Toxic Effects And Biomarkers Of Esophageal Cancer Induced By Nitrosamines Based On Metabolomics

Background and objectives:Esophageal cancer is a common digestive tract malignant tumor.In China,about 90%of esophageal cancer cases are esophageal squamous cell carcinoma(ESCC).Due to the occult early symptoms,late clinical diagnosis,and poor prognosis,ESCC has become a heavy disease burden threatening people’s life and health.Epidemiological studies indicated that the distribution of ESCC shows obvious regional differences,and environmental factors are closely related to the occurrence and development of ESCC.Although extensive studies,risk factors leading to the sustained high incidence of ESCC in some areas have not been clarified.Nitrosamines are a group of the most likely environmental carcinogens associated with ESCC.At present,the evidence of human ESCC caused by nitrosamines is not sufficient.In this study,from the perspective of exposure assessment,the exposure characteristics of nine common nitrosamines in drinking water and human urine in a high incidence area of ESCC in Huai’an were investigated to preliminarily explore the relationship between nitrosamines exposure and the risk of ESCC.Then,the toxic effects in target cells—the human esophageal epithelial Het-1A cells,induced by a mixture of the nine common nitrosamines at the environmentally relevant,human-internal-exposure,and genotoxic concentrations,were investigated on the base of the metabonomics technology.Combined with ICR mice model,the possible adverse health outcomes of co-exposure to low-level nitrosamine were explored.Further,the ESCC in rats was induced by N-methylbenzylnitrosamine(NMBA)to explore the dynamic molecular changes in the carcinogenesis of rat ESCC and the potential mechanisms.On this basis,the dynamic changes of serum metabolic spectrum during the development of human ESCC were identified based on the case-control study in the high incidence area of esophageal cancer.The key metabolic pathways were screened on the combination of the animal experiments and the population-based study.And the mediating mechanisms of association between nitrosamines exposure and human ESCC from the candidate effect biomarkers were analyzed.The objective of this study was to comprehensively screen potential specific effect biomarkers to provide scientific data support for the screening,prevention,and control of human esophageal cancer in the high incidence area.Methods:1.Five representative drinking water treatment plants(DWTPs)were selected in a high EC incidence area(Huai’an)and a low EC incidence area(Nanjing)respectively.The source water and finished water samples in each DWTP were collected.Distributions of nine common nitrosamines in drinking water in the two areas were investigated based on an SPE-GC-MS method.The morning urine samples of 262 healthy residents in Nanjing and 268 healthy residents in Huai’an were collected.The levels of nine nitrosamines in urine were analyzed by the SPE-GC-MS method.The SPE-LC-MS/MS method was used to analyze the nine nitrosamines in urine of 115 healthy controls,89 patients with esophageal precancerous lesions[69 patients with reflux esophagitis with basal cell hyperplasia(RE/BCH),20 patients with esophageal dysplasia(DYS)],and 73 ESCC patients in Huai’an area.And the risk associated with nitrosamines exposure was analyzed by logistic regression.2.Metabolic perturbations in human esophageal epithelial Het-1A cells induced by the co-exposure of nine common nitrosamines in drinking water at environmentally relevant(low concentration group,0.05μg/L),human-internal-exposure(medium concentration group,20μg/L),and genotoxic concentrations(high concentration group,8000μg/L)were investigated by an untargeted metabolomics technology.Then,targeted metabolomics and traditional molecular biology methods were used to verify the toxic mechanisms in vitro,especially the adverse effects of co-exposure of nitrosamines at the low concentration.To further verify the toxic effects in vivo,ICR male mice were exposed to the mixed nitrosamines solutions orally at low(0.0075μg/kg),medium(3μg/kg),and high(1200μg/kg)doses,which were equivalent to the daily intake of nitrosamines through freely drinking water polluted by nitrosamines at 0.05,20,and 8000μg/L.3.Induced ESCC model in the F344 rats was established by intraperitoneal injection of NMBA with high esophageal affinity at the dose of 0.4 mg/kg.The serum,urine,and tissue samples in the healthy rats and rats with different esophageal lesions[BCH,light dysplasia(L-DYS),mild dysplasia(M-DYS),severe dysplasia(S-DYS),carcinoma in situ(CIS),invasive carcinoma(IC)]were collected.Dynamic molecular changes in the occurrence and development of rats ESCC induced by NMBA were explored by an untargeted metabolomics technology.And the potential mechanisms of ESCC induced by NMBA were identified by the expressions of regulation enzymes and related proteins in key metabolic pathways.4.Serum and urine samples were collected from 91 healthy controls,99 patients with esophageal precancerous lesions(60 patients with RE/BCH,39 patients with DYS)and 51 patients with ESCC.Dynamic changes of metabolic spectra in serum from patients with different esophageal lesions were explored.The key metabolic pathways were screened on the combination of the animal experiments and the population-based study.Then,the levels of important metabolites in the key metabolic pathways were investigated through the targeted metabolomics technology.And the mediating mechanisms of association between nitrosamines exposure and human ESCC were analyzed to comprehensively screen the specific potential effect biomarkers of human ESCC related to nitrosamine exposure.Results:1.Exposure characteristics of nitrosamines in the high incidence area of esophageal cancer in Huai’an1.1 Distribution of nitrosamines in drinking water from different regionsThe pollution of total nitrosamines in Huai’an(135.14±98.1 vs.68.65±64.9 ng/L,p<0.01)was higher than that in Nanjing.Among the six predominant nitrosamines,the average concentrations of NDPA(34.91±32.7 vs.15.96±19.23 ng/L,p<0.01),NDBA(16.47±17.58 vs.5.44±10.95 ng/L,p<0.01),NPyr(6.88±8.05 vs.3.1±5.32 ng/L,p<0.05),NMor(32.64±53.72 vs.14.6±31.96 ng/L,p>0.05),NDEA(36.53±65.31 vs.18.00±37.06 ng/L,p>0.05)and and the detection rates of NDPA(72.5%vs.57.5%),NDBA(60%vs.25%),NPyr(50%vs.32.5%),NMor(42.5%vs.35%)and NDEA(37.5%vs.27.5%)were higher in drinking water in Huai’an.While the detection rate(35.0%vs.20.0%)and average exposure level(3.69±8.58 vs.1.78±5.47 ng/L,p>0.05)of NDMA were higher in drinking water in Nanjing.1.2 Levels of nitrosamines in human urine from different regionsSignificant differences of four nitrosamines in the urine between the two areas were found.The detection rates of NDEA,NDBA,NPip,and NDph A in human urine from Huai’an were 2.4(76.49%vs.32.06%),1.53(29.10%vs.19.08%),17.8(27.24%vs.1.53%),and 4.34(38.06%vs.8.78%)times higher than those in Nanjing,respectively.And the concentrations of NDEA,NDBA,NPip,and NDph A in the urine in Huai’an were 2.1(1.31±0.81 vs.0.62±1.06 ng/m L,p<0.01),1.6(0.26±0.71 vs.0.16±0.35 ng/m L,p<0.05),56.0(0.56±1.03 vs.0.01±0.08 ng/m L,p<0.01)and 3.7(0.63±0.94 vs.0.17±0.81 ng/m L,p<0.01)times higher than those in Nanjing,respectively.The urinary excretion of total nitrosamines in Huai’an was 1.8(3.54±1.91 vs.1.94±1.85 ng/m L,p<0.01)times higher than that in Nanjing.1.3 Case-control analysis of urinary nitrosamines in Huai’an areaCompared with the control,NMEA(median,0.22 vs.0.16 ng/m L)in the urine of RE/BCH patients was higher.NMEA(median,0.43 vs.0.16 ng/m L,p<0.05)and NDBA(median,0.11 vs.0.07 ng/ml,p<0.05)in the urine of DYS patients increased.NMEA(median,0.26 vs 0.16 ng/ml,p<0.05),NDBA(median,0.08 vs 0.07 ng/ml,p<0.05),NPyr(median,0.43 vs 0.22 ng/m L,p<0.05),NMor(range,N.D.～0.11 ng/m L vs.N.D.),and the total nitrosamines(median,2.61 vs.2.17 ng/m L,p<0.05)also increased in the urine of ESCC patients.These showed that the urinary excretion of nitrosamines was higher in patients with esophageal diseases.Logistic regression analysis showed that NMEA,NPyr,and NDBA exposure significantly increased the risk of ESCC(p<0.05).At the same time,NMEA and NDBA could also significantly increase the risk of RE/BCH(p<0.05).2.Toxic effects of co-exposure to nitrosamines at low concentrations2.1 Changes of metabolic spectrum of human esophageal epithelial cells exposed to nitrosamines Compared with the control,nitrosamines altered the metabolic spectrum of esophageal epithelial Het-1A cells,and the deviation degree was probably related to the dose.Inflammation-associated pathways,namely,cysteine(Cys)and methionine(MET)metabolism,and nicotinate(NA)and nicotinamide(NAM)metabolism,showed disorders under the action of nitrosamines at the environmentally relevant level,suggesting that inflammatory response could be an important toxic effect of nitrosamines co-exposure at low-dose.2.2 Inflammatory response and the mechanisms in Het-1A cells induced by nitrosaminesNitrosamines stimulated ROS production,increased the oxidative damage,up-regulated the expressions of IL-1β,TNF-αand IL-6,and ptomoted the inflammatory response in cells.For one thing,nitrosamines promoted histone methylation of H3K4me3 and H3K36me3 through Cys and MET metabolism to support the intracellular inflammatory response.For another,nitrosamines decreased the NAD~+/NADH ratio,inhibited NF-кB p65 deacetylation mediated by SIRT1 to activate NF-кB signaling to promote the release of inflammatory cytokines.2.3 Inflammation and the mechanisms in vivo induced by nitrosamines co-exposureUnder the current conditions,obvious inflammation in the liver of mice was induced by nitrosamines at the high dose,but there were no significant changes in esophageal tissue.The western blot assay in the liver showed that nitrosamines at the environmentally relevant concentration did not cause obvious changes of inflammatory related indicators including the histone methylation at H3K4me3and H3K36me3,the level of acetyl p65,and the expression of inflammatory cytokines,but the significant fluctuations of related indicators were clearly observed in vivo with the dose increasing.3.Metabolomic characteristics and potential mechanisms of ESCC in F344 rat induced by nitrosamines3.1 ESCC model in rat induced by nitrosaminesCompared with the control,after continuous exposure to NMBA for 8 weeks,the surface of rat esophageal tissue gradually became rough,with irregular vegetations or white bulges,or cord like,nodular or flat bulges.The number,scope and degree of esophageal lesions were gradually progressed with time.Pathological results showed that NMBA could induce the proliferation of rat esophagus from simple epithelial basal cells,progressive irregular thickening,and gradually form mild,moderate and severe esophageal dysplasia and papilloma,and finally develop into invasive cancer.3.2 Serum metabolic changes during the development of ESCC in rats induced by nitrosamines During the development of ESCC induced by NMBA,the serum metabolic spectrum gradually shifted.The metabolic spectrum of rats in vivo changed significantly when the esophageal lesions developed to L/M-DYS,and rats in the S-DYS group got similar metabolic characteristics as that of the CIS and IC group,indicating that the metabolic changes became irreversibe as the lesions developed into S-DYS.In this process,lipid metabolism and amino acid metabolism were affected.NA and NAM metabolism,sphingolipid metabolism,and tryptophan(TRP)metabolism were the top three key metabolic pathways in the occurrence and development of NMBA induced ESCC in rats.3.3 Regulatory mechanisms during the development of ESCC in rats induced by nitrosamines The inflammatory response was accompanied by the process of the occurrence and development of NMBA induced ESCC in rats.For one thing,NMBA activated TRP-KYN-NAD pathway to promote NAD~+synthesis,activated AHR receptor through IDO1/TDO2-KYN-AHR pathway to induce CYP1A1 expression,and finally promoted the inflammatory response and immune tolerance.At the same time,NMBA amplified the inflammatory response and anti-tumor immune escape in vivo via the"SPHK1-S1P-S1PR1"axis to promoted the malignant transformation of esophageal epithelium.4.Correlation analysis of nitrosamines exposure and human esophageal cancer4.1 Changes of serum metabolic spectrum in people with different esophageal diseasesCompared with the control,the serum metabolic spectrum of RE/BCH,DYS and ESCC patients changed significantly,but the degree of change was different.The serum metabolic spectrum of ESCC patients changed most significantly,mainly including the disorders of lipid metabolism and amino acid metabolism.Considering the dynamic metabolic changes of the ESCC model in rats,sphingolipid metabolism and TRP metabolism probably became the key metabolic pathways in the progression of ESCC.4.2 Mediating effects and the relationship between nitrosamines exposure and human ESCC NPyr exposure significantly increased the risk of ESCC and NMEA exposure increased the risk of RE/BCH,DYS,and ESCC.Serum S1P significantly increased the risk of DYS and ESCC(p<0.05),while TRP and SPH reduced the risk of RE/BCH,DYS,and ESCC(p<0.05).The mediating mechanisms showed that S1P and 5-HTP/TRP(%)had partial mediating effects on the correlation between NMEA exposure and the outcomes of RE/BCH and ESCC.At the same time,TRP had partial mediating effects on the correlation between NPyr and ESCC.The area under curve(AUC),sensitivity,and specificity of combined potential biomarkers composed of S1P,TRP,SPH/S1P,and DH-S1P were 0.90(95%CI 0.85～0.95,p<0.05),0.84,and 0.80,which may be the potential biomarkers of human ESCC related to nitrosamines exposure.Preliminary conclusions:1.Nitrosamines exposure in the high incidence area of esophageal cancer was higher than that in the low incidence area.2.NMEA,NDBA and NPyr were positively correlated with the risk of ESCC,and NMEA and NDBA were positively correlated with RE/BCH risk.3.Inflammatory response was an important toxic effect of nitrosamines co-exposure at the low concentration.On the one hand,nitrosamines induced the up-regulation of Cys and MET metabolism,providing methyl donors for histones methylation to support the inflammatory response.On the other hand,nitrosamines down regulated the NAD~+/NADH ratio,inhibited SIRT1 mediated deacetylation of NF-кB p65,and activated the NF-кB signaling pathway to promote the release of inflammatory cytokines.4.NA and NAM metabolism,sphingolipid metabolism,and TRP metabolism were the key metabolic pathways in the occurrence and progression of NMBA-induced ESCC in rats.They could interfere with energy metabolism,inflammatory microenvironment and immunosuppression to promote malignant transformation of esophageal epithelium.5.Changes of TRP metabolism and sphingolipid metabolism were important metabolic characteristics of human ESCC.6.S1P,TRP,SPH/S1P,and DH-S1P may be potential specific biomarkers of human ESCC related to nitrosamines exposure.