Kidney cell DNA damage caused by combined exposure to volatile anaesthetics and 1 Gy or 2 Gy radiotherapy dose in vivo

The study was designed in accordance with the Animal Protection Act (17), Ordinance on the Protection of Animals Used for Scientific Purposes (18), EU Directive (19), and OECD guidelines (14) and approved by the Ethics Committee of the University of Zagreb Faculty of Science, Zagreb, Croatia (approval No. 251-58-508-11-9).

The 60±5 days old Swiss albino mice with body weight of 22.4±0.3 g were taken from the breeding unit of the Department of Biology, University of Zagreb Faculty of Science. Due to sex differences in DNA damage induced by ionising radiation (female mice suffer higher DNA damage due to hormonal influence and can lose the X chromosome much more easily) (20, 21), we chose only male mice.

The housing conditions of the animals were 22±1 °C, 50–70 % humidity, 12/12 h light/dark cycle, and free access to water and standard laboratory diet (4RF 21, Mucedola, Settimo Milanese, Italy).

The animals were divided into 48 subgroups with five animals each as illustrated in Table 1. Time points were based on our previous experiments (7, 9).

If not specified otherwise, chemicals, reagents, and other materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The inhalation anaesthetic sevoflurane (Sevorane^®), isoflurane (Forane^®), and halothane (Halothane^®) were provided by Abbott Laboratories Ltd. (Queenborough, UK).

Anaesthetic dosing was similar as in earlier studies (9, 22, 23), as it was shown to ensure deep and constant anaesthesia over two hours. Concentrations of each anaesthetic used in animals corresponded to the concentrations used in humans to maintain deep anaesthesia during a single radiotherapy treatment. The doses in this study followed the Guedel’s guide to laboratory animal anaesthesia (24), in which the anaesthetic concentrations corresponding to human dosages are adjusted to animal species, age, weight, and type (inhalation or injection) and duration of anaesthesia (7, 22, 23, 24, 25, 26, 27).

Each anaesthetic – sevoflurane (2.4 % v/v), isoflurane (1.7 % v/v), or halothane (2.4 % v/v) – was added to a 50:50 mixture of oxygen and air (3 L/min) using a specially designed induction chamber connected to an anaesthetic machine (Sulla 800, Sulla, Dräger, Velbert, Germany). This machine for semi-closed rebreathing system was equipped with a Ventilog for automatic ventilation, a Barlolog A ventilation pressure meter, an Oxydig for O₂ measurement, and a compatible evaporator. Mice anaesthesia with either of the three anaesthetics was maintained at a continuous gas flow for 2 h. A satisfactory anaesthesia depth was considered to have been achieved when the mice were sleeping calmly, breathing spontaneously, and not wiggling their tail.

Immediately (about 10 min) after the two-hour anaesthesia, the mice assigned for irradiation (and those that were not anaesthetised) were irradiated with 1 Gy or 2 Gy (⁶⁰Co source, Theratron Phoenix teletherapy unit, Atomic Energy Ltd., Ontario, Canada), at Sveti Duh Clinical Hospital (Zagreb, Croatia) at a dose rate of 1.88 Gy/ min. These radiation doses are common in diverse radiotherapy procedures (brachytherapy, intraoperative, fractionated, and hypofractionated radiotherapy) (16).

Immediately or 2, 4, 6, or 24 hours after anaesthetic and/or radiation exposure was completed, the animals were sacrificed by cervical dislocation according to laboratory animal legislation (18). Resected mouse kidney cortex samples of each animal were taken from a similar position and placed into a chilled homogenisation buffer (0.075 mol/L NaCl and 0.024mol/L Na₂EDTA) at a ratio of 1 g of tissue to 1 mL of buffer. Samples were immediately homogenised at 4 °C by mincing and passing through a stainless-steel mash to obtain a single-cell suspension at 4 °C and immediately take 10 μL to mix with the agarose gel for the alkaline comet assay.

The comet assay was carried out according to a standard procedure described in our previous papers (24, 25). Reporting of the results was in line with the Minimum Information for Reporting on the Comet Assay (MIRCA) and new technical recommendations (28).

Microscopic slides (Vitrognost, Biognost, Zagreb, Croatia) for the comet assay were precoated on the same day with 1 % normal melting point (NMP) agarose and then with a layer of 0.6 % low melting point (LMP) agarose. After solidification, slides were kept at 4 °C in humidified conditions to avoid gel drying. When the kidney cortex single cell suspensions were prepared, a mixture of cell suspension with 100 μL of 0.5 % LMP agarose was immediately layered on the top, and after solidification, a new layer of only 0.5 % LMP agarose was added. When this upper layer turned solid (after 10 min at 4 °C), slides were vertically immersed in Copling jars (Sigma-Aldrich) filled with freshly prepared ice-cold lysis solution (2.5 mol/L NaCl, 100 mmol/L Na₂EDTA, 10 mmol/L Tris-HCl, 1 % sodium sarcosinate, pH 10) with 1 % Triton X-100, and 10 % dimethyl sulphoxide (Kemika, Zagreb, Croatia) and kept there protected from light at 4 °C for 2 h. Slides were then washed with distilled water (Yasenka, Vukovar, Croatia) and immersed into a freshly cold denaturation solution (300 mmol/L NaOH and 1 mmol/L Na₂EDTA, pH 13) and kept protected from light at 4 °C for 20 min. Under a dim light slides were randomly placed in a new cold electrophoretic solution (the same as the denaturation one) in a horizontal gel-electrophoresis unit facing the anode, and electrophoresis was carried out at 25 V (300 mA, 0.8 V/cm). After 20 min, followed neutralisation of the slides with 0.4 mol/L Tris-HCl buffer (pH 7.5) in the dark, repeated three times at five-minute intervals. All slides were immediately dehydrated by immersion into absolute ethanol (99.6 %, Kemika) for at least 5 min, allowed to air-dry, and then stored at room temperature protected from humidity. Before analysis, slides were rehydrated with distilled water for 10 min, stained with ethidium bromide (20 μg/mL) for another 10 min, and examined under an epifluorescence microscope at 200× magnification (BX40, Olympus, Tokyo, Japan) connected with a camera with a charge-coupled device sending images to a computer-based image analysis system (Comet Assay IV software, Instem, London, UK). For each sample (animal) we analysed the images of 40 randomly selected cells, 200 comets in total for each time point as described earlier (29, 30).

DNA damage was determined as tail length (TL, distance between migrated DNA breaks and the nucleus expressed in μm) and tail intensity (TI, % of DNA in comet tail) using the same software.

Cellular DNA repair efficiency can be quantified by determining cellular DNA repair index (CRI), which is defined as decrease in initial value of the parameter due to repair, expressed in percentage. We calculated the CRI for TL and TI according to the formula by Nair and Nair (31) as follows:

Data were analysed with Statistica 14.0.0.15 (TIBCO Software Inc., Palo Alto, CA, USA). Descriptive statistics (mean, median, and standard deviation) was calculated for TL and TI. The data were statistically compared using the Mann Whitney U-test, with statistical significance set at p<0.05.

Tail intensities for all three anaesthetics at all time points were significantly higher than control, except for isoflurane at 2 and 6 h. Tail lengths for halothane were significantly higher than control at 2 and 6 h and lower at 24 h. With sevoflurane TL values were significantly higher than control at 0 and 2 h and significantly lower at 6 and 24 h. With isoflurane damage was lower than control at 0, 2, and 6 h. Halothane caused the highest DNA damage at all time points, significantly higher than sevoflurane and isoflurane, while the two did not differ significantly, except at 2 h (Figure 1, Table 3).

Both TL and TI in 1 Gy irradiated mice and mice exposed to combined treatments were significantly higher than in non-irradiated controls. The highest TL in 1 Gy irradiated mice was observed 24 h after irradiation. The lowest TL was observed 6 h from exposure. At this time point, TI values were the highest (Figure 2). Halothane combined with 1 Gy significantly increased both parameters compared to irradiated-only counterparts. Sevoflurane yielded TL values similar to irradiated-only groups at all time points, except for 24 h, in which significantly lower value was observed. TI values, in turn, were significantly lower after 6 h compared to samples from the irradiated-only mice. Isoflurane significantly lowered both TL and TI after 24 h compared to irradiated-only groups at the same time points (Table 3). Halothane again had significantly higher TI than sevoflurane and isoflurane at corresponding time points. The latter two VAs differed significantly in TI, except at 2 h.

The highest TL in mice irradiated with 2 Gy was observed 2 h after irradiation, while TI was the highest at 6 h after irradiation. Halothane again had the most damaging effect, that was significantly higher for both parameters than in their irradiated-only counterparts at corresponding time points. Sevoflurane also increased both parameters, which were significantly higher at the first three time points, while TI decreased at 24 h and was even lower than in the irradiated-only group. At 2 h, isoflurane had significantly lower TL and TI than irradiated-only counterparts, which significantly rose at 6 h and 24 h and were almost higher than those in the halothane groups at 0 h (Figure 3 and Table 3). Halothane TI values were significantly higher than sevoflurane at 0 h and 24 h and isoflurane at 0 h and 2 h. Sevoflurane and isoflurane differed significantly only at 2 h.

As we observed DNA repair in the 1 Gy and 2 Gy combined treatment with sevoflurane and isoflurane, we compared CRI for TL and TI as shown in Table 2.

In non-irradiated mice exposure to halothane and isoflurane increased the level of DNA repair by hour 24 from exposure, while it remained below control values for sevoflurane.

In mice irradiated with 1 Gy, all three VAs in general demonstrated higher repair rate than the corresponding control, with the highest level of repair triggered by isoflurane.

In mice irradiated with 2 Gy, CRI visibly dropped in all groups, but improved by hour 24. Only isoflurane had higher repair level than control 2 h from exposure, and then the repair rate dropped.