Despite its widely known genotoxic and cytotoxic action, ionising radiation (IR) remains standard cancer treatment (1). Through ionisation of molecules, IR generates toxic free radicals, which can result in single and double-strand DNA breaks, incur oxidative damage to sugar and base residues, and cause chromosomal aberrations and mutations without distinguishing between tumour and normal tissues (1, 2). Even though cancer cells are more vulnerable to IR than normal cells due to rapid cell division and inefficient DNA repair, both sustain DNA damage. For this reason, exposure of normal tissues is limited in terms of maximum deliverable dose, which also limits the efficacy of IR therapy (3).
According to the latest version of the guidelines issued by the Royal College of Radiologists (2), a typical IR dose in different radiotherapies (intraoperative, brachytherapy, fractionated and hypofractionated radiotherapy) is 1 or 2 Gy. To optimise the therapeutic effects of these doses and minimise the damage to normal tissue with precision targeting, some patients such as children or those with anxiety issues or claustrophobia are immobilised (4, 5) or put under the general anaesthesia. Anaesthesia is in some cases also used during intraoperative radiotherapy or brachytherapy.
Standard general anaesthesia involves volatile anaesthetics (VAs), such as isoflurane (I), sevoflurane (S), and halothane (H) as an alternative to intravenous application. They have similar mechanisms of action as they enhance the inhibitory activity of postsynaptic channels [gamma-aminobutyric acid (GABA) and glycine] and inhibit the excitatory activity of synaptic channels [glutamate,
Earlier studies (7, 10–12) have already described DNA damage and repair in human blood samples for each of the three anaesthetics and IR doses usually applied in therapy, but none has yet addressed single-dose exposure to a combination of VAs and IR, even though IR at sufficient doses has a similar mechanism of action as VA (13) and their combinations might have synergistic effects.
Therefore, the aim of this preliminary study was to investigate the effects of combined exposure to a single treatment with isoflurane, sevoflurane, or halothane alone or in combination with IR at 1 and 2 Gy in terms of liver tissue DNA damage and repair immediately and 2, 6, and 24 h after exposure, considering that the dynamics of DNA damage and repair also depend on the target organ tissue and time since exposure (7, 11–14).
The inhalation anaesthetics, namely sevoflurane (Sevorane®), isoflurane (Forane®), and halothane (Halothane®) were procured from Abbott Laboratories (Queenborough, UK). Chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), unless specified otherwise.
The study was approved by the Ethics Committee of the University of Zagreb, Faculty of Science, Zagreb, Croatia (approval No. 251-58-508-11-9) and was designed in accordance with the Croatian Animal Protection Act (15), Ordinance on the protection of animals used for scientific purposes (16, 17), and the EU Directive 2010/63/EU (18).
The study included 240 male Swiss albino mice, aged 60±5 days and weighing 20–25 g. The mice were kept in standard breeding conditions (22±1 °C ambient temperature, 50–70 % humidity, 12-hour photoperiod) and had free access to a standard laboratory diet (Standard Diet GLP, 4RF 1, Mucedola, Settimo Milanese MI, Italy) and tap water. We opted for male mice only, as females are more prone to X-chromosome loss and higher DNA damage after IR (19).
On the experiment day, the animals were randomly divided into the following 12 groups: control (not exposed to either VA or IR); IR treatment alone with either 1 or 2 Gy; anaesthetic treatment alone with S, I, or H; and combined treatments with either of the three anaesthetics and either IR dose (S+1 Gy, S+2 Gy, I+1 Gy, I+2 Gy; H+1 Gy; H+2 Gy). Each group consisted of four subgroups of five animals according to the time points at which their liver was taken: 0 h (immediately after the exposure), 2 h, 6 h, and 24 h after exposure (Figure 1).
Mouse anaesthesia with S (2.4 vol %), I (1.7 vol %), or H (2.4 vol %), was maintained with an anaesthetic machine (Sulla 800; Dräger, Drägerwerk AG & Co. KGaA, Lübeck, Germany) and a compatible evaporator in an induction chamber with the even oxygen-to-air ratio at a continuous flow of (3 L/min) for 2 h. Anaesthesia was considered achieved when the mice fell into a calm sleep, breathed spontaneously, and stilled their tail.
The mice planned for irradiation alone and irradiation combined with anaesthesia received a single dose of either 1 or 2 Gy (dose rate of 1.88 Gy/min) (60Co source, Theratron Phoenix teletherapy unit, Atomic Energy Ltd., Ottawa, Ontario, Canada). Anaesthesia was given to the animals prior the IR treatment to reflect actual radiotherapy conditions. In other words, the animals were treated with anaesthetics prior to IR.
Before liver sampling, the animals were killed by cervical dislocation in compliance with relevant national and EU legislation (16–18). Small pieces of freshly resected mice liver tissue taken from the right lobe of each animal in each group were dissected and homogenised mechanically with a freshly prepared and chilled (4 °C) homogenisation buffer (0.075 mol/L NaCl and 0.024 mol/L Na2EDTA) in the proportion of 1 g of tissue per 1 mL of buffer. Single-cell suspension was prepared by mincing and passing the homogenised sample through a stainless-steel mesh with a pore diameter of 40 µm. The whole procedure of sample preparation was done at 4 °C in order to stop DNA repair in the sample.
We chose the Comet assay to assess DNA damage and repair because it is a standard method used in human biomonitoring studies after
The assay was performed under alkaline conditions as described elsewhere (25, 26) with four agarose layers in total. Briefly, 10 µL of liver cell suspension was mixed with 100 µL of 0.5 % low melting point (LMP) agarose and layered as the third layer onto precoated microscope slides [the first layer consisted of a 1 % normal melting point (NMP) agarose and the second of 0.6 % LMP agarose]. The slides were then covered with coverslips and kept horizontally at 4 °C for 10 min. Then another layer of 0.5 % LMP agarose was placed on top. After solidification and coverslip removal, the slides were immersed in a freshly prepared ice-cold lysis solution (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, 1 % pH 10 sodium sarcosinate with 1 % Triton X-100, and 10 % dimethyl sulphoxide) at 4 °C for 2 h. In a freshly prepared electrophoresis solution (300 mmol/L NaOH, 1 mmol/L Na2EDTA, pH13) at 4 °C, denaturation first (for 20 min, vertical Coplin jars) and then electrophoresis [for another 20 minutes, with changed electrophoresis solution, in a horizontal gel-electrophoresis unit at 25 V (300 mA, 0.8 V/cm)] took place under dim light at 4 °C. Following the electrophoresis, the slides were neutralised three times at five-minute intervals by adding 0.4 mol/L Tris-HCl buffer, pH 7.5, stained with ethidium bromide (20 µg/mL), and examined under a 200× magnification epifluorescence microscope (BX40, Olympus, Tokyo, Japan) linked with a CCD camera to a computer-based image analysis system (Comet Assay IV software, Instem, London, UK). For each animal (sample), 40 randomly selected images of cells were analysed.
A total of 200 comets per group (40 per animal) were analysed for each test point. Tail length (TL, distance of DNA migration from the centre of the nuclear core in µm), tail intensity (TI, the percentage of genomic DNA migration from the nuclear core to the tail during electrophoresis), and tail moment (TM, calculated as the product of TL and TI, in arbitrary units) were the Comet assay parameters used to determine DNA damage.
To calculate cellular DNA repair index (CRI) we used the formula described elsewhere (27), as follows:
where the “comet parameter” refers to either TL or TI.
Statistical analysis was run on Statistica 9.0 (Statsoft, Tulsa, USA) and Statistica 188.8.131.52 packages (TIBCO Software Inc., California, Palo Alto, USA). Before the analysis of 200 comets per each point, we used the analysis of variance (ANOVA) to compare all results for all animals in a group for one measured point. Since the results between them did not significantly differ, they were all combined and the analysis done on 200 comets per group. Means, medians, and standard deviations (SD) were calculated as part of descriptive statistics for TL, TI, and TM. We compared the data for each group with respective controls at 0 h using the Mann-Whitney
In mice exposed to the anaesthetics alone, all three parameters (TL, TI, and TM) were higher than control at nearly all time points (Figures 2a–c). In general, S had the strongest effect on TL; while H and S had the strongest effect on TI and TM. We saw no time-dependent trend for any of the three parameters.
In combination with 1 Gy irradiation, all damage parameter values increased, but with S (in the early hours) and I were generally lower than irradiation alone by the end of measurements, whereas H generally worsened the irradiation effects (Figures 3a–c).
The same is true for H combined with 2 Gy irradiation until hour 24, when TI and TM dropped, whereas all three parameters significantly increased with S by that time (compared to 2 Gy irradiation alone at 0 h). Isoflurane, again, turned out to be the least damaging in such combination.
Considering that we observed some lowering of DNA damage with S and I in combination with 1 and 2 Gy irradiation, we wanted to see whether it would reflect on the level and speed of DNA repair (CRI). Compared to irradiation alone, CRI with I was higher for both TL and TI after 1 Gy exposure at all time points, while a similar effect was seen after 2 Gy exposure in the first six hours. With S, CRI was in general lower for TL and TI regardless of irradiation dose and sampling time. Repair with H was also generally lower after 1 Gy exposure for both TL and TI. Even though CRI it was higher for TL and TI after 2 Gy exposure, the combination of H and 2 Gy IR caused higher DNA damage than radiation alone, which is important for the evaluation of its effects.
Our findings show that a single exposure to any of the three anaesthetics alone caused varying DNA damage in liver cell samples of Swiss albino male mice and that, in general, H caused the highest damage. In combined exposure with IR, S and I demonstrated a slightly protective effect, which was more evident for 1 Gy exposure. For 2 Gy exposure, damage levels were similar to those in irradiated control samples by hour 24, after which they increased significantly.
Although this is a preliminary study and its results need to be explored further as well as include other methods, it gives new evidence about different effects of volatile anaesthetics and their combined use with IR.
It seems that pre-treatment with any of the three VAs can cause some damage to the DNA, most notably halothane. DNA damage always triggers (different) repair mechanisms (in general, there are five) (28). If we consider all TL, TI, TM, and CRI results together, isoflurane lowered the levels of DNA damage in combination with IR, and repair was more efficient than with the other two anaesthetics. Sevoflurane also lowered the DNA damage, but it was followed by lower repair, while halothane caused the highest DNA damage alone and combined with IR, followed by varying repair.
Considering our CRI results and the results of other authors, it seems that sevoflurane and isoflurane have different mechanisms of DNA damage and repair. Sevoflurane has been reported to increase and directly induce more complex DNA damage and is associated with longer and slower DNA repair (more than 24 hours) (11), while isoflurane increases systemic antioxidative status, protecting cells from DNA damage
Respiration is the pathway that eliminates most VAs, but a small amount is metabolised by the liver and then excreted by the kidneys. Of the three VAs investigated in this study, halothane’s liver metabolism is 15–20 %, sevoflurane’s around 5 %, and isoflurane’s up to 0.2 % (39–41). The presence of halothane in the liver this high can also be responsible for its damaging effects. The use of halothane in humans or animals has become less frequent since we learned about its side effects, mostly about its hepatotoxicity through reactive oxidative species (ROS) (42) and its ability to induce enzymes that metabolise it (43). Taking this into account, our findings for halothane were expected. Earlier studies with halothane, isoflurane, and sevoflurane (7, 11, 14, 36, 37) demonstrated a similar pattern in the formation of DNA damage in other organs such as blood, kidney or brain, with at different time points after
Future studies should include combined VA+IR effects on healthy cells surrounding a repeatedly exposed tumour at IR doses lower than the standard 1 or 2 Gy. In addition, they should include both sexes to establish potential differences in DNA damage and repair. We also need to learn more about the mechanisms and modes of action (using histology, oxidative stress biomarkers, Western blotting) and expression of different proteins and enzymes involved in DNA damage and repair, changes in cell proliferation, and the frequency of apoptotic and necrotic processes in the cell. A better insight would also result from a long-time follow up after a single- and multiple-dose exposure to see if DNA damage is completely repaired or eliminated and to determine the sensitivity of different organs after such exposure.
In conclusion, single exposure to VAs alone has led to different DNA damage and repair in the liver cells of male mice. In combination with ionising radiation, sevoflurane and isoflurane had slightly protective effects, mostly at 1 Gy exposure. Our findings are in line with current literature regarding DNA damage and repair in the liver and other organ systems, and suggest that both sevoflurane and isoflurane are suitable for combined treatment with IR, but isoflurane is preferable, where its use is possible.