Study On Biomakers For 4-nonylphenol Exposure Based On Metabolomic Approaches

4-Nonylphenol(4-NP),a kind of environmental persistent organic pollutants with oestrogen-like effect,is highlighted recently due to its toxicity.Therefore,studies on the exposure levels and health effects are critical for its risk assessment.Metabolomic research with high performance liquid chromatography time of flight mass spectrometry(HPLC-QTOF-MS)was conducted in this study by analyzing the urine and plasma of rats exposed to 4-NP on both positive and negative ion modes.Both targeted and non-targeted metabolomic methods were used to achieve complementary information of biological samples for the reliability of biomarkers screened and identified.Quantitative exogenous stimulations and variation of endogenous metabolites were integrated by metabolomics approaches to acquire the biochemical information in the early exposure and dynamic information through the whole exposure process.As a result,ten endogenous biomarkers,including glycine,glycerophosphocholine,5-hydroxytryptamine,malonaldehyde,tryptophan were identified as the potential biomarkers for 4-NP exposure.Biological validation was also applied for the oxidative stress and tryptophan metabolism pathways of the eight biomarkers,which successfully identified the accuracy of the biomarkers we selected.An analytical method of urinary and plasmic metabolomic was established by HPLC-QTOF-MS.During the urinary non-targeted metabolimic process,quantitative method of peak area calibrated by creatinine value was selected,compared to four-time dilution method and the urine volume calibrating method by creatinine value.Under the positive analysis iron mode,10 m M of formic acid was added to improve the detection range,sensitivity and resolution of the analysis,as well as 10 m M of ammonium acetate when under the negative analysis iron mode.The HPLC-QTOF-MS method was validated to satisfy urinary non-targeted metabonomic analysis.In addition,protein removal by organic solvent in the non-targeted metabonomic analysis of plasma was used,which was identified to be superior to solid phase extraction method.The pretreatment and analysis methods were also identified to satisfy requirements of plasmic non-targeted metabonomic analysis.Rats were exposed to 4-NP at 0,50,and 250 mg/kg/day for 4 consecutive days.The contents of 4-NP in urine and plasma were quantified by HPLC-MS/MS.The exposure levels of 4-NP were obtained to validate the successful establishment of the exposure model.A urinary and plasmic non-targeted metabolomic strategy was originally implemented by HPLC-QTOF-MS to explore the toxicological effects of 4-NP and determine the overall alterations in the metabolite profiles.In order to obtain effective biomarker information from massive set of data,variable analysis methods such as principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA),independent sample t-test were used in the study.36 and 29 potential different ions were seperately indentified from urine and plasmas.The qualitative primary and secondary mass spectra obtained by HPLC-QTOF/MS were compared with the PeakView,Massbank and METLIN databases,lysis and standard validation methods,and 12 and 14 potential biomarkers were confirmed from urine and plasma,respectively.In order to verify the effectiveness of potential biomarkers screened from non-targeted metabolomics,a HPLC-MS/MS targeted metabolomics method for urine and plasma samples was established.A total of 12 urine markers and 14 plasma markers were screened for further quantitative analysis.Eight metabolites with a significant difference between the control group and the 4-NP exposure group(independent sample t test test,p <0.05),were identified as the ultimate biomarkers.The five markers in the urine are serotonin,tryptophan,glycine,malondialdehyde and glycerophosphorylcholine,while the five markers in plasma are serotonin,tryptophan,kynurenine,L-tyrosine and arachidonic acid,in which serotonin and tryptophan are markers in both substrates.The biological significance of ten biomarkers indicate that malondialdehyde,glycerophosphorylcholine and arachidonic acid are the result of oxidative stress induced by 4-NP exposure,where serotonin,tryptophan,glycine and kynurenine act as a neurotransmitter substance in the tryptophan metabolic pathway.The results of 8-hydroxy-2-deoxyguanosine(8-OHd G)and malondialdehyde(MDA)in the urine showed that oxidative stress did occur in rats,and the level of 4-NP in vivo was positively related to 8-OHd G and MDA content.The analysis of the seven metabolites in the tryptophan pathway showed that tryptophan metabolism disorder occurred in rats,and 5-hydroxytryptophan and serotonin were positively correlated with the level of exposure to 4-NP.The above results showed that the eight biomarkers selected in this study were effective and 4-NP exposure can lead to oxidative stress injury and neurotransmitter injury.In this study,ten biomolecules of 4-NP exposure in rat urine and plasma were screened by the combination of targeted and non-targeted metabolomics.4-NP exposure can result in oxidative stress injury and metabolic disorders of tryptophan,and then lead to nerve damage.The results provide a reference for more accurate research on risk assessment and toxicity mechanism of 4-NP exposure.