Effects of the ambient fine particulate matter(PM2.5)exposure on urinary metabolic pro files in rats using UPLC-Q-TOF-MS

Lin Zhng,Tengfei Xu,Zifeng Pi,*,Meizhu Zheng,Fengrui Song,Zhiqing Liu,*

a Changchun Institute of Applied Chemistry,Chinese Academy of Science,Changchun Center of Mass Spectrometry,Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry,Changchun 130022,China

b School of Pharmaceutical Sciences,Jilin University,Changchun 130021,China

c The Central Laboratory,Changchun Normal University, Changchun 130032,China

Keywords:Ambient fine particulate matter UPLC-Q-TOF-MS Metabolomics Urine

ABSTRACT In order to study the damage of ambient fine particulate matter(PM2.5)to organism in the environment,the method of urinary metabolomics was used to detect the potential endogenousmetabolites and their related metabolic pathways to clarify the toxicity mechanisms.Particles of PM2.5 collected from the fi brous membrane were extracted ultrasonically and dissolved in physiological saline to prepare PM 2.5 suspension.The solution of saline and fine particular matter was injected into the trachea of rats respectively.The rats were injected tw o times a week for four weeks.Samples of 24-h urine were collected at the 14th day after the end of exposure,and the ultra performance liquid chromatography coupled with quadrupole time-of-fl ight mass spectrometry(UPLC-Q-TOF-MS)was used for the metabolomics detection.Principal component analysis(PCA)was used to investigate the global metabolomic alterations and a clear separation in the scatter diagram were observed.17 potential endogenous metablites were identi fied from urinary samples in rats by UPLC-Q-TOF-MS.The primary metabolism pathw ays involved pentose and glucuronate interconversions,starch and sucrose metabolism,tryptophan metabolism,tyrosine metabolism,phenylalanine metabolism,purine metabolism,acetaminophen metabolism pathway,retinol metabolism and valproic acid metabolism pathway.The results are helpful to understand the toxicological mechanisms of PM2.5 and screened out potential biomarkers of rats which exposure to PM2.5.

Ambient fine particulate matter(PM 2.5)refers to the inhalable particle aerodynamic diameter less than or equal to 2.5 m m,also is know n as fine particles or particles which can be inhaled into the lungs[1,2].PM2.5 derives from both natural and anthropogenic sources,resulting in a complex chemical composition.At present,the main ingredients of PM 2.5 include sulfates,nitrates,ammonium salts,carbonaceous particles(including elemental carbon and organic carbon,in which,elemental carbon is mainly produced in high temperature combustion processes,organic carbon is mainly derived from the relatively low temperature combustion process),metal particles,minerals and so on[3,4].The research showed that PM2.5 had obvious toxic effect,which could cause extensive damage of many aspects of the body such as respiratory system,immune system,endocrine system,cardiovascular system,etc.However,the exact pathogenic mechanism of PM2.5 is not know n as yet.

The physiological or pathological perturbation in a living system w ill cause the perturbations of concentration or fl ux of endogenous metabolites.Metabonomics has been proved to be an important and valuable tool in analyzing biosamples.Due to the complexity of molecular species and its concentration in the range of metabolic group contains,any single technique is not suf fi cient to conduct a comprehensive study of a particular object.With the development of the new analysis techniques,a series of technologies such as gas chromatography-time of fl ight mass spectrometry(GC-TOF-MS)and ultra performance liquid chromatography coupled with quadrupole time-of-fl ight mass spectrometry(UPLC-Q-TOF-MS),and other analytical chemistry techniques for comprehensive metabolomics analysis were used these days.Among these techniques,UPLC-Q-TOF-MSwas w idely used in the analysis of metabolomics due to the availability of accurate mass numbers of compounds,accurate isotopic abundance and abundant mass spectral information[5–7].

In this study,a urinary metabonomics method based on UPLCQ-TOF-MScombined with CG principal component analysis(PCA)was developed to fi nd the effect of PM2.5 on rat urinary metabolism pro fi le.By detecting the changes in urinary metabonomics of rats which were exposured ambient PM 2.5 for shortterm,the impacts on possible metabolic pathw ays and early biological markers were studied to explore for the toxic mechanism which ambient PM2.5 damage to the body.The research results have provided the useful information for the effective prevention and controlling environmental pollution which is caused by fine particles damage to the human body.

Thirty male SD rats with weights ranged from 200 g to 240 g,obtained from Experimental Animal Center of Jilin University(China),were randomly divided into the control group(CG)and PM2.5 model group(MG).The solution of saline and fine particular matter were injected into the trachea of rats respectively,tw ice a week,after the injection,the rats were upright and rotated immediately so as to make the particulate matter as evenly to be distributed as possible.They were injected tw o times a week for four weeks.The animal model established method of PM 2.5 exposure is based on relevant literature[1].

Sam ple collection and preparation process protocol were described as fellow:Samples of 24-h urine were collected on the 14th day after injection and stored at?80?C.Prior to UPLC-MS analysis,urine samples were thawed at 4?C.After centrifuging at 10,000 rpm for 10 min,the supernatant was diluted at a ratio of 1:2 with w ater,and then fi ltered through a 0.22 m m fi lter membrane.

The UPLC-MSconditions were performed using a Waters Acquity UPLCsystem coupled with a Q-TOFSYNAPTG2 High De fi nition Mass Spectrometer(HDMS)(Waters,UK).Separation was performed on a Waters ACQUITYUPLCBEH C18 Column(1.7 m m,2.1 mm?50 mm)kept at 40?Cand at a fl ow rate of 0.5 m L/min.0.1%aqueous formic acid(v/v)(A)and acetonitrile(B)were used as mobile phase.The gradient elution of Bwasperformed asfollow s:5%–20%Bat 0–3 min,20%–40%Bat 3–4 min,40%–100%Bat 4–6 min,100%B at 6–7 min,100%–5%Bat 7–7.1 min and then kept at 5%Bfor 4 min.The sample inject volume was 5 m L.The ESIsource in both positive and negative ion mode was used in MS analysis.The source temperature was 120?C,and desolvation gas temperature was 350?C.Nitrogen was used as cone and desolvation gas.The fl ow rates of cone and desolvation gaswereset at 50 L/h and 600 L/h,respectively.Capillary,cone and extraction cone voltages were set at 3.0 k V,30 V,and 5.0 V in positive ion mode,respectively,while at 2.0 k V,30 V,and 5.0 V in negative ion mode,respectively.MSdata were collected in full-scan mode in the mass range of 100–1000 Da.MSEwas applied for the MSZ analyses with the low collision energy of 5 eV and the high collision energy of 25 eV–35 eV.

The UPLC-MS raw data were processed by MassLynx V4.1(Waters Corporation,Milford,USA)for peaks detection and alignment.EZinfo 2.0 softw are was applied for principal component analysis(PCA)and orthogonal projection to latent structures squares-discriminant analysis(OPLS-DA).The statistical difference for the ion intensities of selected metabolites and urinary biochemical parameters between model and saline control groups was determined using paired-sample t-tests(P<0.05 or P<0.001).Biochemical databases were used to identify potential markers,such as HMDB(http://w w w.hmdb.ca/),METLIN(http://metlin.scripps.edu/),Mass Spectrometry(http://masspec.scripps.edu/index.php),Massbank(http://w w w.massbank.jp/)and KEGG(http://w w w.kegg.com/).

In this study,we have used the quality control sample(QC)for monitoring the repeatability and stability of the analysis system,which was prepared by equally mixing the tested urine samples.Before the analysis of the sample,we fi rst run QCsample 5 times to balance the system.The system performance was evaluated based on the relative standard deviation(RSD)of the peak area and retention time of the selected substance.The evaluation criteria was that peak area RSD<10%and retention time RSD<1%.In the assessment of analytical method stability,the data acquired from the QC sample showed good system stability.The RSD values of retention times and peak areas of the six ions in the QC samples were 0.72%–0.80%and 8.34%–8.95%in positive mode,0.30%–0.36%and 2.88%–9.76%in negative mode,respectively.

UPLC-Q-TOF-MS is w idely used in metabonomic studies[6,12,13].The typical urinary base peak intensity(BPI)chromatograms of the rat urine obtained in ESI positive mode(A)and ESI negative mode(B)based on UPLC-ESI-QTOF/MS are show n in Fig.S1(Supporting information).

In this paper,both positive and negative ion modes were applied to obtain adequate information of metabolites.Because there were hundreds of metabolites detected in urine,the multivariate analysis methods,such as PCA and OPLS-DA,were used to distinguish the groups.The PCA/OPLS-DA was taken as a pattern recognition method using the scatter diagram in thispaper.Pareto scaling was used to reduce disturbances from noise and artifacts in the tw o models.The MG could be clearly separated from CGin PCAscore plotsin both positive and negative ion modes(Fig.1),which indicated that the endogenousmetabolite difference in urine was remarkable.An OPLS-DA model was established to maximize class discrimination.In the OPLS-DA model,variable importance in the projection(VIP)value was applied to fi nd the potential biomarkers which made the greatest contribution to group separation.The ions with VIP value above 1.0 were considered as potential biomarkers.By comparing with MG,17 endogenous metabolites in urine were considered to be potential biomarkers.The identi fi cation of these metabolites was carried out as follow s.

The possible molecular formulas of the potential biomarkers were calculated by high-accuracy quasi-molecular ion within mass error of 10 ppm and fractional isotope abundance detected by QTOF-MS.The structure information was obtained by searching freely accessible databases mentioned before.MS/MSfragmentation patterns also provided necessary information for the structures of biomarkers.Take the biomarker at m/z 187.0076 in negative ion mode as an example(Fig.S2 in Supporting information).Suppose the m/z is[M-H]?,and the possible formula is C7H8O4S.Its major fragment ions in this work were m/z 107.0542 and m/z 80.9532,which represent the fragment of[M-H-SO3]?and[M-H-SO3-C2H2]?.Finally,it was identi fied as p-cresol sulfate by comparing its quasi-molecular ion and fragment ions in the accessible databases.The information of potential biomarkers is summarized in Table 1.

PM2.5 isassociated with the development of many diseases.But the pathogenic mechanisms remain unknow n.In this study,17 significantly changed metabolites were de fined between CG and MG.The primary metabolism pathw ays involved tryptophan metabolism,pentose and glucuronate intercoversions,tyrosine metabolism, phenylalanine metabolism, acetaminophen metabolism pathw ay,pentose phosphate pathw ay,retinol metabolism and valproic acid metabolism pathw ay.The network of the main potential biomarkers is show n in Fig.2.

Fig.1.PCA score plots of urine metabolic pro fi ling of CG(~),MG(&)in positive mode(A)and negative modes(B).

Table 1 Identi fi cation of signi fi cant endogenous metabolites in the rats urine.

Fig.2.Correlation networks of main potential biomarkers in response to PM 2.5 exposure in rats.The red marked metabolites denotes the identi fied metabolites.

For the metabolic pathw ay analysis,we have used the Met PA(http://metpa.m etabolom ics.ca),which is a web-based metabonomic tool used to p erform p athw ay analysis and visualization of quantitative metabonomic data.The relative intensities of p otential biom arkers in the tw o group s are show n in Fig.3.

5-Hydroxy-6-methoxyindole glucuronide is a metabolite of 5-hydroxy-6-methoxyindole,it is metabolized by the liver glucuronosyltransferase in the liver.Glucuronidation is commonly used for the detoxi fi cation and excretion of toxic substances.It combines with chemicals to form glucuronide,which makes it more w ater-soluble and is finally excreted by the kidneys.In this work,the level of 5-hydroxy-6-methoxyindole glucuronide decreased in the group MG,which indicated that the detoxi fi cation of glucuronidation may be reduced.

Both xanthurenic acid and kynurenic acid are important metabolites of the tryptophan metabolism.Up-regulation of xanthurenic acid and kynurenic acid may be induced by activation of tryptophan 2,3-dioxygenase(TPHD)and in metabolic pathw ay generating kynurenine after the exposure to PM 2.5,which w ill cause the abnormality of tryptophan metabolism directly[8].Research has show n that the content of xanthurenic acid and kynurenic acid in rats with renal insuf fi ciency w ill increase,and kynurenine pathw ay is involved in a variety of central nervous system diseases,some products and enzymes play a different pathological role in the process,abnormalities of kynurenine pathw ay may involve the occurrence of diseases such as depression,epilepsy,schizophrenia and other disease[9–11].3-Hydroxyanthranilic acid is an oxidation product of tryptophan metabolism,that is neurotoxic and can induce the formation of free radicals such as hydroxyl radicals and hydrogen peroxide,and improve the level of oxidative stress as well as cause neuronal death through the apoptotic mechanism[11,12].In this study,they were all up-regulated and caused the abnormality of tryptophan metabolism directly in the group MG comp ared with the group CG.

Fig.3.Relative intensities of potential biomarkers in the tw o groups(*P<0.05,**P<0.01).

As for the purine metabolism,7-methylguanine is the product of DNA methylation and depurination,which is considered an important biomarker of lung cancer,and it had been reported that its content was higher in the urine of the smoker than that in the urine of the non-smoker[14].In the group MG,the content of 7-methylguanine increased which showed that the effect of PM 2.5 on rats was similar to that of nicotine on human,while the normal physical condition of the model group had been affected.

As for the phenylalanine metabolism,p-cresol,even at very low concentration,has adverse effects on the central nervous system,cardiovascular system,lungs,kidney,resulting in central nervous system depression[15].p-Cresol sulfate is a typical refractory protein binding toxin,w hen the toxin binds to the protein,it can lead to the changes of protein’s ow n molecular structure,charge and even function,and even cause the damage of other tissue and organs[16].

p-Cresol sulfate and p-cresol glucuronide are uremic toxins,they are p-cresol derivatives.p-Cresol is usually produced by tyrosine or phenylalanine in the intestinal anaerobic fl ora.In this research,compared with the CG group,the level of p-cresol,pcresol sulfate and p-cresol glucuronide in the group MG increased obviously,up-regulation of them could indicated that PM 2.5 might caused the damage of rats’central system and organs.

Besides,there have other metabolism pathw ays.Acetaminophen glucuronide is a natural human metabolite of acetaminophen generated in the liver by UDP glucuonyltransferase.Glucuronidation is used to assist the excretions of toxic substances,drugs or other substances that cannot be used as an energy source.Glucuronic acid is attached via a glycosidic bond to the substance,and the resulting glucuronide,which has a higher w ater solubility than the original substance,is eventually excreted by the kidneys.In the group MG,the level of acetaminophen glucuronide was decreased,which indicated that PM2.5 on the kidney detoxi fi cation had a certain inhibitory effect.

4-Hydroxyphenylacetaldehyde is a by-product of tyrosine metabolism.In this study,the levels of 4-hydroxyphenylacetaldehyde was signi fi cant up-regulated in the MG compared with the CG,which indicated that PM 2.5 caused the disturbance of rats’tyrosine metabolism[17].

Global metabolic pro fi ling on PM2.5 rats using UPLC-QTOF-MS is useful to investigate signi fi cant impact of PM2.5 on metabolism and physiology[18,19].In this paper,a UPLC-Q-TOF-MS-based urinary metabonomics method has been established and used for the fi rst time to screen out potential biomarkers of rats which exposure to PM 2.5,a total of 17 potential biomarkers were identi fied as being primarily involved in pentose and glucuronate interconversions,tryptophan metabolism,purine metabolism,phenylalanine metabolism,tyrosine metabolism and acetaminophen metabolism pathw ay.Among them,the content of 12 kinds of metabolites decreased and 5 kinds of metabolites increased.These changessuggest that PM2.5 had a different degree of damage to the physiological functions,tissues and organs of rats,which provided a reference for clarifying the complex mechanism of PM2.5 induced body injury.In this study,the explored effect of PM2.5 on metabolic pathw ays in rats provided some theoretical support for the further research of PM 2.5 toxic effects and pathogenic mechanisms.

Ack now led gm ent

This research was supported by the National Natural Science Foundation of China(No.31400453).

Appendix A.Supplem entary data

Supplementary data associated with this article can be found,in the online version,at 10.1016/j.cclet.2017.11.019.