Generation of free radical metabolites and superoxide anion by the calcium indicators arsenazo III, antipyrylazo III, and murexide in rat liver microsomes.

At the concentrations usually employed as a Ca2+ indicator, arsenazo III undergoes a one-electron reduction by rat liver microsomes to produce an azo anion radical as demonstrated by electron spin resonance spectroscopy. Either NADH or NADPH can serve as a source of reducing equivalents for the production of this free radical by rat liver microsomes. The steady state concentration of the azo anion radical is proportional to the square root of the protein concentration, suggesting that the radical decays through a nonenzymatic second order process. The steady state concentration of the azo anion radical is not altered in the presence of metyrapone or CO, and is decreased in the presence of NADP+ or p-hydroxymercuribenzoate. These observations suggest that the formation of arsenazo III anion radical is mediated through NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Under aerobic conditions, addition of arsenazo III to rat liver microsomes produces an increase in electron flow from NAD(P)H to molecular oxygen, generating both superoxide anion and hydrogen peroxide. The steady state concentration of the azo anion radical, but neither oxygen consumption nor superoxide anion formation, is enhanced by calcium and magnesium, suggesting an enhanced azo anion radical-stabilization by complexation with the metal ions. Accordingly, the arsenazo III anion radical signal is abolished in the presence of paramagnetic metal ions (Fe3+, Gd3+, and Ni2+) and enhanced in the presence of other diamagnetic metal ions (La3+). Antipyrylazo III is less effective than arsenazo III in increasing superoxide anion formation by rat liver microsomes, and gives a much weaker ESR spectrum of an azo anion radical. Murexide is reduced to the monodehydro-5,5'-iminobarbituric acid radical by rat liver microsomes, and its efficiency as a superoxide anion generator is intermediate between arsenazo III and antipyrylazo III.