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Protective role of isoflurane after combined exposure with 1 or 2 Gy of ionizing radiation assessed with alkaline comet assay in vivo

Cancer yearly increase is connected also with radiotherapy increase, and younger patients such as children are also not spared from the yearly cancer and radiotherapy increase. To ease their pain and to make them comfortable during the radiotherapy, and also the lower the number of the dose treatment, by precise localization of the treatment, sometimes it is necessary to use the volatile anaesthetics as part of general anaesthesia. In past, those anaesthetics were considered safe, but there are new findings demonstrating that this is not always true. We decided to use the most used VA and to examine in vivo on animal model whether there is synergistic effect in combined exposure to isoflurane and to exposure to 1 or 2 Gy of ionizing radiation, usually used in fractioned radiotherapy. Using healthy Swiss male albino mice 60 days old, cca. 25 grams of body weight, we exposed them for 2 hours to 1.7% of inhaled isoflurane mixture with oxygen and air and afterwards some of them were also exposed to 1 or 2 Gy of ionizing radiation and were sacrificed immediately after exposure, 2, 6 and 24 hours from the exposure. We had 5 animals per group and radiation source was cobalt, with the dose rate of 1.88 Gy/minute on the Theratron Phoenix teletherapy unit, Atomic Energy Ltd., in at the Clinical Hospital ʺSveti Duhʺ, Zagreb, Croatia. Study was approved by Ethical Committee of the Faculty of Science (University of Zagreb, Croatia) and was designed in accordance with the relevant Croatian guidelines: The Animal Protection Act (NN 102/17.) and the Ordinance on the protection of animals used for scientific purposes (NN 55/13 ; 39/17). We measured primary DNA damage level using alkaline comet assay, well established method for in vivo genotoxicity assessment according to OECD guidelines on 200 comets per each time point and treatment. DNA damage was assessed using fluorescence microscope Olympus BX 40 connected with CCD camera with Comet Assay IV software and with parameters of tail length, tail intensity and tail moment. We took blood samples and liver samples for the alkaline comet assay. The results demonstrated that both single exposure to isoflurane and ionising radiation caused significantly higher DNA damage levels when compared to the control samples, with isoflurane and 1 Gy ionizing radiation having similar levels, while 2 Gy exposure caused the highest DNA damage levels. Surprisingly, combined exposure of isoflurane and ionizing radiation caused lower levels of DNA damage when compare to only radiated samples. These findings implicate that isoflurane can cause protective effect and cause lower level of DNA damage and should be used in radiotherapy. The mechanism by which isoflurane caused this effect is probably adaptive response, by activating DNA repair mechanisms and the levels of scavengers of reactive free oxygen radicals, covering on that way both types of which IR exposure can cause DNA damage effects (indirect and direct IR effect on DNA).

isoflurane ; ionizing radiation ; comet assay ; Swiss albino mice

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