Quantification Of Liver N6-methyladenosine MRNA Level In Crizotinib-induced Liver Injury In Mice By Using LC-MS/MS

Background and ObjectiveCrizotinib is a multi-target tyrosine kinase inhibitor for anaplastic lymphoma kinase(ALK)gene recombination,c-MET gene amplification and ROS gene recombination.It has been widely used as the standard first-line treatment for ALK-positive patients with locally advanced or metastatic non-small cell lung cancer.However,liver injury caused by crizotinib is one of the main challenges faced by clinicians,and its mechanism is not yet clear.RNA methylation is another epigenetic modification besides DNA methylation and histone modification.More than 150 RNA modifications have been identified,and N6-methyladenosine(m6A)is the most abundant modification in eukaryotic mRNA,and it plays an important role in various biological processes and disease development.Some studies have shown that epigenetic modification also plays an important role in liver injury.To date,there has been no report on the mechanistic link between m6 A and crizotinib-unduced liver injury.Therefore,it is necessary to quantify the level of m6A methylation in the crizotinib-induced liver injury mouse model.The purpose of this study was to investigate the method of establishing a crizotinib-induced liver injury mouse model;to develop and verify a reliable ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method for the determination of five nucleosides in mRNA;to investigate the changes of m6A methylation modification level in the liver tissue of crizotinib-induced liver injury mouse model.Methods1 Establishment of the crizotinib-induced liver injury mouse modelThe mice were randomly divided into model groups and control groups.The mice in the model groups were gavaged with different doses crizotinib(100 mg/kg,300 mg/kg,and 500 mg/kg)every day,using 0.5%CMC-Na as the vehicle.The mice in the control groups were given appropriate 0.5%CMC-Na.All mice were given normal feed,observed and weighed every day.Mouse serum and liver tissues were collected at different times.Two biochemical indicators of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected,and the changes of liver morphology were also observed.2 Determination of m6A in liver tissue mRNA of crizotinib-induced liver injury mice by LC-MS/MSIn addition,a LC-MS/MS methods for quantifying five nucleosides(adenosine,uridine,cytidine,and guanosine and m6A)in mRNA were developed.Liver tissue mRNA was extracted,and the mRNA was digested into single nucleosides.After enzymatic digestion of messenger ribonucleic acid,the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02%formic acid water,and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode.The method was validated over the concentration ranges of 4-800 ng/mL for adenosine,uridine,cytidine,and guanosine and 0.1-20 ng/mL for m6A.The method was fully validated and used to analyze the m6A methylation level in the liver tissue mRNA of crizotinib-induced liver injury mice model.Results1 Establishment of the crizotinib-induced.liver injury mouse model1.1 The effect of crizotinib on serum biochemical indexes in miceCompared with the control group,the serum ALT and AST were significantly increased when ICR mice were given crizotinib 500 mg/kg by intragastric administration for 4 consecutive days.In this study,the levels of ALT and AST in the serum of six groups of ICR mice were compared between male and female mice.The results showed that there were no gender differences in the serum levels of ALT and AST in the mice of this experiment.1.2 The effect of crizotinib on body weight and liver tissue structure in miceCompared with the control group,the body weight of the mice which were given 500 mg/kg crizotinib by gavage for 4 consecutive days began to decrease significantly on the 3rd day.Pathological examination of liver tissue showed that hepatocytes showed extensive to moderate to severe edema,and swollen cells and tiny circular vacuoles could be seen in the cytoplasm,and the cytoplasm was loose and lightly stained or vacuoles-like.2 Determination of m6A in liver tissue mRNA of crizotinib-induced liver injury mice by LC-MS/MS2.1 Optimization of the treatment method of enzymatically hydrolyzed mRNA to single nucleosideThe optimal amouts of S1 nuclease and alkaline phosphatase were 180 U(1?L,180 U/?L)and 2 U(2 ?L,1 U/?L),respectively.The mRNA enzymatic hydrolysis reaction was almost completed within 6 h at 37?,and the amount of enzymatic hydrolysis products remained constant with time(6-12 h).Therefore,the optimal time for enzymatic incubation is 6 h.2.2 Optimization of mass spectrometry conditions and chromatographic conditionsOptimized the main ions of A,U,C,G and m6A protonated molecular ions[M+H]+are m/z 268.1,m/z 245.1,m/z 244.2,m/z 284.2 and m/z 282.1,respectively,and the product ions are m/z 136.1,m/z 113.0,m/z 112.0,m/z 152.1 and m/z 150.1.In addition,the MRM ion pair used for quantitative analysis of the internal standard is m/z243.1? m/z 127.0.An Acquity UPLC HSS T3 column(1.8 ? m,100 mm ×2.1 mm i.d,Waters)was used to separate five nucleosides.The mobile phase was methanol and 0.02%formic acid in water,and gradient elution was used.2.3 Validation of LC-MS/MS method for determination of five nucleosides in mouse liver tissue mRNAThe concentration range of adenosine,uridine,cytidine and guanosine was 4?800 ng/mL,and the concentration range of m6A is 0.1?20 ng/mL.The five nucleosides have a good linear relationship and specificity within their respective concentration ranges.The results of precision and accuracy,matrix effects,residual effects,and sample stability could meet the measurement requirements of biological samples.2.4 Determination of nucleoside levels in mouse liver tissue mRNAThe levels of five nucleosides in the liver mRNA of crizotinib-induced liver toxicity model mice were determined.The content of methylated modified nucleoside m6A in mouse liver mRNA is expressed as the ratio of the concentration of m6A and A(m6A/A%=Cm6A/CA ×%).The results showed that the m6A/A%value in the liver mRNA of the crizotinib-induced liver injury group was significantly lower than that of the control group(p<0.001).Conclusion1.This study successfully established a crizotinib-induced liver injury mouse model.The method was to give 500 mg/kg crizotinib to ICR mice by intragastric administration for 4 consecutive days.2.Developed and validated an LC-MS/MS method for the determination of five nucleosides(adenosine,uridine,cytidine,guanosine and m6A)in mRNA,which is accurate and reliable.3.Compared with the control group,the m6A methylation level in the liver tissue mRNA of crizotinib-induced liver injury mice was significantly decreased.