Use of a potentiometric vital dye to determine the effect of the herbicide bromoxynil octanoate on mitochondrial bioenenergetics in Chlamydomonas reinhardtii

Abstract

The aims of the present study were to validate a vital mitochondrial potentiometric staining method in Chlamydomonas reinhardtii and to utilise this method to examine the effect of the herbicide bromoxynil octanoate on mitochondrial potential in this species. A range of stains was investigated, including Rhodamine 123, DASPMI, Mitotracker Green, Mitotracker Orange and JC-1. Rhodamine 123 (R123) had the highest utility of several candidate stains. Incubation with both 5 and 10 µM carbonyl cyanide 3-chlorophenylhydrazone caused significant fluorescence collapse [Dunn's post test (40.00, P < 0.01) and (45.49, P < 0.01) respectively], demonstrating that the R123 fluorescence reported mitochondrial potential. The effect of the herbicide bromoxynil octanoate was examined. Exposure to 0.1 mM of bromoxynil resulted in a significant increased mitochondrial fluorescence compared with the baseline (Mann–Whitney U = 222, P < 0.002), while concentrations of 1 mM and greater resulted in significant, almost complete loss of mitochondrial potential [mean fluorescence ratio = 1.193–1.289 (where a ratio of 1 represents total potential loss), Mann–Whitney U = 0.0, P < 0.001 (1 mM), 0.0, P < 0.0001 (2 mM), 0.0, P < 0.0001 (5 mM)]. EC50 of the collapse in mitochondrial potential owing to bromoxynil incubation occurred at 0.72 mM, and the mean t50 of bromoxynil octanoate action was 93 s. R123 is a sensitive potentiometric dye in C. reinhardtii that may find further use in investigations of both mitochondrial bioenergetics in plants and environmental toxicology.