Protective Effects Of Kojic Acid On Radiation Damage In Beagle Dogs And Molecular Mechanisms Of Its Anti-radiation

Exposure to ionizing radiation (IR) at work, some harmful effects would be occur when protective measures are improper, violating the operating rules, or its intensity and radiation dosage exceed certain limit. Early in the 1940s, it began for human beings to search for a kind of ideal radioprotective agent, however, during this period only WR-2721, an ammonia and sulfhydryl compound, is approved by the US FDA on clinical resistance to radiation. Although it is significant in effectiveness, some severe side effects, such as hypotension, vomiting, nausea greatly limits its widespread use. Therefore, a novel radioprotector with more efficiency and less toxicity is urgently necessary for human beings.Kojic acid (KA) is produced by many species of Penicillium, Aspergillus, and Acetobacter. It is reported that KA can protect skin from pigmentation, and is widely used in cosmetics and food additives. KA easy to preparation, manufacture, high efficiency, low toxicity, less adverse reactions and convenient route of administration, and other excellent characteristics. However, the potential effects of radiation protection of KA and whether it can be a novel radioprotector is our concern. It has been indicated that KA has properties of radioprotectors and antioxidants and can stimulate the neutrophilic granulocyte phagocytosis and lymphocyte proliferation. Based on their findings, we found that KA not only could increase the 30-day survival rates of male C57BL/6 mice but also markedly protect the hematopoietic systems, immune systems, and DNA against radiation, and has significant radioprotective effects on Chinese hamster ovary (CHO) cells.However, it is not sufficient for us to determine the efficiency of anti-radiation agents when we put rodent animals (mice) as the experiment object. Therefore, in vivo investigations, beagle dogs as the experiment object were used to further observe the anti-radiation effect of kojic acid. In addition, in order to illustrate how Kojic acid is to play its anti-radiation effects, we taken the irradiated CHO cells as experimental model to investigate the effects of kojic acid on mitochondrial DNA damage repair and apoptosis signaling pathways, to lay a good foundation for further clinical trials of kojic acid. The experiment was divided into three parts, and the main findings are as follows:In the first part, as the experiment object, the beagle dogs were used to further observe the anti-radiation effect of kojic acid. Dogs in kojic acid or irradiation group were subcutaneously pretreated with a single dose of 63 mg/kg body weight of kojic acid or equal volume of sterile distilled water at 27 h prior to 3 Gy of lethal dose of gamma-irradiation. The arm widths of the dogs were 12 cm, and the source-to-skin distance was 400 cm with a dose rate of 61.08 cGy/min. We observed and tested the 51-day survival rates, general sign’s, haematological studies, bone marrow DNA contents and micronuclear rates of dogs. In this study, dogs in the irradiation group were all dead within 16 days, and the average survival days for dead animals were 14.5± 0.58. However, two dogs died in kojic acid group during 51-day observation period. Meanwhile, the body weight/temperature of animals pretreated with kojic acid reduced/elevated sharply two weeks after exposed to radiation, and then reverted to normal after 51-day observation period. For the hematopoietic system, we found that the white blood cell counts of dogs in kojic acid group were sharply decreased at 23 days post-irradiation, and recovered to normal at day 29 compared with normal values of animals in different groups prior to radiation. And it was found that the RBC count, HCT, and hemoglobin content of dogs pretreated with kojic acid were maximum decreased from day 21 to day 25 post-irradiation compared with the normal value of this corresponding group 3 days before irradiation, and then recovered gradually. These cells of animals in kojic acid group reverted to normal after 51-day observation period. In addition, the micronuclear rates of dogs treated with kojic acid were significantly declined (p<0.05) compared with that of the dogs in irradiation group while the DNA contents of dogs in kojic acid group were elevated markedly (p<0.05) in comparison with that of animals in irradiation group, which means kojic acid could markedly decrease DNA damage induced by ionizing radiation and have good radioprotective effects on DNA.In the second part, we found that pretreaded under KA, irradiated CHO cells reached a decreased level of percent of tail length, DNA in tail, tail moment, and olive tail moment comet parameters in comparison to the irradiated group (p<0.01). Pretreatment with KA significantly effects the mRNA expression of RAD50, ATM, BRCA1 and RAD51. Briefly, the expression levels of RAD50, ATM, BRCA1 and RAD51 genes were found to be significantly up-regulated in KA with dose-effect dependent compared to the irradiated group (p<0.05, p<0.01). The expression levels of MRE11, RAD50 and NBS1 proteins were found to be significantly up-regulated and mobilization in KA with dose-effect dependent compared to the irradiated group (p<0.01). The expression levels of p-ATM was found to be significantly activation in KA with dose-effect dependent compared to the irradiated group (p<0.01), and the total amount of ATM remained constant in all groups. BRCA1 and RAD51 proteins were found to be significantly enhanced in KA groups with dose-effect dependent compared to the irradiated group (p<0.01). The significant findings were the pretreatment of KA could inhibit γ-ray-induced DNA Double-stranded breaks (DSBs) in CHO cells, and the radioprotective effect might be related to the sequential mobilization MRN complex, activation ATM, up-regulation BRCA1 and RAD51, and then enhancement of the activity of DNA repair. In conclusion, KA could inhibit the γ-ray-induced DNA DSBs by activing the ATM and regulating the downstream effectors of it.In the third part, we found that the apoptotic fraction significantly increased (p<0.01) by irradiation and decreased (p<0.01) in a dose-dependent manner by KA pretreatment. The levels of the intracellular ROS increased markedly (p<0.01) in CHO cells exposed to y-rays, and KA pretreatment significantly attenuated the increase (p<0.01) in DCF fluorescence in a dose-dependent manner. And y-rays induced MMP reduction (p<0.01), and KA pretreatment significantly maintained this effect in a dose-dependent manner (p<0.05, p<0.01). The expression levels of p53 gene did not observed significantly change among the all groups. And we found that the mRNA expression level of Bcl-2 was decreased (p<0.01) in y-rays induced CHO cells and Bax was increased (p<0.01), while pretreatment with KA partly reversed this tendency (p<0.05, p<0.01). The mRNA expressions of Cytochrome c, caspase-3 and PARP were increased in y-rays (p<0.01), and the relase or activation were inhibited in a dose-dependent manner by KA pretreatment (p<0.01). Bcl-2 proteins were decreased (p<0.01) in y-rays induced CHO cells and p53 and Bax proteins were increased (p<0.01), and the ratios of Bcl-2/Bax were inverted after irradiation, while pretreatment with KA partly reversed these tendencies. From the Western blot band density analysis cytochrome c release, caspase-3 activation and PARP cleavage were increased in the y-rays group (p<0.01) compared to the control. And in the presence of KA reduced the cytochrome c release compared to y-rays group and decreased cleaved caspase-3 and PARP (p<0.05, p<0.01). Our study reveals that in the present of KA could inhibit y-rays induced mitochondria-mediated apoptosis. The radioprotective effect may be mediated by scavenging ROS, stabilising mitochondrial transmembrane potential, depressing p53, modulating Bcl-2/Bax, and the sequential inhibiting of Cytochrome c release and caspase-3 activation and its downstream target PARP cleavage.From the present investigation, it can be concluded that kojic acid not only has marked anti-radiation effect on rodent, but also protects beagle dogs from ionizing radiation induced damage. Moreover, this experiment demonstrates that kojic acid could stimulate hematopoietic function of bone marrow in beagle dogs irradiated by radiation. According to the field as we know, Kojic Acid, for the first time, was tested for the mechanisms of its radioprotective effect. KA has great potential as a new class of radioprotector.