title: Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation.
abstract: BACKGROUND
  In cellular viability assays the sole determination of a single parameter might not give precise information on the extent of toxicity. In our study we worked out a multiparametric microplate assay based on bioluminescent ATP quantification, esterase activity-related fluorescence, nucleic acid staining and total intracellular protein measurement from the same sample in MDCK and HepG2 tissue cultures.
  
  
  METHODS
  Dose-response analyses were done after ATP depletion by metabolic poisons (NaF, NaN3) and by ochratoxin A (OTA) mycotoxin treatments. A novel perchloric acid fixation/extraction technique was applied in order to obtain intracellular ATP levels, esterase activity, DNA content and protein data simultaneously. Esterase activity was assessed by a fluorogenic staining. Estimation of cell number was done by DAPI fluorescence. Our results were expressed as ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein and ATP/DAPI ratios. Apoptosis/necrosis rates were measured by Annexin V-propidium iodide and 7-aminoactinomycin D flow cytometric assays and effects of OTA on actin cytoskeleton were also studied by using labeled phalloidin for visualization of actin.
  
  
  RESULTS
  We could verify that the esterase assay was not an energy driven (true viability) process. ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein ratios, DAPI fluorescence and protein levels together with morphological and apoptosis/necrosis parameters deciphered subtle changes in cell viability with good between-run precision. Dose dependent loss in cell number and decreased protein levels were observed in all cases, while disorganization of actin microfilaments was seen in OTA treated cells. The two cell lines did not respond uniformly to the same treatments.
  
  
  CONCLUSIONS
  ATP/protein ratio proved to be a useful viability parameter however, the suppression and/or loss of intracellular protein could cause difficulty in interpreting ATP/protein data. We conclude that correct assessment of cellular viability should be done by measuring multiple parameters related to the specific mode of action of the tested toxic compound.
authors.affiliation: Department of Laboratory Medicine, University of Pécs, H-7624 Pécs, Ifjuság u. 13., Hungary. Electronic address: niki26@gmail.hu.
authors.forename: Nikolett
authors.initials: N
authors.lastname: Sali
authors.affiliation: Institute of Pharmaceutical Technology and Biopharmacy, University of Pécs, H-7624 Pécs, Rókus u. 2., Hungary. Electronic address: sandor.nagy@aok.pte.hu.
authors.forename: Sándor
authors.initials: S
authors.lastname: Nagy
authors.affiliation: Department of Laboratory Medicine, University of Pécs, H-7624 Pécs, Ifjuság u. 13., Hungary. Electronic address: poor.miklos@pte.hu.
authors.forename: Miklós
authors.initials: M
authors.lastname: Poór
authors.affiliation: Department of Laboratory Medicine, University of Pécs, H-7624 Pécs, Ifjuság u. 13., Hungary; János Szentágothai Research Center, H-7624 Pécs, Ifjuság u. 20., Hungary; Melius Foundation, H-7691 Pécs, Mázsaház u. 24., Hungary. Electronic address: tamas.koszegi@aok.pte.hu.
authors.forename: Tamás
authors.initials: T
authors.lastname: Kőszegi
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008954:Models, Biological; D009183:Mycotoxins / Q000633:toxicity*; D009793:Ochratoxins / Q000633:toxicity; D011506:Proteins / Q000378:metabolism
chemical_list: D017304:Annexin A5; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009183:Mycotoxins; D009793:Ochratoxins; D011506:Proteins; C025589:ochratoxin A; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D000255:Adenosine Triphosphate; C007740:fluorexon
keywords: ATP depletion; ATP/protein ratio; Cell cultures; DAPI staining; Luminescence viability assays; Perchloric acid extraction