Screening And Validation Of Radiosensitive Metabolites In Rat Plasma And Tissues Based On Lipidomics Analysis

ObjectiveStudies have revealed that certain lipid metabolites change induced by ionizing radiation,having the potential to become radiation biomarkers.However,the reported metabolites have limitations of insufficient accuracy,lack of dose evaluation model and verification.This study is to screen radiosensitive metabolites in rat plasma and tissues based on targeted lipidomics.Radiosensitive lipid metabolites in plasma were explored for the feasibility of early triage and biological dose estimation.Radiosensitive lipid metabolites in tissues were screened for potential radiation injury biomarkers,which will provide scientific basis for the research of radiation injury mechanism.Method1.Targeted lipidomics was employed to screen radiosensitive lipids after 0,1,2,3,5 and 8 Gy uniform total-body irradiation(TBI)at 4,24,72 h post irradiation in rat plasma.Ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was utilized.Radiosensitive metabolites with good dose-response relationship were obtained by unpaired two-sample t test and linear regression method and a novel dose evaluation model was established by multiple linear stepwise regression method.The metabolic pathways involved with radiosensitive lipid metabolites were explored and analyzed as well.An independent set of rats was utilized as a validation cohort,which was uniformly total-body irradiated with doses of 0,2.5,and 6 Gy.Plasma samples were obtained at 4,24 and 72 h post irradiation.The dose evaluation model was verified for its radiation triage ability and biological dose estimation effect.2.Targeted lipidomics was employed to screen radiosensitive lipids after 0,1,2,3,5 and 8 Gy uniform TBI at 72 h post irradiation in rat small intestine and plasma samples simultaneously.Radiosensitive metabolites with good dose-response relationship were obtained by unpaired two-sample t test and linear regression method.Correlation of radiosensitive metabolites in the tissue and plasma were compared to explore potential biomarkers of radiation-induced intestinal injury.The performance of radiation injury classification was evaluated by ROC analysis.An independent set of rats was utilized as a validation cohort,which was uniformly total-body irradiated with doses of 0,4,and 6 Gy.Small intestine and plasma samples were obtained at 72 h post irradiation.The radiation injury classification ability of potential intestinal injury biomarkers was verifed.3.Targeted lipidomics was employed to screen radiosensitive lipids after 0,5 and 10 Gy uniform TBI at 1,7,14 d post irradiation in rat radiosensitive tissues(small intestine,spleen,testis)and plasma samples simultaneously.Dynamic change rules of metabolites induced by ionizing radiation with dose and post-irradiation time were explored to screen radiosensitive metabolites in different tissues.The performance of injury classification will be evaluated for potential radiation injury biomarkers.Results1.Significant differences were found in the lipid levels of rat plasma induced by 0-8 Gy cobolt-60 gamma rays uniform TBI and 213,233 and 314 lipid metabolites significantly perturbed at 4,24,72 h among 416 individual lipids from 18 subclasses(FA,DG,TG,SM,Cer,Hex2Cer,PC,PC-O,PE,PE-O,PI,PA,PG,PS,LPC,LPC-O,LPE,CE).Glycerophospholipid metabolism,linoleic acid metabolism and alpha-linolenic acid metabolism were significantly perturbed pathways after irradiation.Panel A consisted of 56 lipids exhibited significant triage characteristics(significant decrease in all radiation groups,no significant difference between different radiation groups)at 4 h post irradiation was screened.Panel B-Panel D consisted of 13,69,139 lipids with good dose-response within a dose range of 0-8 Gy(R2>0.80)at 4,24,72 h post irradiation were screened respectively.Panel A exhibited area under the curve(AUC)of 0.982 for triaging non-exposed and exposed groups.Panel B-Panel D exhibited AUC of 0.814-1.000 for triaging different radiation dose groups:0 Gy vs.>0 Gy,≤ 1 Gy vs.>1 Gy,≤ 2 Gy vs.>2 Gy,≤ 3 Gy vs.>3 Gy,≤5 Gy vs.>5 Gy,respectively.The equations for dose estimation were established by stepwise regression analysis for three time points.A dose assessment model for radiation early triage and dose estimation was established and validated using panels of lipids.In the validation cohort,Panel A-Panel D exhibited AUC of 0.820-1.000 for triaging different radiation dose groups:0 Gy vs.>0 Gy,≤2.5 Gy vs.>2.5 Gy,respectively.Thirty-five radiosensitive metabolites with both good dose-response relationship and time response range were screened.Combined compounds exhibited AUC of 0.905-1.000 for triaging different radiation dose groups;0 Gy vs.>0 Gy,≤1 Gy vs.>1 Gy,≤2 Gy vs.>2 Gy,≤3 Gy vs.>3 Gy,≤5 Gy vs.>5 Gy,respectively.In the validation cohort,combined compounds exhibited AUC of 0.920-1.000 for triaging different radiation dose groups:0 Gy vs.>0 Gy,≤2.5 Gy vs.>2.5 Gy,respectively.2.Significant differences were found in the lipid levels of rat small intestine and plasma induced by 0-8 Gy cobolt-60 gamma rays uniform TBI at 72 h and 93,314 lipid metabolites significantly perturbed,respectively.In rat small intestine and plasma,28 and 139 lipids with good dose-response within a dose range of 0-8 Gy(R2>0.80)were screened,respectively.Through comparative analysis,7 common radiosensitive lipid metabolites were screened.Combined 7 radiosensitive lipid metabolites exhibited AUC of 0.852-1.000 for triaging different radiation dose groups(5 Gy vs.0 Gy,8 Gy vs.0 Gy,5 Gy vs.8 Gy)in small intestine,while exhibited AUC of 0.975-1.000 in plasma.In the validation cohort,combined 7 radiosensitive lipid metabolites exhibited AUC of 0.920-1.000 for triaging different radiation dose groups(4 Gy vs.0 Gy,6 Gy vs.0 Gy,4 Gy vs.6 Gy)in small intestine,while had AUG of 0.840-1.000 in plasma.3.Significant differences were found in the lipid levels of rat small intestine induced by 0-10 Gy cobolt-60 gamma rays uniform TBI and 23,94,39 lipid metabolites significantly perturbed at 1,7,14 d post irradiation,respectively.At the three time points,there were 7,55 and 15 differential metabolites shared in the small intestine and plasma,respectively.In rat small intestine,10 radiosensitive lipid metabolites that changed significantly at more than two time points were screened.Combined 10 radiosensitive lipid metabolites exhibited AUC of 0.600-1.000 for triaging different radiation dose groups(5 Gy vs.0 Gy,10 Gy vs.0 Gy,5 Gy vs.10 Gy)in small intestine,while exhibited AUC of 1.000-1.000 in plasma.Comprehensive analysis of diffferential metabolites at 1,3,7,and 14 d post irradiation revealed 11 metabolites may continue to change within 7 days post irradiation.Among them,PC(0-16:0/18:1)and SM(d18:1/13:0)had good dose-effect relationship and time response range at the same time.4.Significant differences were found in the lipid levels of rat spleen induced by 0-10 Gy cobolt-60 gamma rays uniform TBI and 184,185,52 lipid metabolites significantly perturbed at 1,7,14 d post irradiation,respectively.At three time points,there were 63,104 and 18 differential metabolites shared in the spleen and plasma,respectively.In rat spleen,10 radiosensitive lipid metabolites that changed significantly at more than two time points were screened.Combined 10 radiosensitive lipid metabolites exhibited AUC of 0.920-1.000 for triaging different radiation dose groups(5 Gy vs.0 Gy,10 Gy vs,0 Gy,5 Gy vs.10 Gy)in spleen,while exhibited AUC of 0.600,1.000,1.000 in plasma,respectively.5.Significant differences were found in the lipid levels of rat testis induced by 0-10 Gy cobolt-60 gamma rays uniform TBI and 35,105,96 lipid metabolites significantly perturbed at 1,7,14 d post irradiation respectively.At three time points,there were 12,61 and 33 differential metabolites shared in the testis and plasma,respectively.In rat testis,18 radiosensitive lipid metabolites that changed significantly at more than two time points were screened.Combined 18 radiosensitive lipid metabolites exhibited AUC of 0.920-1.000 for triaging different radiation dose groups(5 Gy vs.0 Gy,10 Gy vs.0 Gy,5 Gy vs.10 Gy)in testis,while exhibited AUC value of 1.000-1.000 in plasma.6.Comprehensive analysis of differential metabolites were conducted in rat plasma samples among three parts:(1)0-8 Gy at 4,24,and 72 h post irradiation;(2)0-6 Gy at 4,24,72 h post irradiation;(3)0-10 Gy at 1,7,and 14 d post irradiation.LPC(18:2),PG(18:2/18:3),TG(20:1/18:1/18:2)were significantly down-regulated within 7 days after exposure among different batches of animal experiments and different dose ranges,and the trend of decrease remained unchanged.Comprehensive analysis of differential metabolites in the tissues of small intestine,spleen and testis were implemented.The types of differential metabolites shared by small intestine,spleen and testis included SM,PC,and PC-O.There were 6,15,and 23 potential intestinal,spleen and testicular specific lipid biomarkers,respectively.Conclusion1.A dose assessment model for radiation early triage and dose estimation was established and was validated for potential application value in triaging different radiation exposure levels.Thirty-five radiosensitive metabolites with both good dose-response relationship and time response range had more value in convenient practical application.LPC(18:2),PG(18:2/18:3)and TG(20:1/18:1/18:2)were more potential radiation biomarkers.2.Seventeen potential biomarkers of radiation-induced intestinal injury were screened.PC(O-16:0/18:1)and SM(d18:1/13:0)were more potential radiation-induced intestinal injury biomarkers.Ten potential biomarkers of radiation-induced spleen injury and 18 potential biomarkers of radiation-induced testicular injury were screened.It has the potential to predict the risk and severity of radiation-induced intestinal,spleen and testicular injury by detecting the above radiosensitive metabolites in rat plasma,respectively.3.The types of differential metabolites shared by small intestine,spleen and testis included SM,PC,and PC-O.There were 6,15,and 23 potential intestinal,spleen and testicular specific lipid biomarkers,respectively.Innovations1.Systematic and in-depth exploration focusing on the the feasibility of radiosensitve lipid metabolites as radiation biomarkers was implemented.Targeted lipidomics was employed to screen radiosensitive lipids,having the advantages of rapid,sensitive,high-throughput and high coverage.The study has a certain degree of innovation in research strategy.2.A dose assessment model using panels of radiosensitve lipid metabolites was established and validated for the potential of early triage,having certain research significance and application value.3.Radiosensitve lipid metabolites in tissues were screened.It has the potential to predict radiation-induced tissue-specific injury by detecting corresponding metabolites in plasma.The present study can provide certain scientific basis for the research of radiation injury mechanism.