Non-targeted Metabolomics Study On Toxicity Of Carbon Black Nanoparticles In Rat/Lung Cancer Cells

Objective:In this study,the endogenous metabolites in rat/lung cancer cells were profiled using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry?UHPLC-Q-TOF-MS?-based metabolomics approach.Therefore,potential biomarkers of subchronic toxicity endured by CBNPs and related molecular mechanisms could be found.Methods:In vivo research,eighteen rats were divided into 3 groups:CB exposure group,recovery group and control group.The rats in the exposed group were given CBNPs by oral and nasal exposure.After the end of the exposure,the blood of the rats was collected,and then the serum samples were obtained.Three times the amount of acetonitrile was added into the 100?L of the serum sample to get the mixture which was broken to extract the endogenous components.Metabolites were analyzed using IDA techniques in ESI source.The raw data was imported into the Progenesis QI software?Waters,USA?which was used for peak alignment,deconvolution and selection of the adduct ion.In vitro study,the A549 cells were treated with different concentrations of CBNPs for 48 h.Then the cytotoxicity was evaluated by Cell Counting Kit-8?CCK-8?assay under dual wavelength conditions.The CBNPs concentration of IC50 value was selected for further mechanism studies.After A549 cells incubated with CBNPs at a dose of 70?g/mL for 48 h,metabolites were profiled using a comprehensive non-targeted metabolomics strategy based on UHPLC-Q-TOF-MS/MS platform with SWATH^TM data acquisition to analysis the metabolic profiles of CBNPs in A549 cells.The obtained LC-MS raw data was analyzed by the important data mining tools in MarkerView for peak detection,peak alignment and normalization,and t-test for significant analysis.At the same time,the interference of the unrelated variable group in the LC-MS peak spectrum was excluded by using PCVG filtering technology.The data set in vivo and vitro was analyzed in order to get the P value by independent Student's t test in SPSS Statistics 21 software?IBM,USA?.Meanwhile,the processed data sets were imported into SIMCA-P 14.0 software?Umetrics,Uppsala,Sweden?for further multivariate statistical analysis.The OPLS-DA is a kind of supervised multivariate statistical method which can get the VIP score.In this study,compounds that satisfy both P<0.05 and VIP>1 were considered as differential compounds.The potential biomarkers were identified through some online databasessuchasHumanMetabolomeDatabase?HMDB,http://www.hmdb.ca?and Metlin?https://metlin.scripps.edu?.The pathway enrichment analysis was processed by an online software MetaboAnalyst 4.0?http://www.metaboanalyst.ca/?and the metabolic pathways for all the potential biomarkers were screened by the online database KEGG?http://www.genome.jp/kegg?.Results:After multivariate statistical analysis,39 potential biomarkers were identified in rat serum.The metabolic pathways involved mainly hormone metabolism,amino acid metabolism,nucleotide metabolism and lipid metabolism.Thirty-two potential biomarkers were also identified in A549 cell.The metabolic pathways contained energy metabolism,amino acid metabolism and lipid metabolism.It is worth noting that prolonged exposure to CBNPs may increase the risk of infertility in vivo experiments.Conclusions:In this study,a practical UHPLC-Triple-TOF-MS/MS method was developed for the subchronic toxicity of carbon black nanoparticles in A549 cells using a novel IDA and SWATH^TM mode.The potential biomarkers and metabolic pathways were finally identified.These important pathways may be the key molecular events in the CBNPs toxicity.These findings provided data to further explain the biological mechanisms and recovery mechanisms of the toxicity of carbon black nanoparticles.And these also offered new analytical methods for the toxicity studies of other nanomaterials.