Tissue-Specific Expression of p53 and ras Genes in Response to the

Sep 7, 2011 - Marine mussels can develop hemeic and gonadal neoplasia in the natural environment. Associated with these diseases are the tumor suppres...
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Tissue-Specific Expression of p53 and ras Genes in Response to the Environmental Genotoxicant Benzo(α)pyrene in Marine Mussels Yanan Di,† Declan C. Schroeder,‡ Andrea Highfield,‡ James W. Readman,§ and Awadhesh N. Jha*,† †

School of Biomedical and Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, U.K. Marine Biological Association of the United Kingdom (MBA), Citadel Hill, Plymouth PL1 2PB, U.K. § Plymouth Marine Laboratory (PML), Prospect Place, The Hoe, Plymouth PL1 3DH, U.K. ‡

bS Supporting Information ABSTRACT: Marine mussels can develop hemeic and gonadal neoplasia in the natural environment. Associated with these diseases are the tumor suppressor (TS) p53 and the proto-oncogene ras coded proteins, both of which are highly conserved among molluscs and vertebrates. We report, for the first time, tissuespecific expression analysis of p53 and ras genes in Mytilus edulis by means of quantitative RT-PCR. A tissue-specific response was observed after 6 and 12 days exposure to a sublethal concentration of a model Polycyclic Aromatic Hydrocarbon (PAH), benzo(α)pyrene (B(α)P). This sublethal concentration (56 μg/L) was selected based on an integrated biomarker analysis carried out prior to gene expression analysis, which included a ‘clearance rate’ assay, histopathological analysis, and DNA strand break measurements. The results indicated that the selected concentration of B(α)P can lead to the induction of DNA strand breaks, tissue damage, and expression of tumor-regulating genes. Both p53 and ras are expressed in a tissue-specific manner, which collaborate with tissue-specific function in response to genotoxic stress. The integrated biological responses in Mytilus edulis strengthen the use of this organism to investigate the fundamental mechanism of development of malignancy in invertebrate which could be translated to other organisms including humans.

’ INTRODUCTION Mutational inactivation of tumor suppressor (TS) genes and activation of oncogenes are two of the most frequently observed and thoroughly studied molecular pathways in human cancer research.1 p53, as a representative TS gene, serves as the major cellular barrier against cancer development. DNA damage or cellular stress can activate the p53 protein via the orchestrated action of multiple post-translational modifications which either increase the stability of p53 or directly enhance its DNA-binding affinity, leading to the arrest of cell cycle progression to allow DNA repair.2 The ras proto-oncogene encodes low molecular weight guanine nucleotide-binding proteins that cycle between inactive GDP-bound and active GTP-bound forms.3 Activated ras oncogenes are frequently detected in human and other animal tumors with point mutations occurring within exons 1 and 2: codons 12, 13, and 61.4 When this gene suffers a mutation in one of the above-mentioned codons, the encoded protein inhibits GTPase activity, leading to altered cell proliferation, differentiation, and cell cycle checkpoint control.5 Recent studies concerning the development of carcinogenesis in mammals and fish reported mutant forms of ras in a large proportion and wide variety of tumors.6 Due to the worldwide distribution and ecological habitat, mussels are extensively used for environmental studies7 and could serve as a model organism for elucidation of neoplastic disorders as leukemia (hemeic neoplasia) and gonadal neoplasia r 2011 American Chemical Society

have been reported in the blue mussel Mytilus sp. around the world.8 In this context, occurrence of malignancies in bivalve molluscs has also been correlated with higher incidence of tumors in human populations who were exposed to increased use of herbicides.9 Defining common mechanisms of tumor formation in mussels, therefore, could provide valuable information for human cancer research. Furthermore, both the gene sequences for p53 and ras have recently become available for Mytilus edulis and show regions of highly conserved sequences across different groups of organisms, including humans.10 Therefore, a study to elucidate expression of tumor-regulating genes in mussels is both timely and warranted, especially when seeking to develop a nonhuman model to explore the underlying mechanisms of neoplasia. Benzo(α)pyrene (B(α)P), a representative polycyclic aromatic hydrocarbon (PAH), is the most extensively studied genotoxicant and carcinogen in aquatic organisms.11 Various groups of aquatic organisms collected from PAH contaminated sites, including the European flounder Platichthys flesu, the oyster Crassostrea virginica, and the marine mussel Mytilus edulis, have been reported to have a greater incidence of neoplastic disease Received: May 6, 2011 Accepted: September 7, 2011 Revised: September 2, 2011 Published: September 07, 2011 8974

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Figure 1. A: Clearance rates of Isochrysis galbana in Mytilus edulis following 6 and 12 days B(α)P exposure under in vivo condition. Error bars present SEM. B: Induction of DNA strand break (represented as % Tail DNA) in Mytilus edulis. hemolymph following 6 and 12 days in vivo B(α)P exposure. * indicates significant increase of % Tail DNA in treated groups compared with control group (p < 0.05). # indicates significant differences of % Tail DNA between day 6 and day 12 within each treated groups (p < 0.05).

compared to samples collected from uncontaminated sites.1214 This study, therefore, aims to elucidate the gene expression profiles of p53 and ras in M. edulis following exposure to B(α)P. An integrated approach with biomarkers at different levels of biological organization were analyzed prior to gene expression analysis. Gene expression was analyzed in different tissues based on previous studies where neoplasia has been found with different susceptibilities among humans15 and other marine bivalve tissues.16 To our knowledge, this is the first study to evaluate tissue-specific expression profile of tumor-regulating genes (p53 and ras) in the blue mussel M. edulis. Attempts have also been made to link biomarker responses at different levels of biological organization following B(α)P exposure.

’ MATERIAL AND METHODS Mussels Sampling and B(α)P Exposure Setups. Mussels were collected from Trebarwith Strand (North Cornwall, UK,

50°380 N; 4°450 W) and were immediately transported to the laboratory where they were maintained in the aerated tanks with filtered ( mantle > adductor muscle (Figure 3B). 8977

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Environmental Science & Technology Significantly lower ras expression was detected in the adductor muscle and mantle. The expression level of ras showed no significant difference in all the tissues for both incubation periods in untreated control conditions except for adductor muscle which showed significantly increased ras expression abundance after 12 days incubation. Relative Quantification of p53 and ras Expression in Tissues Induced by B(α)P. Tissues from mussels exposed to 56 μg/L B(α)P were selected for gene expression analysis as this concentration induced the highest response for feeding behavior (i.e., clearance rate) and significant DNA strand breaks at cellular level. The relative quantification of p53 and ras expressions were normalized in different tissues by the 2ΔΔCt method20 (Figure 3C-D). Wide interindividual variation of p53 expression was detected in all tissues. After 6 days exposure, a significant increase in p53 expression was detected in the adductor muscle, approximately 2.34 ( 0.89-fold higher than the control. A small but insignificant decrease in p53 expression was found in the other three tissues. After 12 days exposure, significantly increased p53 expression was detected in both the mantle and adductor muscle with 5.93 ( 0.83- and 3.28 ( 1.23-fold higher expression levels, respectively. Lower induction of p53 expression in gill and digestive gland was observed, but it was not significantly different compared to the control. p53 expression also increased with longer B(α)P exposure period. Approximately 6-fold higher p53 expression was detected in the mantle after 12 days exposure compared to 6 days. The other three tissues also showed a slightly increased p53 expression after a longer exposure time, but the differences were not statistically significant (Figure 3C). There were no significant differences in ras expression in gill, digestive gland, and mantle after 6 days exposure (Figure 3D). A significant 2.29 ( 1.01-fold higher ras expression was only seen in the adductor muscle. After 12 days exposure, the mantle showed significantly (2.18 ( 1.14-fold) higher ras expression, but the adductor muscle and digestive gland showed no significant increase in expression. The gill tissue showed a slight decrease in ras expression but was not statistically significant. Increased ras expression was found in all tissues apart from the adductor muscle after longer B(α)P exposure. Significantly increased ras expression, approximately 2.22-fold, was found in the mantle after 12 compared to 6 days exposure. On the contrary, no significant difference in ras expression was seen in the digestive gland and gill after 12 days compared to 6 days exposure. Slightly decreased expression was seen in the adductor muscle, again indicating tissue specific expression.

’ DISCUSSION The concentration ranges selected in this study have previously shown to induce cellular responses in mussels.21,22 They do, however, exceed the documented solubility of the compound (2.3 μg/L).23 The presence of dissolved organic matter has been shown to enhance solubility 24 and owing to the high Kow (6.04),25 the B(α)P rapidly partitions onto particulates and the mussels themselves. This is consistent with the comparatively low GC-MS measured concentrations, their variability, and the rapid depletion of the B(α)P from the aqueous phase. In the higher B(α)P exposure concentrations (56 and 100 μg/L), there were no observed differences in clearance rates between untreated control and exposed mussels. This suggests that mussels are relatively tolerant to these types and concentration range of organic chemicals and can metabolize them without causing

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marked physiological changes at individual level.17 It is reported that blue mussels exposed to 1 μg/L B(α)P for 6 h, followed by 6 h in clean seawater, had tissue concentrations 7080% of the initial B(α)P concentration in the water,26 suggesting the ability of mussels to accumulate B(α)P in the tissues. However, the clearance rates of mussels after 12 days exposure were significantly lower than 6 days, suggesting other conditions, for example starving stress, rather than direct B(α)P-induced stress may have affected the physiological condition of the mussels. Only a small decrease in clearance rates were observed at 56 μg/L B(α)P exposure. This effect may have been caused by the direct toxic effect of B(α)P as it has been shown to induce blood cell lysosomal membrane damage in mussels.27 Histopathological results carried out on 6 mussels showed changes in the mussel tissues; however, the prevalence of these changes was not concentration-dependent. This sample size was perhaps not large enough to confirm concentration-dependent relationship.28 No evidence of neoplasia was found in any of tissues sampled. Only necrosis was observed in some of the exposed tissues, which could be considered as very early stage of tumor development.29 Neoplasia in invertebrate species is relatively rare compared to incidences reported for vertebrates.30 Attempts to induce neoplasia in vivo in mussels has as of yet been unsuccessful suggesting that its etiology in invertebrates under laboratory conditions potentially involves factors other than exposure to the selected carcinogenic chemicals alone.16 Nonetheless, the histopathological results provide fundamental evidence of the physiological effects of B(α)P that could lead to disease development, such as neoplasis and immune suppression.31 Significant increases in DNA strand breaks were also observed with increasing B(α)P concentrations, demonstrating the capability of B(α)P to induce a genotoxic response. Interestingly, the level of DNA damage showed a significant decrease in DNA strand breaks after 12 days exposure. This suggests the potential involvement of DNA repair, recombination, and cell cycle checking mechanisms32 during the process of mussels responding to the B(α)P exposure. Excessive damaged cells could be lost through apoptotic pathway leading to apparent reduction in the level of DNA damage.33 Toxicant concentrations via dosage or exposure period is required to reach a threshold level before DNA repair systems are initiated.34 Since the in vivo control values were relatively high (30% of Tail DNA) to begin with compared to an in vitro study using H2O2,35 it is likely that the mussels used in our study were already stressed and, therefore, were more sensitive to B(α)P lowering their toxicity threshold. Although both DNA strand breaks and histopathological effects suggest that B(α)P can indeed cause stress in mussels, gene expression studies on key neoplasia associated genes could initiate the interrelated process of mutagenesis and carcinogenesis through common pathways seen in other organisms.36 In normal conditions (i.e., without any induced stress), p53 and ras expressions showed a tissue-specific expression pattern. The highest expression abundance was obtained in gill followed by digestive gland, mantle and adductor muscle tissues. The gill and digestive gland of bivalve molluscs play an important role in food collection, absorption, and digestion,28 and cells in these two tissues are likely to be more susceptible with stresses. Elevated expression of p53 has been shown in mammalian studies where brain was treated by adrenalectomy and excitotoxic treatment.24,37 Increased expression has also been shown with apoptosis of antral ovarian follicles in rats.38 Moreover, a significant 8978

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Environmental Science & Technology up-regulation of p53 expression in neoplastic hemocytes compared to normal hemocytes has also been reported in Mytilus trossulus.39 All these results confirm that p53 expression changes can be an indicator of cells responding to stress. Interestingly, in our study not all the tissues showed increased p53 and ras expression after exposure to B(α)P. Only the adductor muscle for both exposure periods and the mantle after 12 days exposure showed obvious increased expression for both the genes (i.e., p53 and ras). The adductor muscle is the main tissue to preserve hemolymph and consists of less cell types compared to other tissues. Mantle, as the main tissue to produce sperm, has been found to show gonadal neoplasia (solid tumor) in mussels collected from polluted sites,40 but no evidence of neoplasia occurred in other tissues in the same individuals. These results support that mantle cells or sperm produced during spermatogenesis could be more sensitive to chemicals. A physiologically based pharmacokinetic research on eastern oyster (Crassostrea virginica) found tissue-specific bioaccumulation of dioxin after 28-days post exposure to 2,3,7,8-tetrachlorodibenzo-F-dioxin (TCDD), suggesting the toxico-kinetics of chemicals are variant in tissues.41 Using fish as models for environmental carcinogenesis, it has been reported that different tissues respond differently to carcinogens generating different types of tumor.42 In common with other organisms, it is important to elucidate the metabolic kinetics of B(α)P in mussel tissues to link the tissue-specific expressions of tumor related genes. The high interindividual variation within our samples is likely to be caused by intrinsic and extrinsic factors.7,43,44 Other physiological factors such as feeding and reproduction could also affect the pollutant uptake by the organisms. As a consequence, the body burden of organic pollutants in individuals inhabiting the same site as well as the resultant metabolic and antioxidative responses may vary considerably.45 Therefore, B(α)P uptake and accumulation in different tissues will also show the interindividual variability. The result of longer exposures increased p53 expression which complements the finding of decreased DNA damage after 12 days exposure, indicating potential involvement of DNA repair mechanisms. This is supported by the fact that both p53 and ras function in association with DNA repair and apoptosis processes.46,47 Increased p53 expression suggests that the concentration of 56 μg/L of B(α)P can induce the p53 expression to halt the cell for repair. ras, as pro-oncogene, will be activated too when the cells undergo this stress. Absence of neoplastic cells in the experimental animals suggests that no up-regulation of ras expression caused the response to the ras related pathways which could lead to uncontrolled cell growth over the exposure period. Combining DNA strand break and gene expression results, B(α)P significantly induce DNA damage in hemocytes and increases expression of p53, but this was not significant for all four different tissues. It is consistent with previously documented report that B(α)P induced quantitatively different levels of DNA adducts in different tissues of M. edulis.48 Significant increases in DNA damage are not necessarily related to increased p53/ras expression in our study and is similar to research in human cells, where increased micronuclei formation had no obvious relationship with p53 expression following exposure to a range of genotoxic chemicals under in vitro condition.49 In addition, Banni et al. (2009)50 also found up-regulation of p53 in digestive gland tissue, a large down regulation in hemocytes and no modulation for p53 expression in other tissues following exposure of mussels to B(α)P and crude oil. However, a significant increase in DNA

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strand breaks was observed only in hemocytes. These results further suggest that gene expression in single cell types may give significantly different results in different tissues, bearing in mind that genotoxicity is a cell specific process. In addition, since different tissues have different turnover rates and the fact that B(α)P could cause cell cycle stage specific changes in gene expression,51 it is also likely that tissue-specific gene expression pattern in our study could be related to cell cycle durations. Apart from alteration of p53/ras expression reflecting their functional change in cells, the mutation and post-transcriptional modification of these two genes have also been documented in response to DNA damage.52,53 The localization of p53 in cells also plays an important role in relation to its function in apoptosis process and leukemia formation. The maintenance of tumor phenotype has been reported to require nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes.54 Vassilenko et al. (2010) also reported an increased number of allelic variants in the leukemic mussels Mytilus trossulus which may arise from separate somatic mutation events in hemocyte precursors or from additional p53-like gene copies in polyploidy.55 In conclusion, while B(α)P induced tissue and DNA damage confirms its action as genotoxicant, it also induces expression of tumor-regulating genes (i.e., p53 and ras) following 12 days exposure but with a high level of interindividual variation. Normal expression of p53 and ras occurred in a tissue-specific manner, reflecting normal physiological function of tissues. Concurrent induction of DNA damage and tissue-specific gene expression has been found in our study. However, the multiple sites targeted by an integrated network of signaling pathways which are highly sensitive to genotoxic stresses must be modified to yield a functional p53 and/or ras.52 The work at translational levels and related pathways of p53 and ras are required to further elucidate the mechanism. Our study goes some way toward achieving these goals and sheds light on tissue-specific expression of tumorregulating genes at whole organism level which should also complement research into human health area.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed methods and materials, detailed prevalence of histopathological change result, detailed analysis of p53 and ras expression efficiency, Tables S1 and S2, and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +44 (0) 1752584633. Fax: +44 (0) 1752584605. E-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by grants from Overseas Research Students Awards Scheme (ORSAS) and European Regional Development Fund, INTERREG IVA (Grant No. 4059). ’ REFERENCES (1) Solomon, H.; Brosh, R.; Buganim, Y.; Rotter, V. Inactivation of the p53 tumor suppressor gene and activation of the Ras oncogene: cooperative events in tumorigenesis. Disc. Med. 2010, 9 (48), 448–454. 8979

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dx.doi.org/10.1021/es201547x |Environ. Sci. Technol. 2011, 45, 8974–8981