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Abstract |
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We tested the influence of atracurium and cisatracurium (final concentrations: 0, 0.96, 3.2, 9.6, 32, and 96 µM) on proliferation of human cells (hepatoma HepG2 cells and human umbilical vein endothelial cells) in vitro. In additional experiments, glutathione, N-acetylcysteine, or carboxyl esterase was added before the addition of either relaxant. The number of cells counted after 72 h of incubation was expressed as a percentage of the mean cell number in wells incubated without additives. Atracurium and cisatracurium progressively decreased cell proliferation in a concentration-dependent pattern. With human umbilical vein endothelial cells, atracurium or cisatracurium (3.2 µM) decreased the cell count to 67.7 % (SD, 14.8%) and 50% (SD, 8.6%), respectively. Cell proliferation was not inhibited by mivacurium. The results were similar to those with HepG2 cells. Glutathione, N-acetylcysteine, and carboxyl esterase partially reversed the effects of atracurium and cisatracurium. When incubated in a buffer with glutathione, atracurium decreased the number of glutathione-sulfhydryl groups. The findings that atracurium and cisatracurium inhibit proliferation of human cell lines in vitro, but that mivacurium does not, and that this effect is alleviated by glutathione and N-acetylcysteine, as well as by the carboxyl esterase, indicate that the inhibition may be caused by the reactive acrylate metabolites.
IMPLICATIONS: We tested the influence of atracurium and cisatracurium on proliferation of human cells (hepatoma HepG2 cells and human umbilical vein endothelial cells) in vitro. Atracurium and cisatracurium progressively decreased cell proliferation in a concentration-dependent pattern, whereas cell proliferation was not inhibited, even by the largest concentration of mivacurium.
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Introduction |
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Atracurium and one of its isomers, cisatracurium, are degraded and inactivated as nondepolarizing muscle relaxants via two pathways: a base-catalyzed elimination reaction (Hofmann elimination) and, as a minor pathway, enzyme-catalyzed ester hydrolysis. The products of Hofmann elimination are laudanosine and an acrylate ester. The products of hydrolysis are a quaternary acid and a quaternary alcohol. The presence of relatively large concentrations of laudanosine in plasma and urine in patients medicated with atracurium may be interpreted (1) as evidence that, in quantitative terms, degradation via Hofmann elimination predominates over the hydrolytic degradation.
Electrophilic acrylate esters are chemically reactive, and their generation in vivo raises a question of their possible adverse effects. Different acrylate esters are irritants to the eyes, the skin, or the nasal mucosa (2–5), and special precautions are recommended to reduce occupational exposure to these compounds (6).
Attempts to experimentally estimate possible adverse effects of acrylate esters generated from atracurium yielded contradictory results. In vitro incubation of rat hepatocytes indicated that large concentrations (>250 µM) of atracurium are deleterious to the cells. The damage was documented by the release of the intracellular enzyme lactic dehydrogenase into the incubation medium (7,8). However, no cellular damage was evident after perfusion of the isolated rat liver with atracurium (9–11).
We decided to test whether those muscle relaxants that metabolize via acrylate esters impede proliferation of two human cell lines in vitro. We postulated that if an inhibition of cell proliferation could be demonstrated, then the nucleophilic scavengers glutathione (GSH) and N-acetylcysteine (NAC) might reduce the inhibitory effect of the electrophilic metabolites on cell proliferation. Similarly, an improved cell proliferation might be expected from an enhanced enzymatic hydrolysis of the electrophilic acrylate esters to an alcohol and acrylic acid, which are markedly less electrophilic and reactive compounds.
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Methods |
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Atracurium (Tracrium®, 10 mg/mL), cisatracurium (Nimbex®, 2 mg/mL) and mivacurium (Mivacron®, 2 mg/mL) were purchased from Glaxo Wellcome (Research Triangle Park, NC). L-GSH was purchased from Fluka Chemie (Buchs, Switzerland [49750]); NAC (A7250), Ellman’s reagent (D-8130), glutathione-S-transferase (G-6511), collagenase type II (C 6885), and carboxyl esterase (E 3128) from Sigma-Aldrich (St. Louis, MO); and Tris-buffer (708976) from Boehringer (Mannheim, Germany).
The human hepatoma cell line HepG2 was obtained from the American Type Culture Collection (ATCC No. HB 8065) and was cultured and passaged in Dulbecco’s modified minimum essential medium (Biochrom KG, Schöller Pharma, Vienna) containing 10% fetal bovine serum, 1% L-glutamine, 0.2% penicillin, 0.2% streptomycin, and 1% nonessential amino acids (all from PAA Laboratories GmbH, Linz, Austria). Endothelial cells from six human umbilical veins (HUVEC) were isolated as described previously (12). Briefly, cells were harvested from umbilical cords by incubation with collagenase type II for 30 min and plated into 25-cm2 culture flasks (Falcon; Becton Dickinson, Franklin Lakes, NJ) previously coated with 0.2% gelatin (Sigma). Cells were identified as endothelial cells by immunofluorescence staining for factor VIII antigen production and by their specific cobblestone-like morphology. For our experimental setup, we cultured Passages 2 and 3 in endothelial growth medium (Bio Whittaker, Walkersville, MD).
Cells (2 x 104 HepG2 cells and 3 x 104 HUVEC cells) were plated to each of the 24 wells on a tissue culture plate (Nunc, Roskilde, Denmark), and the cells were allowed to attach overnight. After exchanging the medium, we performed four series of experiments by varying the additives to the wells. In the first series, a single muscle relaxant (atracurium, cisatracurium, or mivacurium) was added in various concentrations to separate wells. In the second series, the additives were either a single scavenger (GSH or NAC) or the esterase, with each added to separate wells in various concentrations. In the third series, GSH or NAC (final concentration 3.2 mM) was added to the incubation medium before the addition of either atracurium or cisatracurium in various concentrations. In the fourth series of experiments, esterase (final concentrations either 0 or 0.4 U/mL) was added to the incubation medium before the addition of cisatracurium (final concentrations either 0 or 96 µM). The final concentrations of the muscle relaxants in the first and third series were 0, 0.96, 3.2, 9.6, 32, or 96 µM. The final concentrations of each scavenger in the second series were 0 or 3.2 x 10-6, 10-5, 10-4, or 10-2 M. The wells not containing an additive were labeled control wells. Each combination of additives was tested in eight wells. Once the compounds were added to the culture medium, the medium was not changed during the subsequent 72 h of incubation at 37°C in an atmosphere of air with 5% CO2. At the end of incubation, the cells in each well were counted with an electronic particle counter (Coulter, Dunstable, UK).
A final volume of 1 mL of Tris buffer (0.05 M, pH 8) contained 1.2 x 10-7 mol GSH and 5.9 x 10-9 g glutathione-S-transferase (13). Incubation was initiated by the addition of atracurium (0, 1.2, 12, or 36 x 10-7 mol) and was performed in duplicates for 60 min in nitrogen atmosphere and at room temperature. At the end of the incubation, 20 µL of Ellman’s reagent was added, and the extinction at 412 nm was measured 30 min later in a spectrophotometer (Beckmann DU 650; Beckman, Munich, Germany). The absorption in atracurium-containing solutions was averaged and expressed as a percentage of the absorption measured in the solutions containing only GSH.
The number of cells in each well counted on Day 3 was expressed as a percentage of the average cell number in the simultaneously incubated wells without additives (control wells). After establishing that the distributions of the percentages did not deviate from the normal distribution, an analysis of variance was performed for a given cell line exposed to different concentrations of one muscle relaxant. If the analysis demonstrated significant differences among the groups, then a multiple t-test with Bonferroni’s correction was performed comparing the percentage of cell numbers in the wells exposed to a given concentration of a muscle relaxant with the percentage of cell numbers in the simultaneously incubated control wells. Similarly, percentage of cell numbers in wells containing a given concentration of a muscle relaxant were compared with percentage of cell numbers obtained from wells containing the same concentration of the relaxant and an additive (either a scavenger or the enzyme). Because the working hypothesis specified a treatment-induced decrease in cell proliferation, a single-tailed t-test was used. A decrease in cell proliferation was considered to be significant if an equally large or a larger decrease could have arisen by chance in fewer than 1 of 20 observations (P < 0.05). Data are presented as mean ± SD.
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Results |
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Figures 1 and 2 present the results obtained in HepG2 cells. On Day 3, the average number of cells in the control wells without additives was 12.3 (SD 3.1) times more than the number of cells added to the wells at the start of incubations. Whereas atracurium and cisatracurium inhibited cell proliferation in a concentration-dependent manner, mivacurium did not (Fig. 1). The minimal concentration that significantly (P < 0.05) inhibited cell proliferation was 9.6 µM for both atracurium and cisatracurium. GSH or NAC in a final concentration of 3.2 mM (Fig. 2) improved cell proliferation at larger concentrations of the relaxants. Proliferation of HepG2 cells incubated only with either GSH or NAC was not different from that in control wells for scavenger concentrations up to and including 3.2 mM, whereas the largest concentration of either scavenger inhibited cell proliferation (Table 1).
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The results obtained with the incubation of HUVEC cells are presented in Figures 3 and 4. The cell count in control wells on Day 3 varied among experiments; the cell number increased 5.8 (SD 1.3) times in comparison with the number of cells added to the wells at the start of incubations. Mivacurium did not decrease the proliferation of these cells (Fig. 3), but atracurium and cisatracurium decreased the cell numbers significantly (P < 0.05). The minimal concentration of either drug that significantly (P < 0.05) decreased the cell number was 0.96 µM. The inhibitory effect on cell proliferation was consistently alleviated by GSH or NAC (3.2 mM) (Fig. 4). When the cells were incubated only with the scavengers in concentrations up to and including 3.2 mM, cell proliferation was not altered. The largest concentration of either scavenger inhibited cell proliferation (Table 1).
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The influence of carboxyl esterase was tested in both cell lines. By themselves, the three smaller concentrations of the enzyme had no influence on proliferation of HUVEC cells, whereas the two largest concentrations decreased the cell number (Table 1). The influence of esterase (final concentration 0.4 U/mL) on cell proliferation was tested in both cell lines in the presence of cisatracurium (final concentration 96 µM). As was observed earlier, this concentration of cisatracurium markedly decreased the cell number in either cell line (Table 1). Addition of esterase significantly (P < 0.01) improved cell proliferation and, in the case of the HepG2 cells, restored it to control levels.
Incubation of GSH with atracurium for 60 min demonstrated a markedly diminished concentration of the sulfhydryl groups after 60 min of incubation with three concentrations of atracurium (atracurium:GSH molar ratios 1:1, 10:1, and 30:1). By setting the final concentration of the sulfhydryl groups in the solutions not containing atracurium to 100%, the final concentrations of the sulfhydryl groups in the atracurium-containing solutions decreased to 75% (SD 2%), 6% (SD 0.5%), and 0.6% (SD 0.01%), respectively (duplicate measurements).
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Discussion |
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The principal result of our study is the finding of a concentration-dependent inhibition of proliferation of two human cell lines in vitro by those isoquinoline nondepolarizing muscle relaxants that have a reverse ester group and are potentially degraded via Hofmann elimination. The muscle relaxant with a true ester group and of similar chemical structure that is degraded via ester hydrolysis, i.e., mivacurium, does not inhibit cell proliferation. A confirmation that the inhibition may be attributed to the electrophilic acrylate metabolites produced in the elimination reaction was provided by the observation that the nucleophilic scavengers GSH and NAC, as well as carboxyl esterase, partially attenuate the proliferation inhibition that is induced by atracurium and cisatracurium. Disappearance of sulfhydryl groups on incubation of GSH with atracurium further buttresses this notion.
Several methodological details of our study should be considered. Cell proliferation in vitro is highly variable and varies not only among cell lines, but also with the number of passages before the cells are used in experiments. Therefore, each experiment was performed on cells from the same passage, and each experiment included control wells without additives. This method minimizes differences between the control and treated wells insofar as the proportions of resting and proliferating cells. Altogether, cell proliferation was measured in approximately 1500 wells, including approximately 250 control wells. The incubation period was fixed to three days after preliminary experiments; this demonstrated that cell counts after two or four days of incubation provided results similar to those after three days. The drawback of a fixed incubation period is that it provides static information. Even so, the presented results document a previously little-known effect of benzylisoquinoline muscle relaxants containing a reverse ester group.
To ensure that differences in cell proliferation are caused by experimental interventions and not by differences in the proportion of resting cells, an additional experiment was performed in which HepG2 cells were growth arrested (by using only 0.1% fetal bovine serum instead of 10%) before experimentation. No significant difference in proliferation behavior was found between growth-arrested and nonarrested HepG2 cells (data not shown). We could not achieve growth arrest with HUVEC cells because during the incubation in serum-deprived medium, endothelial cells tend to detach from the bottom of the culture flasks. Furthermore, because both the control and treated cells were obtained from the same cell pool, no difference in proportions of resting and proliferating cells was expected.
Cell proliferation in vitro is an intricate process subject to interference at many steps involved in cell division. It is, therefore, not surprising that by using this sensitive index to estimate cell function, a reduced proliferation was demonstrated even in the presence of small concentrations of either atracurium or cisatracurium (Figs. 1 and 3). The minimal inhibitory concentrations of atracurium and cisatracurium were estimated to be 10 µM for the HepG2 cells and approximately 1 µM for the HUVEC cells. For a comparison, the initial plasma concentrations of either drug may be estimated by postulating that the intubating doses (atracurium 0.5 mg/kg and cisatracurium 0.2 mg/kg) (14,15) are diluted in plasma (volume 0.04 L/kg). The resulting concentrations are 10 µM for atracurium and 3 µM for cisatracurium. Much larger concentrations of atracurium or cisatracurium may arise after a bolus IV injection. Our results indicate that proliferation of HepG2 cells is inhibited by doses of atracurium in routine clinical use, whereas HUVEC cells are already inhibited by plasma concentrations of either atracurium or cisatracurium that are smaller than those that result from intubating doses. The results indicate that the HUVEC cells are more sensitive to the attack by the electrophilic acrylate metabolites than are the hepatoma cells (HepG2).
Addition of GSH or NAC to the incubation solutions partially alleviated the inhibitory effect of either atracurium or cisatracurium (Figs. 2 and 4). By themselves, these compounds in the concentrations used (3.2 mM) neither enhanced nor reduced the proliferation in either cell line, whereas the largest concentration of either scavenger inhibited cell proliferation. We attribute the partial protective effect of these compounds to their known physiologic role as scavengers of chemically reactive intermediates and free radicals.
The tripeptide GSH (L-glutamyl-L-cysteinylglycine) is the main intracellular low-molecular-weight thiol. It acts as a nucleophilic scavenger and as a cofactor in enzymatic reactions protecting cells and tissues from oxidative injury or from an attack by free radicals. NAC is a precursor of GSH and, when used for therapeutic purposes, it may act by itself either as an antioxidant or as a scavenger of free radicals (16,17). The concentration of GSH in whole blood is approximately 0.8 mM (18), whereas its plasma concentrations are in the micromolar range (3 or 0.3 µM) (18,19). Because the half-life of GSH in plasma is short, e.g., about 1.6 minutes in human plasma (19), we used a relatively large concentration of 3.2 mM of either scavenger for all the concentrations of atracurium or cisatracurium. Our results indicate that the reactive acrylate metabolites of atracurium or cisatracurium may reduce the plasma concentrations of GSH in vivo. However, preexisting small plasma or tissue levels of GSH, e.g., during preoperative fasting (20,21), may delay the detoxification of these metabolites. NAC is a precursor of GSH and is used therapeutically as a scavenger of free radicals. Typical clinical doses used are approximately 1.8 g/d. If this dose of NAC would be immediately, exclusively, and completely transferred to plasma, the corresponding concentration in plasma would be roughly 3.9 mM. Here we took an NAC concentration for our experiments identical to the chosen concentration of GSH, i.e., 3.2 mM.
The protective effect of the nucleophilic scavengers GSH and NAC, both containing a sulfhydryl group, may be ascribed to the formation of adducts with the acrylate esters. The interaction is irreversible, and it may be postulated that a similar reaction, i.e., a covalent binding, occurs with cellular constituents. The irreversible interaction may be represented as follows by using a sulfhydryl-containing nucleophile:
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The first species in the scheme represents an acrylate ester characterized by the double bond vicinal to the ester group. The second species is a nucleophilic scavenger containing a sulfhydryl group. Both species interact irreversibly to form an adduct, the right-most species in the scheme. The notion that sulfhydryl groups are consumed in the formation of an adduct is supported by the finding that the sulfhydryl groups disappear during the incubation of GSH with atracurium. Endogenous constituents that possibly interact with acrylates may contain other nucleophilic groups, e.g., primary, secondary, or tertiary amino groups, or other groups, in place of the sulfhydryl group. We propose that the scavengers protect the cells by covalently binding to acrylates and so decrease acrylate concentration around the cells.
Chemical reactivity of acrylate esters is greatly reduced either by ester hydrolysis or by a substitution of a methyl group for the hydrogen on the carbon atom vicinal to the ester. Hydrolysis of an acrylate ester as catalyzed by an esterase produces an alcohol and acrylic acid. Acrylic acid is much less reactive than the acrylic ester.
Previous experiments examining the influence of atracurium and its metabolites yielded contradictory results. The use of a much coarser index of cell injury, such as cell death documented by the extrusion of intracellular enzymes, required both much larger concentrations of atracurium and an extended lag period before the damage became evident (7,8). These conditions, especially the time period necessary for the effects to become manifest, were not always observed. Presumably this methodological detail explains why the other experiments (9–11) yielded contradictory results. Although it is likely that in these experiments the interaction between the reactive acrylate ester metabolites and cell constituents occurred within minutes to hours after the addition of either atracurium or cisatracurium to the incubation medium, the evidence of this interaction was evident days later.
Although the results of our study suggest that the likely mechanism of inhibition of cell proliferation is an interaction of the reactive ester metabolites with certain cell constituents, the kinetic aspects of the inhibition remain unsolved. A reduced cell number on Day 3 may have resulted from lethal injury to a large fraction of the cells at the start of incubation and the remaining surviving cells dividing at the normal rate. However, the alternative that all the cells initially survive but that the cell division proceeds at a slower rate cannot be excluded. Further studies are required to define precisely both the cell constituents involved and the kinetics of inhibition.
The proposition that the electrophilic acrylate esters are responsible for the inhibition of cell proliferation could not be tested directly. The reason is that acrylate esters of the type generated from atracurium or cisatracurium are not commercially available. However, the two findings, namely no effect of mivacurium on cell proliferation and a relief brought about by the nucleophilic scavengers GSH and NAC, as well as by carboxyl esterase, provide a strong albeit indirect indication that it is the reactive electrophilic acrylate esters that cause inhibition of cell proliferation.
Extrapolation of these findings obtained with proliferating cells in vitro to the clinical conditions in vivo might be premature at the present state of our knowledge. Although adverse effects of atracurium or cisatracurium potentially attributable to the reactive acrylate metabolites have not been reported in the literature, the postulated purely chemical processes are conceivable in vivo as well as in vitro. The extracellular distribution of both muscle relaxants makes it likely that the drugs come in contact with both proliferating and nonproliferating cells in vivo. Should the postulated interaction with endogenous nucleophiles occur in vivo, then the manifestations of the interaction may not be immediately evident clinically.
In conclusion, atracurium and cisatracurium reduce the proliferation of two human cell lines in vitro even in concentrations (<10 µM) expected in plasma after injections of endotracheal intubating doses of these muscle relaxants.
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Acknowledgments |
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Supported, in part, by the research funds of the Department of Anesthesiology and Critical Care Medicine, University of Innsbruck.
We thank Harald Sparr, MD, for a critical reading of the manuscript.
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Footnotes |
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Presented in part at the Anaesthesia Research Society meeting in Liverpool, United Kingdom, March 23–24, 2000 (abstract published in Br J Anaesth 2000;85:159P), and the European Society of Anaesthesiologists 2000 Congress in Vienna, Austria, April 1–4, 2000.
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