title: Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™.
abstract: In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose. The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10(-3)M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10(-5)M, and only 19 (38%) of these compounds appeared to be toxic in both assays. In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds were identified, whereas MitoXpress was more sensitive for a few compounds. A species specific difference was clearly found with digoxin, a human specific potassium channel inhibitor. Thus both assays are valuable identifiers of early toxicity with discrimination in time, compounds and species.
authors.affiliation: Toxicology & Drug Disposition, Merck Research Laboratories, P.O. Box 20, 5340 BH Oss, The Netherlands. willem.schoonen@gmail.com
authors.forename: Willem G E J
authors.initials: WG
authors.lastname: Schoonen
authors.forename: Joe C R
authors.initials: JC
authors.lastname: Stevenson
authors.forename: Walter M A
authors.initials: WM
authors.lastname: Westerink
authors.forename: G Jean
authors.initials: GJ
authors.lastname: Horbach
mesh_terms: D000818:Animals; D002460:Cell Line; D064420:Drug-Related Side Effects and Adverse Reactions; D005470:Fluorometry; D006019:Glycolysis / Q000187:drug effects; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009249:NADP / Q000187:drug effects / Q000378:metabolism; D010078:Oxazines / Q000737:chemistry; D010101:Oxygen Consumption / Q000187:drug effects*; D004364:Pharmaceutical Preparations / Q000008:administration & dosage; D051381:Rats; D013045:Species Specificity; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D010078:Oxazines; D004364:Pharmaceutical Preparations; D014966:Xanthenes; C005843:resazurin; D009249:NADP