Registration Dossier

Administrative data

Endpoint:
additional ecotoxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
non-guideline study assessing effects of DP on various proteins as basic research. Technically, well documented but hardly a regulatory background can be seen, as most effects canot be associated directly to biological effects in fish. No dose response information on altered protein concentrations was provided except of Hsp70 and Annexin A4, which did not follow a dose-response curve as expected.

Data source

Reference
Reference Type:
publication
Title:
Effects of dechlorane plus on the hepatic proteome of juvenile Chinese sturgeon (Acipenser sinensis)
Author:
X. Liang, W. Li, C.J. Martyniuk, J. Zha, Z. Wang, G. Cheng, J.P. Giesy
Year:
2014
Bibliographic source:
Aquatic Toxicology 148 (2014) 83 – 91
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
DP was i.p. injected to juvenile Chinese sturgeon in doses of 0, 1, 10 and 100 mg/kg ww in one single application. After 14 days liver proteomes were analysed by 2-dimensional electrophoresis coupled matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI–TOF/TOF–MS).
GLP compliance:
not specified
Type of study / information:
Study investigating the effects of i.p. injected dechlorane plus in Chinese Sturgeon to alter various proteine (liver proteomes) levels and concentrations.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Dechloranes Plus (CAS no. 13560-89-9; M.W. 653.7; purity > 95%) was purchased from Wellington Laboratories Inc. (Guelph, Ontario, Canada). Due to its extremely lipophilic character, DP was dissolved in corn-oil for intraperitoneal injections according to Wu et al. (2012).

Results and discussion

Any other information on results incl. tables

Hepatic proteome profiles: Representative 2-DE gels of hepatic proteins from control and DP-treated groups are shown in Fig. 1 (see attachment). Quantitative spot comparisons were performed with image analysis software and approximately 740 spots were detected on each gel. Among these proteins, 39 protein spots were found to be altered in abundance (>2-fold) in one or more DP-treated groups compared to that of controls. According to the ratio value, there were 12, 24, and 15 significantly altered spots at the 1, 10, and 100 mg/DP/kg wet weight, respectively. Seven spots (spots 10, 86, 125, 224, 333, 517, 520; shown in Fig. 1) displayed consistent directional changes (increasing/decreasing) in response to the DP dose response. Altered protein spots were submitted for identification using MALDI–TOF–TOF analysis and searches for protein homology the NCBI nr database. There were 27 proteins that were successfully identified (Table 1).

Differentially expressed proteins and pathway analysis: In general, the identified proteins were involved in metabolism, signal transduction, calcium ion binding, protein folding, structure stabilizing, as well as other functions (Table 1). Following a more general description of proteins based upon gene ontology, pathway analysis was performed to further integrate protein data and to determine if there were common cell processes affected by altered proteins (Fig. 2). Based upon protein responses and interactions (e.g. expression, binding, regulation), the processes of cell structure (actin organization, microtubule assembly), transcription regulation (protein folding, mRNA degradation, RNA processing and metabolism) and cell metabolism (glucose metabolism and the tricarboxylic acid cycle) were increased while the process of organelle transport (intra-golgi transport, endoplasmic reticulum and golgi transport) was decreased based on protein responses. Proteins that were altered in abundance by DP were also related to processes such as apoptosis, cell differentiation, and cell death. Interestingly, GAPDH and heat shock cognate protein 70 (also known as HSPA8) were significant hubs within the network, being involved in multiple cell processes and having many interactions with the other proteins regulated by DP. Below we describe in more detail the different proteins associated with these cell processes. The abundance of many metabolism-associated proteins was increased by DP, indicating that the metabolic process was a main target of DP. Most of these proteins were associated with carbohydrate metabolism and included glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase B, isocitrate dehydrogenase [NADP] cytoplasmic (IDH), malate dehydrogenase, mitochondrial, and alpha-enolase (Table 1). Three proteins associated with amino acid metabolic process were markedly down-regulated, including beta-ureidopropionase, alanine-glyoxylate aminotransferase a, and betaine–homocysteine S-methyltransferase 1 (Table 1). In the pathway analysis, the process of serine glycine metabolism was decreased, consistent with the suggestion that amino-acid related processes are suppressed by DP. Four significantly altered protein spots were involved in signal transduction. Ras-related protein Rab-6B (RAB6B) and BAI1- associated protein 2-like 1b were significantly down-regulated in liver samples exposed to DP (Table 1). Expression of GDP dissociation inhibitor 2 (GDI2) was inhibited in 10 mg/kg group and the protein predicted: diacylglycerol kinase delta-like, partial (DGKD) was induced in 100 mg/kg group. Annexin A4 (ANXA4), predicted: hypothetical protein LOC100536704 (partial) (CDHR2) and hippocalcin-like protein 1 are proteins associated with calcium ion binding; each of these proteins were also altered in abundance by DP. ANXA4 was down-regulated in the 1 mg/kg and 10 mg/kg groups while exposure to DP increased the abundance of CDHR2 in the 1 mg/kg and 100 mg/kg treatment groups (Table 1). Hippocalcin-like protein 1 exhibited a 2 - 4 fold increase in 10 mg/kg and 100 mg/kg DP-treated groups (Table 1). Heat shock cognate protein 70 (HSC70) and T-complex protein 1 subunit epsilon (CCT5), proteins involved in stress responses, showed opposite directional changes in abundance levels. HSC70 was up-regulated after exposure to 10 mg/kg DP while CCT5 was down-regulated in all three DP-treated groups (Table 1). Structural proteins (intraflagellar transport protein 172 homolog, keratin type I cytoskeletal 18, and bactin1 protein) were also significantly altered in abundance by DP, and proteins changes ranged from 2 to 14 fold (Table 1).

Validation by Western blot: To further confirm changes in the abundance of proteins identified in the proteomic analysis and to further investigate the toxicity pathways of DP, two proteins (HSP70 and ANXA4) were analyzed using Western blot (Fig. 3). Anti-HSP70 mouse monoclonal anti-body was used to measure the expression level of HSC70. HSP70 was significantly up-regulated following DP exposure (p < 0.05), a result that was consistent with 2-DE observation. However, ANXA4 was not significantly altered in response to DP, which was not in agreement with the 2-DE results. Data generated from 2-DE determined that this protein was decreased approximately 2.5 fold at 1 and 10 mg/kg DP.

Discussions :To gain insight into the mechanisms of toxicity of DP, 2-DE coupled MALDI–TOF–TOF was used to study the hepatic proteome response of juvenile Chinese sturgeon injected intraperitoneally (i.p.) with DP. A total of 39 significantly altered proteins were detected and 27 of these proteins were identified by mass spectrometry. These proteins were primarily involved in metabolism, signal transduction, calcium ion binding, protein folding, and structure stabilizing. Previous studies have mainly focused on PBDEs (Alm et al.,2006; Chiu et al., 2012; De Wit et al., 2008; Kling and Förlin, 2009; Kling etal., 2008), and molecular data are limited for DP. However there are some data for PBDEs that can be compared to proteomic data collected in the liver of Chinese sturgeon. For example, the hepatic proteome of zebrafish exposed to tetra-bromobisphenol A (TBBPA) was analyzed by differential in-gel electrophoresis (DIGE) and 12 proteins were found to be significantly altered. These proteins were also associated with stress response, metabolism, and the stabilization of cell structure (De Wit et al., 2008). In addition, mussels exposed to PBDE-47 had proteomic responses that were related to xenobiotic stress, amino acid metabolism, oxidative stress and effects on the cytoskeleton (Apraiz et al., 2006). Thus, these cell processes and associated proteins may be useful toxicological signatures for the general class of flame retardants. Pathway analysis suggested that differentially expressed proteins were involved in carbohydrate and amino acid metabolic process. In the present study, GAPDH was identified in two spots, most likely due to post translational modifications of the protein, resulting in different pI focusing and electrophoresis. In previous studies, oxidative modification of GAPDH was found to cause significant inhibition of GAPDH dehydrogenase activity in Alzheimer’s disease brain (Butterfield et al., 2010). Sheng and Wang (2009) also detected several isoforms of GAPDH in T cell with higher molecular mass and more basic pI. Increases of GAPDH and IDH, two proteins that are involved in carbohydrate metabolism, have also been reported in zebrafish exposed to hexabromocyclododecane (HBCD) and TBBPA (Kling and Förlin, 2009). It was hypothesized in the study that an over-expression of these proteins may contribute to increased cytotoxicity and the production of cellular defense systems involved in xenobiotic metabolism and oxidative stress. In addition, betaine homocysteine methyltransferase (BHMT) is an enzyme responsible for remethylation of homocysteines to form methionine. Suppression of this enzyme can result in increased generation of homocysteine and an increased activity of antioxidant enzymes (Kharbanda et al., 2005; Moat et al., 2000). In a previous study, De Wit et al. (2008) found that BHMT was down-regulated in the liver of zebrafish after TBBPA exposure, and concluded that this was a result of oxidative stress. Our results are consistent with these previous observations, and it is hypothesized that DP may affect energy production and induce oxidative stress in Chinese sturgeon. Two stress-related proteins, HSC70 and CCT5 were significantly altered with DP treatment. The protein HSC70 is a constitutively expressed molecular chaperone which belongs to the heat shock protein 70 family. It plays an important role in facilitating protein folding and maintaining their structure and function (Liu et al., 2012). It is also involved in many clinical diseases such as cancer, cardiovascular, neurological, and hepatic diseases; thus it is a significant target for therapeutic treatments (Liu et al., 2012). HSC70 is therefore a ubiquitous protein that is multi-functional and is responsive to a multitude of internal and external signals. In the present study, HSC70 was found to be significantly up-regulated following DP treatment. Western blot analysis of HSP70 further supported our proteomic results. In previous proteomic studies with PDBEs, many HSP70 family members such as HSP70 protein 5, HSP70 protein 8, and HSP70 protein 9B were quantified in the livers of zebrafish (De Witet al., 2008; Kling and Förlin, 2009). DeWit et al. (2008) reported that HSP70 protein 5 was markedly up-regulated in response to TBBPA, whereas, Kling and Förlin (2009) found that HSP70 protein 8 and HSP70 protein 9B were down-regulated in exposures using TBBPA and a mixture of HBCD and TBBPA, respectively. These results imply that different flame retardants might induce divergent downstream pathways related to HSP70 proteins. The protein CCT5 plays an important role in maintaining cellular homeostasis by assisting the folding of many proteins involved in cytoskeleton organization and cell cycle (Huang et al., 2012). In addition, CCT5 in the cell nucleus might play unexpected roles in biological processes including RNA processing, apoptosis, and cell metabolism (Huang et al., 2012). In this study, the expression of CCT5 was consistently decreased in individuals from all three DP treatments. In HBCD exposures, CCT5 subunit 6A was decreased (1.5 fold) in the liver cells of zebrafish (Kling and Förlin, 2009). Our results are consistent with previous studies, which suggest that DP may affect protein folding and biological processes related to CCT5. Our pathway analysis suggested that proteins related to cell cytoskeleton were affected as well and this could be due, in part to disruptions in CCT5 expression. The abnormal expression of HSC70 and CCT5 by DP implies that DP may induce a series of biological responses, with some leading to changes in cell metabolism and apoptosis. Proteomic analysis also suggested that DP may affect small G-protein signaling cascades, as there was a decrease of RAB6B as well as GDI2 and an increase of DGKD. RAB6B is a small G-protein (GTPase) of the Ras oncongene family. Small G-proteins regulate a wide variety of cell functions including gene expression, intracellular vesicle trafficking, and the cell cycle (Matozaki et al., 2000). GDI inhibits GDP dissociation and keeps the small G-protein in the inactive form (Matozaki et al., 2000). Dysfunction in the regulation of Rab GTPases and GDIs can lead to a variety of cancers and neurological diseases (Harding and Theodorescu, 2010; Hutagalung and Novick, 2011) and proteins related to this signaling pathway can be affected by flame retardants. For example, up-regulation of G-protein subunit α was observed in the tubificid (Monopylephorus limosus) exposed to BDE-183 for 8 weeks. Moreover, mitogen-activated protein kinase 12 (MAPK12), which is a downstream signal protein of Ras, was significantly decreased in response to BDE-47 and BDE-183 (Chiu et al., 2012). In the present study, the consistent down-regulation of RAB6B, a protein which mediates gene expression and affects cellular proliferation, may suggest that this protein is sensitive to DP exposure. In addition, DGKD, a type II DGK, was found to be increased in response to DP. DGK may indirectly regulate protein kinase C (PKC) and small GTPases levels or their activated state through phosphorylation of diacylglycerol (DAG). It is hypothesized that this may affect downstream biological processes such as cell proliferation, cell differentiation, and cytoskeletal rearrangements (Topham and Prescott, 1999; van Blitterswijk and Houssa, 2000); processes that were identified in our pathway analysis. Our findings and that of previous studies indicate that small G-protein signaling cascades may be impaired by DP and other flame retardants. Proteomic data and pathway analysis indicated that calcium ion signaling pathways may also be affected by DP, as Ca2+ was a small molecule that contained numerous interactions with differentially expressed proteins in the protein network. Therefore, we hypothesize that DP may result in adverse effects on aquatic organisms via impaired Ca2+ signaling. One of the impacted proteins with an integral role in Ca2+ signaling is ANXA4, and this protein was down-regulated in individuals from two of the three doses of DP. ANXA4 is a member of the annexin protein family which can bind to membrane phospholipids in a Ca2+-dependent manner, providing a bridge between Ca2+ signaling and membrane functions (Gerke et al., 2005). However, in addition to regulating Ca2+ signaling and membrane functions, ANXA4 is also a modulator of chloride (Gerke and Moss, 2002). Noteworthy is that previous studies suggest that one of the underlying molecular mechanisms of the adverse effects of polychlorinated biphenyls (PCBs) and PBDEs have been perturbations in intracellular signaling, including Ca2+ homeostasis and PKC translocation (Kodavanti and Ward, 2005). Our results suggest that DP may influence Ca2+ homeostasis by regulating the expression of ANXA4, and we hypothesize that Ca2+ signaling may be affected by DP in a similar way to that of PCBs and PBDEs. Lastly, we point out that in the present study, 2-DE revealed that the expression of ANXA4 was decreased; however Western blot results showed that the abundance of ANXA4 was not significantly affected after DP treatment. The discrepancy between the two methods may be due to a low affinity antibody for ANXA4 as Chinese sturgeon are quite evolutionary divergent than other teleost fishes and mammal (Ma et al., 2011). Despite the lack of technical congruence, previous literature in flame retardants and pathway analysis of all differentially expressed proteins suggests that DP affects the regulation of Ca2+ signaling. Regulation of Ca2+ signaling can also occur via different cellular mechanisms independent of annexins. For example, calcium can regulate Ras activation, which activates MAPK kinase (MEK) (Cullen and Lockyer, 2002). The activated MAPK translocates to the nucleus and stimulates the activity of transcription factors through phosphorylation, which in turn regulates cell proliferation and apoptosis (Matozaki et al., 2000). Moreover, Fan et al. (2010) showed that PBDE mixtures and congeners activate the MAPK pathway, which may be involved in the initiation of events that lead to adverse effects that are associated with these persistent chemicals. Proteomic results from the present study indicated that small G-protein signaling cascades may be regulated by DP. Therefore, we hypothesize that DP may affect gene expression by activating MAPK pathway. Based on our data and principles of cell signaling, we generate a model for how DP may act in the liver of aquatic organism (Fig. 4). Initially, DP may impair the movements of Ca2+ from the extracellular compartment to the intracellular compartment by affecting intermediate ANXA4 function, followed by effects on the Ras signal cascade and protein folding process. Gene expression and protein synthesis may be impacted by upstream signals from GTPase pathways and mis-folded proteins accumulated by abnormal protein folding process. At the same time, the xenobiotic system may be evoked to degrade DP. These signaling events may affect the processes of cell proliferation and apoptosis, and may lead to adverse effect. We point out that this is only a model of DP action based upon proteomics data, and generates a general framework for future studies. Similar to other flame retardants, it is expected that DP affects multiple signaling cascades within the teleost liver and these molecular events must be further validated experimentally. In conclusion, 39 protein spots were significantly altered in abundance with different doses of DP and of these, 27 proteins were successfully identified using MS. Differentially expressed proteins and pathway analysis indicated that DP exposure may induce oxidative stress, cell proliferation and apoptosis. Meanwhile, these responses may be mediated through the stress response, small G- protein signaling cascades, calcium ion binding and carbohydrate metabolism. The underlying mechanisms of DP appear comparable to PBDEs, impacting calcium homeostasis and activation the Ras signal cascade. Future studies should continue to validate these proteins as potential biomarkers for the exposure to DP in fish.

Applicant's summary and conclusion

Conclusions:
Analysis of different proteins showed that some protein concentrations in Chinese sturgeon were altered upon exposure to 1, 10 and 100 mg/kg ww applied by single intraperitoneal injection. 39 of 740 identified proteins were significantly altered in concentration and 27 of those could be identified. There main functions were associated with oxidative stress, cell proliferation (of Ca2+) and apoptosis.
Executive summary:

In conclusion, 39 protein spots (out of 740) were significantly altered in abundance with different doses of DP and of these, 27 proteins were successfully identified using MS. Differentially expressed proteins and pathway analysis indicated that DP exposure may induce oxidative stress, cell proliferation and apoptosis. Meanwhile, these responses may be mediated through the stress response, small G- protein signaling cascades, calcium ion binding and carbohydrate metabolism. The underlying mechanisms of DP appear comparable to PBDEs, impacting calcium homeostasis and activation the Ras signal cascade. Whether such proteins can be used as biomarkers, remains unclear and is subject to further research.