Paraquat-induced Reactive Oxygen Species Production in the Brain

“…These studies point out the involvement of several mitochondrial (30), microsomal (31,32), and cytosolic (23,33) could be considered a potential site for PQ 2ϩ reduction (34). Additionally, in Candida albicans, the inhibition of respiration almost completely suppressed PQ 2ϩ -induced generation of ROS (35). However, the relevance of mitochondria in mammalian systems has remained relatively unexplored.…”

Evidence for Electron Equilibrium between the Two Hemes bL in the Dimeric Cytochrome bc1 Complex

“…MCLA has a high sensitivity for O 2 . in the neutral pH range (34,35). The MCLA chemiluminescence method, which has been widely used to detect O 2 .…”