Visualization Of Oxidative Stress-induced Hepatotoxicity By Real Time Imaging

Background:Drug induced liver injury(DILI)is a very common side effect in clinical medicine.As the main organ to metabolism the exogenous nutrients and medicine,the liver is very vulnerable to be injured.DILI is responsible for approximately 50%of all cases of acute liver failure(ALF)and belongs to the most common causes of drug withdrawal,boxed warnings or denial of approval.Therefore,it is important to know the risk of DILI of a new medicine and finding the effective way to screen and test the liver toxicity of a new medicine before clinical.Although there have been many methods widely used in the clinical and pre-clinical to test DILI,they can only detectable when the damage has happened and the cell is dead.We still lack methods that can detect the injury before the results come.Oxide stress is the imbalance of the reduction-oxidation system when the reactive oxygen species(ROS)and reactive nitrogen species(RNS)cannot be deleted in time.In many cases,OS contribute the DILI of many medicine.So,the OS status in the liver is a very good marker to reflect DILI before cell death.However,the most widely used methods to detect the ROS and RNS is to test the redox levels from the tissue only after killing the experiment animals.A new in vivo method that can detect OS in the liver real time caused by drug toxicity is deeply needed.Purpose:To establish a mouse model that can test the OS caused by DILI in vivo through bioluminescent imaging(BLI)and PCL-1,a probe that can react with ROS and RNS to produce luciferin.Methods:1.PCL-1 was synthesized by a chemical method.2.A cell line that can express luciferase was established by Lipofectamine transfection3.A mouse line that can express luciferase specifically in the liver was established by microinjection and embryo transfer.4.PCL-1 was used to detect exogenous free radical in culture cell and tumor model.5.PCL-1 was used to detect free radical in vivo in mice liver treated with APAP and CC14.6.HE,immunostaining and TUNEL were used to evaluate the liver injury caused by APAP and CCl4.Results:1.PCL-1 was synthesized successfully.HepG2-Luc cell line which can express luciferase was established.We have created a new transgenic mouse model,the Alb-Luc mice,in which luciferase is expressed specifically in the hepatocytes.2.We observed BLI signal in HepG2-Luc after H2O2 added,we also observed BLI signal in mouse tumor model after treated with H2O2 through PCL-1.3.We observed BLI signal in HepG2-Luc after APAP added,we also observed BLI signal in HCR mice after treated with APAP through PCL-1.4.The results showed that strong BLI signals were detected in both cohorts of the APAP-treated mice.However,mice treated with the minimum toxic dose of APAP exhibited a much higher peak BLI signal intensity than those treated with the non-toxic dose of APAP.We found that the administration of glutathione could indeed effectively reduce the BLI signal intensities in the mice that were treated with the toxic dose of APAP.The results showed that significant increases in necrosis,tyrosine nitrosylation,and TUNEL-positive cells were evident in samples derived from the mice treated with 300 mg/kg APAP,but not those treated with 200 mg/kg of APAP.Also,co-treatment with glutathione resulted in the reduction of all these parameters and was apparently not toxic.5.The results showed that CCl4 at both dosing levels could induce a significant BLI signal aging,with the toxic dose giving rise to BLI signals with higher intensities than those treated with the non-toxic dose.Post-mortem analyses also showed that only animals treated with the toxic dose of CCl4 exhibited signs of necrosis,tyrosine nitrosylation and cell death.6.Similar BLI experiments with Alb-Luc mice that were treated with a non-toxic dose as well as a sub-lethal dose of isonicotinylhydrazide(INH)failed to detect any BLI signals.7.We found that indeed,strong BLI signals were still detected in only those animals that had been treated with the presumed toxic dose of APAP,but not from those that had been treated with the non-toxic dose of APAP,despite that strong peak BLI signals were detected in both cohorts in the first series of BLI imaging.Similar results were obtained when CCl4-treated Alb-Luc mice were examined.We found that the hepatotoxicity-specific BLI signal that is caused by 300mg/kg of APAP,a toxic overdose of APAP,was effectively eliminated by the glutathione treatment.The results of this experiment showed that the addition of a non-toxic dose of CCl4 in conjunction with a non-toxic dose of APAP was indeed sufficient to give rise to a positive BLI signal.In addition,the post-mortem histo-pathological analysis showed that the co-treatment was indeed sufficient to cause hepatocyte necrosis that is indicative of OS-induced acute hepatotoxicity.Conclusion:In this study,we first showed that the Alb-Luc mice,in which the luciferase reporter is expressed specially in the liver,could be used to detect the OS in the liver induced by medicine through BLI imaging and using PCL-1,a reporter for OS,this finding establishes a new model for the detection DILI in live mice by BLI,a well-established and widely used technology.With this new model,we observed the high BLI signal,on behalf of ROS and RNS induced by APAP and CCl4,especially at the high injury dose.Furthermore,the BLI signal is consistent with the pathological analysis and serum biomarkers,demonstrating that this model can be effectively used for the rapid assessment of the potency of individual agents in causing the elevation of OS in the liver and diagnosis liver injury before cell death.Besides,we found that the decay of the signal is related to the decree of injury and a second time of PCL-1 injection can be used to judge the injury.