Apoptosis and mitochondrial dysfunction in human chondrocytes following exposure to lidocaine, bupivacaine, and ropivacaine.
BACKGROUND
Several mechanisms have been proposed to explain toxicity of local anesthetics to chondrocytes, including the blockade of potassium channels and mitochondrial injury. The purposes of this investigation were to study the effects of lidocaine, bupivacaine, and ropivacaine on human chondrocyte viability and mitochondrial function in vitro and to characterize the type of cell death elicited following exposure.


METHODS
Primary chondrocyte cultures from patients with osteoarthritis undergoing knee replacement were treated with saline solution and the following concentrations of local anesthetics: 2%, 1%, and 0.5% lidocaine, 0.5% and 0.25% bupivacaine, and 0.5% and 0.2% ropivacaine for one hour. Cell viability and apoptosis were measured by flow cytometry at twenty-four hours and 120 hours after treatment. Nuclear staining and caspase 3 and 9 cleavage assays (Western blot) were used to further establish the induction of apoptosis. Mitochondrial dysfunction was evaluated by the accumulation of mitochondrial DNA damage (quantitative Southern blot), changes in adenosine triphosphate production (bioluminescence kit), and mitochondrial protein levels (Western blot analysis).


RESULTS
Exposure of primary human chondrocytes to a 2% concentration of lidocaine caused massive necrosis of chondrocytes after twenty-four hours, 1% lidocaine and 0.5% bupivacaine caused a detectable, but not significant, decrease in viability after twenty-four hours, while 0.5% lidocaine, 0.25% bupivacaine, and both concentrations of ropivacaine (0.5% and 0.2%) did not affect chondrocyte viability. Flow cytometry analysis of chondrocytes 120 hours after drug treatment revealed a significant decrease in viability (p < 0.05) with a concomitant increase in the number of apoptotic cells at all concentrations of lidocaine, bupivacaine, and ropivacaine analyzed, except 0.2% ropivacaine. Apoptosis was verified by observation of condensed and fragmented nuclei and a decrease in procaspase 3 and 9 levels. Local anesthetics induced mitochondrial DNA damage and a decrease in adenosine triphosphate and mitochondrial protein levels.


CONCLUSIONS
Lidocaine, bupivacaine, and ropivacaine cause delayed mitochondrial dysfunction and apoptosis in cultured human chondrocytes.
Grishko
Valentina
V
Department of Orthopaedic Surgery, University of South Alabama, 3421 Medical Park Drive, Mobile, AL 36693, USA. vgrishko@jaguar1.usouthal.edu
Xu
Min
M
Wilson
Glenn
G
Pearsall
Albert W
AW
D000577:Amides / Q000633:toxicity*; D000704:Analysis of Variance; D000779:Anesthetics, Local / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002045:Bupivacaine / Q000633:toxicity*; D002358:Cartilage, Articular / Q000187:drug effects* / Q000201:enzymology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008012:Lidocaine / Q000633:toxicity*; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D000077212:Ropivacaine
D000577:Amides; D000779:Anesthetics, Local; D000077212:Ropivacaine; D008012:Lidocaine; D002045:Bupivacaine