Investigation of the Effect of Amniomax on Lidocaine-Induced Toxicity in Healthy Colon Cell Culture

Abstract

Background: Lidocaine (LIDO) toxicity is a critical concern in regional anesthesia, with no specific antidote currently available. While lipid emulsions are commonly used as rescue agents in cases of local anesthetic systemic toxicity (LAST), their efficacy is inconsistent, and their safety remains controversial. AmnioMax^® (AMX), a specialized cell culture medium enriched with growth factors and bioactive molecules, has the potential to offer cytoprotective effects. This study aims to investigate the therapeutic efficacy of AMX in mitigating lidocaine-induced cytotoxicity and to explore its protective mechanisms at the cellular level. Methods: Healthy colon cells (CCD-18Co) were used in this study. Four experimental groups were established as follows: control, LIDO, AMX, and LIDO + AMX. Cellular viability in the control group was set at 100%. LIDO was administered at concentrations ranging from 0.06 to 10%, AMX at 0.625-100%, and LIDO + AMX at 60% of the half-maximal effective concentration (EC₅₀) combined with LIDO (0.06-10%). Cells were incubated for 24 h, after which cellular viability, DNA damage, apoptosis, intracellular reactive oxygen species (iROS), intracellular calcium (Ca), mitochondrial membrane potential (MMP), and glutathione (GSH) were evaluated. Results: LIDO exposure led to a concentration-dependent decrease in viability compared to the control group (p < 0.001), while AMX significantly increased viability (p < 0.001). In the LIDO + AMX group, viability was also reduced (p < 0.001); however, cytotoxicity was significantly lower than in the LIDO group (p < 0.05). Both the LIDO and LIDO + AMX groups showed increased iROS levels, DNA damage, and apoptosis (p < 0.001), along with the decreased MMP and GSH levels (p < 0.001) compared to the control. However, in the LIDO + AMX group, iROS, DNA damage, and apoptosis were significantly lower than in the LIDO group (p < 0.01), MMP levels were increased (p < 0.001), and no significant difference was observed in GSH levels. Conclusions: AMX demonstrated cytoprotective effects against LIDO-induced cytotoxicity, suggesting its potential as an alternative therapeutic agent for LAST.

Keywords: AmnioMax®; apoptosis; cell culture; cytotoxicity; lidocaine; viability.

Figure 1

The effects of increasing concentrations of (A) lidocaine (LIDO), (B) AmnioMax^® (AMX), and (C) combined treatment (LIDO + AMX) on the viability of healthy colon CCD-18Co cells. Statistical significance: p < 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. All experimental data are presented as the mean ± standard deviation (SD), calculated from four independent experiments conducted under the same conditions.

Figure 2

The effects of lidocaine (LIDO) and the combination of LIDO with 60% AmnioMax^® (AMX) on (A) intracellular ROS, (B) intracellular calcium levels, (C) DNA damage in CCD-18Co cells, and (D) fluorescence microscopy images (scale bar: 100 μm) of DNA damage levels in CCD-18Co cells treated with LIDO (1–10%) and the combination of LIDO with AMX (60%). Differences in CCD-18Co cells: * p < 0.05; ** p < 0.01; *** p < 0.001, differences between both groups are considered statistically significant at # p < 0.05; ## p < 0.01; ### p < 0.001. Data are represented as mean ± SD of four independent experiments.

Figure 3

The effects of lidocaine (LIDO) and the combination of LIDO with 60% AmnioMax^® (LIDO + AMX) on intracellular (A) GSH, (B) mitochondrial membrane potential, (C) apoptosis in healthy colon cells, and (D) fluorescence microscopy images (scale bar: 100 μm) of apoptosis levels in CCD-18Co cells treated with lidocaine (1–10%) and the combination of lidocaine with AMX (60%). Differences in CCD-18Co cells: * p < 0.05; ** p < 0.01; *** p < 0.001; differences between both groups are considered statistically significant at # p < 0.05; ## p < 0.01; ### p < 0.001. Data are represented as mean ± SD of four independent experiments.