The Protective Effects Of Metallothionein On Cyclophosphamide-induced Immune Toxicity

Cyclophosphamide was a cancer treatment drug that widely used in clinical, it played an important role in the treatment of multiple myeloma, lung cancer, cervical cancer, ovarian cancer and other malignancies. However, it also brought a number of side effects on the skin, respiratory, urinary and reproductive systems and the immune system, which severely limited its treatment effects as a clinical anti-cancer drug. The immune system was the defense system of body, which played an important role in the resistance to exogenous bacteria and virus, furthermore, it was also meaning for the immune system reconstruction of tumor patients. Considering this, it was necessary to find out the ways to weaken its immune toxicity. Metallothionein was a small molecular proteins that could be induced by kinds of stimuli, and had various physiological functions, including the heavy metal detoxification, the control of trace elements and radiation. Furthermore, metallothionein could reduce the immunosuppression to some extent. According the physiological function of metallothionein, we suspected that it was able to decrease oxidative damage and promote DNA damage repair to reduce the inhibition of immune system caused by immune inhibitors. Metallothionein could protect body from damage caused by exogenous factors, this function may be by reducing the degree of oxidative damage of the body, and promoting the realization of DNA damage repair. As we know, CP can bring cell cytotoxicity by DNA alkylation. In order to improve the clinical application of CP in experimental foundation and theoretical basis, we tried to research the function of MT in protecting body from immune toxicity caused by CP.In this study, we took the MT-/- and MT+/+ mice as animal model to observe the damage of peripheral blood, bone marrow, thymus and spleen caused by cyclophosphamide. We investigated the protective effects of metallothionein on cyclophosphamide-induced immune inhibition by oxidative stress and DNA damage repair.Our results showed that after the treatment of cyclophosphamide, it happened a series of injuries in peripheral blood, bone marrow, spleen and thymus. We can know that the low white blood cell counts, the increase of micronucleus rate in bone marrow, the decrease of the ratio of viscera and brain, and MT-/- mice are more obvious than MT+/+mice in these results. Further study showed that the level of MDA in MT-/- mice was higher than that of in MT+/+ mice; and the activity of GSH-Px in spleen and thymus showed a trend of lower, and the enzyme activity reduced significantly in MT-/- mice.In addition, the activity of SOD increased in spleen and MT-/- mice seemed endure more changes; while in the thymus, the activity of SOD increased in the MT+/+ mice but decreased in the MT-/- mice. The above results showed that the oxidative damage was more serious in MT-/- mice. Nrf2/ARE signal pathway was an important way to protect body from oxidative damage, in our study, we payed attention on spleen and thymus to detect the expression of Nrf2 protein、HO-1 protein and NQO-1 protein in molecular level. The results indicated that the infection of cyclophosphamide led to the increase of Nrf2 protein, especially in MT-/- mice, and HO-1 and NQO-1, as its downstream protein has the similar changes an Nrf2 protein. These results suggested that MT could protect the immune toxicity of CP in changing the oxidative damage by changing related enzyme activity, which by activatiing Nrf2 signaling pathways.In this study, we also studied the DNA damage in the molecular level. Comet assay in peripheral blood showed that cyclophosphamide induce severe DNA damage. γH2AX protein was one of the sign of double-stranded DNA fracture, our results suggested that exposing CP can increase the level of yH2AX protein, and the protein in MT-/- mice are higher than that in MT+/+mice. Further study on related proteins of DNA damage repair, such as ATM, P53 and P21, showing that given cyclophosphamide can lead the activation of ATM, and increase the expression of P53 and p-P53, and the MT-/-mice change more significantly. As the downstream of P53, the expression of P21 also increased, but there was no differences between MT+/+ and MT-/- mice. These results suggest that MT could protect the immune toxicity of CP in changing the expression of ATM, P21 and P53 protein involved in DNA damage repair pathways.According all the results, CP can generate immunosuppression toxicity in mice, and MT can reduce the toxicity of immunosuppression caused by CP. we speculated that MT can exert its functions by two ways.One reason was that MT can reduce oxidative damage caused by CP, MT can affect the expression of proteins in Nrf2/ARE signaling pathways and the expression of related antioxidant enzymes; The other was that MT can repair DNA damage caused by CP, which may be the reason that MT can effect the expression of proteins in DNA damage repair.