Cisatracurium is an intermediate acting, non-depolarizing neuromuscular blocking agent. Due to its major elimination via the Hofmann pathway, occurring independently of kidney and liver function, it is used during anaesthesia in patients with multiple organ failure or liver transplantation . In a recent study, growth-inhibitory effects of atracurium and cisatracurium, but not mivacurium, on human hepatoma (HepG2) and umbilical vein endothelial cells (HUVEC) could be demonstrated in vitro . Notably, these events were already observed at concentrations similar to those encountered in human plasma after a single-bolus injection of a respective intubating dose. Although no exact mechanism of action could be demonstrated, anti-proliferative effects of both atracurium and cisatracurium could be antagonized by either esterases or glutathione. These observations strongly suggest that formation of acrylate esters elicits oxidative stress, thereby precipitating the growth-inhibitory effect of atracurium and cisatracurium. Oxidative stress has been demonstrated as a potent inducer of apoptotic cell death in various cells and tissues .
The aim of the present study was to investigate whether oxidative stress caused by cisatracurium induces apoptosis in HUVEC cells in vitro.
Materials and methods
HUVEC were cultured in endothelial cell growth medium (PromoCell GmbH, Heidelberg, Germany) containing 0.4% endothelial cell growth supplement/heparin, 2% fetal calf serum, 0.1 ng mL−1 epidermal growth factor, 1 μg mL−1 hydrocortisone, 1 ng mL−1 basic fibroblast growth factor, 50 ng mL−1 amphoteracin B, and 50 μg mL−1 gentamicin. Passages 2-5 of HUVEC were used for the present experiments. Cells were seeded onto 6-well plates at a density of 5 × 105 cells per well and allowed to attach for 2 days under standard culture procedures. For experiments, cells were incubated with cisatracurium (Glaxo-Smith-Kline) at concentrations of 0.96, 3.2, 9.6, 32 and 96 μmol either with or without the addition of glutathione (3.2 mmol). In order to exclude a potential influence of benzene sulphonic acid (BSA) which is used as an additive to stabilize cisatracurium preparations, HUVEC were incubated with 116.5 mmol of BSA (corresponding to the BSA content of the highest concentration of cisatracurium used in our study). Additional studies were performed with mivacurium (0.96, 3.2, 9.6, 32 and 96 μmol) as well as the cisatracurium breakdown product laudanosine (10, 30 and 100 μM).
Measurement of DNA fragments
Following 24 h incubation, supernatants were removed and retained, since cells growing as monolayers tend to become detached during apoptosis. The adherent cells were harvested with trypsin- EDTA and combined with the supernatant. Following centrifugation at 400 g for 5 min, the pellets were suspended in lysis buffer (20 mmol EDTA, 100 mmol Tris pH 8.0, 0.8% sodium lauryl sarcosinate). DNA fragmentation was measured using the Cell Death Detection ELISA Plus (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. In brief, cell lysates were placed into streptavidin-coated 96-well microtitre plates and incubated with both biotin-conjugated mouse monoclonal anti-histone and horseradish peroxidase-conjugated mouse monoclonal anti-DNA antibodies (sandwich ELISA principle). Quantification of DNA fragments was done by photometric determination of horseradish peroxidase using 2,2′-azino-bis 3-ethyl benzthiozoline-6-sulphonic acid as substrate. Results of Cell Death Detection ELISA Plus are given as optical density (OD) values. It should be noted that increasing OD values indicate increasing DNA fragmentation and, therefore, apoptosis observed in vitro.
As the working hypothesis focused on alterations induced by treatment with cisatracurium, data was analysed using a two-tailed t-test. Data are represented as mean ± SEM. Statistical significance was assumed at P < 0.05.
Figure 1 represents results obtained in HUVEC cells incubated with different concentrations of cisatracurium either with or without glutathione. Data are given as OD values in relation to unstimulated controls. Following a 24-h incubation period, cisatracurium caused a dose dependent increase in apoptotic cell death. While OD values in control incubations were 100 ± 36%, this parameter was significantly augmented following cisatracurium at a concentration of 0.96 (232 ± 52%), 3.2 (280 ± 12%), 9.6 (348 ± 92%), 32 (379 ± 79%) and 96 μmol (490 ± 91%); P < 0.05 as compared to controls, respectively. Glutathione (3.2 mmol) attenuated the pro-apoptotic effect of cisatracurium with OD values averaging 95 ± 33% following single incubation of HUVEC with glutathione, 81 ± 23% following glutathione plus 0.96 μmol cisatracurium, 87 ± 12% following glutathione plus 3.2 μmol cisatracurium, 75 ± 13% following glutathione plus 9.6 μmol cisatracurium, 97 ± 34% following glutathione plus 32 μmol cisatracurium, as well as 178 ± 39% following glutathione plus 96 μmol cisatracurium; P < 0.05 as compared to the respective incubation without glutathione.
No significant rise in OD values could be observed after 24 h incubations with mivacurium at concentrations of 0.96 (102 ± 5), 3.2 (103 ± 9), 9.6 (95 ± 6), 32 (105 ± 23) and 96 μmol (91 ± 14) as compared to controls (100 ± 6). Similarly, treatment of HUVEC with laudanosine at concentrations of 10 (101 ± 6), 30 (90 ± 13) and 300 μmol (89 ± 10) did not result in a dose-dependent induction of apoptosis. Finally, BSA (116.5 mmol) did not elicit a rise in OD values after a 24-h incubation period (88 ± 19) as compared to control measurements (100 ± 22%).
The major finding of the present study was the induction of apoptosis by cisatracurium, but not mivacurium, in vitro. This may, in principle, explain the growth-inhibitory effect of cisatracurium upon human cell lines previously reported . Hofmann decomposition mainly yields laudanosine and monoacrylate esters  in an equimolar fashion. The presence of large concentrations of laudanosine in the urine of patients treated with atracurium  suggests that also high amounts of acrylate esters are concomitantly formed. Nigrovic and colleagues reported cytotoxic effects of atracurium at high doses in in vitro models using isolated rat liver or rat hepatocytes, respectively . Electrophilic acrylate esters are chemically reactive and may exert cytotoxic adverse effects, e.g. genomic damage [7,8].
The question remains whether the in vitro observations of the present study are of significance in vivo. Initial plasma levels following routine intubation doses of cisatracurium (0.2 mg kg−1 body weight) have been reported to be 3 μmol , whereas others determined concentrations below 1 μmol . With respect to these data, cytotoxic effects of cisatracurium elicited in our study were already achieved at concentrations approaching those observed in human plasma after single-bolus application of cisatracurium. In the collective of intensive care patients requiring long-term relaxation, formation of even higher amounts of acrylate esters may be assumed.
The possible involvement of the second degradation product, laudanosine, in the pro-apoptotic and anti-proliferative actions of cisatracurium in HUVEC could be excluded by direct incubation with laudanosine. Increasing concentrations of laudanosine did not result in an increased rate of apoptotic cell death, thus pointing towards acrylate esters as the predominant mediator of the cytotoxic effects of cisatracurium. In the present study, the detrimental results of cisatracurium on cell growth and survival could be antagonized by the simultaneous application of glutathione. Since detoxification of acrylates is performed via conjugation to glutathione , this further emphasizes the hypothesis of acrylate esters being the principal initiators of programmed cell death following incubation with cisatracurium. Since the stabilizing agent BSA was ineffective in triggering apoptosis in HUVEC, the most probable substance to elicit the adverse effects observed in our study are acrylate esters formed during breakdown of cisatracurium.
Mivacurium, the second non-depolarizing muscle relaxant investigated, did not induce apoptosis in HUVEC in the present study. Mivacurium does not decompose spontaneously via Hofmann reaction but is degraded via choline esterases . This further underlines the assumption that Hofmann reaction breakdown products are the main precipitators of apoptosis. Recent forensic evidence has highlighted preferential accumulation of atracurium and its breakdown products in lungs, kidneys and myocardium . These findings were attained indirectly, i.e. measuring the second breakdown product of the Hofmann reaction, laudanosine. However, they may offer an alternative explanation to several of the conversant side-effects of atracurium. These have been described in lungs and the cardiovascular system, while sufficient evidence to support atracurium toxicity in kidneys is lacking [12,13].
Previous studies dealing with potential cytotoxic effects of atracurium mainly focussed on the isolated liver and hepatocytes. In vitro, Nigrovic and co-workers demonstrated a deleterious effect of atracurium on rat hepatocytes. In contrast to these findings, perfusion of isolated rat livers did not result in organ damage in a number of subsequent studies [14-16]. However, incubation with atracurium in the latter studies was short lasting (≈10 min), and organ function was monitored 1-2 h later. Apoptosis and subsequent growth inhibition is a time-consuming process, i.e. extended incubation periods of 6-12 h have been described as necessary to obtain evidence of apoptosis .
However, the clinical implications of these in vitro findings remain to be ascertained in vivo. Only case reports on suspected toxicity of atracurium have been published. In particular, tachycardia, hypotension and hypoxia were noted in a compilation of case reports by Clarkson and colleagues . In addition to the acknowledged side-effects of atracurium and its isoform (e.g. histamine liberation), oxidative stress elicited by acrylate esters might represent an alternative explanation for short-term adverse effects of cisatracurium and its isoform atracurium[2,19]. In summary, the present investigation demonstrates the induction of apoptosis by cisatracurium in HUVEC in vitro. These actions are most likely due to the reactive nature of acrylate metabolites generated during Hofmann elimination.
We acknowledge the kind assistance of Albert Amberger PhD in providing the HUVEC cells for the experiments. The authors would like to thank Ms M. Czechowski and Ms M. Schloesser for expert technical assistance. The first author J. Rieder received travel fees to present an abstract of this manuscript at the ESA congress in Nice, France, 2002.
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