In Vivo Metabolic Characteristics Analysis And Internal And External Exposure Correlation And Toxic Effect Pathways Of Heterocyclic Aromatic Amines

Heterocyclic aromatic amines（HAAs）as a class of heterocyclic aromatic chemical contaminants with harsh carcinogenicity and mutagenicity,widely exist in high-temperature processed protein-and creatine-rich meat products.Because of their strong mutagenic,carcinogenic and other physiological and toxicological effects,the risk of human health have always been concerned caused by long-term intake of HAAs.At present,studies on some metabolic pathways and exposure biomarkers of HAAs are still not clear,leading to the incomplete description of their in vivo exposure spectrum,and correlation between internal and external HAAs exposure haven’t reported before.In addition,the molecular pathway of metabolic toxicity remains to be further explored,and the full spectrums of metabolic fingerprint are still unclear.In this study,base on rat animal models and human experiments,we systematically revealed typical HAAs dietary exposure levels and their metabolic pathways in vivo,improved and reconstructed HAAs exposure profiles in vivo,and further constructed physiological toxicokinetic models.Finally,based on specific fingerprints,the molecular pathway of metabolic toxicity induced by HAAs exposure were revealed.Firstly,an efficient method for simultaneously analyzing 7 kinds of heterocyclic aromatic amines（HAAs）in thermal processing meat foods was developed based on isotope dilution ultra-performance liquid chromatography coupled to tandem mass spectrometry（UPLC-MS/MS）.This method was applicable to simultaneouly screen and analysis of multiple HAAs in large batches of thermal processed meat products.The results showed that the types of HAAs exposed in daily diet were mainly Ph IP and Me IQx,and detected include Ph IP,Me IQx,4,8-Di Me IQx,etc.The rates of detection are 70.0%,61.3%and 9.7%respectively,and the content ranges are from 0.11-48.34 ng/g,0.11-9.22 ng/g,0.29-0.45 ng/g,respectively.Next we developed an advanced strategy that adopts chemical isotope labeling ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry for urinary nontargeted metabolomics analysis to gain new insight into screening and identification of exposure biomarkers.Rats were orally administered with a single dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine（Ph IP）or2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline（Me IQx）（1 and 10 mg/kg bw）and their D₃-isotopic compounds,respectively,and urine samples were then continuously collected within 36 h.We monitored 23 and 37 urinary metabolites as the biotransformation products of Ph IP and Me IQx,respectively,and first identified N¹-demethylated metabolites of Ph IP,tentatively named 2-amino-6-phenylimidazo[4,5-b]pyridine,and further clarified the demethylation metabolic pathway of Ph IP and Me IQx,and dihydroxylation products of classical HAAs was another new detoxification pathway of liver cytochrome P450 in vivo.We combined various detailed metabolic pathways under the action of different metabolic enzymes,and finally improved and reconstructed the HAAs in vivo exposure spectrums.Then,UPLC-MS/MS methods for simultaneous quantitative monitoring of HAAs and its metabolites in rat urine and human urine samples were established and optimized respectively,and methodological validation was carried out to ensure the overall reliability of the approaches.To further explore the characteristics of urine metabolism of HAAs within 0-36hours after oral gavage,the results showed that the total excretion of urine after 36 hours by exposure to high doses of Ph IP（10 mg/kg bw）accounted for 25.90±9.62%of the total intake dose,among which,demethylated Ph IP,unchanged Ph IP,hydroxylated Ph IP,hydroxymethyl Ph IP,and glucuronized Ph IP accounted for 0.503±0.204%,6.95±2.80%,2.10±0.89%,0.227±0.075%,and 16.12±5.64%,respectively,In the low-dose group（1 mg/kg bw）,the proportion of total excretion was 20.10%±5.24%,and the proportion of other metabolites was0.780±0.303%,5.31±1.70%,2.43±0.771%,0.112±0.029%,4.98±0.591%,6.50±1.86%,respectively.On the other hand,after 36 hours of exposure to Me IQx at high dose and low dose,the total urinary excretion accounted for 15.81±6.51%and 10.92±4.90%of the total intake dose,respectively.The results of the human clinical trial showed that the total urinary excretion of Ph IP in 20 volunteers within 0-24 hours after a single intake of roast steak ranged from3.8-14.5 ng,accounting for 0.7-2.6%of the total intake dose.At last,the multi-species physiologically based toxicokinetic（PBTK）model of HAAs（multi-chamber PBTK model exposed to Ph IP and the single-chamber PBTK model exposed to Me IQx）were constructed in rats and humans.The applicability of the models was verified by comparing the predicted results with the independent experimental datasets of rats and humans respectively,and finally completed internal and external exposure correlation.Finally,a non-targeted metabonomic analysis method based on high-throughput high-resolution mass spectrometry combined with classical multivariate statistical models was established,which was applied to the study of serum and urine metabonomics and effect pathways in rats exposed to HAAs.The results of serum metabonomics showed that 23 and 18differential key pivotal metabolites were screened in the serum of rats exposed to Ph IP and Me IQx,respectively.Further by analysis of metabolic pathways,we found that both HAAs significantly interfered with the metabolism of tricarboxylic acid cycle,aminoacyl t RNA biosynthesis,multiple amino acids metabolism and energy metabolism,and here arginine and proline metabolism,arginine biosynthesis and pyruvate metabolism were the specific metabolic pathways affected by Ph IP exposure,while Me IQx was more sensitive to bile acid metabolism and phenylalanine metabolism.Finally,we elucidated the characteristic changes of these endogenous effect biomarkers in vivo and revealed the potential molecular pathway of tumorigenesis,such as colorectal induce by carcinogenic HAAs.Exposure to HAAs may increase the risk of colitis,colorectal cancer and other diseases by disrupting the metabolism of polyamines,amino acids or energy metabolism in the body.Similar results were verified in urine metabonomics analysis.The findings showed that 20 and 12 different metabolites were screened in rat urine,respectively.In addition,our findings from metabolic pathway analysis confirmed that both HAAs could significantly disturb histidine metabolism,arginine and proline metabolism,tryptophan metabolism,pyrimidine metabolism,and tricarboxylic acid cycle,etc.Furthermore,we found histamine,methionine,alanine,and 4-guanidinobutanoic acid could be considered as potential characteristic biomarkers for the oncogenicity or carcinogenicity of both Ph IP and Me IQx.Individually,tryptamine,malic acid,and taurine may serve as specific biomarkers of Ph IP exposure,while proline,hydroxyprolyl-methionine and thymine may be considered as signature metabolites of Me IQx exposure.The human clinical trial results that people with high HAAs exposure to roasted steak intervention showed that the metabolism of histidine metabolism and taurine and hypotaurine metabolism were the two main metabolic pathways affected by HAAs exposure in vivo,and L-Histidinal,L-Histidinol,Acetylhistamine,1-Methylhistidine and taurine could be considered as specific effect biomarkers of HAAs based on grilled steak matrix.We speculated that HAAs-induced upregulation of histamine and taurine increased the risk of tumorigenesis due to the changes in the activation of histamine receptors and regulation of TUG1/mi R-421/KDM2A/ERK axis,respectively.In conclusion,the research provided technical support for a comprehensive understanding of the exposure assessment and in vivo metabolic characteristics of dietary carcinogenic HAAs,and theoretical basis for the internal exposure assessment and internal and external exposure correlation by improving the in vivo exposure spectrum and building physiological toxicokinetic models.In addition,the description of metabolic characteristic fingerprints coursed by the exposure of HAAs based on differential biomarkers had important reference values for providing new insight into causal relationship between dietary intake of HAAs and cancer risk..