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Journal ArticleDOI

A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. Application to Mus musculus mice under arsenic/cadmium exposure

TL;DR: The results show that As/Cd exposure produces interactions in the distribution of both toxics between organs and plasma of mice and antagonistic interactions with selenium containing proteins in the bloodstream, and these toxic elements have important influence in the levels of seleno-proteins in the plasma.
About: This article is published in Journal of Proteomics.The article was published on 2014-06-02 and is currently open access. It has received 38 citations till now. The article focuses on the topics: Arsenic.

Summary (5 min read)

1. Introduction

  • Arsenic (As) and cadmium (Cd) are important inorganic co-pollutants in the environment, which are the origin of numerous environmental issues.
  • Additionally, in experimental systems, arsenic and cadmium exhibit a great influence on metabolic cell functions [4,6].
  • These approaches require the use of high sensitivity atomic detectors mainly ICP-MS [9], generally coupled to a chromatographic module (in single or multidimensional arrangements), and mass spectrometry for parallel biomolecule identification in an integrated workflow [9,10].
  • For this reason, metallomics provides a good alternative to deep insight into the fate of elements in exposed organisms to metals, and provides information about metals trafficking, interactions and homeostasis [11].
  • Statistical analysis of the results allowed us to compare the different metabolic profiles, establishing the metabolites altered by the presence of these contaminants.

2.1. Instrumentation

  • A cryogenic homogenizer SPEX SamplePrep (Freezer/Mills 6770) was used for solid tissue disaggregation.
  • Disaggregated tissues were subsequently disrupted with a glass/teflon homogenizer.
  • Chromatographic separations were performed by using a Model 1100 HPLC pump with detector UV (Agilent, Wilmington, DE, USA) as delivery system.
  • The parameters for QqQ-TOF system were optimized to obtain the higher sensitivity with minimal fragmentation of molecular ions, both in positive and negative ionization mode.

2.2. Standard solutions and reagents

  • All reagents used for sample preparation in the metallomic approach were of the highest available purity.
  • The void volume was determined by using blue ferritin (440 kDa).
  • Standard solutions of 1000 mg L−1 of Se stabilized with 5% (v/v) nitric acid Suprapur and of 1000 mg L−1 of Br- stabilized with 5% (v/v) nitric acid Suprapur were purchased fromMerck (Darmstadt, Germany).
  • Methanol and chloroform were purchased from Aldrich (Steinheim, Germany),while dichloromethane and formic acidwere supplied by Merck (Darmstadt, Germany).
  • Alanine, valine, isoleucine, proline, glycine, serine, threonine, glutamic acid, phenylalanine, fructose, galactose, glucose, tyrosine, tryptophan, urea, aspartic acid, glutamine, cholesterol, α-ketoglutarate, isocitric acid, citric acid, lactic acid and uric acid were purchased from SigmaAldrich to be used as standard substances in gas chromatography quantification.

2.3. Animal handling

  • M. musculus (inbred BALB/c strain) mice were obtained from Charles River Laboratory .
  • Individual organs were excised, weighed in Eppendorf vials, cleaned with 0.9% NaCl solution, frozen in liquid nitrogen and stored at −80 °C until their use for extract preparation.
  • Plasma collection from five mice of each group was carried out by centrifugation (4000 g, 30 min, 4 °C), after addition of heparin as anticoagulant for separation into plasma and red blood cells (RBCs).
  • The investigation was performed after approval by the Ethical Committee of the University of Huelva .

2.4. Measurement of the clinical parameters in blood and histopathology in liver from mice under As/Cd exposure

  • Blood activity of alanine transferase, alkaline phosphatase, amilase, lipase and aspartate transferase and concentrations of bilirubin, albumin, ferritin, LDL, HDL, triglycerides and creatinine were determined.
  • Standard controls were run before each determination, and the values obtained for the on of metallomics and metabolomics studies to evaluate the (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011.
  • The intra-assay variability of biochemical tests was relative to 12 repeated determinations of the control serum in the same analytical session, whereas inter-assay variability for each parameter was calculated on the mean values of control sera measured during 6 analytical sessions.
  • Both biochemical and histological examinations were utilized to assess liver injury.

2.5. Determination of total metals in plasma, liver and kidney

  • First of all, individual organs were disrupted by cryogenic homogenization.
  • For total metal determination, three samples of plasma, pulverized livers and kidneys of mice from each group were exactly weighed (100 mg) in 5-ml microwave vessels and 500 mg of a mixture containing nitric acid and hydrogen peroxide (4:1 v/v) was added.
  • After 10 min, the PTFE vessels were closed and introduced into the microwave oven.
  • The mineralization was carried out at 400 W from room temperature ramped to 160 °C for 15 min and held for 10 min at this temperature.
  • All the analyses were performed by using two replicates of each sample, using 5 mice per group.

2.6. Metallomic approaches based on ICP-MS detection for analysis of plasma, liver and kidney extracts of mice (M. musculus) under As/Cd exposure

  • Pools of organs from male mice of different groups of exposure were treated following a procedure described elsewhere [11] for later application of size exclusion chromatography with inductively coupled plasma mass spectrometry and octopol reaction system (SEC-ICP-ORS-MS).
  • The quantification of selenium containing proteins and selenium-metabolites in the different chromatographic peaks was carried out by post-column species-unspecific isotopic dilution (SUID) analysis as described by C. Sariego-Muñíz et al. [16].
  • Mathematical treatments were applied to correct BrH+ and SeH+ polyatomic interferences.
  • Mass bias corrections were applied by using the 78Se/74Se and 80Se/74Se isotope ratios, calculated (exponential mode) as previously described by J. Ruiz-Encinar et al.40.

2.7. Metabolomic study of plasma of mice (M. musculus) under As/Cd exposure by DI-ESI(±)-QTOF-MS

  • For metabolomic analysis, metabolite extraction from individual plasma was carried out in a two-step approach following a procedure described elsewhere [4].
  • The polar and lipophilic extracts were reconstituted to 200 μL with (1:1) chloroform/water mixture before the analysis by ESI-MS.
  • The supernatant was carefully collected avoiding contamination with the precipitated proteins, transferred to another Eppendorf tube and the resulting supernatant was taken to dryness under nitrogen stream and stored to −80 °C until analysis.
  • For data acquisitions by positive ionization, 0.1% formic acid was added to polar extract and 30 mM of ammonium acetate to lipophilic extract.
  • In the case of negative ionization intact extracts were directly infused to the mass spectrometer.

2.8. Metabolomic study of plasma of mice (M. musculus) under As/Cd exposure by GC–MS

  • The supernatant was transferred to another Eppendorf tube and dried under nitrogen stream.
  • TMCS participates in the derivatization of amides, secondary amines and hindered hydroxy groups.
  • The injector temperature was kept at 280 °C.
  • For mass spectrometry detection, ionization was carried out by electronic impact (EI) with a voltage of 70 eV, using full scan mode in the m/z range 35–650, with an ion source temperature of 200 °C.
  • The identification of endogenous metabolites was based on comparison with the corresponding standards according to their retention times and mass spectra characteristics by searching on NIST Mass Spectral Library (NIST 02).

2.9. Histopathological study of liver from mice under As/Cd exposure

  • Liver sample animals were excised as described above and immediately fixed in 4% neutral buffered formalin followed by dehydration in increasing grades of alcohol, clearing in xylene, and embedding in paraffin wax.
  • Liver sections (4 mm thickness) obtained in a Leica Leitz 1512 precision rotary microtome (Leitz, Wetzlar, Germany) were stained with hematoxylin and eosin (H&E).
  • The slides were blinded and analyzed by light microscopy for liver injury [18].

3.1. Biochemical parameters in blood of mice under controlled exposure to As/Cd

  • Blood sampling work was performed by the same skilled technician for all samples, and all manipulations performed before and after blood collection were accurately settled, so that variability caused by blood sampling was negligible.
  • Therefore, differences in the values assessed reflect factors directly associated with the blood samplingmethod, including handling stress, anesthesia, hemolysis, and tissue damage.
  • In the present study, the level of hemolysis in all serum samples was scored by direct observation.
  • The results obtained in the last day of the exposure experiment (12th day) are shown in Table 1.
  • Please cite this article as: García-Sevillano MÁ, et al, A combinati effects of metal interactions in mammals.

M. musculus under both toxic metals exposure

  • The presence of arsenic and cadmium in the organs (liver and kidney) and plasma of M. musculus subjected to controlled exposure to As/Cd was evaluated by using ICP-ORS-MS, and the results are shown in Table 2.
  • The results are also shown in Table 2 and confirm quantitative recoveries in all the cases.
  • Instrumental detection limits are also given in this table.
  • The distribution of arsenic and cadmium in liver, kidney and plasma samples from mice exposed to As/Cd can be observed.
  • Similar results are obtained for Cd concentrations in plasma.

M. musculus under As/Cd exposure by SEC-ICP-ORS-MS

  • To check the presence and potential interactions of metalbiomolecules in liver of M. musculus exposed to As/Cd the coupling SEC-ICP-MS was used, obtaining As and Cd-traced peaks from cytosolic fractions of liver (Fig. 1).
  • This fact can be related to the interaction of As with enzymes such as carbonic anhydrase (CA) and superoxide dismutase (Cu/Zn-SOD) with molecular masses of 35 kDa and 32 kDa, respectively.
  • The increase of this peak is more pronounced when As is administered alone in comparison with the joint administration As/Cd.
  • This peak presents higher intensity in mice exposed to the mixture As/Cd during 6 days (Fig. 2A).

3.4. Speciation of selenium in plasma of mice (M. musculus) under cadmium exposure by SEC-AF-HPLC-SUID-ICP-ORS-MS

  • Quantification of Se containing proteins (selenoprotein P – SeP, extracellular glutathione peroxidase – eGPx and selenoalbumin – SeAlb) and low molecular weight.
  • Se species has been performed inmice plasma using the proposed speciationmethod.
  • Se concentrations determined by IDA-ICP-ORS-MS after acid digestion (Table 3).
  • The effect of mice independent exposure to As or Cd on selenium containing proteins present in plasma is similar, decreasing the concentration of SeP, SeAlb and Se-metabolites and increasing the level of eGPx (Table 3).

3.5. Metabolomic study of plasma from mice (M. musculus) under As/Cd exposure by DI-ESI(±)-QTOF-MS and GC–MS

  • In order to discriminate between the groups of mice differentially exposed to As/Cd, a partial least squares discriminant analysis (PLS-DA) was performed employing the intensities of the m/z signals in the polar and lipophilic extracts from mice plasma, using positive and negative ionization mode of acquisition by DI-ESI-QTOF-MS.
  • The models built with polar and lipophilic metabolites allow a good classification of samples in different groups, which are shown by the respective score plots (Fig. 2).
  • To identify which variables were responsible for this separation, the Variable Influence on the Projection (VIP) parameter was used.
  • 1 – Quantification of mice plasma metabolites (Mus musculus) exposed to arsenic and cadmium by GC–MS, also known as Table 5t5.
  • This process induces degradation of membrane phospholipids and cell apoptosis.

Cd exposure

  • The pathological changes in response to As2O3 and CdCl2 exposure were examined and compared among different experimental groups in the liver.
  • The liver is a primary defense organ that detoxifies drugs and xenobiotics, which increase the probability to injury in this organ.
  • Normal morphology of liver histological sections from mice CONTROL GROUP is shown in Fig.
  • Arsenic exposure originates important hepatic damage, such as steatosis, inflammation, significant fibrosis in periportal areas and necrosis (Fig. 4, As GROUP).
  • Cadmium administration resulted in sinusoidal congestion, Mallory bodies' appearance and multifocal hepatic necrosis after 12 days of exposure (Fig. 4, Cd GROUP).

4. Discussion

  • Experiences in living organisms conducting exposure to multiple toxics, as is the case of As and Cd, reveal the interest of this kind of studies due to the interactions occurring between them along the complex biological processes, from toxic exposure to excretion and their toxicological consequences.
  • This fact explains the decreased levels of SeP in mice plasma under Cd exposure (Table 3).
  • Please cite this article as: García-Sevillano MÁ, et al, A combinati effects of metal interactions in mammals.
  • In the present study, blood chemistry clearly shows toxic cirrhosis induced by Cd, which is aggravated with the joint exposure toAs (see also Fig. 3).
  • The final consequence is the accumulation of highly cross-linked undegradable aggregates such as lipofuscin, which can be considered as the long-term result of a decreased degradation of oxidized proteins and increase of intracellular free radical formation.

5. Conclusion

  • This work illustrated the potential of combined use of a metabolomic approach, based on organic mass spectrometry for the study of biochemical effects induced by As/Cd exposure, with a metallomic approach, based on inorganic mass spectrometry for metals/metalloids-biomolecules and metabolites characterization inmice exposed to both elements.
  • Uin the distribution and accumulation of arsenic and cadmiumwere obtained when both toxic metals are administered together.
  • In addition, antagonistic interactions with selenium containing proteins (mainly SeP) in the bloodstream have been observed when both xenobiotics are ingested at the same time.
  • Administration of heavy metal and metalloid, together or separately, resulted in differential liver injury, which has been characterized by the predominance of Please cite this article as: García-Sevillano MÁ, et al, A combinati effects of metal interactions in mammals.

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Q1. What are the contributions in "A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. application to mus musculus mice under arsenic/cadmium exposure" ?

On the other hand, bothdirect infusion mass spectrometry ( DIMS ) and gas chromatography–mass spectrometry ( GC–MS ) provided information about changes in metabolites caused by metals.