title: Cytotoxic effects of the radiocontrast agent iotrolan and anesthetic agents bupivacaine and lidocaine in three-dimensional cultures of human intervertebral disc nucleus pulposus cells: identification of the apoptotic pathways.
abstract: BACKGROUND
  Discography and discoblock are imaging procedures used to diagnose discogenic low back pain. Although needle puncture of the intervertebral disc (IVD) itself induces disc degeneration, the agents used in these procedures may also have harmful effects on IVD cells. The purpose of this study was to analyze whether radiocontrast agents and local anesthetic agents have detrimental effects on human nucleus pulposus (NP) cells.
  
  
  METHODS
  Healthy human NP cells were cultured for 7 days in three-dimensional (3D) cell-alginate bead composites, and were then exposed to clinically relevant doses of a radiocontrast agent (iotrolan) or local anesthetic (lidocaine or bupivacaine). Cell viability and apoptosis were measured by confocal microscopy and flow cytometry. On the basis of caspase expression profiles, the apoptotic pathways activated by the agents were identified by Western blot analysis.
  
  
  RESULTS
  The radiocontrast agent iotrolan did not affect NP cell viability or induce apoptosis. In contrast, both the anesthetic agents significantly decreased cell viability and increased the apoptotic cell number in a time- and dose-dependent manner. After 120 min, 2% lidocaine and 0.5% bupivacaine decreased percent live cells to 13% and 10%, respectively (p<0.05). The number of apoptotic cells was doubled by increasing lidocaine dosage from 1% to 2% (23% and 42%) and bupivacaine from 0.25% to 0.50% (25% and 48%) (p<0.05). Western blot analysis revealed that both anesthetic agents upregulated cleaved caspase-3 and caspase-8, whereas only bupivacaine upregulated cleaved caspase-9.
  
  
  CONCLUSIONS
  The present study demonstrates that iotrolan does not affect the viability of healthy human NP cells. In contrast, the two anesthetic agents commonly used in discography or discoblock may cause extensive damage to IVDs by inducing apoptotic cell death.
authors.affiliation: Department of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Iwasaki
authors.affiliation: Department of Advanced Medicine for Spine and Spinal Cord Disorders, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sudo
authors.affiliation: Department of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Department of Advanced Medicine for Spine and Spinal Cord Disorders, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Ito
authors.affiliation: Department of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Norimasa
authors.initials: N
authors.lastname: Iwasaki
mesh_terms: D000293:Adolescent; D000779:Anesthetics, Local / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D002045:Bupivacaine / Q000633:toxicity*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002648:Child; D003287:Contrast Media / Q000633:toxicity*; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology; D008012:Lidocaine / Q000633:toxicity*; D015398:Signal Transduction; D014283:Triiodobenzoic Acids / Q000633:toxicity*; D055815:Young Adult
chemical_list: D000779:Anesthetics, Local; D003287:Contrast Media; D014283:Triiodobenzoic Acids; C033300:iotrolan; D008012:Lidocaine; D020169:Caspases; D002045:Bupivacaine