e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Short communication
Gender-specific metabolic responses in gonad of mussel Mytilus galloprovincialis to 2,2 ,4,4 -tetrabromodiphenyl ether Chenglong Ji a,b , Jianmin Zhao a , Huifeng Wu a,∗ a
Key Laboratory of Coastal Zone Environmental Processes, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS); Shandong Provincial Key Laboratory of Coastal Zone Environmental Processes, YICCAS, Yantai 264003, PR China b The University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Polybrominated diphenyl ethers (PBDEs) are widely used as a class of brominated flame-
Received 19 December 2013
retardants. As a congener of PBDEs, 2,2 ,4,4 -tetrabromodiphenylether (BDE 47) is the most
Received in revised form
toxic congener to animals. In this study, we applied metabolomics to characterize the
3 April 2014
gender-specific metabolic responses in mussel Mytilus galloprovincialis exposed to BDE 47
Accepted 6 April 2014
for 30 days. Results indicated the apparent gender-specific responses in M. galloprovincialis
Available online 18 April 2014
with BDE 47 exposures (1 and 10 g/L) at metabolite level. Basically, BDE 47 induced disruption in osmotic regulation and altered energy metabolism in mussels, via differential
Keywords:
metabolic pathways. In addition, the hormesis phenomenon was observed in both male
BDE 47
and female mussel samples exposed the two concentrations of BDE 47, indicated by the con-
Mytilus galloprovincialis
trarily altered metabolites from two BDE 47 treatments (1 and 10 g/L), respectively. Overall,
Toxicological effects
this study confirmed the gender-specific responses to BDE 47 exposures in mussels and
NMR
suggested the gender differences should be considered in marine ecotoxicology.
Metabolomics
1.
Introduction
As a family of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs) are widely used in numerous industrial products, such as plastics, electronic products, textiles and upholstery foam (de Wit, 2002). PBDE pollutions have been found in aquatic environments and animals because of the usage of PBDE products and discharge of industrial
∗
Corresponding author. Tel.: +86 535 2109190; fax: +86 535 2109000. E-mail address: [email protected] (H. Wu).
http://dx.doi.org/10.1016/j.etap.2014.04.007 1382-6689/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
wastewaters (de Wit, 2002; Hites, 2004). Although tetra-, penta, hexa- and hepta-BDEs have been banned, PBDEs have been increasingly detected in the environments and organisms because of the increasing usage of other congeners. Additionally, PBDEs can be broken down to lower brominated PBDEs with higher toxicities, leading to potential ecotoxicological risk (Soderstrom et al., 2004). PBDEs can induce diverse toxicities, including hepatotoxicity, carcinogenecity, neurotoxicity and immunotoxicity, in animals (de Wit, 2002;
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
1117
Fig. 1 – A representative 1-dimensional 500 MHz 1 H NMR spectrum of gonad tissue extracts of mussel M. galloprovincialis from male control group, (A) original and (B) generalized log-transformed forms. Keys: (1) branched chain amino acids: valine, leucine and isoleucine, (2) threonine, (3) alanine, (4) arginine, (5) lysine, (6) glutamate, (7) glutamine, (8) proline, (9) acetoacetate, (10) succinate, (11) -alanine, (12) hypotaurine, (13) aspartate, (14) asparagine, (15) malonate, (16) choline, (17) betaine, (18) taurine, (19) glycine, (20) homarine, (21) -glucose, (22) ␣-glucose, (23) glycogen, (24) unknown (5.95 ppm), (25) ATP, (26) tyrosine, (27) histidine and (28) phenylalanine.
Dingemans et al., 2007; Birnbaum and Cohen Hubal, 2006; Branchi and Capone, 2003; Barber et al., 2006). In addition, PBDEs are disruptors in the sex steroid and thyroid endocrine systems (Jiang et al., 2009). In aquatic environment, 2,2 ,4,4 -tetrabromodiphenylether (BDE 47) is one of the main congeners of PBDEs (Ikonomou et al., 2002), and is the most toxic congener to animals (Jin et al., 2008). Marine mussel Mytilus galloprovincialis is often used as a marine environmental bioindicator in marine ecotoxicology and biomonitoring programs due to its high tolerance and accumulation of contaminants (Ciacci et al., 2012). In routine biomonitoring programs or marine ecotoxicology, researchers do not often differentiate the male or female individuals when using the M. galloprovincialis as the experimental animal. However, the gender differences may introduce undesirable variations into induced toxicological effects and distort the interpretation of toxicological mechanisms, when both male and female individuals are used for exposures in same groups. As a system biology approach, metabolomics measures all the small molecular weight metabolites (metabolome) involved in all metabolic pathways in biological samples (Wu and Wang, 2010; Liu et al., 2011). The comparison of the metabolite profiles from control and a contaminant treatment can indicate the perturbation and toxicological effects induced
by contaminant in organisms. Therefore, metabolomics has been widely used in environmental toxicology (Williams et al., 2009; Santos et al., 2010; Liu et al., 2011). In this work, metabolomics was applied to investigate the differential metabolic changes in male and female mussels in response to BDE 47 exposure.
2.
Materials and methods
2.1.
Animals and experimental design
Sexually matured mussels M. galloprovincialis (shell length: 5.5–6.0 cm, n = 60) were collected from a pristine site (Yantai, China). After acclimatization in laboratory for 7 days, the animals were randomly divided into four tanks each containing 15 individuals. The mussels cultured in the normal filtered seawater (FSW) and FSW containing 0.002% DMSO (v/v) were used as control and solvent control groups, respectively. The other two groups of mussels were exposed to two sublethal concentrations (1 and 10 g/L) of BDE 47, respectively. After exposure for 30 days, all the mussels were dissected for gonad tissues.
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
Fig. 2 – O-PLS-DA scores derived from 1 H NMR spectra of gonad tissue extracts from solvent control ( ) and male mussel groups ( ) with BDE 47 exposures, (A) 1 g/L and (C) 10 g/L, and corresponding coefficient plots (B) and (D). The color map shows the significance of metabolite variations between the two classes (solvent control and BDE 47 treatment). Peaks in the positive direction indicate metabolites that are more abundant in t BDE 47-exposed groups. Consequently, metabolites that are more abundant in the control group are presented as peaks in the negative direction. Abbreviations: ACA, acetoacetate; Asp, aspartate; -Ala, -alanine; Glc, glycogen; Gln, glutamine; Gly, glycine; His, histidine; HMR, homarine; HPT, hypotaurine; Tyr, tyrosine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.2.
Sex determination
The gonad tissues were carefully fixed in Bouin’s fixative solution after dissection from the mussels. After fixation for 24 h, the tissues were then dehydrated in a progressive series of ethanol and embedded in paraffin. Histological sections (6m thickness) were cut from the paraffin embedded tissues and mounted on slides which were stained with hematoxylineosine (HE) and observed under a light microscope (Olympus BX61, Tokyo, Japan) at ×200.
2.3.
Metabolite extraction
Polar metabolites in gonad tissues of mussels were extracted using a modified methanol/water/chloroform system, as described previously (Lin et al., 2007; Wu et al., 2008). Briefly, the gonad tissue (ca. 100 mg) was homogenized and extracted in 4 mL/g of methanol, 5.25 mL/g of water and 2 mL/g of chloroform. The methanol/water layer with polar metabolites was transferred to a glass vial and dried in a centrifugal concentrator. The tissue extracts were subsequently dissolved in 600 L phosphate buffer (100 mM Na2 HPO4 and NaH2 PO4 with 0.5 mM TSP, pH 7.0) in D2 O. The mixture was vortexed and
then centrifuged at 3000 × g for 5 min at 4 ◦ C. The supernatant substance (550 L) was then pipetted into a 5 mm NMR tube before NMR analysis.
2.4.
1
H NMR spectroscopy
Extracts of gonad tissues were analyzed on a Bruker AV 500 NMR spectrometer performed at 500.18 MHz (at 298 K) as described previously (Zhang et al., 2011a). All 1 H NMR spectra were phased, baseline-corrected, and calibrated (TSP at 0.0 ppm) manually using TopSpin (version 2.1, Bruker). Metabolites were identified following the tabulated chemical shifts (Fan, 1996) and using the software, Chenomx (Evaluation Version, Chenomx Inc., Canada).
2.5. Spectral pre-processing and multivariate data analysis All NMR spectra were processed using custom-written ProMetab software in Matlab (version 7.0; The MathWorks, Natick, MA, USA), as described previously (Parsons et al., 2007; Liu et al., 2011). Data analysis was performed using the software SIMCA-P+ (V11.0, Umetric, Sweden). The
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
1119
Fig. 3 – O-PLS-DA scores derived from 1 H NMR spectra of gonad tissue extracts from solvent control ( ) and female mussel groups ( ) with BDE 47 exposures, (A) 1 g/L and (C) 10 g/L, and corresponding coefficient plots (B) and (D). The color map shows the significance of metabolite variations between the two classes (solvent control and BDE 47 treatment). Peaks in the positive direction indicate metabolites that are more abundant in t BDE 47-exposed groups. Consequently, metabolites that are more abundant in the control group are presented as peaks in the negative direction. Abbreviations: ACA, acetoacetate; Arg, arginine; Asn, asparagine; ATP, Adenosine triphosphate; BCAA, branched chain amino acids; Bet, betaine; -Ala, -alanine; Cho, choline; Glc, glycogen; Gly, glycine; HPT, hypotaurine PC, phosphocholine; Phe, phenylalanine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
supervised multivariate data analysis methods, partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA), were sequentially used to uncover and extract the statistically significant metabolite responses induced by BDE 47 exposures. The metabolic differences responsible for the classifications between control and BDE 47-exposed groups could be detected in the coefficient-coded loadings plots. A hot color (i.e., red) corresponds to the metabolites being highly positive/negative significant in discriminating between groups, while a cool color (i.e., blue) corresponds to no significance. The details of data analysis were described previously (Feng et al., 2013).
3.
Results and discussion
Fig. 1 shows a typical 1 H NMR spectrum of gonad tissue extracts from male control group in original (Fig. 1A) and generalized-transformed forms (Fig. 1B). The metabolites were identified by Chenomx software and labeled in Fig. 1, including amino acids (branched chain amino acids (valine, leucine and isoleucine), alanine, threonine, arginine, lysine, glutamate,
glutamine, -alanine, aspartate, glycine, tyrosine, phenylalanine and histidine), organic osmolytes (hypotaurine, taurine, homarine and betaine), an intermediate in Krebs cycle (succinate) and energy storage compounds (ATP, glucose and glycogen). Our previous work confirmed that there were intrinsic (gender-specific) metabolite differences between male and female mussels, which suggested that the gender differences could result in gender-specific responses to toxicant exposures (Ji et al., 2013). O-PLS-DA indicated that there was no significant metabolic difference between seawater control and solvent control groups from either male or female mussels (data not shown). Then, only the solvent control groups were used in further metabolomic analysis. After exposed to BDE 47 for 30 d, O-PLS-DA analysis exhibited that both treatments (1 and 10 g/L) of BDE 47 induced significant metabolic responses in both male and female mussel samples, with reliable Q2 values (>0.4) (Figs. 2 and 3). Fig. 4 summarized the disturbed metabolic pathways related to the metabolic responses of M. galloprovincialis to BDE 47 treatments. The low concentration (1 g/L) mainly caused increases in hypotaurine, homarine and -alanine, and decreases in aspartate, glycine, histidine and tyrosine (Fig. 2B).
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
HPT Cys
Gly
3
Ser
4 5 Glc
Glycolysis ADP
Asn
β-Ala
Cho
1 2 PC
Val, Leu
Pyruvate
ATP
Asp
7 8
Bet
10
6
Acetyl-CoA
16
Oxaloacetate
9
Acetoacetyl-CoA 15 ACA Citrate Arg
Krebs Cycle Fumarate ATP
Pyruvate Phe
Tyr
Succinate
11
ADP Succinyl-CoA
Glycolysis
Acetoacetyl-CoA
Val, Ile
2-Oxoglutarate 14 13 Glu 12 Gln
His
Fig. 4 – The metabolic pathways in gonad tissues of mussel M. galloprovincialis exposed to BDE 47. Arrowheads indicate the changes of metabolites in male (blue) and female (red) individuals from 1 g/L BDE 47 (hollow) and 10 g/L BDE 47 (solid) treatment. The green circles with number inside represent the enzymes involved in the metabolic pathways. Abbreviations: ACA, acetoacetate; ADP, adenosine diphosphate; Arg, arginine; Asp, aspartate; Asn, asparagine; ATP, Adenosine triphosphate; -Ala, -alanine; Bet, betaine; Cho, choline; Cys, cysteine; Glc, glycogen; Gly, glycine; Gln, glutamine; Glu, glutamate; His, histidine; HPT, hypotaurine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; PC, phosphocholine; Ser, serine; Tyr, tyrosine; Val, valine. Enzymes: (1) choline kinase; (2) phosphocholine phosphatase; (3) glycine hydroxymethyltransferase; (4) l-serine ammonia-lyase; (5) threonine ammonia-lyase; (6) pyruvate carboxylase; (7) aspartate–ammonia ligase; (8) asparagine synthase; (9) aspartate 4-decarboxylase; (10) aspartate aminotransferase, cytoplasmic; (11) phenylalanine-4-hydroxylase; (12) glutamine synthetase; (13) glutamate synthase (NADPH/NADH); (14) glutamate dehydrogenase; (15) 3-oxoacid CoA-transferase; (16) acetyl-CoA C-acetyltransferase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Among these metabolic biomarkers, hypotaurine and homarine are known organic osmolytes in marine mussels. The increased levels of hypotaurine and homarine clearly indicated the osmotic stress induced by BDE 47 (1 g/L) in male mussels. In marine mussels, amino acids play important roles in both osmotic regulation and cellular energy metabolism (Viant et al., 2003). However, no energy metabolism-related metabolites were altered in 1 g/L of BDE 47-exposed male mussel samples. The elevation of -alanine probably meant the osmotic stress, together with the elevated hypotaurine and homarine. However, the decreased amino acids (aspartate, glycine, histidine and tyrosine) were decreased to compensate the increases of betaine and hypotaurine in low concentration (1 g/L) of BDE 47-treated female mussel samples. After exposed to the high concentration (10 g/L) of BDE 47, the metabolic changes were different to those in the low concentration (1 g/L) of BDE 47-treated male mussel samples, except decreased glycine. As shown in the loading plot (Fig. 2D), acetoacetate and glutamine were elevated, while hypotaurine and glycogen were depleted. In particular, the depleted hypotaurine was contrary to that in the low concentration (1 g/L) of BDE 47-treated male mussel samples, which probably meant the hormetic effects induced by BDE 47 in mussels. The similar hormetic phenomenon was observed in benzo[a]pyrene (BaP)-treated clam
Ruditapes philippinarum (Zhang et al., 2011b). As shown in Fig. 4, acetoacetate is synthesized from acetyl CoA and can be reduced to -hydroxybutyrate by -hydroxybutyrate dehydrogenase in a NADH-requiring reaction which is related to fatty acid oxidation of fat metabolism. Therefore the elevation of acetoacetate could be the indicator of increased demand of energy, as well as the decreased glycogen, by accelerating the consumption of glycogen. The alteration of amino acids including glycine and glutamine could be related to osmotic regulation, as indicated by hypotaurine. In female mussel samples, the significant metabolic responses were also discovered in both BDE 47 exposed-groups (Fig. 3A and C), which were different to those in BDE 47 exposed-male mussel samples. For the low dose (1 g/L) of BDE 47 treatment, branched chain amino acids, -alanine, hypotaurine, asparagine, phosphocholine, choline, betaine and glycine were elevated, while arginine and ATP were depleted (Fig. 3B). The elevation of two organic osmolytes, hypotaurine and betaine, suggested that 1 g/L of BDE 47 induced osmotic stress. Betaine is biosynthesized from the oxidation of choline that is involved in the synthesis of phosphocholine (Fig. 4). The elevated choline meant the increased demand of betaine synthesis in female mussel gonad. As shown in Fig. 4, phosphocholine is made in a reaction, catalyzed by choline kinase converting ATP and choline into
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
phosphocholine and ADP. In another metabolic pathway, ADP and phosphoarginine can be converted to ATP and arginine, which is catalyzed by arginine kinase (Watts and Bannister, 1970). Hereby, the depletion ATP and arginine indicated the reduced energy-regenerating system in female mussel samples with 1 g/L of BDE 47 treatment. The elevated branched chain amino acids, asparagine and glycine demonstrated the osmotic stress combined with the elevated osmolyte, hypotaurine. After exposure of BDE 47 at 10 g/L, branched chain amino acids, hypotaurine and ATP were contrarily altered in female mussel gonads, compared with those from the low concentration (1 g/L) of BDE 47 treatment (Fig. 3D), which implied the hormesis effects of BDE 47 to female mussel samples, as mentioned above. The increases of ATP and glycogen implied the decreased energy demand in female mussel samples with 10 g/L of BDE 47 exposure, and therefore these two energy storage compounds were accumulated. Acetoacetate was reduced, which suggested the increased fatty acid oxidation from fat metabolism to compensate for the energy supply. The decreased branched chain amino acids and phenylalanine might be related to osmotic regulation combined with the decreased hypotaurine in 10 g/L of BDE 47-exposed female mussel samples. In summary, our results indicated that the gender-specific metabolic responses in male and female mussel M. galloprovincialis to BDE 47 exposures (1 and 10 g/L) for 30 days. The low concentration (1 g/L) caused osmotic stress in male mussel gonads, while the high concentration (10 g/L) induced disruption in osmotic regulation and increased demand of energy. In female mussel gonads, the low concentration of BDE 47 caused osmotic stress as well via the metabolic pathway by synthesize more betaine, which was different from that in male mussel gonads. However, the exposure of low concentration of BDE 47 caused a reduced energy-regenerating system in female mussel samples, as indicated by decreased ATP and arginine, which was not observed in male mussel gonads. BDE 47 at 10 g/L induced disruption in osmotic regulation demonstrated by the reduced hypotaurine. In addition, the increased fatty acid oxidation from fat metabolism was found to compensate for the energy supply in 10 g/L of BDE 47treated female mussel samples. Overall, this study confirmed the gender-specific responses to BDE 47 exposures in mussels and suggested the gender differences should be considered in marine ecotoxicology.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgment This work was supported by The Key Deployment Program of Chinese Academy of Sciences (KZZD-EW-14-03).
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Short communication
Gender-specific metabolic responses in gonad of mussel Mytilus galloprovincialis to 2,2 ,4,4 -tetrabromodiphenyl ether Chenglong Ji a,b , Jianmin Zhao a , Huifeng Wu a,∗ a
Key Laboratory of Coastal Zone Environmental Processes, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS); Shandong Provincial Key Laboratory of Coastal Zone Environmental Processes, YICCAS, Yantai 264003, PR China b The University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Polybrominated diphenyl ethers (PBDEs) are widely used as a class of brominated flame-
Received 19 December 2013
retardants. As a congener of PBDEs, 2,2 ,4,4 -tetrabromodiphenylether (BDE 47) is the most
Received in revised form
toxic congener to animals. In this study, we applied metabolomics to characterize the
3 April 2014
gender-specific metabolic responses in mussel Mytilus galloprovincialis exposed to BDE 47
Accepted 6 April 2014
for 30 days. Results indicated the apparent gender-specific responses in M. galloprovincialis
Available online 18 April 2014
with BDE 47 exposures (1 and 10 g/L) at metabolite level. Basically, BDE 47 induced disruption in osmotic regulation and altered energy metabolism in mussels, via differential
Keywords:
metabolic pathways. In addition, the hormesis phenomenon was observed in both male
BDE 47
and female mussel samples exposed the two concentrations of BDE 47, indicated by the con-
Mytilus galloprovincialis
trarily altered metabolites from two BDE 47 treatments (1 and 10 g/L), respectively. Overall,
Toxicological effects
this study confirmed the gender-specific responses to BDE 47 exposures in mussels and
NMR
suggested the gender differences should be considered in marine ecotoxicology.
Metabolomics
1.
Introduction
As a family of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs) are widely used in numerous industrial products, such as plastics, electronic products, textiles and upholstery foam (de Wit, 2002). PBDE pollutions have been found in aquatic environments and animals because of the usage of PBDE products and discharge of industrial
∗
Corresponding author. Tel.: +86 535 2109190; fax: +86 535 2109000. E-mail address: [email protected] (H. Wu).
http://dx.doi.org/10.1016/j.etap.2014.04.007 1382-6689/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
wastewaters (de Wit, 2002; Hites, 2004). Although tetra-, penta, hexa- and hepta-BDEs have been banned, PBDEs have been increasingly detected in the environments and organisms because of the increasing usage of other congeners. Additionally, PBDEs can be broken down to lower brominated PBDEs with higher toxicities, leading to potential ecotoxicological risk (Soderstrom et al., 2004). PBDEs can induce diverse toxicities, including hepatotoxicity, carcinogenecity, neurotoxicity and immunotoxicity, in animals (de Wit, 2002;
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
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Fig. 1 – A representative 1-dimensional 500 MHz 1 H NMR spectrum of gonad tissue extracts of mussel M. galloprovincialis from male control group, (A) original and (B) generalized log-transformed forms. Keys: (1) branched chain amino acids: valine, leucine and isoleucine, (2) threonine, (3) alanine, (4) arginine, (5) lysine, (6) glutamate, (7) glutamine, (8) proline, (9) acetoacetate, (10) succinate, (11) -alanine, (12) hypotaurine, (13) aspartate, (14) asparagine, (15) malonate, (16) choline, (17) betaine, (18) taurine, (19) glycine, (20) homarine, (21) -glucose, (22) ␣-glucose, (23) glycogen, (24) unknown (5.95 ppm), (25) ATP, (26) tyrosine, (27) histidine and (28) phenylalanine.
Dingemans et al., 2007; Birnbaum and Cohen Hubal, 2006; Branchi and Capone, 2003; Barber et al., 2006). In addition, PBDEs are disruptors in the sex steroid and thyroid endocrine systems (Jiang et al., 2009). In aquatic environment, 2,2 ,4,4 -tetrabromodiphenylether (BDE 47) is one of the main congeners of PBDEs (Ikonomou et al., 2002), and is the most toxic congener to animals (Jin et al., 2008). Marine mussel Mytilus galloprovincialis is often used as a marine environmental bioindicator in marine ecotoxicology and biomonitoring programs due to its high tolerance and accumulation of contaminants (Ciacci et al., 2012). In routine biomonitoring programs or marine ecotoxicology, researchers do not often differentiate the male or female individuals when using the M. galloprovincialis as the experimental animal. However, the gender differences may introduce undesirable variations into induced toxicological effects and distort the interpretation of toxicological mechanisms, when both male and female individuals are used for exposures in same groups. As a system biology approach, metabolomics measures all the small molecular weight metabolites (metabolome) involved in all metabolic pathways in biological samples (Wu and Wang, 2010; Liu et al., 2011). The comparison of the metabolite profiles from control and a contaminant treatment can indicate the perturbation and toxicological effects induced
by contaminant in organisms. Therefore, metabolomics has been widely used in environmental toxicology (Williams et al., 2009; Santos et al., 2010; Liu et al., 2011). In this work, metabolomics was applied to investigate the differential metabolic changes in male and female mussels in response to BDE 47 exposure.
2.
Materials and methods
2.1.
Animals and experimental design
Sexually matured mussels M. galloprovincialis (shell length: 5.5–6.0 cm, n = 60) were collected from a pristine site (Yantai, China). After acclimatization in laboratory for 7 days, the animals were randomly divided into four tanks each containing 15 individuals. The mussels cultured in the normal filtered seawater (FSW) and FSW containing 0.002% DMSO (v/v) were used as control and solvent control groups, respectively. The other two groups of mussels were exposed to two sublethal concentrations (1 and 10 g/L) of BDE 47, respectively. After exposure for 30 days, all the mussels were dissected for gonad tissues.
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Fig. 2 – O-PLS-DA scores derived from 1 H NMR spectra of gonad tissue extracts from solvent control ( ) and male mussel groups ( ) with BDE 47 exposures, (A) 1 g/L and (C) 10 g/L, and corresponding coefficient plots (B) and (D). The color map shows the significance of metabolite variations between the two classes (solvent control and BDE 47 treatment). Peaks in the positive direction indicate metabolites that are more abundant in t BDE 47-exposed groups. Consequently, metabolites that are more abundant in the control group are presented as peaks in the negative direction. Abbreviations: ACA, acetoacetate; Asp, aspartate; -Ala, -alanine; Glc, glycogen; Gln, glutamine; Gly, glycine; His, histidine; HMR, homarine; HPT, hypotaurine; Tyr, tyrosine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.2.
Sex determination
The gonad tissues were carefully fixed in Bouin’s fixative solution after dissection from the mussels. After fixation for 24 h, the tissues were then dehydrated in a progressive series of ethanol and embedded in paraffin. Histological sections (6m thickness) were cut from the paraffin embedded tissues and mounted on slides which were stained with hematoxylineosine (HE) and observed under a light microscope (Olympus BX61, Tokyo, Japan) at ×200.
2.3.
Metabolite extraction
Polar metabolites in gonad tissues of mussels were extracted using a modified methanol/water/chloroform system, as described previously (Lin et al., 2007; Wu et al., 2008). Briefly, the gonad tissue (ca. 100 mg) was homogenized and extracted in 4 mL/g of methanol, 5.25 mL/g of water and 2 mL/g of chloroform. The methanol/water layer with polar metabolites was transferred to a glass vial and dried in a centrifugal concentrator. The tissue extracts were subsequently dissolved in 600 L phosphate buffer (100 mM Na2 HPO4 and NaH2 PO4 with 0.5 mM TSP, pH 7.0) in D2 O. The mixture was vortexed and
then centrifuged at 3000 × g for 5 min at 4 ◦ C. The supernatant substance (550 L) was then pipetted into a 5 mm NMR tube before NMR analysis.
2.4.
1
H NMR spectroscopy
Extracts of gonad tissues were analyzed on a Bruker AV 500 NMR spectrometer performed at 500.18 MHz (at 298 K) as described previously (Zhang et al., 2011a). All 1 H NMR spectra were phased, baseline-corrected, and calibrated (TSP at 0.0 ppm) manually using TopSpin (version 2.1, Bruker). Metabolites were identified following the tabulated chemical shifts (Fan, 1996) and using the software, Chenomx (Evaluation Version, Chenomx Inc., Canada).
2.5. Spectral pre-processing and multivariate data analysis All NMR spectra were processed using custom-written ProMetab software in Matlab (version 7.0; The MathWorks, Natick, MA, USA), as described previously (Parsons et al., 2007; Liu et al., 2011). Data analysis was performed using the software SIMCA-P+ (V11.0, Umetric, Sweden). The
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
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Fig. 3 – O-PLS-DA scores derived from 1 H NMR spectra of gonad tissue extracts from solvent control ( ) and female mussel groups ( ) with BDE 47 exposures, (A) 1 g/L and (C) 10 g/L, and corresponding coefficient plots (B) and (D). The color map shows the significance of metabolite variations between the two classes (solvent control and BDE 47 treatment). Peaks in the positive direction indicate metabolites that are more abundant in t BDE 47-exposed groups. Consequently, metabolites that are more abundant in the control group are presented as peaks in the negative direction. Abbreviations: ACA, acetoacetate; Arg, arginine; Asn, asparagine; ATP, Adenosine triphosphate; BCAA, branched chain amino acids; Bet, betaine; -Ala, -alanine; Cho, choline; Glc, glycogen; Gly, glycine; HPT, hypotaurine PC, phosphocholine; Phe, phenylalanine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
supervised multivariate data analysis methods, partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA), were sequentially used to uncover and extract the statistically significant metabolite responses induced by BDE 47 exposures. The metabolic differences responsible for the classifications between control and BDE 47-exposed groups could be detected in the coefficient-coded loadings plots. A hot color (i.e., red) corresponds to the metabolites being highly positive/negative significant in discriminating between groups, while a cool color (i.e., blue) corresponds to no significance. The details of data analysis were described previously (Feng et al., 2013).
3.
Results and discussion
Fig. 1 shows a typical 1 H NMR spectrum of gonad tissue extracts from male control group in original (Fig. 1A) and generalized-transformed forms (Fig. 1B). The metabolites were identified by Chenomx software and labeled in Fig. 1, including amino acids (branched chain amino acids (valine, leucine and isoleucine), alanine, threonine, arginine, lysine, glutamate,
glutamine, -alanine, aspartate, glycine, tyrosine, phenylalanine and histidine), organic osmolytes (hypotaurine, taurine, homarine and betaine), an intermediate in Krebs cycle (succinate) and energy storage compounds (ATP, glucose and glycogen). Our previous work confirmed that there were intrinsic (gender-specific) metabolite differences between male and female mussels, which suggested that the gender differences could result in gender-specific responses to toxicant exposures (Ji et al., 2013). O-PLS-DA indicated that there was no significant metabolic difference between seawater control and solvent control groups from either male or female mussels (data not shown). Then, only the solvent control groups were used in further metabolomic analysis. After exposed to BDE 47 for 30 d, O-PLS-DA analysis exhibited that both treatments (1 and 10 g/L) of BDE 47 induced significant metabolic responses in both male and female mussel samples, with reliable Q2 values (>0.4) (Figs. 2 and 3). Fig. 4 summarized the disturbed metabolic pathways related to the metabolic responses of M. galloprovincialis to BDE 47 treatments. The low concentration (1 g/L) mainly caused increases in hypotaurine, homarine and -alanine, and decreases in aspartate, glycine, histidine and tyrosine (Fig. 2B).
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HPT Cys
Gly
3
Ser
4 5 Glc
Glycolysis ADP
Asn
β-Ala
Cho
1 2 PC
Val, Leu
Pyruvate
ATP
Asp
7 8
Bet
10
6
Acetyl-CoA
16
Oxaloacetate
9
Acetoacetyl-CoA 15 ACA Citrate Arg
Krebs Cycle Fumarate ATP
Pyruvate Phe
Tyr
Succinate
11
ADP Succinyl-CoA
Glycolysis
Acetoacetyl-CoA
Val, Ile
2-Oxoglutarate 14 13 Glu 12 Gln
His
Fig. 4 – The metabolic pathways in gonad tissues of mussel M. galloprovincialis exposed to BDE 47. Arrowheads indicate the changes of metabolites in male (blue) and female (red) individuals from 1 g/L BDE 47 (hollow) and 10 g/L BDE 47 (solid) treatment. The green circles with number inside represent the enzymes involved in the metabolic pathways. Abbreviations: ACA, acetoacetate; ADP, adenosine diphosphate; Arg, arginine; Asp, aspartate; Asn, asparagine; ATP, Adenosine triphosphate; -Ala, -alanine; Bet, betaine; Cho, choline; Cys, cysteine; Glc, glycogen; Gly, glycine; Gln, glutamine; Glu, glutamate; His, histidine; HPT, hypotaurine; Ile, isoleucine; Leu, leucine; Phe, phenylalanine; PC, phosphocholine; Ser, serine; Tyr, tyrosine; Val, valine. Enzymes: (1) choline kinase; (2) phosphocholine phosphatase; (3) glycine hydroxymethyltransferase; (4) l-serine ammonia-lyase; (5) threonine ammonia-lyase; (6) pyruvate carboxylase; (7) aspartate–ammonia ligase; (8) asparagine synthase; (9) aspartate 4-decarboxylase; (10) aspartate aminotransferase, cytoplasmic; (11) phenylalanine-4-hydroxylase; (12) glutamine synthetase; (13) glutamate synthase (NADPH/NADH); (14) glutamate dehydrogenase; (15) 3-oxoacid CoA-transferase; (16) acetyl-CoA C-acetyltransferase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Among these metabolic biomarkers, hypotaurine and homarine are known organic osmolytes in marine mussels. The increased levels of hypotaurine and homarine clearly indicated the osmotic stress induced by BDE 47 (1 g/L) in male mussels. In marine mussels, amino acids play important roles in both osmotic regulation and cellular energy metabolism (Viant et al., 2003). However, no energy metabolism-related metabolites were altered in 1 g/L of BDE 47-exposed male mussel samples. The elevation of -alanine probably meant the osmotic stress, together with the elevated hypotaurine and homarine. However, the decreased amino acids (aspartate, glycine, histidine and tyrosine) were decreased to compensate the increases of betaine and hypotaurine in low concentration (1 g/L) of BDE 47-treated female mussel samples. After exposed to the high concentration (10 g/L) of BDE 47, the metabolic changes were different to those in the low concentration (1 g/L) of BDE 47-treated male mussel samples, except decreased glycine. As shown in the loading plot (Fig. 2D), acetoacetate and glutamine were elevated, while hypotaurine and glycogen were depleted. In particular, the depleted hypotaurine was contrary to that in the low concentration (1 g/L) of BDE 47-treated male mussel samples, which probably meant the hormetic effects induced by BDE 47 in mussels. The similar hormetic phenomenon was observed in benzo[a]pyrene (BaP)-treated clam
Ruditapes philippinarum (Zhang et al., 2011b). As shown in Fig. 4, acetoacetate is synthesized from acetyl CoA and can be reduced to -hydroxybutyrate by -hydroxybutyrate dehydrogenase in a NADH-requiring reaction which is related to fatty acid oxidation of fat metabolism. Therefore the elevation of acetoacetate could be the indicator of increased demand of energy, as well as the decreased glycogen, by accelerating the consumption of glycogen. The alteration of amino acids including glycine and glutamine could be related to osmotic regulation, as indicated by hypotaurine. In female mussel samples, the significant metabolic responses were also discovered in both BDE 47 exposed-groups (Fig. 3A and C), which were different to those in BDE 47 exposed-male mussel samples. For the low dose (1 g/L) of BDE 47 treatment, branched chain amino acids, -alanine, hypotaurine, asparagine, phosphocholine, choline, betaine and glycine were elevated, while arginine and ATP were depleted (Fig. 3B). The elevation of two organic osmolytes, hypotaurine and betaine, suggested that 1 g/L of BDE 47 induced osmotic stress. Betaine is biosynthesized from the oxidation of choline that is involved in the synthesis of phosphocholine (Fig. 4). The elevated choline meant the increased demand of betaine synthesis in female mussel gonad. As shown in Fig. 4, phosphocholine is made in a reaction, catalyzed by choline kinase converting ATP and choline into
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1116–1122
phosphocholine and ADP. In another metabolic pathway, ADP and phosphoarginine can be converted to ATP and arginine, which is catalyzed by arginine kinase (Watts and Bannister, 1970). Hereby, the depletion ATP and arginine indicated the reduced energy-regenerating system in female mussel samples with 1 g/L of BDE 47 treatment. The elevated branched chain amino acids, asparagine and glycine demonstrated the osmotic stress combined with the elevated osmolyte, hypotaurine. After exposure of BDE 47 at 10 g/L, branched chain amino acids, hypotaurine and ATP were contrarily altered in female mussel gonads, compared with those from the low concentration (1 g/L) of BDE 47 treatment (Fig. 3D), which implied the hormesis effects of BDE 47 to female mussel samples, as mentioned above. The increases of ATP and glycogen implied the decreased energy demand in female mussel samples with 10 g/L of BDE 47 exposure, and therefore these two energy storage compounds were accumulated. Acetoacetate was reduced, which suggested the increased fatty acid oxidation from fat metabolism to compensate for the energy supply. The decreased branched chain amino acids and phenylalanine might be related to osmotic regulation combined with the decreased hypotaurine in 10 g/L of BDE 47-exposed female mussel samples. In summary, our results indicated that the gender-specific metabolic responses in male and female mussel M. galloprovincialis to BDE 47 exposures (1 and 10 g/L) for 30 days. The low concentration (1 g/L) caused osmotic stress in male mussel gonads, while the high concentration (10 g/L) induced disruption in osmotic regulation and increased demand of energy. In female mussel gonads, the low concentration of BDE 47 caused osmotic stress as well via the metabolic pathway by synthesize more betaine, which was different from that in male mussel gonads. However, the exposure of low concentration of BDE 47 caused a reduced energy-regenerating system in female mussel samples, as indicated by decreased ATP and arginine, which was not observed in male mussel gonads. BDE 47 at 10 g/L induced disruption in osmotic regulation demonstrated by the reduced hypotaurine. In addition, the increased fatty acid oxidation from fat metabolism was found to compensate for the energy supply in 10 g/L of BDE 47treated female mussel samples. Overall, this study confirmed the gender-specific responses to BDE 47 exposures in mussels and suggested the gender differences should be considered in marine ecotoxicology.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgment This work was supported by The Key Deployment Program of Chinese Academy of Sciences (KZZD-EW-14-03).
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