Download Changes in urine metabolites following a single oral

Changes in urine metabolites following a single oral dose of selenomethionine,
methylmercury or their combination in juvenile white sturgeon
Susie S.Y.
1,2
Huang ,
Jonathan P.
1Centre
2
Benskin ,
John R.
2
Cosgrove ,
Silas S.O.
3
Hung ,
Laurie H.M.
1
Chan
for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, ON, Canada.
2AXYS Analytical Services Ltd., Sidney, BC, Canada.
3Department of Animal Science, University of California, Davis, CA, USA.
Email: [email protected]
RESULTS
INTRODUCTION
Selenium (Se) interacts with various elements in organisms, not only
mitigating the toxicity of the interacting elements but also that of its
own. The interaction between Se and mercury (Hg) is well
documented, however, the underlying molecular mechanisms are still
not well understood, particularly in aquatic organisms. As urinary
excretion is the major pathway for Se elimination, metabolomic
analysis of the urine can offer considerable insights towards key
molecular events involved in Se and Hg interaction.
A
10000
B
Control
MeHg
Se+Hg
1000
100
MATERIAL AND METHODS
10
Experimental Design
Details on experimental design and handling can be found in Huang et
al. (2013) (Aquat. Toxicol. 126, 274-282). In brief, a combined technique of
stomach intubation and urinary catheterization was used to orally
intubate and collect urine from juvenile white sturgeon (Acipenser
transmontanus; 0.863 ± 0.04 kg) subjected to a single dose of either 0
(Control), SeMet only (6.34 µM), MeHg only (4.24 µM), or their
combination (Se+Hg; 7.48 µM and 4.47 µM, respectively). Urine
samples were collected at the 6, 24, and 48h post-intubation periods
from a subgroup of 4 fish/treatment and stored at -80◦C until
extraction. Animal husbandry, operation, and sampling procedures
complied with protocols approved by the Campus Animal Care and
Use Committee at the University of California, Davis.
1
Ala
Samples were thawed at room temperature and centrifuged at 21,000
RCF, at 4◦C, for 10 min. Supernatant (300 µL) was aliquoted to a
maximum recover HPLC vial and lyophilized overnight. The dried
samples were then reconstituted with 200 µL of solvent consisted of:
50% acetonitrile, 40% methanol, and 10% water with 0.1% formic acid
(all LC/MS grade) and filtered through a 0.2 micron PTFE filter.
Extracts were stored at -80◦C until analysis.
Fig 1. PLS-DA score and loadings plots of urine metabolites at 6h (A, B) and 48h (C, D) post-intubation in white sturgeon
intubated with a single dose of Control, MeHg or Se+Hg. Important metabolites are encircled in the loadings plots.
AXYS Targeted Metabolomic Platform
Hexose
Lipids
Bile Acids
Fatty Acids
• Over 216 metabolites from 8
classes including amino acids (AA),
biogenic amines (BA), bile acid,
acylcarnitines (ACY),
glycerophospholipids (GPL),
sphingolipids, fatty acids, and
∑hexose were measured using the
ISO17025 accredited targeted
metabolomic platform by AXYS
Analytical Services Ltd. (Benskin et al.
(2014) Environ Sci. Technol. 48(19),
11670-11678)
• Instrumentation: 5 analytical
methods using LC- of FI-MS/MS.
• Quantifications validated using
isotope dilution or internal standard
approaches and a multipoint
calibration curve.
Data Analysis
1. Data processing: AA and BA were normalized against [creatinine]
and data log transformed and Pareto scaled when necessary
2. Multivariate analysis: PLS-DA  externally validated using cross
validations (Q2>0.5) and permutation tests (p<0.05)
3. Statistical analysis: non-parametric volcano plots (p<0.05)
4. Pathway analysis: KEGG Zebrafish (Danio rerio) Database
All data processing and statistical analyses were performed using
MetaboAnalyst 2.5. (Xia et al. (2012). Nucl. Acids Res. 40, W127-W133)
Leu
Orn
Phe
Ser
Val
Table 3. Significant metabolic pathways affected by MeHg, at 48 postintubation period, in juvenile white sturgeon.
Metabolite Extraction
Amino Acids
Ile
Fig 2. Select urine AA and their concentrations (ng/mL; log scaled), at
48h post-intubation, that were significantly different (p<0.05)
between treatment groups.
D
C
Glu
Table 1: Changes in concentrations (≥2-fold ) of significant urine metabolites in white sturgeons at 6, 24, and 48h post-intubation.
Amino Acid
Biogenic Amines
6h
24h
48h
↓
↓
↓
↓
↓
↓
6h
24h
48h
↑
↑
Acylcarnitines
Phosphatidylcholine
MeHg vs. Control
↑
↑
↑
↑
↑
Se+Hg vs. MeHg
↓
↓
↓
↓
↓
Sphingomyelins
↑
↑
↓
↓
↓
Note: ↑= increase; ↓= decrease; ↑<↑<↑; ↓<↓<↓.
Table 2.Common metabolites, over a 48h post-intubation period, that exhibited a significant (p<0.05) fold change against the
MeHg group in urine of white sturgeon orally intubated with a single dose of Control or Se+Hg.
Compound Name
Class
Control
Se+Hg
Valine
AA
0.04
3.20
Leucine
AA
0.04
4.70
Phenylalanine
AA
0.05
5.59
Alanine
AA
0.02
3.75
Dodecanoylcarnitine
Acylcarnitines
1.98
0.01
Hexadecadienylcarnitine
Acylcarnitines
2.76
1.56E-03
PC aa C42:2
Phosphatidylcholine diacyl
3.12
0.01
PC aa C42:4
Phosphatidylcholine diacyl
2.58
6.40E-03
PC ae C30:2
Phosphatidylcholine acyl-alkyl
1.75
1.79-03
PC ae C38:1
Phosphatidylcholine acyl-alkyl
2.77
7.50E-03
SM C24:1-OH
Sphingomyeline
5.62
0.012
SMC26:1
Sphingomyeline
2.84
0.04
Pathway Name
Aminoacyl-tRNA biosynthesis
Gly, Ser, and Thr metabolism
Val, Leu, and Ile biosynthesis
Arg and Pro metabolism
Sphingolipid metabolism
Glutathione metabolism
Phe, Try, and Typ biosynthesis
Phe metabolism
Glycerophospholipid metabolism
Cys and Met metabolism
Arachidonic acid metabolism
p
<0.01
0.01
0.01
0.02
0.03
0.05
0.05
0.03
0.05
0.03
0.03
Impact
0.10
0.53
0.67
0.14
0.20
0.20
0.50
0.41
0.17
0.12
0.12
SUMMARY OF RESULTS
 Intubation of MeHg and Se+Hg resulted in significant
separation of metabolite groups over time in the urine of
juvenile white sturgeon.
 MeHg  ↓[AA], ↑[ACYS] and [GPL] compared with the
Control, where the magnitude of change correlated with time.
 Changes in [metabolite]s in MeHg exposed fish are indicative of
↑fatty acid oxidation, altered lipid metabolism and
↓membrane stability/integrity.
 ↓[Ala] and [Ser] suggest modification of selenoamino acid
metabolism.
 Se+Hg ↑[AA], ↓[ACYS] and [GPL] compared with the MeHg
group.
 SeMet co-intubation modified lipid catabolism, fatty acid
oxidation, and AA metabolism/excretion compared with the
MeHg group.
 Se may mitigate Hg toxicity through the reduction of oxidative
stress and modification of energy metabolism in juvenile white
sturgeon.
ACKNOWLEDGEMENT
The support for this project was provided by NSERC funds to
L.H.M.C. and Calfed #SP2006-035 to S.S.O.H. We thank B.
Chandramouli and J. Liu for their technical assistance and AXYS
Analytical Services Ltd. for providing the metabolomic
measurements.