5-Fluorouracil Triggers Drug-induced Liver Injury By Activating The Ferroptotic Pathway

Objectives: Drug-induced liver injury(DILI)is a common adverse drug reaction caused by drugs and metabolites that directly impair liver cells or induce allergic reactions.Severe DILI can lead to liver failure or even death.In recent years,the global incidence of cancer has been increasing year by year,and chemotherapy has been applied more and more widely in the comprehensive treatment of cancer.Most drugs are metabolized by the liver,which greatly increases the burden on the liver.DILI induced by antitumor drugs is one of the common side effects.5-Fluorouracil(5-FU)is a widely used chemotherapeutic drug and one of the common drugs that cause DILI.5-FU has been shown to trigger liver function impairment in humans and model animals.However,the exact mechanism of liver injury induced by 5-FU exposure has not been fully elucidated.Ferroptosis is a newly discovered form of non-apoptotic cell death triggered by iron accumulation and lipid peroxidation.Ferroptosis involves a variety of pathological processes,including neurodegeneration,cancer,ulcerative colitis,liver and kidney ischemia-reperfusion injury,and plays a key role in the occurrence and development of various diseases.However,the ferroptosis has not been reported in liver injury induced by 5-FU.Therefore,the purpose of this study was to verify the role of the ferroptotic pathway in DILI induced by 5-FU.Methods: 1.Wild-type C57BL/6J mice were intraperitoneally injected with 5-FU(100mg/kg)for 5 consecutive days,while the control group was injected with the same volume of normal saline(10 m L/kg).The mice were euthanized 5 days later.Plasma and liver were collected.During the experiment,the body weight,food intake and water intake related basic indicators were detected.2.H&E staining was used to observe the morphological changes of mouse liver,and TUNEL staining was used to detect the number of apoptotic bodies in liver.Immunohistochemistry was used to detect the indicator of oxidation.3.Western blot and quantitative real-time PCR were used to detect the levels of ferroptosis and liver injury related factors in liver.4.Superoxide anion fluorescence probe(dihydroethidium,DHE)was used to detect ROS levels in tissues.5.Human normal hepatocytes L-02 were cultured in vitro,and the changes of oxidative stress,iron metabolism and cell death related indicators were detected after the exposure of 5-FU.6.L-02 cells were pretreated with Fer-1(1 μM),DFO(100 μM),GSH(1 m M),NAC(1 m M)or Zn PP(1 μM)and then treated with 5-FU(100 μM).ROS levels were detected by DCFHDA probe or DHE probe to evaluate the oxidative stress of cells under different treatment conditions.The levels of ferroptosis related factors in cells were detected by western blot and quantitative real-time PCR.Results: 1.C57BL/6J mice after the injection of 5-FU for 5 days showed diarrhea,depression and weight loss,increased ALT and AST contents in plasma,reduced liver tissue size and volume.2.5-FU significantly increased the iron levels in plasma and liver.5-FU activated the ferroptosis pathway,which was manifested by excessive intake of iron and abnormal ROS levels.3.In L-02 cells,5-FU can also induce ferroptosis,which is manifested by the increased levels of iron in cells and the imbalance of oxidation/antioxidant.4.The application of various ferroptosis inhibitors alleviated cell death induced by 5-FU.The specific ferroptosis inhibitor Fer-1 alleviated the abnormal expression of ferroptosis related proteins caused by 5-FU.5.HO-1 inhibitor can slow down5-FU-induced L-02 cell death and rescue the abnormal expression of 5-FU-induced ferroptosis related proteins.Conclusion: 5-FU may induce drug-induced liver injury.It could be caused by an increase in iron levels and a build-up of lipid peroxidation in liver.5-FU increased the intracellular iron content by upregulating genes and proteins related to iron intake,which accompanied by dysregulation of the intracellular oxidation/antioxidant system.In addition,5-FU intensified iron accumulation by upregulating HO-1 levels,which further increased intracellular lipid peroxidation.These promote cell death.Our results provide new insights for further study of 5-FU toxicity and better understanding of the mechanism of liver injury.