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British Journal of Anaesthesia 103 (5): 711–18 (2009) Advance Access publication August 22, 2009 Apoptosis induction by different local anaesthetics in a neuroblastoma cell line R. Werdehausen1, S. Fazeli1, S. Braun1, H. Hermanns1, F. Essmann3, M. W. Hollmann4, I. Bauer2 and M. F. Stevens5* 1Department of Anaesthesiology and 2Department of Experimental Anaesthesiology, University of Du¨sseldorf,Du¨sseldorf, Germany. 3Institute of Biochemistry, University of Tu¨bingen, Tu¨bingen, Germany. 4Department of Experimental and Clinical Experimental Anaesthesiology and 5Department of Anaesthesiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1100 DE Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] Background. Local anaesthetics are known to induce apoptosis in clinically relevant concen-trations. Hitherto, it is unknown what determines the apoptotic potency of local anaesthetics.
Therefore, we compared apoptosis induction by local anaesthetics related to their physico-chemical properties in human neuronal cells.
Methods. Neuroblastoma cells (SHEP) were incubated with eight local anaesthetics, two of the ester and six of the amide types. At least, five concentrations of each local anaestheticwere evaluated. After incubation for 24 h, rates of cells in early apoptotic stages and overallcell death were evaluated by annexin V and 7-amino-actinomycin D double staining by flowcytometry. The concentrations that led to half-maximal neurotoxic effects (LD50) were calcu-lated and compared for all local anaesthetics.
Results. All local anaesthetics were neurotoxic in a concentration-dependent manner. Alldrugs induced similar rates of early apoptotic cell formation at low concentrations, whereas athigh concentrations, late apoptotic or necrotic cell death predominated. Comparison of LD50 values of the different local anaesthetics resulted in the following order of apoptotic potencyfrom high to low toxicity: tetracaine.bupivacaine.prilocaine¼mepivacaine¼ropivacaine.
lidocaine.procaine¼articaine. The toxicity correlated with octanol/buffer coefficients and alsowith experimental potency of the local anaesthetic, but was unrelated to the structure (esteror amide type).
Conclusions. All commonly used local anaesthetics induce neuronal apoptosis in clinicallyused concentrations. The neurotoxicity correlates with lipid solubility and thus with the con-duction blocking potency of the local anaesthetic, but is independent of the chemical class(ester/amide).
Br J Anaesth 2009; 103: 711–18 Keywords: measurement techniques, flowmetry; model, neuroblastoma cells; toxicity, localanaesthetics; toxicity, neurotoxicity Accepted for publication: July 21, 2009 Local neurotoxicity after neuraxial application of local although incidental neuronal damage has been described anaesthetics is rare. Nevertheless, it is a severe problem for other local anaesthetics as well. In animal studies, neu- when it occurs. Clinical profiles of neurotoxicity have rotoxicity of many local anaesthetics has been demon- been based on the reported incidence of cauda equina syn- strated.4 – 8 Experimental drome or transient neurologic syndrome (TNS) after spinal mechanism of local anaesthetics-induced neurotoxicity anaesthesia.1 – 3 Clinically, lidocaine is the local anaesthetic which has most often been linked to local neurotoxicity, sodium channel or electrical inactivation of a nerve.7 9 # The Author [2009]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved.
For Permissions, please email: [email protected] Werdehausen et al.
Comparison of the neurotoxic potency (concentration- were grown in Roswell Park Memorial Institute (RPMI) dependency) for different local anaesthetics is difficult in 1640 medium with L-glutamine, supplemented with 10% animals for biometric reasons.
heat-inactivated fetal calf serum and 50 mg ml21 each of Local anaesthetics induce elevations of intracellular penicillin and streptomycin. All cells were cultured under calcium concentration through external influx or release equal conditions including a humidified atmosphere con- from intracellular stores.10 – 12 Furthermore, local anaes- taining carbon dioxide 5% at 378C.
thetics activate certain kinases and inhibit the energy pro-duction in the mitochondria.13 – 18 In this process, apoptosis Exposure to local anaesthetics and experimental has been shown to be one mechanism of neurotoxicity in vitro, especially in marginally toxic concentrations.19 – 22Recent studies have delineated the subcellular mechanism Before the experiments, cells were cultured overnight in of apoptosis induction by local anaesthetics in neuronal cell complete medium at a density of 4105 cells ml21 to cultures.22 23 Hence, this model seems suitable for the allow logarithmic growth. Adherent cells were cultured in investigation of the neurotoxic and neuroapoptotic potential 3 ml samples with fresh medium alone as negative of different local anaesthetics.
control, or one added local anaesthetic for 24 h at concen- The investigation of the toxicity of different local anaes- trations as indicated. Addition of local anaesthetics did not thetics may help to evaluate which properties of local alter the pH value of the medium (7.39, range 7.35 – 7.43).
anaesthetics are responsible for their toxic effects. Doesthe chemical structure, that is, ester or amide type, influ- Apoptosis detection assay ence their toxicity? Ester-type local anaesthetics have been The fraction of cells in an early state of apoptosis was considered to be somewhat more neurotoxic in comparison with amides.10 24 Are there certain physicochemical prop- isothiocyanate-conjugated (FITC) annexin V and counter- erties that determine the toxic potential of a local anaes- staining with 7-amino-actinomycin D (7-AAD). Annexin thetic like lipophilicity, pKa value, protein binding, or V binds to phosphatidylserine on the outer leaflet of the molecular weight? plasma membrane. 7-AAD is excluded by cells with intact Therefore, we compared the concentration-dependent membranes. Therefore, 7-AAD staining reveals membrane neuroapoptotic and neurotoxic potencies of the amide-type disintegration and is a marker for primary or secondary local anaesthetics bupivacaine, lidocaine, mepivacaine, necrotic cell death. Cells staining with annexin V-FITC prilocaine, and ropivacaine and also the ester type local (below referred to as annexin V), but not with 7-AAD, are anaesthetics procaine and tetracaine in our human neuronal defined as early apoptotic.28 Briefly, for annexin V/7-AAD cell culture model.
staining, cell culture medium including detached cells wastransferred from sample wells to analysing tubes. Adherentcells were trypsinized for 3 min with 1.5 ml 0.05% trypsin with 1 mM EDTA per sample. Detached cells were addedto corresponding analysing tubes to pool all cells fromeach sample. Subsequently, cells were washed twice with cold PBS and resuspended in annexin binding buffer 97 ml Unless stated otherwise, reagents were purchased from (10 mM N-[2-hydroxyethyl]piperazine-N0-3[ propanesulfo- Sigma Aldrich (St Louis, MO, USA). All local anaesthetics nicacid]/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at were obtained in the highest commercially available con- a concentration of 1106 cells ml21. Next, annexin centration as their hydrochloride salts. Bupivacaine, lido- V-FITC 5 ml and 7-AAD 2 ml (50 mg ml21) were added caine, mepivacaine, and prilocaine were obtained from and samples were incubated for 15 min in the dark at AstraZeneca (London, UK). Procaine was purchased from room temperature. Subsequently, annexin binding buffer Jenapharm (Jena, Germany), and articaine and tetracaine 150 ml was added, cells were resuspended and analysed were acquired from Sanofi Aventis (Paris, France). None of the commercially available solutions contained preservatives.
All fluorescence-activated cell sorting analyses were Ropivacaine was kindly provided by AstraZeneca, Research performed on a FACScalibur (Becton Dickinson, Franklin and Development (So¨derta¨lje, Sweden). Phosphate-buffered Lakes, NJ, USA) using CellQuest analysis software (BD saline (PBS) without calcium and magnesium was purchased Biosciences, Franklin Lake, NJ, USA). For each determi- from Gibco, Invitrogen (Carlsbad, CA, USA).
nation, a minimum of 10 000 cells were analysed.
Statistical analysis Human neuroblastoma cells (SHEP) are a subclone All experiments were performed at least in triplicate.
derived from the human neuroblastoma cell line SK-N-SH Results are expressed as means (SD). All calculations were and have been characterized before.25 – 27 All cell lines made with the SPSS program version 15.0 (SPSS Inc., Local anaesthetic induced neuroapoptosis Chicago, IL, USA). Concentration – response curves of the The percentage of early apoptosis (annexin Vþ, different local anaesthetics were determined by probit 7-AAD-) of all cells first increased in a concentration- regression under the guidance of our statistics department.
dependent manner, reaching a maximum with local anaes- The LD50 was obtained from probit analysis and compared thetic concentrations well below the LD50 values (Fig. 4).
by means of analysis of variance (ANOVA) with Tukey's The highest percentage of cell death by early apoptosis post hoc test. Correlations between LD50 values with lipid varied between the different local anaesthetics between solubility, pKa, blocking potency, non-ionized fraction, 14% and 28% after 24 h of incubation. With a further protein binding, molecular weight, and experimental increase in concentration, the percentage of early apoptotic blocking potency were made by means of Spearman's cell formation decreased and returned to baseline for rank correlation test. P,0.05 was considered significant.
almost all local anaesthetics (Fig. 4).
Concentrations of local anaesthetics inducing cell death in All local anaesthetics investigated were neurotoxic at con- ,50% of analysed cells resulted in a considerable fraction centrations observed intrathecally after spinal anaesthesia.
of cell positive for annexin V only. This indicates an early They induced in a concentration-dependent manner apop- stage of apoptosis in at least a fraction of the analysed totic and with higher concentrations necrotic cell death.
cells. Nevertheless, also at these concentrations, cells The toxicity of the local anaesthetics correlated with their staining positive for annexin V staining and 7-AAD were octanol/buffer partition coefficient and thus their relative present (Fig. 1), indicating late apoptosis or necrosis.
clinical potency.
Higher concentrations (leading to cell death in more than Several publications demonstrated that lidocaine and 50% of analysed cells) of all investigated local anaes- other local anaesthetics can induce apoptosis in neuronal thetics led to increased fractions of cell staining positive and non-neuronal cells.13 14 19 – 21 33 – 37 Most of these both for annexin V and for 7-AAD. This indicates a late studies investigated one drug only.13 20 21 33 – 37 Therefore, stage of apoptosis or primary necrosis. Thus, all investi- toxic potency of various local anaesthetics from different models cannot be compared for methodological reasons.
concentration-dependent manner (Fig. 2). The toxic con- Boselli and colleagues19 compared the toxic and apoptotic centrations inducing early stages of apoptosis and late potential of lidocaine and ropivacaine in human lympho- stages of cell death varied over a wide range (almost cytes. Unfortunately, only one concentration of lidocaine hundred-fold) for the different local anaesthetics.
and a different—not equipotent—concentration of ropiva- In order to compare the toxicity of all eight local anaes- caine were investigated. These shortcomings are discussed thetics, the concentration-toxicity functions derived from by the authors themselves and they advocate studies with regression analysis were used to calculate the concentrations more concentrations and local anaesthetics in human neur- that induced 50% overall cell death (LD50). Overall cell onal cell lines, as presented here.
death was defined as the sum of cells staining positive for More recently, Perez-Castro and colleagues38 compared annexin V only and cells staining positive for annexin V and the cytotoxic effects of short (10 min) exposure to procaine, 7-AAD. The different LD50 values are displayed in Figure 3.
mepivacaine, lidocaine, chloroprocaine, ropivacaine, or Overall ANOVA of LD50 values revealed significant differ- bupivacaine in human SH-SY5Y neuroblastoma cells and ences between the eight local anaesthetics (P,0.001).
found the same order of toxicity as seen in the data pre- Tukey's post hoc test comparison yielded the following sented here. In contrast to the results presented here, they found apoptosis (non-quantitative caspase activation) only after exposure to high concentrations of lidocaine and bupi- vacaine. Probably, exposure to very high concentrations for In order to identify factors affecting toxicity, the LD50 a very short time leads to a greater predominance of necro- values were correlated to a number of known physico- tic cell death, whereas during long-term exposure of neur- chemical properties as displayed in Table 1. LD50 values ones, apoptosis is one major mechanism of cell death.
correlated well with octanol/buffer distribution coeffi- Lirk and colleagues14 compared the neurotoxic poten- cients. Spearman's rank correlation coefficient was 20.88 tials of lidocaine, bupivacaine, and ropivacaine in equipo- (P,0.01), that is, the higher the local anaesthetic solubi- tent concentrations in primary cell cultures of rat dorsal lity in oil, the lower was the observed toxic concentration.
root ganglia. They evaluated their blocking potential on In contrast, LD50 values did not correlate with pKa values, voltage-gated sodium channels NaV1.1– 3 and NaV1.6 in fraction of non-ionized local anaesthetic, or protein rat pituitary cells, and applied equipotent concentrations of binding. Finally, LD50 values correlated positively with each of the three local anaesthetics on primary cell cul- experimental effective anaesthetic concentrations (corre- tures of rat dorsal root ganglia for 24 h. At equipotent con- lation coefficient: 0.81, P,0.05).29 – 32 centrations, the percentage of cell death did not differ

Werdehausen et al.
7-AAD fluorescence Annexin V fluorescence Fig 1 Flow cytometric analysis of neuroblastoma cells incubated for 24 h with local anaesthetics. Dot plots represent typical results after incubationwith medium alone (control), lidocaine, bupivacaine, or tetracaine. Intensity of red fluorescence by 7-AAD-stained cells is indicated on the ordinate,whereas intensity of green fluorescence emerging from cell-bound annexin V-FITC is indicated on the abscissa. Unstained and therefore vital cells leadto a population of dots in the lower left quadrants as in controls. Cells in the lower right quadrants are stained positive for annexin V-FITC only andare therefore in an early apoptotic stage, whereas cells in the upper right quadrants were stained positive for annexin V-FITC and 7-AAD indicatinglate apoptosis or primary necrosis. Note that vital cells and early apoptotic cells can be found at lower concentrations of all local anaesthetics, whereasat high concentrations, almost all cells show signs of lost cell membrane integrity.
Local anaesthetic induced neuroapoptosis Concentration (mM) Fig 2 Concentration-dependent neurotoxicity of local anaesthetics. Sigmoid-shaped curves for concentration – response relationship were calculated byprobit regression analysis using the results from triplicate experiments with five different concentrations of each local anaesthetic. Single symbolsrepresent results from single experiments.
Ropivacaine Mepivacaine Local anaesthetic Fig 3 Half-maximal neurotoxic concentrations (LD50) of investigated local anaesthetics. LD50 values were calculated from probit analysis and aremean (SD). The LD50 values of two local anaesthetics adjacent to a significance sign were different, as were LD50 values of all local anaesthetics ondifferent sides of a significance sign. Thus, the LD50 value of lidocaine was significantly higher than the values for mepivacaine, ropivacaine,prilocaine, bupivacaine, and tetracaine. Furthermore, this value was significantly lower than that of procaine or articaine. ANOVA with Tukey's post hoctest. *P,0.05; **P,0.01.
Table 1 Correlation between physicochemical properties and toxicity of local anaesthetics. Concentrations of local anaesthetics (mM and % solution ofhydrochloride salts) leading to half-maximal neurotoxic effects (LD50) after 24 h of incubation were calculated by probit regression analysis of concentration –response relationship experiments in SHEP neuroblastoma cells. Values are mean (SD). Partition coefficients (O/B PQ) with H-octanol/buffer at pH 7.4 and258C,30 – 32 ionization constants ( pKa),29 31 32 unionized fractions at pH 7.4 (LAb pH 7.4),29 31 32 protein binding (PB),29 31 32 molecular weight (MW) ofthe local anaesthetics' base in Dalton units30 – 32 and relative experimentally effective anaesthetic concentrations (EAC) from rat sciatic nerve blockingprocedures30 – 32 were used to calculate Spearman's rank correlation coefficients (Spearman's r) Spearman's coefficient between the three local anaesthetics. Their results are The concentrations of local anaesthetics that induced reconfirmed and generalized to more local anaesthetics apoptosis in our model are within the same range as those through the results of the present study.
observed intrathecally after single-shot spinal anaesthesia Werdehausen et al.
xin-V +/7-AAD cells (%) Concentration (mM) Fig 4 Concentration-dependent induction of early apoptotic cells. The fractions of cells undergoing an early stage of apoptosis were identified bypositive annexin V-FITC and negative 7-AAD staining after 24 h incubation with the different investigated local anaesthetics. Note that induction of early apoptosis increases within the first three concentrations of each local anaesthetic, and at higher concentrations, the fraction of cells with signs ofearly apoptosis decrease again revealing a change in the mechanism of cell death from apoptosis to necrosis.
in primates and in sciatic nerves of rodents during nerve All drugs induced swelling, but 2-chloroprocaine and tetra- blockade.39 40 Therefore, concentrations inducing neurotoxi- caine significantly more than lidocaine or bupivacaine.
city could be reached clinically.41 However, after a single- However, the effects of different local anaesthetics were shot spinal anaesthesia or peripheral nerve block, these compared at a single concentration, and thus equipotency concentrations are only reached for about 1 h, whereas in could not be ensured. The same group investigated the our cell culture model, concentrations were kept constant blocking and toxic concentrations of etidocaine, lidocaine, for 24 h. It is well known that beyond the concentration, the 2-chloroprocaine, and procaine on the sciatic nerve of rats time of exposure to a local anaesthetic is important for the 2 days after a single-shot injection. They found a perfect development of neurotoxicity; therefore, neurotoxicity after correlation between nerve blocking concentrations and single application is a rare complication clinically. Thus, toxic concentrations.44 Unfortunately, they instituted only intraneural injection of lidocaine 2% (78 mM) for single- two commonly used drugs. Nevertheless, their findings shot sciatic nerve block did not lead to any functional nerve regarding procaine and lidocaine are in accordance with damage, although this concentration is more than eight the data presented here.
times the LD50 concentration observed here.42 Sakura and colleagues7 reported that equipotent concen- Although we used human tumour cells which might gen- trations of lidocaine and bupivacaine resulted in an equal erally be resistant to apoptosis induction, they were actually sensory deficit after 4 days in a model of spinal anaesthe- more sensitive to apoptosis induced by local anaesthetics sia in rats. In a subsequent study, Sakura and colleagues45 than primary rodent dorsal root cell cultures and hybrid found an increased functional and morphological neuro- immortalized dorsal root ganglia.14 20 Obviously, the cell toxicity of lidocaine in comparison with bupivacaine, culture model used for the presented study has several probably because this study was more appropriately limitations in translating data to the in vivo situation.
powered. Yamashita and colleagues46 in a similar model Human SHEP neuroblastoma cells are growing and divid- found bupivacaine and ropivacaine to be significantly less ing during the local anaesthetic exposure rather than being toxic than lidocaine and tetracaine. Recently, Umbrain and mature terminally differentiated neurones. They are derived colleagues47 demonstrated that intrathecal administration from malignant neural crest cells which would ordinarily of equipotent doses levobupivacaine and lidocaine led to differentiate into the sympathetic chain, adrenals, or dorsal release of glutamate and prostaglandin E2, while on root ganglia. Nevertheless, despite those limitations, our repeated injection, lidocaine but not levobupivacaine led model of a human neuronal cell line seems even more sen- to enhanced prostaglandin release.
sitive in detecting minor differences between different To summarize, most in vivo animal studies have found local anaesthetics compared with in vivo studies.
rank orders of toxicity in accordance with our results. The Previous animal studies compared the neurotoxic, only difference is that lidocaine seems to be more toxic in but not the apoptotic, potency of different local anaes- some studies. This varying toxicity of lidocaine might be thetics.4 5 7 43 No study has compared a wide range of related to other mechanisms not detected in all models.
local anaesthetics in varying concentrations, but a few Thus, lidocaine could release inflammatory substances like studies compared different local anaesthetics in equipotent CGRP as recently reported.48 The release of inflammatory concentrations. Myers and colleagues4 investigated the substances only under certain circumstances could explain effect of 2-chloroprocaine, tetracaine, lidocaine, and bupi- why the incidence of TNS varies within a wide range vacaine in high concentrations on the sciatic nerve of rats.
between 4% and 33%.1 Local anaesthetic induced neuroapoptosis The high degree of correlation between the toxicity and octanol/buffer coefficient is remarkable. Since the lipophi- We acknowledge the expert statistical guidance by Reinhart Willers from lic properties of local anaesthetics correlate with their our institutional statistics department.
potency, a correlation between toxicity and potency at thesodium channel is obvious. That might suggest that thetoxicity is mediated via the sodium channel. Studies inducing a long-term blockade with tetrodotoxin7 49 or This work was supported by institutional sources only.
functionally inactivating a nerve9 have shown that a con-duction block per se does not lead to nerve fibre degener-ation. Furthermore, apoptosis induction and toxicity havealso been shown in cells not expressing voltage-gated sodium channels.22 33 35 36 This implies that local anaes- 1 Hodgson PS, Neal JM, Pollock JE, Liu SS. The neurotoxicity of thetics developed on the basis of the known structures are drugs given intrathecally (spinal). Anesth Analg 1999; 88: 797 – 809 predictably neurotoxic depending on the lipophilic proper- 2 Zaric D, Christiansen C, Pace NL, Punjasawadwong Y. Transient ties. Therefore, one could argue that we have to develop neurologic symptoms (TNS) following spinal anaesthesia with local anaesthetics with a completely different structure in lidocaine versus other local anaesthetics. Cochrane Database Syst order to completely avoid this toxicity, for example, tetro- Rev 2005; CD003006 3 Denny NM, Selander DE. Continuous spinal anaesthesia. Br J dotoxin which displays no local toxicity.7 Disappointingly, Anaesth 1998; 81: 590 – 7 the structurally different tricyclic antidepressant amitripty- 4 Myers RR, Kalichman MW, Reisner LS, Powell HC. Neurotoxicity line which also inhibits the voltage-dependent sodium of local anesthetics: altered perineurial permeability, edema, and channel induces apoptosis and local neurotoxicity.50 More nerve fiber injury. Anesthesiology 1986; 64: 29 – 35 recently, lipophilic effects of a series of amino-amide 5 Kalichman MW, Powell HC, Myers RR. Pathology of local local anaesthetics on human potassium channels have been anesthetic-induced nerve injury. Acta Neuropathol (Berl) 1988; 75: reported.51 This interaction might also be involved in toxic 6 Seitz RJ, Lipfert P, Willrich A, Himmelmann F. Toxic effects of actions of local anaesthetics, and again lipophilic proper- ties seem to play an important role in determining the nerve fibers and blood – nerve barrier in the mouse. Exp Brain toxic potential.
Res 1989; 74: 293 – 302 Some studies have attributed the cytotoxicity of local 7 Sakura S, Bollen AW, Ciriales R, Drasner K. Local anesthetic neu- anaesthetics and amitriptyline to an unspecific membrane rotoxicity does not result from blockade of voltage-gated sodium effect as a detergent.52 53 However, these physicochemical channels. Anesth Analg 1995; 81: 338 – 48 effects occur at concentrations approximately 10-fold higher.
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Lidocaine toxicity in primary afferent neurons from the rat.
Furthermore, findings that cells are protected against local J Pharmacol Exp Ther 1998; 285: 413 – 21 anaesthetic induced apoptosis by Bcl-2 overexpression, 9 Stevens MF, Novotny GE, Lipfert P. Inactivation of baroafferents caspase-9 deficiency, caspase-inhibitors, and p38 mitogen- leads to loss of barosensitivity without changes in nerve mor- activated-kinase-inhibitors argue against a detergent-like phology. J Auton Nerv Syst 1998; 68: 125 – 34 effect of local anaesthetics as the principal cause of cell 10 Hogan QH. Pathophysiology of peripheral nerve injury during death.14–16 22 54 Nevertheless, it is conceivable that higher regional anesthesia. Reg Anesth Pain Med 2008; 33: 435 – 41 concentrations of local anaesthetics, which induce necrosis, 11 Johnson ME, Saenz JA, DaSilva AD, Uhl CB, Gores GJ. Effect of local anesthetic on neuronal cytoplasmic calcium and plasma might produce a less specific membrane effect.
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