Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

Abstract

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by ICP-MS. Measurement of the relative ATP and ADP concentrations indicates an initial 3-fold depletion of ATP suggesting an alteration in the ATP:ADP ratio. At the 6h time point the lidocaine exposed population contained ATP concentrations roughly 85% that of the negative control suggesting the surviving population adapted its metabolic pathways to, at least partially restore cellular bioenergetics. Metabolite analysis indicates an increase of intermediates in the pentose phosphate pathway, the preparatory phase of glycolysis, and NADPH. Oxidative stress produced by lidocaine exposure targets aconitase decreasing its activity with an observed decrease in isocitrate and an increase citrate. Similarly, increases in α-ketoglutarate, malate, and oxaloacetate imply activation of anaplerotic reactions. Antioxidant molecule glutathione and its precursor amino acids, cysteine and glutamate were greatly increased at later time points. Phosphatidylserine externalization suggestive of early phase apoptosis was also observed. Genetic studies using metacaspase null strains showed resistance to lidocaine induced cell death. These data suggest lidocaine induces perpetual mitochondrial depolarization, ROS/RNS generation along with increased glutathione to combat the oxidative cellular environment, glycolytic to PPP cycling of carbon generating NADPH, obstruction of carbon flow through the TCA cycle, decreased ATP generation, and metacaspase dependent apoptotic cell death.

Keywords: Apoptotic cell death pathways; Flow cytometry; Local anesthetic toxicity; Mass spectrometry; Metabolomics profiling; Oxidative stress.

Graphical abstract

Fig. 1

Xenobiotic Induced Cell Death and Mitochondrial Depolarization: A. Bar graph representing the mean percentage non-vital cells (Q1)±SEM of three independent experiments; B. Bar graphs representing the mean ratio of red (J-aggregates) to green (J-monomers)±SEM of three independent experiments normalized to the negative control.

Fig. 2

Xenobiotic Induced Oxidative Stress and Intracellular Calcium Accumulation: A.-C. : Assessed using general and species specific ROS/RNS probes and FACS;A. Cellular oxidative environment, bar graphs representing the geometric mean fluorescence intensity measured in the FITC channel±SEM of three independent experiments; B. Superoxide generation, bar graphs representing the FlowJo exported geometric mean fluorescence intensity measured in the PE channel±SEM of three independent experiments; C. Hydroxyl radial and peroxynitrite generation, bar graphs representing the geometric mean fluorescence intensity in the FITC channel±SEM of three independent experiments of HPF fluorescence; D. Intracellular calcium measured in WCL. Performed using ICP-MS. First normalized to internal standard Ga, then normalized to phosphate levels for cell density.

Fig. 3

Temporal assessment of Xenobiotic Induced Alterations in Metabolite Concentrations, Carbohydrate Metabolism Metabolite Pathways and HeatMap: Displayed as fold change to non-treatment negative control. Preferential use of the PPP for NADPH generation. Enzymes shown in boxes were previously reported to be oxidatively modified upon lidocaine exposure with diminished activity (OX) or increased in abundance of transketolase (TKL); see text for citation. Cellular energetics and redox cofactors alterations compared to negative control shown in inset box at bottom right. Color legend with fold change shown in upper left. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (F16BP), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), phosphyenolpyruvate (PEP), pyruvate (Pyr), alanine (Ala), citrate (Cit), isocitrate (IsoCit), Alpha-ketoglutarate (α-KG), malate (Mal), oxaloacetate (OAA), D-6-phospho-glucono-δ-lactone (6PGL), 6-phosphogluconate (6PG), seduheptulose-7-phosphate (S7P), and erythrose-4-phosphate (E4P).

Fig. 4

Line Plots of Temporal Assessment of Phosphatidylserine Externalization Along with Membrane Permeabilization; An Indicating Feature of Apoptosis: A. Hydrogen peroxide; B. Lidociane; . In the line plots for both hydrogen peroxide and lidocaine the AnnexinV- PI- (non-stained) and AnnexinV+ PI- (PS externalization) merge together at similar time points followed by an increase in AnnexinV+ PI+ population. PS externalization occurs prior to membrane permeabilization.

Fig. 5

KO mutant Sensitivity: KO mutants of proteins in pathways of apoptosis (Yca1, Nuc1, Aif) are resistant to hydrogen peroxide and lidocaine, most notably in the Yca1 mutant. KO mutant sensitivity is increased with Atg8, and not altered with Atg12 or Atg32 mutants suggesting that some components of autophagy may play a cytoprotective role.