title: The myotoxic effect of bupivacaine and ropivacaine on myotubes in primary mouse cell culture and an immortalized cell line.
abstract: BACKGROUND
  The 2 local anesthetics (LAs) bupivacaine and ropivacaine have acute cytotoxic effects on different tissues. In this respect, LA-induced myotoxicity has been subject to various studies; however, the exact mechanisms are still not fully understood. Most in vitro studies use immortalized cell lines because of feasibility. Thus, establishing a primary cell line might result in more accurate results. In this study, we examined the effects of immortalization on bupivacaine- and ropivacaine-induced myotoxicity in vitro.
  
  
  METHODS
  An immortalized (N = 6) and a primary cell line (N = 8) of the same tissue and species were established, and differentiation in myotubes was induced. Cells were exposed to increasing concentrations of bupivacaine and ropivacaine for 1 or 2 hours, respectively. Twenty-four and 48 hours after treatment, the fractions of dead and vital cells were measured using flow cytometry. Significance was tested through 1-way analysis of variance with post hoc Dunnett T3 test. Medians of dataset pairs were compared by T test.
  
  
  RESULTS
  In both cell lines, increasing concentrations of both LAs resulted in decreased cell survival (e.g., P < 0.001 for 5000 ppm bupivacaine, 1 or 2 hours of incubation, and 24 hours recovery in both cell lines). For the same LA concentrations, survival was significantly higher in the immortalized cell culture (e.g., P < 0.001 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). In addition, equal concentrations of bupivacaine resulted in significantly fewer vital cells compared with ropivacaine (e.g., P = 0.032 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). Two hours of incubation resulted in a significantly higher rate of dead cells compared with 1 hour of incubation (e.g., P = 0.004 for C2C12 cells, 2500 ppm bupivacaine, and 24 hours recovery).
  
  
  CONCLUSIONS
  Primary skeletal muscle cells are more vulnerable to LAs than immortalized cells. The higher myotoxic potential of bupivacaine compared with ropivacaine in vivo can be reproduced in vitro. Incubation time has an influence on cell survival.
authors.affiliation: From the Department of Anesthesiology, University Hospital Regensburg, Regensburg; and Department of Anesthesiology and Intensive Care Medicine, Klinikum Ludwigshafen, Ludwigshafen, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Hofmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Metterlein
authors.forename: Gabriele
authors.initials: G
authors.lastname: Bollwein
authors.forename: Michael
authors.initials: M
authors.lastname: Gruber
authors.forename: Christoph
authors.initials: C
authors.lastname: Plank
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Graf
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Zink
mesh_terms: D000577:Amides / Q000633:toxicity*; D000779:Anesthetics, Local / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002045:Bupivacaine / Q000633:toxicity*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects*; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects; D009135:Muscular Diseases / Q000139:chemically induced*; D032446:Myoblasts / Q000187:drug effects; D009336:Necrosis; D061251:Primary Cell Culture; D000077212:Ropivacaine
chemical_list: D000577:Amides; D000779:Anesthetics, Local; D003470:Culture Media; D000077212:Ropivacaine; D002045:Bupivacaine