Bupivacaine causes cytotoxicity in mouse C2C12 myoblast cells: involvement of ERK and Akt signaling pathways

Abstract

Aim: The adverse effects of local anesthetics (LAs) on wound healing at surgical sites have been suggested, and may be related to their cytotoxicity. This study was aimed to compare the cellular toxicity of bupivacaine and lidocaine (two well-known LAs), and to explore the molecular mechanism(s).

Methods: Toxicity of bupivacaine and lidocaine was assessed in cultured mouse C2C12 myoblasts by cell viability and apoptosis assays. Effects of LAs on extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) activation, which are essential for cell proliferation and survival, were evaluated by immunoblotting.

Results: Both LAs, especially bupivacaine, prevented cell growth and caused cell death in a dose-dependent manner. The half maximal inhibitory concentrations (IC(50)) for bupivacaine and lidocaine were 0.49+/-0.04 and 3.37+/-0.53 mmol/L, respectively. When applied at the same dilutions of commercially available preparations, the apoptotic effect induced by bupivacaine was more severe than that of lidocaine in C2C12 cells. Furthermore, bupivacaine significantly diminished the ERK activation, which may underlie its anti-proliferative actions. Both LAs suppressed Akt activation, which correlated with their effects on apoptosis.

Conclusion: Our study demonstrated that, when used at the same dilutions from clinically relevant concentrations, bupivacaine is more cytotoxic than lidocaine in vitro. Anti-proliferation and cell death with concomitant apoptosis mediated by bupivacaine may offer an explanation for its adverse effects in vivo (eg slowing wound healing at the surgical sites). A less toxic, long-acting anesthetic may be needed.

Figure 1

Myotoxicity of bupivacaine in vitro. (A) Representative images of C2C12 myoblasts cultured in the absence (control) or presence of bupivacaine (BPV) for 0−2 d. (B) Percentages of viable and dead cells from total cells. (C) Dose-dependent inhibition of cell growth. C2C12 cells were exposed to bupivacaine for 24 h and then subjected to MTT assays. Cell growth rate (%) was normalized to the control. Data are mean±SEM (n=3). ^bP<0.05 vs control.

Figure 2

Bupivacaine is more toxic than lidocaine in vitro. (A) Dose-dependent inhibition curves were determined by MTT assays using a series of concentrations of bupivacaine (BPV) and lidocaine (LID) (0.1, 0.316, 1, 3.16, and 10 mmol/L). The IC₅₀ values are 0.49±0.04 mmol/L (BPV) and 3.37±0.53 mmol/L (LID). Data are mean±SEM (n=3). (B) Conversions between absolute concentrations expressed in Molarity (mmol/L) and dilution folds of the LA stocks in Percentile (%).

Figure 3

Apoptotic effects of bupivacaine and lidocaine. (A) C2C12 cells were grown in the absence (control) or presence of bupivacaine or lidocaine (1:20 dilution from 0.5% BPV and 1% LID stocks, respectively) for 24 h and then stained with Hoechst 33258 (a, d, and g) and PI (b, e, and h). Total cells (c, f, and i) were displayed as black and white images converted from Hoechst staining images. Magnification: ×100. (B) Cell death rates were plotted as mean±SEM (n=5) of the percentages of bright blue (apoptotic) cells or red (late apoptotic and necrotic) cells from the total cells in the same field as in (A). ^cP<0.01.

Figure 4

Effects of bupivacaine and lidocaine on ERK (p44/p42 MAPK) activation. (A) C2C12 cells were grown in the absence (control) or presence of bupivacaine (BPV) or lidocaine (LID) for 24 h and subjected to immunoblotting with anti-pERK (top panel) and anti-total ERK antibodies (bottom panel), respectively. (B) Densitometric analysis of anti-pERK signals (normalized to total ERK levels) (each LA used at 1:20 dilution) from three independent experiments. Data are relative ERK activation levels with control set as 100%, presented as mean±SEM. ^cP<0.01; NS, not statistically significant.

Figure 5

Effects of bupivacaine and lidocaine on Akt activation. (A) C2C12 cells were grown in the absence (control) or presence of bupivacaine (BPV) or lidocaine (LID) for 24 h and subjected to immunoblotting with anti-pAkt (top panel), anti-total Akt (middle panel), and anti-β-actin antibodies (bottom panel), respectively. (B) Densitometric analysis of anti-pAkt signals (normalized to total Akt levels) (each LA used at 1:20 dilution) from four independent experiments. Data are relative Akt activation levels with control set as 100%, presented as mean±SEM. ^bP<0.05, ^cP<0.01.