Sevoflurane and propofol are widely used anaesthetic agents and are often used clinically in combination.1 Sevoflurane has a low incidence of adverse effects and is used in more than 2 million cases annually. Although the incidence is low, hepatotoxicity is seen in a small proportion of patients following anaesthesia with sevoflurane,2–6 especially after prolonged use and low flow anaesthesia.6–11 Many factors could induce or contribute to liver damage during sevoflurane anaesthesia, such as decreased liver blood flow or increased intracellular calcium content.10,12–14 Additionally, 3 to 5% of sevoflurane undergoes dose-dependent hepatic biotransformation to hexafluoroisopropanol, inorganic fluoride ions and carbon dioxide, which could contribute to hepatotoxicity. However, the mechanisms whereby sevoflurane induces hepatotoxicity remain largely unelucidated.
Gap junctions act as intercellular protein channels, directly connecting adjacent cells and facilitate synchronous functioning of diverse cells.15,16 They play an important role in maintaining homeostasis and in mediating cellular proliferation.17,18 Gap junction channel proteins are encoded by a supergene family composed of more than 20 members.19 Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis.20 Hepatocellular gap junctions are composed of two hemichannels of adjacent cells that are built up by connexin proteins, such as Cx32, Cx26 and Cx43, the latter accounting for 90% of total hepatic connexin proteins.21 Mice deficient in Cx32 were protected against liver damage, acute inflammation and death caused by hepatotoxic drugs22 and mice carrying the dominant-negative mutant Cx32 V139M gene showed high susceptibility to chemical hepatocarcinogenesis,23 thus implicating these gap junction proteins in protection against chemical-mediated hepatotoxicity.
Propofol has been shown to alleviate hepatic ischaemia reperfusion injury24,25 and to significantly reduce radiation-induced cytotoxicity in vitro by reducing Cx32 expression.26 We hypothesised that sevoflurane-induced hepatotoxicity was mediated by gap junctions and propofol could attenuate sevoflurane-induced cytotoxicity by its action on intercellular gap junctions. In the present study, we investigated the cytotoxicity of sevoflurane against immortalised normal rat liver cells and whether this effect was mediated by gap junction intercellular communications. We also investigated the effect of propofol on sevoflurane-induced cytotoxicity and the underlying mechanism.
Materials and methods
Cells and drug treatments
The immortal rat liver cell line BRL-3A, which naturally expresses Cx32/43, was purchased from American Type Culture Collection (ATCC; Manassas, Virginia, USA) and grown at 37°C in Dulbecco's modified Eagle's medium (DMEM)-F12 supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, New York, USA).
For drug treatments, all exposures to sevoflurane (Abbott Laboratories, Green Oaks, Illinois, USA) were performed in airtight containers (Lock & Lock, Seoul, South Korea) for 4 h through an anaesthesia apparatus (Datex Ohmeda 7100; GE Healthcare, Little Chalfont, UK) containing 0.5 l min−1 oxygen. Oleamide (Sigma, St Louis, Missouri, USA) was dissolved in dimethyl sulfoxide (DMSO) and freshly diluted before drug treatment to a final working concentration of 2.3 μg ml−1 in culture medium. In addition, the propofol used was prepared in lipid emulsion (AstraZeneca, London, UK).
Colony-forming assays were performed as previously described.27 For high-density culture in which each cell was in contact with three to five other cells on average, with substantial opportunity for formation of gap junctions, BRL-3A cells were seeded at 10 × 103 cells per cm2 in six-well plates. For low-density culture in which cells had little opportunity to form gap junctions, cells were seeded at 5 × 102 cells per cm2. Cells were treated as indicated elsewhere and 5 to 7 days later, colony formation was assessed by staining with crystal violet. An aggregation of at least 50 cells was considered a colony and the fraction of surviving cells was calculated as the number of colonies from treated cells divided by the number of colonies from untreated cells.
Parachute dye-coupling assays
To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays as described previously.18,19 Donor and receiver cells were grown to confluence. After double-labelling with 5 μmol l−1 CM-DiI (a membrane dye that does not spread to coupled cells) and 5 μmol l−1 calcein-acetoxymethyl ester, which is converted into the gap junction-permeable dye calcein intracellularly (both from Molecular Probes; Invitrogen, Carlsbad, California, USA), donor cells were harvested and seeded onto the receiver cells at a donor:receiver ratio of 1 : 150. Donor cells were allowed to attach to the monolayer of receiver cells and form gap junctions for 4 h at 37°C. The average number of receiver cells containing dye per donor cell was visually determined using a fluorescence microscope and normalised to that of controls.
Western blotting assays
Whole-cell lysates of BRL-3A cells were prepared as previously described.28 The immunoblotting procedure was performed as depicted previously29 and the following antibodies were used: mouse anticonnexin32; connexin43; and β-actin antibodies (all from Sigma). The protein bands were visualised by using Western Lightning chemiluminescence reagents (PerkinElmer Life and Analytical Sciences, Boston, Massachusetts, USA). Densitometry was performed using Quantity One software with a GS-800 densitometer (Bio-Rad Laboratories, Hercules, California, USA).
Data were statistically analysed using unpaired Student's t-test at a significance level of P <0.05 and were expressed as mean (SD) using Sigma Plot 10.0 software (Systat Software, San Jose, California, USA).
Sevoflurane exerts cell density-dependent cytotoxicity
To investigate whether sevoflurane was cytotoxic against BRL-3A cells and whether this cytotoxicity was dependent on cellular density, we grew these cells in low and high density as described earlier. These cells were then treated with clinically relevant doses of sevoflurane. Colony formation assays revealed that, in low-density culture, low doses of sevoflurane (1.1%, approximately 1.3 minimum alveolar concentration) caused no apparent inhibition of BRL-3A cells with 97.1 (0.0126)% of the cells still viable (Fig. 1). In addition, high doses of sevoflurane (4.4%) failed to noticeably suppress the clonogenic growth of BRL-3A cells with 83.4 (0.0352)% of the cells still viable (P >0.05 vs. controls). In high-density culture, 2.2 to 4.4% sevoflurane caused markedly greater inhibition of the clonogenic growth of BRL-3A cells with 67.6 (0.0342)% and 61.2 (0.0172)% of the cells viable, respectively (P = 0.0003 and 0.00028, vs. the same doses of sevoflurane at low-density culture), whereas 1.1% sevoflurane caused no apparent inhibition of BRL-3A cells with a viability rate of 90.5 (0.07)% (P >0.05 vs. controls). These findings indicated that sevoflurane caused cell density-dependent cytotoxicity against BRL-3A cells in vitro.
Density-dependent cytotoxicity by sevoflurane is mediated by gap junctional intercellular communication
The cell density dependence of sevoflurane-induced cytotoxicity suggested the involvement of gap junctional intercellular communications. To examine the role of gap junctional intercellular communications in sevoflurane-induced cytotoxicity, we pharmacologically facilitated or blocked gap junction function using oleamide, a sleep-inducing lipid that is also known to block gap junctions. As expected, in high-density culture, 4.4% sevoflurane markedly suppressed the clonogenic growth of BRL-3A cells with a viability rate of 56.6 (0.172)% (Fig. 2). Blocking of gap junctions with 10 μmol l−1 oleamide significantly attenuated the suppression by 4.4% sevoflurane of the clonogenic growth of BRL-3A cells with a viability of 83.6 (0.043)% (oleamide and sevoflurane vs. sevoflurane, P <0.01).
Propofol attenuates sevoflurane-induced cytotoxicity
Propofol has been shown to alleviate radiation-induced cytotoxicity by inhibiting Cx32 expression in vitro.26 Here, we confirmed this finding in BRL-3A cells by western blotting assays, which showed that propofol (3.2 μg ml−1) markedly reduced CX32 levels at 24 h posttreatment while no such effect on CX43 expression was observed (Fig. 3a and 3b). We investigated whether propofol inhibited gap junctional intercellular communications. The parachute dye-coupling assays showed that propofol markedly inhibited gap junctional intercellular communications of BRL-3A cells (Fig. 3c). To further study whether propofol attenuated sevoflurane-induced cytotoxicity, we pretreated BRL-3A cells with 3.2 μg ml−1 propofol for 1 h prior to treatment with sevoflurane. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells [viability: propofol and sevoflurane, 91.5 (0.0387)% vs. sevoflurane, 56.6 (0.0172)%; P <0.01; Fig. 3d)]. The findings indicate that propofol, at clinically relevant doses, inhibits gap junctional intercellular communications in BRL-3A cells and alleviates sevoflurane-induced cytotoxicity in vitro.
Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane.2–6 In the present study, we demonstrated that clinically relevant doses of sevoflurane significantly suppressed the clonogenic growth of rat liver cells in vitro growing in high-density cultures, which may closely mimic the in-vivo scenario in which hepatocytes contact one another and form functioning intercellular gap junctions. This inhibitory effect was associated with changes in the ultrastructure of the rat liver cells including swollen mitochondria and derangement in the reticulum (data not shown). We further demonstrated that sevoflurane-induced cytotoxicity required gap junctions, as sevoflurane-induced cytotoxicity was noticeably attenuated by a gap junction blocker. This implicates gap junctions in the development of sevoflurane-mediated cytotoxicity.
Sevoflurane and propofol are often used together in clinical settings.1 Several studies have shown that propofol could possess hepatoprotective properties.24–26 Consequently, we have demonstrated that propofol markedly attenuated sevoflurane-induced cytotoxicity against immortalised rat liver cells. Furthermore, the attenuation was only observed in high-density cultures. There are two principal mechanisms of intracellular communication: one is indirect communication mediated by secreted growth factors or hormones binding to cognate receptors on other cells and acting as second messengers and the other is direct communication in which small molecules transport between adjacent cells via gap junctions.30 To define whether gap junctions were associated with sevoflurane-induced cytotoxicity of BRL-3A cells, we used pharmaceutical agents to inhibit or facilitate gap junction function and investigated their effects on sevoflurane-induced cytotoxicity. We showed that oleamide-mediated inhibition of gap junctions attenuated sevoflurane-induced reduction of colony formation in high-density BRL-3A cells, whereas retinoic acid activation of gap junctions acted in conjunction with sevoflurane to further suppress colony formation (data not shown). Interestingly, neither oleamide nor retinoic acid significantly influenced sevoflurane cytotoxicity against BRL-3A cells in low-density culture. These data strongly suggest that a gap junction inhibitor may play a protective role against sevoflurane-induced hepatotoxicity.
Propofol inhibits gap junction intercellular communication,26,31,32 but its effect on connexin protein expression has not been completely illuminated. Propofol was shown to reduce radiation-induced cytotoxicity in vitro by reducing Cx32 expression.26 We also observed that propofol reduced the levels of Cx32 in immortalised rat liver cells growing in high-density culture, suggesting that propofol may militate against cytotoxicity by modulating gap junction function. These results indicate that propofol decreases gap junction intercellular communication mainly via downregulation of Cx32 protein level.
There are multiple possible causes for hepatotoxicity following anaesthesia such as hypoxaemia, systemic hypotension, hepatic surgery and toxic metabolites of sevoflurane. Our study is based on in-vitro model and cannot address hepatotoxicity following hypotension or hepatic surgery as it requires an in-vivo approach. Our model, therefore, does not accommodate all the causes for hepatotoxicity following anaesthesia. Instead, our investigation focused on whether gap junction intercellular communication plays any role in sevoflurane hepatotoxicity by using an in-vitro high-density and low-density cell culture model. Our study provides the first direct evidence that propofol alleviates sevoflurane-induced cytotoxicity against BRL-3A cells, which is dependent, at least partially, on gap junction function, possibly through Cx32 homomeric or heteromeric complexes. Gap junctions are complex and dynamic molecular mechanisms, and have the ability to modulate the function of their transmissibility through varied protein expressions in the cell membrane and various compositions of different connexin components. It is reasonable to hypothesise that sevoflurane-induced liver dysfunction is a result of alterations in particular connexin homomeric or heteromeric complex. To our knowledge, no inhibitors are known that specifically target the different connexins. The results from the present study provide evidence for future studies to develop such specific therapeutic agents.
Acknowledgements relating to this article
Assistance with the study: none.
Financial support and sponsorship: this work was supported by the Natural Science Foundation of Guangdong Province, China (No. 9151802904000014).
Conflicts of interest: none.
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