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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

02 Jun 2014-Journal of Proteomics (Elsevier)-Vol. 104, pp 66-79

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.

AbstractArsenic and cadmium are toxic metals of environmental significance with harmful effects on man. To study the toxicological and biochemical effects of arsenic/cadmium in mammals a combined metallomic and metabolomic approach has been developed, complemented with the measurement of biochemical parameters in blood and histopathological evaluation of liver injury in mice Mus musculus under exposure to both xenobiotics. Size-exclusion chromatography (SEC) was combined with affinity chromatography (AF) and ICP-MS detection using species unspecific isotopic dilution analysis (SUID) to characterize the biological effects of As/Cd on selenium containing proteins in the bloodstream of exposed mice. On the other hand, both direct infusion mass spectrometry (DIMS) and gas chromatography–mass spectrometry (GC–MS) provided information about changes in metabolites caused by metals. 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. Interplay with essential metabolic pathways, such as energy metabolism and breakdown of membrane phospholipids were observed, which are more pronounced under As/Cd exposure. In addition, heavy metal and metalloid causes differential liver injury, manifested by steatosis (non-alcoholic fatty liver disease, NAFLD) and infiltration of blood cells into the space of Disse. Biological significance This work presents new contributions in the study of arsenic/cadmium interactions in mice Mus musculus under controlled exposure. With the combination of metallomic and metabolomic approaches the traffic of As and Cd from liver to kidney by means of blood was observed and excretion of As (as arsenic metabolites) or Cd (as MTCd) is inhibited with the simultaneous administration of As/Cd, and these toxic elements have important influence in the levels of seleno-proteins in the plasma. In addition, the metabolomic approach reveals inhibition of different metabolic cycles such as tricarboxylic acid and phospholipid degradation that causes membrane damage and apoptosis that is histopathologically confirmed. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Topics: Arsenic (50%)

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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UNCORRECTED PROOF
1 A combination of metallomics and metabolomics
2 studies to evaluate the effects of metal interactions
3 in mammals. Application to Mus musculus mice
4 under arsenic/cadmium exposure
5 Miguel ÁngelQ1 García-Sevillano
a,b,c
, Tamara García-Barrera
a,b,c,
,
6 Francisco Navarro-Roldán
d
, Zaida Montero-Lobato
d
, José Luis Gómez-Ariza
a,b,c,
⁎⁎
7
a
Department of Chemistry and Materials Science, Faculty of Experimental Science, University of Huelva, Campus de El Carmen, 21007 Huelva, Spain
8
b
International Agrofood Campus of Excellence International ceiA3, University of Huelva, Spain
9
c
Research Center of Health and Environment (CYSMA), University of Huelva, Campus de El Carmen, 21007 Huelva, Spain
10
d
Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen,
11 21007 Huelva, Spain
12
14
ARTICLE INFO15 ABSTRACT
16 Article history:
17 Received 6 December 2013
18 Accepted 10 February 2014
19
20
Arsenic and cadmium are toxic metals of environmental significance with harmful effects on
21 man. To study the toxicological and biochemical effects of arsenic/cadmium in mammals a
22 combined metallomic and metabolomic approach has been developed, complemented with the
23 measurement of biochemical parameters in blood and histopathological evaluation of liver
24 injury in mice Mus musculus under exposure to both xenobiotics. Size-exclusion chromatography
25 (SEC) was combined with affinity chromatography (AF) and ICP-MS detection using species
26 unspecific isotopic dilution analysis (SUID) to characterize the biological effects of As/Cd on
27 selenium containing proteins in the bloodstream of exposed mice. On the other hand, both direct
28 infusion mass spectrometry (DIMS) and gas chromatographymass spectrometry (GCMS)
29 provided information about changes in metabolites caused by metals. The results show that
30 As/Cd exposure produces interactions in the distribution of both toxics between organs and
31 plasma of mice and antagonistic interactions with selenium containing proteins in the
32 bloodstream. Interplay with essential metabolic pathways, such as energy metabolism and
33 breakdown of membrane phospholipids were observed, which are more pronounced under
34 As/Cd exposure. In addition, heavy metal and metalloid causes differential liver injury,
35 manifested bysteatosis (non-alcoholic fattyliver disease, NAFLD) and infiltration of blood cells
36 into the space of Disse.
48 KeywordsQ2 :
49 Metals interactions
50 Arsenic
51 Cadmium
52 Metallomics
53 Metabolomics
54 Mus musculus
55 Mass spectrometry
56 Histopathological evaluation
57
JOURNAL OF PROTEOMICS XX (2014) XXX XXX
This article is part of a Special Issue entitled: Environmental and structural proteomics.
Correspondence to: T. García-Barrera, Department of Chemistry and CC.MM, Faculty of Experimental Science, University of Huelva, Campus
de El Carmen, 21007 Huelva,Spain. Tel.: +34 959219962; fax: +34 959 219942.
⁎⁎ Correspondence to: J.L. Gómez-Ariza, Department of Chemistry and CC.MM, Faculty of Experimental Science, University of Huelva, Campus
de El Carmen, 21007 Huelva,Spain. Tel.: +34 959219968; fax: +34 959 219942.
E-mail addresses: tamara@dqcm.uhu.es (T. García-Barrera), ariza@uhu.es (J.L. Gómez-Ariza).
http://dx.doi.org/10.1016/j.jprot.2014.02.011
1874-3919/© 2014 Published by Elsevier B.V.
Available online at www.sciencedirect.com
ScienceDirect
www.elsevier.com/locate/jprot
JPROT-01710; No of Pages 14
Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the
effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011

UNCORRECTED PROOF
37 Biological significance
38 This work presents new contributions in the study of arsenic/cadmium interactions in
39 mice Mus musculus under controlled exposure. With the combination of metallomic and
40 metabolomic approaches the traffic of As and Cd from liver to kidney by means of blood was
41 observed and excretion of As (as arsenic metabolites) or Cd (as MTCd) is inhibited with the
42 simultaneous administration of As/Cd, and these toxic elements have important influence in
43 the levels of seleno-proteins in the plasma. In addition, the metabolomic approach reveals
44 inhibition of different metabolic cycles such as tricarboxylic acid and phospholipid degradation
45 that causes membrane damage and apoptosis that is histopathologically confirmed.
46 This article is part of a Special Issue entitled: Environmental and structural proteomics.
47 © 2014 Publi shed by Elsev ier B.V.
58
5960
61
62
63
1. Introduction
64 Arsenic (As) and cadmium (Cd) are important inorganic
65 co-pollutants in the environment, which are the origin of
66 numerous environmental issues. Biological systems are ex-
67 posed to environmental complex ecosystems where the chem-
68 ical species of the elements may interact with synergistic or
69 antagonistic effects, and have to be considered in relation to the
70 metabolic processes involved [1]. In addition, these metals are
71 not biodegradable and have a long life in the environment.
72 Accumulation of these toxic metals/metalloids in ecosystems is a
73 major source of human exposure and hence a threat to human
74 health, mainly As and Cd, which are by-products from processing
75 other metals, leading to common exposure in industrial settings.
76 The biochemical effects of independent exposure to As and Cd
77 have been extensively studied in experimental animals [25],
78 however, the biological response of mammals under simulta-
79 neous exposure to both toxicants has been poorly studied. As a
80 result, the toxicological effects provoked by arsenic and cadmium
81 administration remain still unclear. Additionally, in experimental
82 systems, arsenic and cadmium exhibit a great influence on
83 metabolic cell functions [4,6]. There are evidences about the
84 interaction of As/Cd in rats, which is reflected in changes in
85 different biomarkers assays [7 ]. These authors report that
86 combined exposure to As/Cd is more damaging than separate
87 exposure to each elements, inducing lipid peroxidation and both
88 glutathione and metallothionein up-regulation.
89 In this sense, to obtain a representative information
90 about changes in metabolites caused by complex metal
91 exposure, -omics methodologies have been proposed as a good
92 alternative [2,4]. Metallomics is a relatively new field related to
93 metal-biomolecule expression and identification in biological
94 systems, which represent a more than 30% of molecules in cells.
95 In metallomics metals are used as markers or tags to track these
96 molecules in complex biological matrices [8].Theseapproaches
97 require the use of high sensitivity atomic detectors mainly
98 ICP-MS [9], generally coupled to a chromatographic module
99 (in single or multidimensional arrangements), and mass
100 spectrometry for parallel biomolecu le identification in an
101 integrated workflow [9,10]. For this reason, metallomics pro-
102 vides a good alternative to deep insight into the fate of elements
103 in exposed organisms to metals, and provides information
104 about metals trafficking, interactions and homeostasis [11].On
105 the other hand, metabolomics is based on the comprehensive
106 evaluation of metabolites involved in different metabolic
107 processes in organisms, considering the metabolome as the
108entire cellular set of endogenous low molecular mass biomole-
109cules (typically <1000 Da) [12].Massspectrometry(MS)and
110nuclear magnetic resonance (NMR) spectroscopy are major
111analytical tools used in metabolomics approaches [13,14]. Never-
112theless, the performance of DIMS on biological fluids or tissues
113from mice under metal exposure has proved to be a good choice
114for this purpose [4].
115In this work, a metallomic approach based on SEC-ICP-MS has
116been used to achieve a better understanding of the function,
117detoxification processes, interactions and regulation of metals in
118laboratory mouse Mus musculus under controlled exposure to
119arsenic and cadmium. Additionally, 2D-SEC-AF-SUID was per-
120formed to quantify selenium containing proteins in mice plasma
121with ICP-qMS as multielemental detector. On the other hand,
122intended to get as much metabolic information as possible,
123plasma and liver from these animals, after exposure to metals
124during 12 days, were also studied using direct infusion high-
125resolution mass spectrometry (DI-ESI-QqQ-TOF-MS). Statistical
126analysis of the results allowed us to compare the different
127metabolic profiles, establishing the metabolites altered by the
128presence of these contaminants. In addition, several metabolites
129were quantified by gas chromatographymass spectrometry
130(GCMS) in plasma from mice. Finally, the study has been
131complemented with the measurement of conventional bio-
132chemical parameters in blood and the histopathological study
133of liver mice.
134
135
2. Material and methods
1362.1. Instrumentation
137A cryogenic homogenizer SPEX SamplePrep (Freezer/Mills 6770)
138was used for solid tissue disaggregation. Disaggregated tissues
139were subsequently disrupted with a glass/teflon homogenizer.
140The extraction was followed by ultracentrifugation with an
141ultracentrifuge Beckman model L9-90K (rotor 70 Ti). Polycarbon-
142ate bottles of 10 ml with cap assembly (Beckman Coulter) were
143used for this purpose. A microwave oven (CEM Matthews, NC,
144USA, model MARS) was used for the mineralization of extracts.
145Trace elements and heteroelement-containing biomolecules
146were analyzed with an inductively coupled plasma mass
147spectrometer Agilent 7500ce (Agilent Technologies, Tokyo,
148Japan) equipped with an octopole collision/reaction cell. Chro-
149matographic separations were performed by using a Model 1100
150HPLC pump with detector UV (Agilent, Wilmington, DE, USA) as
151delivery system.
2 JOURNAL OF PROTEOMICS XX (2014) XXX XXX
Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the
effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011

UNCORRECTED PROOF
152 Metabolomic experiments were performed in a mass
153 spectrometer QSTAR XL Hybrid system (Applied Biosystems,
154 Foster City, CA, USA) by using the electrospray (ESI) source. The
155 parameters for QqQ-TOF system were optimized to obtain the
156 higher sensitivity with minimal fragmentation of molecular
157 ions, both in positive and negative ionization mode. To acquire
158 MS/MS spectra, nitrogen was used as collision gas.
159 Gas chromatographic analysis was performed in a Trace
160 GC ULTRA gas chromatograph coupled to an ion trap mass
161 spectrometer detector ITQ900, both from Thermo Fisher
162 Scientific, using a Factor Four capillary column VF-5MS
163 30 m × 0.25 mm ID, with 0.25 μm of film thickness (Varian).
164 Blood activity of alanine transferase, alkaline phosphatase,
165 amilase, lipase and aspartate transferase, and concentrations of
166 bilirub in, albumin, ferritin, LDL, HDL, triglyce ride s and creatinine
167 were determined by using an automated analyzer (Selectra Junior
168 Spinlab 100, Vital Scientific, Dieren, Netherlands; Spinreact,
169 Girona, Spain) according to the manufacturers' instructions.
170 2.2. Standard solutions and reagents
171 All reagents used for sample preparation in the metallomic
172 approach were of the highest available purity. Phenylmethane-
173 sulfonyl fluoride (PMSF) and tris(2-carboxyethyl)phosphine
174 hydrochloride (TCEP) (BioUltra grade, >98%) were obtained
175 from Sigma-Aldrich (Steinheim, Germany).
176 Standards used for mass calibration of analytical SEC
177 columns (mass range 703 kDa) were: ferritin (440 kDa) (purity
178 95%), bovine serum albumin (67 kDa) (purity 96%), superoxide
179 dismutase containing Cu and Zn (32 kDa) (purity > 70%),
180 myoglobin (14 kDa) (purity > 98%), metallothionein I containing
181 Cd, Cu and Zn (7 kDa) (purity > 95%) and arsenobetaine (179 Da)
182 (purity > 98%). All these reagents were purchased from
183 Sigma-Aldrich (Steinheim, Germany). The mobile phase used
184 in SEC was 20 mM ammonium acetate (Suprapur grade)
185 purchased from Merck (Darmstadt, Germany), which was
186 prepared daily with ultrapure water (18 MΩcm) from a Milli-Q
187 system (Millipore, Watford, UK). The pH was adjusted at pH 7.4
188 with ammonia solution, this later prepared by dilution of 20%
189 (w/v) ammonia solution (Suprapur, Merck) with ultrapure
190 water. The void volume was determined by using blue ferritin
191 (440 kDa).
192 Human serum certified reference material BCR-637 was
193 purchased from the Institute for Reference Materials and
194 Measurements (IRMM, Geel, Belgium). Standard solutions of
195 1000 mg L
1
of Se stabilized with 5% (v/v) nitric acid Suprapur
196 and of 1000 mg L
1
of Br- stabi lized with 5% (v/v) nitric acid
197 Sup rapur were purchased f rom Merck (Darmstadt, German y).
198 Enriched
74
Se and
77
Se were obtained from Cambridge Isotope
199 Laboratories (Andover, MA, USA) as elemental powder and it
200 was dissolved in the minimum volume of nitric acid (Suprapur
201 grade) and diluted to volume with ultrapure water.
202 All the solvents used in sample preparation for metabolomic
203 study of liver tissue and plasma were of HPLC-grade. Methanol
204 and chloroform were purchased from Aldrich (Steinheim,
205 Germany), while dichloromethane and formic acid were supplied
206 by Merck (Darmstadt, Germany).
207 Derivatizing agents, methoxylamine hydrochloride and
208 N-methyl-N-(tr imethylsilyl) trifluoroacetamide ( MSTFA) con-
209 taining 1% trimethylchlorosilane (TMCS), were obtained from
210Sigma-Aldrich. Alanine, valine, isoleucine, proline, glycine,
211serine, threonine, glut amic acid, phenylalanine, fructose,
212galactose, glucose, tyrosine, tryptoph an, urea, aspartic acid,
213glutamine, cholesterol, α-ketoglutarate, isocitric acid, citric
214acid, lactic acid and uric acid were purchased from Sigma-
215Aldrichtobeusedasstandardsubstancesingaschromatog-
216raphy quantification.
2172.3. Animal handling
218M. musculus (inbred BALB/c strain) mice were obtained from
219Charles River Laboratory (Spain). Mice 7 weeks of age were fed
220ad libitum with maintenance pellets deficient in metals
221content. The animals were allowed to acclimate for 5 days
222with free access to food and water under controlled condition
223(temperature (2530 °C) and a 12 h lightdark cycle) prior to
224start exposure experiment. For the experiment exposure, a
225total o f 64 M. musculus mice were divided in to four groups
226(16 mice per cage): control group (CONTROL GROUP), group
227exposed to arsenic (As GROUP), group exposed to Cd (Cd GROUP)
228and finally, group simultaneously exposed to As and Cd (As/Cd
229GROUP).
230Arsenic (As
2
O
3
) and cadmium (CdCl
2
) were orally adminis-
231trated by using an oral gavage for mice. The control group was
232treated with 100 μL of 0.9% NaCl. In the case of arsenic, daily
233dose was 3 mg/kg of body weight and per day and for
234cadmium 0.1 mg/kg of body weight and per day both together
235in a dose of 100 μL. M. musculus mice were sacrificed after the
236sixth day of the beginning of the experiment (8 mice in each
237group) and 12nd day of the experience to evaluate the effect of
238exposure conditions and diet.
239Mice were individually anesthetized by isoflurane inhala-
240tion and exsanguinated by cardiac puncture, dissected by
241using a ceramic scalpel and finally the organs transferred
242rapidly to dry ice. In parallel a portion of each liver was
243reserved for the histological assessment. Individual organs
244were excised, weighed in Eppendorf vials, cleaned with 0.9%
245NaCl solution, frozen in liquid nitrogen and stored at 80 °C
246until their use for extract preparation. Plasma collection from
247five mi ce of each group was carried out by centrifugation
248(4000 g, 30 min, 4 °C), after addition of heparin (ANTICLOT) as
249anticoagulant for separation into plasma and red blood cells
250(RBCs). In addition, 10 mg of 100 mM of PMSF and 100 mM of
251TCEP mixture were added as proteases inhibitor and reducing
252agent, respectively, for metallomic studies. On the other
253hand, three samples of blood without any anticoagulant
254were used to the measurement of biochemical parameter.
255Mice were handled according to the norms stipulated by the
256European Community. The investigation was performed after
257approval by the Ethical Committee of the University of Huelva
258(Spain).
2592.4. Measurement of the clinical parameters in blood and
260histopathology in liver from mice under As/Cd exposure
261Blood activity of alanine transferase, alkaline phosphatase,
262amilase, lipase and aspartate transferase and concentrations
263of bilirubin, albumin, ferritin, LDL, HDL, triglycerides and
264creatinine were determined. Standard controls were run
265before each determination, and the values obtained for the
3JOURNAL OF PROTEOMICS XX (2014) XXX XXX
Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the
effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011

UNCORRECTED PROOF
266 different biochemical parameters were always within the
267 expected ranges. The intra-assay variability of biochemical
268 tests was relative to 12 repeated determinations of the control
269 serum in the same analytical session, whereas inter-assay
270 variability for each parameter was calculated on the mean
271 values of control sera measured during 6 analytical sessions.
272 Both biochemical and histological examinations were utilized
273 to assess liver injury.
274 2.5. Determination of total metals in plasma, liver and kidney
275 First of all, individual organs were disrupted by cryogenic
276 homogenization. For total metal determination, three sam-
277 ples of plasma, pulverized livers and kidneys of mice from
278 each group were exactly weighed (100 mg) in 5-ml microwave
279 vessels and 500 mg of a mixture containing nitric acid and
280 hydrogen peroxide (4:1 v/v) was added. After 10 min, the PTFE
281 vessels were closed and introduced into the microwave oven.
282 The mineralization was carried out at 400 W from room
283 temperature ramped to 160 °C for 15 min and held for 10 min
284 at this temperature. Then the solutions were made up to 2 g
285 with ultrapure water and the metals analyzed by ICP-MS. The
286 element Rh was added as internal standard (1 ng g
1
). All the
287 analyses were performed by using two replicates of each
288 sample, using 5 mice per group.
289 2.6. Metallomic approaches based on ICP-MS detection
290 for a nalysis of plasma, liver and kidney extracts of mice
291 (M. musculus) under As/Cd exposure
292 Pools of organs from male mice of different groups of exposure
293 were treated following a procedure described elsewhere [11] for
294 later application of size exclusion chromatography with induc-
295 tively coupled plasma mass spectrometry and octopol reaction
296 system (SEC-ICP-ORS-MS). On the other hand, to avoid changes in
297 selenium species, the samples were directly injected into the
298 column, without prior dilution to evaluate the effects of cadmium
299 in selenium containing proteins by in series two-dimensional
300 size exclusion and affinity high performance liquid chroma-
301 tography with ICP-MS detection (2D/SEC-AF-ICP-ORS-MS [15].
302 The fractionation of selenium containing proteins by two-
303 dimensional chromatographic separations, based on SEC
304 prior to the use of a double affinity column, was carried out
305 following a procedure described elsewhere [15].
306 The quantification of selenium containing proteins and
307 selenium-metabolites in the different chromatographic peaks
308 was carried out by post-column species-unspecific isotopic
309 dilution (SUID) analysis as described by C. Sariego-Muñíz et al.
310 [16]. The intensity of different Se isotopes and polyatomic
311 interferences were converted to mass flow chromatogram for
312 the quantification of selenium species in plasma and serum
313 samples. Dead time correction was carried out by using the
314 procedure described by F. Vanhaecke et al. [17], which results
315 in 47 ns in this study. Mathematical treatments were applied
316 to correct BrH
+
and SeH
+
polyatomic interferences. Mass bias
317 corrections were applied by using the
78
Se/
74
Se and
80
Se/
74
Se
318 isotope ratios, calculated (exponential mode) as previously
319 described by J. Ruiz-Encinar et al.
40
. Finally, online dilution
320 equation was applied to each point of the chromatogram and
321 the amount of selenium in each chromatographic peak
322calculated by using the Origin 8.5.1 software (Microcal Software
323Inc., Northampton, MA, USA).
3242.7. Metabolomic study of plasma of mice (M. musculus) under
325As/Cd exposure by DI-ESI(±)-QTOF-MS
326For metabolomic analysis, metabolite extraction from indi-
327vidual plasma was carried out in a two-step approach
328following a procedur e des cribed elsewhere [4].Thepolar
329and lipophilic extracts were reconstituted to 200 μLwith(1:1)
330chloroform/water mixture before the analysis by ESI-MS. For
331DI-ESI(±)-QTOF-MS of plasma samples, proteins were re-
332moved from blood plasma by adding 400 μLof1:1methanol/
333ethanol mixture to 100 μL of plasma in an Eppendorf tube
334followed by vigorous vortex shaking for 5 min at room
335temperature and centrifugation at 4000 g for 10 min at 4 °C.
336The supernatant was carefully collected avoiding contamin a-
337tion with the precipitated proteins, tra nsferred to anothe r
338Eppendorf tube and the resulting supernatant was taken to
339dryness under nitrogen stream and stored to 80 °C until
340analysis. The pellet was homogenized again, with 200 μLofa
341mixture of (2:1) chloroform/methanol mixture, using a pellet
342mixer (2 min), to extract lip ophilic metabolites and centrifuged
343(10,000 g at 4 °C for 10 min). Finally, the resulting supernatant
344was taken to dryness under nitrogen stream and stored to 80 °C
345until analysis.
346The polar and lipophilic extracts were reconstituted to
347200 μL of (1:1) chloroform/water mixture before the analysis
348by ESI-MS. For data acquisitions by positive ionization, 0.1%
349formic acid was added to polar extract and 30 mM of
350ammonium acetate to lipophilic extract. In the case of negative
351ionization intact extracts were directly infused to the mass
352spectrometer.
3532.8. Metabolomic study of plasma of mice (M. musculus) under
354As/Cd exposure by GCMS
355Plasma was thawed at 4 °C and vortex-mixed before use. For
356the extraction of metabolites 100 μL of plasma were mixed
357with 400 μL of 1:1 methanol/ethanol mixture in an Eppendorf
358tube and vortexed for 5 min at room temperature, followed by
359centrifugation at 4000 g for 10 min at 4 °C. The supernatant
360was transferred to another Eppendorf tube and dried under
361nitrogen stream. All the dried samples were derivatized with
36250 μL methoxylamine hydrochloride (20 mg mL
1
in pyridine)
363at 70 °C for 40 min, for protection of carbonyl groups by
364methoximation, followed by treatment with 50 μL of MSTFA
365containing 1% of TMCS at 50 °C for 40 min, to derivatizate
366primary amines and primary and secondary hydroxy groups.
367TMCS participates in the derivatization of amides, secondary
368amines and hindered hydroxy groups. Finally, the derivatized
369samples were vortex-mixed for 2 min and centrifuged at 4000 g
370for 5 min to collect the supernatant for GC analysis.
371Chromatography was performed on a Factor Four capillary
372column VF-5MS 30 m × 0.25 mm ID, with 0.25 μm of film
373thickness (Varian). The injector temperature was kept at
374280 °C. Helium carrier gas was used at a constant flow rate of
3751 mL/min. To acquire a good separation, the column tempera-
376ture was initially maintained at 60 °C for 5 min, and then
377increased from 60 to 140 °C at a rate of 7 °C/min for4 min. Then,
4 JOURNAL OF PROTEOMICS XX (2014) XXX XXX
Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the
effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011

UNCORRECTED PROOF
378 the column temperature was increased to180 °C at 5°°C/min for
379 another 6 min. After that, the temperature was increased to
380 280 °C at 5 °C/min, and held for 2 min. For mass spectrometry
381 detection, ionization was carried out by electronic impact (EI)
382 with a voltage of 70 eV, using full scan mode in the m/z range
383 35650, with an ion source temperature of 200 °C. For analysis,
384 1 μl of sample was injected in splitless mode. The identification
385 of endogenous metabolites was based on comparison with
386 the corresponding standards according to their retention
387 times and mass spectra characteristics by searching on NIST
388 Mass Spectral Library (NIST 02).
389 2.9. Histopathological study of liver from mice under As/Cd
390 exposure
391 Liver sample animals were excised as described above and
392 immediately fixed in 4% neutral buffered formalin followed by
393 dehydration in increasing grades of alcohol, clearing in
394 xylene, and embedding in paraffin wax. Liver sections (4 mm
395 thickness) obtained in a Leica Leitz 1512 precision rotary
396 microtome (Leitz, Wetzlar, Germany) were stained with
397 hematoxylin and eosin (H&E). The slides were blinded and
398 analyzed by light microscopy for liver injury [18].
399
400
3. Results
401 3.1. Biochemical parameters in blood of mice under controlled
402 exposure to As/Cd
403 Blood sampling work was performed by the same skilled
404 technician for all samples, and all manipulations performed
405 before and after blood collection were accurately settled, so that
406 variability caused by blood sampling was negligible. Therefore,
407 differences in the values assessed reflect factors directly associ-
408 ated with the blood sampling method, including handling stress,
409 anesthesia, hemolysis, and tissuedamage.Inthepresentstudy,
410 the level of hemolysis in all serum samples was scored by direct
411 observation. The results obtained in the last day of the exposure
412 experiment (12th day) are shown in Table 1.
4133.2. Total metals distribution of arsenic and cadmium in mice
414M. musculus under both toxic metals exposure
415The presence of arsenic and cadmium in the organs (liver and
416kidney) and plasma of M. musculus subjected to controlled
417exposure to As/Cd was evaluated by using ICP-ORS-MS, and the
418results are shown in Tab le 2. Recovery experiments were
419performed by spiking the extracts with 1, 5, 10 or 50 ng g
1
of
420analytes depending on the relative concentration of either one in
421theextracts.TheresultsarealsoshowninTable 2 and confirm
422quantitative recoveries in all the cases. Instrumental detection
423limits are also given in this table.
424The Q3distribution of arsenic and cadmium in liver, kidney
425and plasma samples from mice exposed to As/Cd can be
426observed. An increased concentration of arsenic is obtained in
427mice plasma exposed to As in comparison with those exposed
428to As/Cd. Similar results are obtained for Cd concentrations in
429plasma. However, in kidney, the highest concentrations of As
430are obtained in mice exposed to As/Cd. In mammals, highly
431toxic inorganic arsenic is mainly metabolized in liver, after
432absorption from gastrointestinal tract, to produce methylated
433species such as MA
V
and DMA
V
, which are excreted by urine
434[19,20]. In this sense, our results show a major excretion of As
435when Cd is administered simultaneously (Table 2). In contrast,
436the major accumulation of Cd in kidney cytosolic extract is
437obtained when this element is administered isolated to mice.
438Since the most important interaction between these elements
439was observed in the liver, the cytosolic extract of this organ was
440used to study the biological response of exposed mice by SEC-
441ICP-ORS-MS.
4423.3. Profiles of As and Cd-containing biomolecules in liver of
443M. musculus under As/Cd exposure by SEC-ICP-ORS-MS
444To check the presence and potential interactions of metal-
445biomolecules in liver of M. musculus exposed to As/Cd the
446coupling SEC-ICP-MS was used, obtaining As and Cd-traced
447peaks from cytosolic fractions of liver (Fig. 1).
448In Fig. 1 can be observed the presence of low molecular
449mass As species (<300 Da) in liver cytosolic extracts analyzed
Table 1t1:1 Clinical parameters in blood from Mus musculus mice under As/Cd controlled exposure after twelve days of
t1:2 exposure.
t1:3
t1:4 Clinical
parameters
(mean ± SD)
Bilirubin
mg/dL
Ferritine mg/dL Albumin gr/dL LDL mg/dL HDL mg/dL Alanine transpherase UI/L
t1:5 CONTROL GROUP 0.07 ± 0.01 207 ± 9 3.4 ± 0.5 64 ± 5 99 ± 8 106 ± 8
t1:6 As GROUP 0.07 ± 0.02 202 ± 11 3.6 ± 0.7 85 ± 6 108 ± 11 61 ± 11
t1:7 Cd GROUP 0.05 ± 0.02 243 ± 15 3.3 ± 0.4 94 ± 6 115 ± 9 103 ± 9
t1:8 As/Cd GROUP 0.02 ± 0.01 250 ± 17 3.5 ± 0.5 82 ± 4 107 ± 14 150 ± 15
t1:9
t1:10 Clinical
parameters
(mean ± SD)
Alkaline phosphatase
UI/L
Amilase
UI/L
Triglycerides
mg/dL
Lipase
UI/L
Creatinine
mg/dL
Aspartate transpherase
UI/L
t1:11 CONTROL GROUP 149 ± 11 3515 ± 251 172 ± 14 32 ± 4 0.24 ± 0.04 384 ± 21
t1:12 As GROUP 131 ± 13 3290 ± 303 203 ± 12 24 ± 3 0.36 ± 0.02 381 ± 32
t1:13 Cd GROUP 157 ± 15 2989 ± 189 173 ± 14 35 ± 5 0.22 ± 0.02 719 ± 24
t1:14 As/Cd GROUP 127 ± 12 3455 ± 225 241 ± 18 20 ± 4 0.25 ± 0.03 952 ± 62
5JOURNAL OF PROTEOMICS XX (2014) XXX XXX
Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the
effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011

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  • ...selenoproteins in liver for later transport to plasma [28], Decreased levels of selenium metabolites in mice plasma after the administration ofAs/Cd, aswell as a little reductionof SeAlb levels along the exposure can be observed in Table 3, which supports the hypothesis that SeAlb and selenometabolites have been transported to liver for the synthesis of required SeP....

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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.