title: Use of a potentiometric vital dye to determine the effect of the herbicide bromoxynil octanoate on mitochondrial bioenenergetics in Chlamydomonas reinhardtii.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Crop and Environment Research Centre, Harper Adams University College Newport, Shropshire, UK. mark@biovital.org.uk
authors.forename: Mark R
authors.initials: MR
authors.lastname: Brickley
authors.forename: Emily
authors.initials: E
authors.lastname: Lawrie
authors.forename: Veronika
authors.initials: V
authors.lastname: Weise
authors.forename: Chris
authors.initials: C
authors.lastname: Hawes
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Cobb
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects*; D005456:Fluorescent Dyes / Q000737:chemistry; D006540:Herbicides / Q000378:metabolism / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000737:chemistry* / Q000187:drug effects; D009570:Nitriles / Q000378:metabolism / Q000494:pharmacology*; D011199:Potentiometry / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D006540:Herbicides; D009570:Nitriles; C006826:bromoxynil