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Sequencing of p53 genes from Mytilus edulis and Mytilus trossulus for use in environmental effects monitoring… Muttray, Annette Friederike 2004

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Sequencing of p53 genes from Mytilus edulis and Mytilus trossulus for use in environmental effects monitoring of primary treated effluent by  ANNETTE FRIEDERIKE MUTTRAY '  Ph.D., The University of British Columbia, 2001 M.Sc., Aberdeen University, U K , 1994  A THESIS SUBMITTED I N P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF  M A S T E R OF APPLIED SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Civil Engineering, Waste Management and Pollution Control Program)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A September 2004  © Annette Friederike Muttray, 2004  THE UNIVERSITY OF BRITISH  COLUMBIA  F A C U L T Y OF G R A D U A T E STUDIES  Library Authorization  >  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Murtray, Annette N a m e of Author (please print)  Title of Thesis:  Date (dd/mm/yyyy)  Sequencing of p53 genes from'Mytilus edulis and Mytilus trossulus for use in environmental  effects monitoring of primary treated effluent  Degree: Department of  MASc  Year: Civil Engineering  The University of British Columbia Vancouver, B C C a n a d a  2004  ABSTRACT This thesis describes the sequencing and potential use of a genetic biomarker for coastal environmental effects monitoring of municipal effluents. The Blue .Mussel {Mytilus edulis) and the Bay Mussel (Mytilus trossulus) are currently investigated for their use in municipal effluent effects monitoring. Both can develop a disease of the haemolymph, called haemic neoplasia or leukemia, albeit at different prevalences. The p53 gene is a tumor suppressor gene that is fundamental in cell cycle control and apoptosis. It is mutated or differentially expressed in about 50 % of all human cancers and has been implicated in leukemia development in clams. Thus, the p53 gene family was chosen as potential biomarkers for haemic neoplasia in Mytilus spp. During this study, the p53 mRNA sequences of both Mytilus species were elucidated and analyzed in detail using several databases. Sequences show 99.8 % similarity on the protein level, but are only 96.5 % similar at the D N A level, and differ especially in their 3' untranslated regions, which are important in the regulation of post-transcriptional events. Future studies are required to show potential linkages of p53 gene family expression patterns with haemic neoplasia in the mussels. During this study, an additional gene sequence was discovered and is submitted as a Mytilin C antimicrobial peptide precursor in M. trossulus. These peptides occur in the haemolymph and haemocytes of mussels and prevent microbial infections of the open circulatory system. This thesis also reviews the current state of knowledge on the use of molecular biomarkers for monitoring effects of endocrine disrupting compounds and surfactants and reviews iri-depth the fate and effects of one group of surfactants, linear alkylbenzene sulfonates (LAS). L A S were identified as a major cause of toxicity in the , effluent of one treatment plant in the Greater Vancouver Regional District, and have been shown to enhance the effects of endocrine disrupting compounds. Based on our current knowledge about increased prevalence of haemic neoplasia in other bivalves in polluted areas, clam p53 gene family expression patterns, as well as environmental carcinogenesis involving the endocrine and p53 systems in humans, it is suggested that the p53 gene family is a good candidate for development of a molecular biomarker.  TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS . . LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS. LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS. ACKNOWLEDGEMENTS  CHAPTER 1 - INTRODUCTORY CHAPTER 1 1 Background and objectives 1 1.1 Problem statement 1 1.2 Marine receiving environment monitoring and molecular biomarkers 2 1.3 The context for design of thesis tasks and objectives 4 2 Technical background 6 2.1 A brief introduction to bivalve molluscs and bivalve blood cells , 6 2.2 Why mussels for receiving environment monitoring? 7 2.3 Brief overview of the G V R D Caged Bivalve Program at the Lions Gate WWTP outfall 8 2.4 Leukemia in bivalves 9 2.5 Genetic.changes during haemic neoplasia 14 2.6 The p53 family of tumor suppressors and their application as genetic biomarkers : ...16 2.7 Genomic Technologies for Monitoring of Microcontarriinants with Potential Endocrine Disrupting Effects - A n Update..... .20 CHAPTER 2 - MANUSCRIPT C H A P T E R -—23 2.1 Manuscript I - Genomic Technologies for monitoring of microcontaminants with potential endocrine disrupting effects - a literature review .24 2.2 Manuscript II - Fate and effects of linear alkylbenzene sulfonates in the aquatic environment ;5 8 2.3 Manuscript III - Identification and phylogenetic comparison of p53 in two distinct mussel species {Mytilus) 73 CHAPTER 3 - ADDITIONAL UNPUBLISHED METHODS, RESULTS AND OBSERVATIONS — 113 1 Microscopic observations and sample selection for PCR.... 113  2 R N A extraction and observations 3 In-depth description of molecular methods and materials 3.1 cDNA synthesis 3.2 Degenerate PCR 3.3 Gel purification of PCR products 3.4 3'A-tailing.... 3.5 Ligation and cloning 3.6 3' and 5' R A C E PCR , 4p63/73 : 5 Mytilus trossulus Mytilin C  114 115 115 115 117 117 117 118 118 .121  CHAPTER4 - CONCLUDING CHAPTER——-—— — 126 1 Discussion and recommendations for future research 126 1.1 p53 expression and environmental monitoring 126 1.2 p53 and the endocrine system 128 1.3 Chemical agents for haemic neoplasia in bivalves and p53 expression 133 1.4 Other causative agents for haemic neoplasia in bivalves and p53 expression .135 1.5 L A S treatability studies and effects on E D C removal 136 2 Major conclusions and recommendations 137  REFERENCES  -.138  LIST OF TABLES Table 1: Summary of the functions of the different isoforms and family members of the p53 family '. '. 19 Table 2: List of suspected endocrine disrupting compounds.....  37  Table 3: A selection o f compounds from the I G C V A M E D W G proposed substances list, based on their capacity to bind to the estrogen receptor (ER) and activate transcription of genes which are .under the control of the E R 40 Table 4: Summarized results for the effects seen after exposure of Xenopus laevis to 10 n M acetochlor  52  Table 5: Range of toxocity values for a variety of species or groups of organisms  67  Table 6 (Table 1 of the submitted manuscript): Primers used in the identification of p53 in Mytilus sp.. 99 Table 7 (Table 2 of the submitted manuscript): List of species, accession numbers, and abbreviations used for the phylogenetic analysis of p53 proteins in Figure 2 Table 8: Catalog of primers used during the course of this project. Table 9: Effects of the interactions of p53 and M D M 2 with GR, A R and E R  100 116  ...........133  LIST OF FIGURES Figure 1: Maps showing locations of mussel sampling stations in the Vancouver Inner and Outer Harbour (Burrard Inlet) (map A ) and Howe Sound (map B)..... 10 Figure 2: Schematic of the moorings deployed by G V S & D D containing duplicate mussel cages at various depths.., 11 Figure 3: Comparison of the domain structure of p53 protein and the six major isoforms encoded by p63 and p73  18  Figure 4: Schematic p53 family member pathways  19  Figure 5: Outline of the mechanism of the steroid hormone action  30  Figure 6: Simplified overview of the method for sample preparation and hybridization to cDNA chips 45 Figure 7 (Figure 1 of the submitted manuscript): Multiple pairwise alignment of amino acid sequences for p53 homologues from Mytilus edulis (Mep53), Mytilus trossulus (Mtp53), Mya arenaria (Map53), Drosophila melanogaster (Dmp53), Xenopus laevis. (Xlp53), Danio rerio (Drp53), Barbus barbus (Bbp53), and Homo sapiens (Hsp53) .. 101 ;  Figure 8 (Figure 2 of the submitted manuscript): Phylogenetic relationship between p53 proteins of diverse species indicating separate lineages for mollusca and annelida 101 Figure 9 (Figure 3 of the submitted manuscript): Clustal X alignment of the 3'UTR of the p53 gene of three variants of M. edulis, Mep53_v\, Mep53_y2 (AY735341), Mep53_v3 (AY735340), M. trossulus (Mtp53) and M. arenaria (Map53) : 101 Figure 10 (Figure 4 of the submitted manuscript): Output for the phylogenetic footprint analysis 102 Figure 11 (Figure 5.of the submitted manuscript): Leukemic haemocytes from mussels. Mytilus spp treated with murine monoclonal antibody 1E10 and analysed with laser scanning confocal microscopy :. 102 Figure 12: Ethidium bromide-stained agarose gels showing result's of 3'Smart™ . R A C E PCR reactions 121  Figure 13: M. galloprovincialis mytilin C antimicrobial peptide precursor - like sequence in M. trossulus. Nucleic acid and putative protein sequence for M. trossulus clone 209-13, direct entry to GenBank, accession number AY730626 122 Figure 14: Results for the Blast search (Discontiguous megablast) conducted May 9, 2004 at, indicating an overall 77 % similarity with the mRNA of Mytilus galloprovincialis mytilin B antimicrobial peptide precursor 123 Figure 15: ClustalW alignment of available mytilin sequences  124  Figure 16: Primary interpretation permutations of response reading of one gene in an array t... 126 Figure 17: Estrogen-responsive protein Efp controls cell cycle and breast tumor growth 132  LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS  aa  amino acid  Ab  antibody  AR  androgen receptor  ARE  adenylate/uridylate-rich element  ATM  ataxia telangiectasia mutated (protein)  bp  base pairs  BLAST  Basic Local Alignment Search Tool  Da  Dalton  cDNA  complementary D N A  DES  diethylstilbestrol  CDK  cyclin-dependent kinase  CHK2  checkpoint kinase 2 (protein)  CPE  cytoplasmic polyadenylation element  CPSF  polyadenylation specificity factor  DBD  D N A binding domain  DNA  deoxyribonucleic acid  E2  estrogen 17-beta-estradiol  EC50  median effective concentration  EDC  endocrine disrupting compound  EEM  environmental effects monitoring ,  ER  estrogen receptor  EST  expressed sequence tag  GC  glucocorticoid hormone  GR  glucocorticoid receptor  GVRD  Greater Vancouver Regional District  GVS&DD  Greater Vancouver Sewerage and Drainage District  HRE  thyroid-stimulating hormone  kb  . kilo base pairs  LAS  linear alkylbenzene sulfonates  LC50  median lethal concentration  LOEC  lowest-observable-effects concentration  Ml3  universal primer sequence based on N-terminal coding sequence of the lacZ gene  MBAS  methylene blue active substances  MDM2  mouse double minute 2 (oncoprotein, p5 3-binding protein)  MLD  million liters per day  mRNA  messenger ribonucleic acid  NA  nucleic acid  NES  nuclear export signal  NLS  nuclear localization domain  NOEC  no-observable effects concentration  nt  nucleotide(s)  ORF  open reading frame  p53  protein 53 (tumor supressor protein), italics denotes nucleic acid  PAH  polyaromatic hydrocarbons  PCB  polychlorinated biphenyls  PCR  polymerase chain reaction  ' poly(A)  poly-adenosin tail  POP  persistent organic pollutants  PXXP  amino  acid  motif  containing proline-variable-variable-proline  residues RACE  rapid amplification of cDNA ends  RT-PCR  Reverse Transcriptase PCR  SDS  sodium dodecyl sulfonate  T3  active thyroid hormone  TAD  transcriptional activation domain  TAE  Tris acetate E D T A  Taq  Taq polymerase (PCR enzyme isolated from Thermus aquaticus)  TH  thyroid hormone  TR  thyroid hormone receptor  TRE  thyroid-hormone response element  TSH  thyroid-stimulating hormone  3'UTR  untranslated region at the 3'end of a gene sequence  WET  whole effluent toxicity  WWTP  wastewater treatment plant  YES  yeast estrogen screen  ACKNOWLEDGEMENTS  I would like to thank Drs. Susan Baldwin and Ken Hall for their advise on technical as well as administrative matters, but especially Dr. Baldwin for her unwavering support and enthusiasm for the project and for providing me with much-needed feedback. A big thank-you goes to Drs. Carol Reinisch and Rachel Cox at the Marine Biological Laboratory, Woods Hole, Massachusetts, for welcoming me warmly at their laboratory, introducing me to the mysteries of bivalve leukemia and eukaryotic genetics, and getting me started in the lab (again). I would also like to thank Dr. Sylvie St-Jean at the National Water Research Institute of Environment Canada, Burlington, for teaching me about mussels, mussel leukemia and mussel sampling, for having me at her field lab at Pictou, Nova Scotia, and for participating in study and experimental designs. A very special thank-you goes to Mr. Paul van Poppelen of the Greater Vancouver Regional District without whom none of this would have happened. I thank him for sharing his profound knowledge and ideas, some of which have materialized in this thesis. I thank him for his trust, his determination, well-placed cynicism, his firm commitment and friendship. A thank-you the lab members of Dr. Jamie Piret's laboratory as well as the Biotechnology Teaching Laboratory at U B C for sharing their precious space with me. Lastly, but very importantly, I thank Dr. Albert van Roodselaar and the Policy and Planning Department of the Greater Vancouver Regional District for funding this project.  1  CHAPTER 1 - INTRODUCTORY CHAPTER 1  BACKGROUND AND OBJECTIVES  1.1 Problem statement In recent years, researchers have voiced concern about endocrine disruption and persistent organic pollutants in our waste streams and in our aquatic environment receiving those wastes. Currently, our wastewater treatment technology is not designed to deal with these chemicals (Tschobanoglous et al., 2003) (p.8), especially as they often occur at low concentrations. However, many of these chemicals are exerting various deleterious effects on all kinds of living beings, including humankind (Chyczewski, 2001). Releases into the environment are often inadvertent, and substances have undergone insufficient toxicity and other effects testing. Especially in the area of endocrine disrupters in the environment, the general belief of the scientific community is that while endocrine disruption certainly exists, more research is needed to clarify mechanisms, the substances responsible, and the extent of the problem, and that no clear cause and effect relationships have been demonstrated to date (Birkett, 2003).  The removal of persistent organic compounds (POPs), surfactants and endocrine disrupting compounds (EDCs) during the wastewater treatment process depends on the inherent physicochemical properties of the compounds and the treatment processes applied. As both, pollutant properties and treatment processes, can vary widely, a general review of the fate of POPs and EDCs in wastewater treatment goes likely beyond the scope of this thesis. However, it is sufficient to say that the nonpolar and hydrophobic nature of many pollutants will cause them to adsorb onto particles, and will therefore be concentrated in sewage sludge (Langford et al., 2003). Other pathways of removal are potentially as follows: Association with fats and oils, aerobic and anaerobic degradation, chemical degradation, and volatilization. Despite these various removal pathways, pollutants and their degradation intermediates are found in treated sewage effluents at concentrations capable of inducing adverse reactions in aquatic biota (Gomes et al., 2003) and ultimately humans through introduction into drinking water and other exposure pathways (Gaterell, 2003).  2  Using advanced treatment technologies, such as flocculation, sedimentation, filtration, ozonation, U V treatment and peroxidation, one can reduce (but likely not completely eliminate) the concentrations of POPs and EDCs in wastewaters. The additional costs involved can be more easily justified if the treated effluent is destined for human use. However, monetary and nonmonetary benefits of reducing the effects of these compounds on the environment are more complex to determine and justify. Therefore, there is a need for accurate monitoring so as to develop a better understanding of potential environmental effects. This knowledge will impact decision-making visa vi the need for more advanced wastewater treatment technologies. Therefore, developing tools for environmental effects monitoring is an important part of improved management of our waste streams and helps to protect the receiving environment for its beneficial and sustainable uses and, increasingly, for its intrinsic values (Gaterell, 2003).  1.2 Marine receiving environment monitoring and molecular biomarkers In British Columbia, municipal (and other) effluent discharges have mainly been regulated based on permits under the B C Waste Management Act and in particular the Water Quality Guidelines. Regulation and enforcement are currently based on Whole Effluent Toxicity (WET) testing, in which organisms such as rainbow trout are exposed to dilutions of the whole effluent for a specified time period. In some instances, Daphnia and Microtox ® toxicity testing is performed. However, WET tests are not required if it can be proven that the environment is protected and for discharges into marine open waters. The onus of proof lies on the discharger. Environment Canada has developed national Environmental Effects Monitoring Programs (EEM) for Pulp and Paper and Metal Mining (URL:, with sewage discharge E E M soon to be developed, in order to determine whether the regulations for these industries are sufficient to meet the requirements of the Fisheries Act and protect fish habitat (Burd, 2002). E E M has addressed questions such as whether contaminants enter the system, whether the contaminants are bioavailable, whether there is a measureable response and whether the contaminants are causing the response (Burd et al., 2003).  3  Many different options exist for receiving environment monitoring. In most cases, different approaches will be used in concert to address cause and effect relationships. Options range from "simple" chemical analysis of the water column and sediments to monitoring biomarkers of indigenous and introduced animal and plant populations. While the former only addresses presence, absence or concentration of selected substances of concern without looking at biological availability or effects, the latter will address biological effects, but potentially without directly linking them to causes. Laboratory studies, for instance toxicity identification and evaluation (TIE) studies will address cause-effect relationships (Coombe et al., 1996), but removed from the environment, and based on a few species commonly used for toxicity determinations.  Biomarkers have the potential ability to quantify actual exposure history, provide a temporal framework for this exposure, and determine a threshold biological response and possibly detrimental biological consequences (Wirgin et al., 1994). The biomarker approach provides several advantages over the measurement of environmental or tissue concentration of contaminants: It integrates the bioavailable sources of contaminant levels (including from sediments and prey species, for instance), notes a possible hypersensitivity of the bioresponse, potential cost reductions when compared to chemical analysis, and most importantly evaluation of an endpoint of a quantifiable biological effect (Wirgin et al., 1994). Biomarkers that have been evaluated in fish range from the community to the molecular level. Biomarkers on the molecular level, based on the changes in D N A and gene expression levels, offer the advantage of rapid and often sensitive responses to xenobiotics and a degree of specificity in the response. While whole animal- and community-based biomarkers in plants and animals (such as reduction in growth, development of cancers, death, reproductive success, etc.) will similarly address biological effects, they can have complex and multiple causes and are usually only observed at later stages of exposure. It is suggested here that measurement of induction of gene expression and initial accumulation of certain mRNAs and proteins can serve as early-warning indicators of environmental stresses if understood in-depth and applied judiciously. The applicability of this approach to aquatic organisms is firstly limited by the number of gene sequences and gene probes available; however, this battery is increasing rapidly thanks to large genome sequencing projects, such as the Genomic Research on Atlantic Salmon project  4  (, the genome sequencing of C. elegans and zebrafish by the Sanger Insititute ( or a variety of prokaryotic genome sequencing projects throughout the world. The second limitation lies with a clear and deep understanding'of the triggers for and regulation of expression of particular genes and their effects on a protein and organism level.  v>  1.3 The context for design of thesis tasks and objectives The Liquid Waste Management Plan was prepared by the G V R D , adopted by all municipalities and the Greater Vancouver Sewerage and Drainage District Board in 2001, and approved by the Province of B.C. under the Waste Management Act in 2 0 0 2 W i t h the approval, a number of. conditions were imposed by the Province onto the G V R D , including the establishment of a program to study endocrine disrupting chemicals, persistent organic pollutants and other microcontaminants such as pharmaceutical drugs found in regional liquid waste, and their potential environmental impacts. "This should include, but not be limited to, effluent characterization to identify and quantify the contaminants and biological assays using new techniques such as gene chip arrays to determine their sublethal impacts." (Ministry of Water Land and Air Protection, G V R D Liquid Waste Management Plan)  A study program was initiated by the G V R D in 2002, and includes different aspects, institutions and research groups. Part of this current thesis became a review of to-date available techniques for toxicogenomics, such as gene chip arrays, their functionality, and an initial evaluation of their potential use for environmental monitoring (Manuscript I). Updates on some sections of this review are included in the introductory chapter of this thesis, because of the fast speed by which this area of research is developing. Concurrently, an environmental monitoring program was developed by the G V R D , which includes the use of wild and caged mussel species to assess environmental effects of effluents in situ in the Burrard Inlet. This program is a departure from traditional monitoring schemes, which rely on biological effects observed in fish and benthic invertebrates. Mussels had been widely  1  used in Mussel Watch Programs (O'Connor et al., 1995) and monitoring for pulp and paper mill effluents (Salazar et al., 1997; St-Jean et al., 2003), but have only recently been introduced to municipal effluent monitoring (St-Jean et al., 2004). A number of advantages over fish have led to that change, such as their more simple physical organization and hence assessment, their filter feeding habits, and their sessile nature. Lastly, in 2001 and 2003, two studies commissioned by the G V R D used TIE approaches to identify the cause of acute toxicity in effluents from some G V R D wastewater treatment plants (WWTP) (EVS Consultants, 2001; E V S Consultants, 2003). In the case of the Lions Gate WWTP, which is a primary effluent treatment plant located on the North Shore of the Burrard Inlet, linear alkylbenzene sulfonates (LAS) were isolated as the cause of the observed toxicities. Because some surfactants have been reported to be endocrine disrupting compounds (such as nonylphenol ethoxylates) it made sense to review current literature on the effects and the environmental fate of L A S . This was done within the specific context of the marine receiving environment and the environmental monitoring scheme involving bivalve species (see above). The results of that review are contained in the report to the Environmental Monitoring Committee of the G V R D (Manuscript II). In brief, L A S have not been shown to have endocrine disrupting effects themselves, but may enhance E D effects from other pollutants present in the effluent. Their removal from sewage effluent is relatively straightforward due to sorption to particles and aerobic biodegradation, and L A S should therefore be targeted when considering treatment plant upgrades.  With the above points in mind, thesis objectives were to investigate and evaluate the availability of certain gene sequences for environmental effects monitoring from the mussel species. It became clear that no D N A array was available for mussels and that, even i f it was available, the number of non-annotated sequences would not allow for useful evaluation of D N A array data in respect to receiving environment effects. Instead, the thesis opted to focus on a small set of genes where one could reasonably expect a connection between gene expression and observable effect. One particular aspect of mussel health that is affected by environmental conditions, the development of a neoplastic disease of the haemolymph, was chosen for further study on a genetic level. The thesis is integrated with the con-current G V R D caged bivalve study, which provides not just a supply of suitable exposed and control animals, but which also provides data  for potential future correlation analysis. In particular, the p53 gene family was chosen based on its frequent involvement in cancerogenesis, its relatively well-known function (in humans and rodents) and its frequent use for analysis of cancerogenesis by assessing mutational changes in the gene or measuring gene expression. The question became whether changes in expression of the p53 gene can simply be correlated to exposures to certain stress factors. A pre-requisite for answering this question must obviously be to determine the existence of a p53 gene (family) in the chosen mussel species. Future studies are required to address potential changes in expression levels of p53 in response to pollutants of concern in the G V R D environment (EDCs, LAS).  2 TECHNICAL BACKGROUND  Note: The following literature review is also presented in a compressed format in Manuscript III. 2.1 A brief introduction to bivalve molluscs and bivalve blood cells A typical bivalve mollusc has a shell, muscular foot, and a visceral mass containing the heart, gut, digestive gland, gonads and kidney. The body is enclosed in a mantle, which forms the mantle cavity filled with water in which the large respiratory organ (gills and plicate organ), anus, nephridiopore(s) and gonadopore(s) are located. The gills are not only used for respiration but more importantly for filter-feeding. Mytilidae have a fused gonad, where the gonad is fused with the mantel tissue. For an excellent and hands-on review of mussel anatomy the reader is referred to (Fox, 2001).  The well-developed blood vascular system is a semi-open circulatory system and consists of the heart and the vessels leading to the visceral mass. There are no capillaries joining the arteries and the veins; instead, haemolymph pumped from the heart into arteries eventually seeps into sinuses. From these spaces the haemolymph enters veins and eventually is carried back to the heart. Major sinuses include the renal sinus, the visceral sinus and the adductor muscle sinus (Cheng, 1981). The bivalve haemolymph is responsible for nutrient absorption, waste disposal, osmosis and defense against trauma (wound repair) and invasive organisms (Cheng, 1981). Haemocytes are the main cell type in the haemolymph. Surprizingly little is known about the origin of haemocytes in molluscs. The generally accepted belief is that haemocytes arise from  7 differentiation of connective tissue cells (Cheng, 1981). This, however, is in need of verification. There are also a variety of interpretations as to how many types of haemocytes occur in bivalve molluscs or as. to the ontogeny of these cells. Cheng reviewed the available literature and concluded that there are two categories of haemocytes common to all bivalves: granulocytes . (cells with large number of granular inclusions such as lysosomes, phagosomes, Golgi and mitochondria) and hyalinocytes (cells with less cytoplasm and no or very little granules). When granulocytes are spread out on a substrate (such as a glass slide) they produce spike-like pseudopods. These pseudopods play a crucial role in phagocytosis of bacterial and other infections.  2.2 Why mussels for receiving environment monitoring? Marine mussels {Mytilus edulis and Mytilus trossulus) have been used widely for marine monitoring purposes and contaminant accumulation studies, mainly in so called "Mussel watch programs" (Widdows et al., 1992), and have been shown to have a considerable potential for > assessment of sub-lethal biological effects. Mussels are amenable to a range of physiological rate measurements, which respond to a variety of contamination types. Their single-biggest advantage is likely that mussels are sessile filterfeeders. Both particulate (bacteria, algae) as well as dissolved (amino acids, sugars) nutrients are taken up by the submerged organism (Hawkins et al., 1992). It allows for easier cause-effect relationship evaluations in time and space when compared to more mobile species such as some fish. When cages are used instead of sampling wild populations, the side effects of caging the animals are also likely reduced when compared to other more mobile species. Mussels filter several liters of water per hour and are therefore directly exposed to contaminants (particulate and dissolved) present in the water column. They can tolerate various water depths, current conditions and salinities and can therefore be used to monitor different locations in respect to effluent plumes arid coastal geography. It was also shown that mussels have relatively low activity of enzymes systems capable of metabolizing organic contaminants, such as PAHs and PCBs (Livingstone et al., 1992).  Several mussel measurement endpoints are currently used. The most common are survival and growth, scope for growth (net difference between energy assimilation and expenditure, energy  8 available for growth and reproduction), the gonado-somatic index, and bioaccumulation of contaminants in shell and tissue. Physiological endpoints based on haemolymph samples-include phagocytotic activity, lysosome retention, protein, glycogen and lipid tissue concentrations.  2.3 Brief overview of the GVRD Caged Bivalve Program at the Lions Gate WWTP outfall Within the establishment of a Receiving Environment Monitoring Program and the development of bioindicators for monitoring effluent effects, the G V R D uses caged and wild bivalve populations to assess the potential effects and the causes of municipal effluent from the Lions Gate WWTP (Figure 1). This WWTP is serving a population of around 180,000 people, covers an area of more than 60 km via a separate sewer system and has an average dry weather flow of 80 M L D (million liters per day) (1999 data). The treatment process is primary; sludge is digested on site. The effluent discharges into First Narrows, under the Lions Gate Bridge and, depending on the state of the tide, either moves into the Inner Harbour or out to the Outer Harbour (Burrard Inlet). In addition to the Lions Gate effluent, these areas are also subject to the influence of the Fraser River. Other important influences on these receiving waters are freshwater discharges (Capilano River and other tributaries), spoil and dredged material dumping, fishing, shipping and industrial activities. Continuous and cyclical monitoring is required to adequately distinguish among these various factors. Year two of the program started in February of 2003 with the deployment of mussels atsix impacted sites and one reference site (Figure 1). Four wild populations (one as reference) are included in the assessment. Overall, the responses of three mussel species will be compared, M. edulis, M. trossulus, and M. californianus. Endpoints that will be assessed are survival, growth, reproduction, immunological endpoints (lysosome retention, reactive oxygen intermediates, phagocytotic activity, cell mortality), and assessment of haemic neoplasia. The mussels will be exposed for a minimum period of six months and up to one year. Mussels will be sampled bimonthly for a period of one year at all sites. A minimum of 30 mussels will be assessed at each sampling. The results of this assessment were not available at the time of this thesis and are therefore not included here. Mussels are contained in net socks that are suspended from frames attached to a chain at various depths, depending on sampling location  9 and anticipated plume depth. The cages are held in place by an anchor weight and an uplift buoy (Figure 2). 2.4 Leukemia in bivalves Recently, tests were developed to assess the progression of haemocytes in the haemolymph toward a disease called haemic neoplasia or leukemia. Haemic neoplasia or leukemia is a common disease in bivalves (15 species are known to be affected (Peters, 1988)) and transforms the haemocytes from a functional to a non-functional prolific state. The disease was first reported in Mytilus edulis by Farley (Farley, 1969). The neoplasia can be transferred to other organs, eventually leading to mortality among affected populations due to the multiple functions that haemocytes normally fulfill in healthy individuals (see above). Leukemic haemocytes are enlarged and take on a rounded appearance. They loose their ability to attach to surfaces or to actively phagocytose. Cells show an enlarged diffuse nucleus and the normal nuclei-cytoplasm ratio decreases (McGladdery et al., 2001a).  A variety of terminology has been used to describe the condition, such as haemocytic and haemic neoplasia, sarcomatoid proliferative disease, disseminated sarcoma, leukemia and others. The variable terminology results from the numerous terms that are used to describe vertebrate cancers, which are primarily based on their tissue of origin. The origin of the diseased haemocytes in bivalves is still unclear, largely due to the fact that the origin of normal haemocytes is still unresolved. Among the different authors there is alsodifferences in histological interpretations of different conditions as well as the potential true heterogeneity in the conditions among, or even within, different species. Leukemia is frequently used as a term because the malignant cells divide in the haemolymph and, as the disease progresses, invade solid tissues (Smolowitz et al., 1989; Harper et al., 1994).  10  s 1 CT  S*65O00mN 9  4-rrrh  -  SQ  > -o o CD  c  OJ  OS its u  a o o  ca  o c_>  a  D  s>  ft S3  £ S  >  —  OJ  —i  co  o  CUJ  u  0  a  3  CO  >  CO  3  E  D  OJ  _  1  W S  D  d ca  a  ft  OJ  c  o  c 3  S  co ft ca  -a  Ill ill  ax.  1  a & o a ft,  o M — id  1  _  >  XI o  3 _ 3  e  CO  OJ  a .  ca  60  O  ca  3G  ca c oj ca a co ca  o  co ca CD C <-!—1 5 ^  OJ  o  T3  M O CO CO  g  C" ft< 2 "§ 2 5  s  co ft  •S Ba « i  —  •:  aa =  3  1)  Ca  ao  on  o  _ —  CO  2  CO  _  a.  ca ft.  <—  •2  G  -1  —  c CO C  u __  CO  r  i s  ft  o  O  c  3 a  O  c  era  2 —•  w  -a -a  _•  CJ c_ ® — _ . 'no CO CD ca > C o  %1 —  V1DH030  —  o  ^  ca  ff  ft 2 ft. .S a ft  - s  Q -S -c ft ca  OJ  g  £ to oj O 3 3o -gc H oo o cj -o e 3 o .tt c OJ > a c £ ca  -  13  cd  0.IVH1S o o  5  id D  h I g* 'I '  1 1  I  111  I  111  I  1 11|•••t 11 I - • 11  11  marker buoy  70-80 m  chain  Duplicate frame top  Duplicate frame bottom  anchor weight  Figure 2: Schematic of the moorings deployed by G V S & D D containing duplicate mussel cages at various depths.  There are more or less distinct stages through which the disease progresses. Sequential haemolymph sampling of affected mussels held in aquaria demonstrated the progressive nature and fatal outcome of the disease (Elston et al., 1988a). In the early stages, morphologically transitional cells were observed with large pleomorphic nuclei with one or more nucleoli, yet retaining abundant cytoplasm capable of pseudopodial movement. Neoplastic cells in later stages were characterized by minimal cytoplasm with remnant pseudopodia and nuclei that were generally spherical, yet exhibited "blebbing" and fragmentation. The disease is fatal within 3 to 6 months, but remission has also been observed. The immune system of certain individual mussels is capable of recognition of neoplastic cells and can retard or reverse the advancement of the disease. This process includes the destruction of neoplastic cells and the formation, of a matrix of extracellular fibrous material and haemocytes. Diseased cells show the expression of a neoplasia-specific epitope on the cell surface (Reinisch et al., 1983) which has been used for  12 diagnosis of the disease in Mya arenaria (Miosky et al., 1989) and, recently, in M. edulis (StJean et al., 2004).  '  1  Elston et al. (Elston et al., 1992) have provided a detailed review on disseminated neoplasias in bivalves and the next paragraphs are largely a summary of that review as it pertains to Mytilus sp. and environmental influences that may contribute to its cause and progression.  The causes for the disease are multiple and it is not yet entirely clear which factor is the prevalent or perhaps initiating versus contributing cause. Environmental factors seem to play a major role, but there are also genetic and seasonal factors as well as infectious triggers (viral) and temperature effects. Clearly, there is a genetic basis for the susceptibility, as exemplified by the three main Mytilus species: the high susceptibility observed in M. trossulus, low prevalence observed in M. edulis and near absence or reports of disseminated neoplasia in M. galloprovincialis (Elston et al., 1992). Casson-Mannevy (Cosson-Mannevy et al., 1984) reported disease prevalences of 0.0 to 29.2% in British Columbia, Canada, where most mussels on nonexposed shores are M. trossulus. Bower (Bower, 1989) conducted field studies on the prevalence of the disease in M. edulis in British Columbia (it may'actually have been M. trossulus based on species distribution and currently accepted nomenclature) and concluded that it was a manifestation of ageing in a bivalve with a relatively short life span. However, other studies seem to reject that concept where infectious and progressive stages of the disease have been found in adult and juvenile clam populations, and Brooks and Elston (Brooks et al., 1991) found high susceptibility in young M. trossulus and the development of "resistance" in the older population through attrition of young susceptible animals and selection of resistance mechanisms.  Transplantation of neoplastic cells from an affected population to healthy population has been shown in a number of laboratory studies and may be of importance in dense populations with little water circulation. While two reports seem to show viral infection in Mya arenaria as a causative agent, no viral agents have been isolated from affected M. edulis (Elston et al., 1992).  Seasonality affects the prevalence of haemic neoplasia in M. trossulus. Elston et al. (Elston et al., 1992) combined the results of six studies in a bar graph, and it appears that haemic neoplasia  13 occurs year-round but has the highest prevalence in the months of November and December, followed by slightly higher than normal prevalence in the spring months. This may be due to a slower progression of the disease in colder water and therefore diseased animals will remain longer in the sampled populations. This hypothesis is confirmed by Bower (Bower, 1989) who reported that the disease leads to higher mortalities in warmer waters.  It seems clear from the studies of bivalves in polluted environments that toxicants and possibly other stresses can increase the prevalence and quite probably the rate of progression of neoplasia if the disease is already present in the population (Elston et al., 1992). Usually, one can observe a background prevalence in populations not (or less) affected by pollution of <5% (McGladdery et al., 2001a). Pollution may simply be an additional stress, rather than an initiating causative factor, on the immune system of the affected animal. This is supported by the notation that the disease exerts a metabolic drain on the energy reserves of affected individuals (Elston et al., 1992). The authors found that during the winter months (when food and energy are limited), individuals with advanced neoplasia consistently failed to show gametogenesis while normal mussels, or those in early stages of the disease, demonstrated active gametogenesis. In contrast,, in the spring and summer months (when food and energy are not limited), individuals with advanced neoplasia, but also showing gametogenic activity can be found. Metabolic studies provide evidence that compounds that are carcinogenic in mammals or fish may not necessarily be carcinogenic in bivalves. However, other compounds appear to be metabolized to proximate carcinogens by bivalves, thus providing a biochemical basis for chemical carcinogenesis.  Field studies by various authors during the 1970s as a result of numerous oil spills off the northeastern US coast (Searsport, ME) could not prove a cause and effect relationship between the oil contamination and various cancer mortalities in Mya arenaria. However, it was concluded that spilled refined petroleum products could be a contributing factor to the neoplasms (Elston et al., 1992). Farley et al. (Farley et al., 1991), studying the disseminated neoplasia in clams from Chesapeake Bay, reported a linear correlation between prevalence of the disease and tissue concentration of chlordane, a compound used in termite control and a known carcinogen. M i x (Mix, 1983) reported that the concentration of polynuclear aromatic hydrocarbons (or polyaromatic hydrocarbons, PAH) in the tissues of Mytilus edulis in Yaquina Bay, OR, were significantly greater at high neoplasia prevalence sites than at the low prevalence site, but also  14 noted that the result did not imply or suggest a direct causal relationship between P A H exposure and neoplasia. Cause and effect relationships can be validated in appropriately designed and tightly controlled laboratory exposure experiments. Gardner (Gardner, 1994) listed a number of studies where bivalve molluscs had been exposed to contaminated sediments containing PAHs, quinones, carbazoles and some heavy metals and developed neoplasms. Gardner also listed a variety of studies of in-situ exposure of mollusc species (including Mytilus) where tumors developed through exposure to environmental agents. Harper et al. (Harper et al., 1994) also observed a 60% prevalence of leukemia in clams from New Bedford Harbor, a site heavily contaminated by PCBs. In contrast, mussels from Puget Sound which have reportedly high prevalence of leukemia (Elston et al., 1988a) did not show a significant increase in the disease when they were fed PCBs and PAHs in their diet and consequently showed high body burdens of the contaminants (Krishnakumar et al., 1999). The analysis of over 8000 mussels for the National Oceanic and Atmospheric Administration's Mussel Watch project from approximately 80 West Coast and East Coast sites showed a significant correlation between haemic neoplasia and arsenic tissue concentrations (East Coast) and haemic neoplasia and PAHs (West Coast) (Hillman, 1993). Other contaminants were either weakly correlated or negatively correlated with haemic neoplasia. The differences between East and West Coast might be attributable to the difference in species abundance and their respective response to different contaminants.  2.5 Genetic changes during haemic neoplasia Of special interest in the context of this thesis are the genetic changes during the progression of haemic neoplasia in bivalves. A number of previous studies have researched changes in cellular D N A content, transformed genotypes and detection of gene expression and shall be reviewed here.  Farley (Farley, 1969) (in.(Elston et al., 1992)) first reported anomalies of mitosis in M. edulis with disseminated neoplasia. He noted that there were at least twice as many chromosomes in atypical cells as in normal cells. Elston et al. (Elston et al., 1990) studied the occurrence of 5n D N A levels compared to the normal haemocytes of diploid (2n) D N A content. In the later stages of the disease, characterized by lOn cells, the cells only had remnant pseudopodia and-large irregular and fragmented nuclei. The disease was usually fatal shortly after the appearance of the  15 lOn cells. Subsequently 4n/8n populations of diseased haemocytes were found, occurring in 29% of examined individuals, compared to 66% of 5n/10n individuals (Moore et al., 1991). Bower (Bower, 1989) also found an increased D N A level in Mytilus neoplastic cells.  Elston (Elston et al., 1992) reported the detection of genes of the ras and myc family by Southern analysis and expression of these genes in Mytilus disseminated neoplasia. Oncogenes fos, myb, and sis were not detected. The genes ras and myc are fairly well characterized oncogenes in humans. In mammalian cells, the highly regulated expression of Myc family transcription factors is closely tied to cell growth and proliferation as well as inhibition of terminal differentiation and induction of apoptosis. Deregulation of myc expression drives progression of many different types of cancer (Secombe et al., 2004). The transcription factor p53 (see below) is positively responsive to Myc (Nilsson et al., 2003). By contrast, expression of myc is required for proliferation; it can over-ride p5 3-induced Gl-cell cycle arrest by inducing an inhibitor of the cyclin kinase inhibitor p21 (Atkin, 2000). The latter normally coordinates S and M phases of the cell cycle. If absent, cells with damaged D N A arrest not in G l but in a G2-like state from which they can pass through additional S phases without intervening normal mitoses. The deformed polyploid cells that result may then die by apoptosis. The uncoupling of S and M phases may contribute to the acquisition of the chromosomal abnormalities manifested by most tumor cells when apoptotic pathways have been circumvented, as was observed by (Farley, 1969), (Bower, 1989), (Moore et al., 1991), and (Elston et al., 1990) in bivalves. Ras proteins are membraneassociated signal transduction proteins. The Ras protein regulates or signals cell division. In most situations the gene is inactive', but in malignant cells the Ras protein is active and signals cells to divide, even though they should not. It is normally only active in presence of a growth factor; however, mutated ras protein results in cell growth in the absence of growth factors.  In his detailed review, Elston (Elston et al., 1992) concluded that further studies on the occurrence and significance of oncogenes in disseminated neoplasia were needed. This thesis and future work based on it may contribute to a small extent to these studies.  16 2.6 The p53 family of tumor suppressors and their application as genetic biomarkers The p53 tumor suppressor gene was first discovered as a suspected oncogene by three independent research groups in 1979 (DeLeo et al., 1979; Lane et a l , 1979; Linzer et al., 1979). Its importance has been illustrated by many studies since which indicate that p53 is either mutated or lost in over 50% of human cancers (Hollstein et al., 1991). It has been termed the "gatekeeper of the genome" as well as a "network hub"(Vogelstein et al., 2000) because of its central role in the molecular networks that decide the fate of cellular life and death. As a transcriptional activator, p53 is normally turned "off, but it will spring into action upon damage to D N A by radiation or chemical treatments, hypoxia or activation of oncogenes. The p53 protein binds to the regulatory regions (promoters) of certain genes involved in cell growth arrest and .cell death in response to D N A damage. Thus, it will prevent cells from passing on the wrong D N A message potentially turning these cells into malignant tumors. p53 is at the intersection of many cellular stress response pathways that lead to either cell cycle arrest, D N A damage repair, apoptosis or senescence (Hofseth et al., 2004).  Since its discovery in humans, p53 homologues have been found in many species that are either of environmental or aquacultural interest. For instance, to my knowledge, the first nonmammalian p53 was isolated from aXenopus laevis c D N A library in 1987 (Soussi et al., 1987). Rainbow trout p53 was discovered in 1992 by cDNA cloning (Caron de Fromentel et al., 1992) and, based on this sequence, from a variety of other fish of this family in 1999 with specific primers to conserved regions of the p53 sequence (Bhaskaran et al., 1999). Screening of an adult zebrafish cDNA library resulted in the sequencing of additional fish p53 (Cheng et a l , 1997). Standard searches of genomic databases have identified similar genes in the invertebrates Caenorhabditis elegans (Derry et al., 2001; Schumacher et al., 2001) and Drosophila melanogaster (Jin et al., 2000; Ollmann et al., 2000), and very recently in Entamoeba histolytica (Mendosa et al., 2003). The first mollusc p53 originated from the squid Loligo forbesi (Ishioka et al., 1995) but has likely more similarity to a p63/73. Kelley et al. were able to isolate both a p53 and a p73 homologue in the bivalve mollusc Mya arenaria (Kelley et al., 2001). A detailed comparative analysis of the highly conserved functional domains in p53 ( Figure 3) as well as their respective functions and binding proteins are provided in Manuscript III.  17  Only in 1997, p73 and p63 were discovered as closely related homologues of p53 ( al., 1997; Yang et al., 1998). The amino acid identities of critical functional domains within the two recently found proteins are strikingly similar to those in p53 not just in humans, but across species: an acidic N-terminal transactivation (TA) domain, a highly conserved core D N A binding domain, and a C-terminal oligomerization domain (Figure 3). But just because proteins have a similar amino acid sequence does not mean they do the same job. Proteins p63 and 73, like p53, are also involved in growth arrest and cell death, albeit using different pathways (Figure 4). However, new studies demonstrate that, despite being evolutionarily conserved, the members of the p53 gene family have distinct, even antagonistic biological functions (Melino et al., 2002; Yang et al., 2002; Courtois et a l , 2004). p63 has been found to have crucial functions for the normal development of epithelial skin tissue and limbs, and stem cell differentiation. p73, on the other hand, has been found to be determinant of cellular differentiation and apoptosis (cell death) in neuronal tissues.  Both p63 and p73 (but so far not p53) have different structural variants expressing different regulatory functions: Two promoters (PI and P2) were found that yield two distinct classes of proteins. The PI promoter leads to isoforms showing T A domain with p53 homology. The P2 promoter gives rise to N-terminal-truncated (AN) isoforms with biological properties opposite to those of p63/p73 T A isoforms. A range of positive and negative feedback loops have been found between p53 and between the T A and A N isoforms of p63/p73 (Figure 4 and (Benard et al., 2003)). There have also been found three different splice variants of p63 and p73 at the protein level, a, f3 and y, and many more splice variants at the R N A level. Splicing refers to a process in which introns of a gene are removed and exons are joined to form a continuous coding sequence ofmessenger R N A , which is then translated to a corresponding protein sequence. Splice variants therefore lead to different proteins which may have different functions, further diversifying the regulatory possibilities for p63 and p73 (Cox et al., 2003).  18  p53  N  TAD  PXXP  ~ 25 % p63/73 N ANp63/ ANt>73  DBD  NLD  ~ 65 %  OD  ~ 35 %  C  % Identity  TAD  PXXP  DBD  NLD  OD  PXXP  SAM  PS  c  N  PXXP  DBD  NLD  OD  PXXP  SAM  PS  c  Figure 3: Comparison of the domain structure of p53 protein and the major isoforms encoded by p63 and p73. T A D , transactivation domain; P X X P , proline-rich regions; D B D , D N A binding domains; N L D , nuclear localization domain; OD, oligomerization domain; S A M , sterile alpha motif domain implicated in protein-protein interactions; PS, post-SAM domain implicated in transcriptional suppression; N , amino-terminal domain (truncated in the ANp63 and 73 isoforms); C, carbon-terminal domain. The S A M region can be differentially spliced to achieve further isoforms and functionality. % Identity refers to the percent amino acid sequence identity between p53 and p63/73. Adapted from (Yang et al., 2002; Melino et al., 2002; Benard et al., 2003).  This diversity of functions in the p53 gene family raises the question whether they are indeed good candidates for genetic markers in response to environmental stress. Evidence exists that they may very well be useful indicators, but there is no doubt that future studies will have to clarify which isoform of which family member is used best. Currently, p53 gene sequences have been put on gene chip arrays for salmonids (G. van Aggelen, personal communication), because it is expected that p53 is expressed (and therefore upregulated?) following D N A damage in cells, or because it is mutated in cancerous cells. Initially, mutated p53 protein was found in leukemic haemolymph cells of the soft-shell clam Mya arenaria (Barker et al., 1997) using an antibody specific to mutant p5 3. Subsequently, it was shown that normal p53 expression levels were unchanged in healthy and leukemic haemocytes. Interestingly, protein p73 was expressed in leukemic haemocytes but normal haemocytes (Kelley et al., 2001; Stephens et al., 2001). The proteins and genes were sequenced for both p53 and p73 in Mya arenaria and were shown to have a nearly identical core region with diverging 3' ends indicating that they may be splice Variants (Kelley et al., 2001). In another clam, Spisula solidissima (surf clam), another related  19  Morphogenesis, Epithelial development/ differentiation  Growth arrest  GD  Apoptosis  Neurogenesis, Neuronal development differentiation  Figure 4: Schematic p53 family member pathways. Besides specific developmental and physiological functions, p63 and p73 participate to p53 "genomic guardian" function. Upon DNA damaging stress the two homologues interplay with p53 to achieve growth arrest and apoptosis. p53 as well as TAp73 induce a direct activation (block arrows) of AN/?73 gene creating thus a feedback loop to control negatively these functions. Modified from (Benard et al., 2003).  Cell-cycle  Apoptosis  Development  +++  +++  -  TAp63  ++  +  +++  ANp6 3  -  -  +  TAp73  ++  ++  +++  ANp73  -  -  ++  arrest p53  Table 1: Summary of the functions of the different isoforms and family members of the p53 family. Adoptedfrom(Melino et al., 2002).  20 p63/73-like protein, pi20 was shown to be temporally expressed during early embryonic development and significantly suppressed upon exposure to PCBs (polychlorinated biphenyls) (Jessen-Eller et al., 2002). Because development of clam leukemia is influenced by anthropogenic disturbances in the environment (McGladdery et al., 2001b) and p73 expression is differential in normal and leukemic haemocytes in Mya (Kelley et al., 2001), it is worthwhile examining whether in addition to p53, or in conjunction with p53, p73 can be used as a genetic tool for the early detection of environmental stressors. Preliminary experiments have shown that ~63 and 73 kDa proteins are expressed approximately four times stronger in leukemic than in normal haemocytes in Mytilus edulis haemolymph (R. Stephens, C. Reinisch and R. Cox, personal communication) using an antibody elicitied against a 23 aa sythetic protein which includes part of the highly conserved D N A binding domain V (p53-73 specific antibody, (Stephens etal., 2001)).  2.7 Genomic Technologies for Monitoring of Microcontaminants with Potential Endocrine Disrupting Effects - An Update In April 2003, an internal report was submitted to the G V R D Policy and Planning Department and the thesis advisors reviewing to date available genomic technologies for monitoring microcontaminants with potential endocrine disrupting effects. The report is included in the Manuscript Chapter, Manuscript I, and also includes a brief description of the endocrine system, a list of EDCs, and effects of EDCs, which will not be repeated here. Since then, a number of updates relevant to this report were noted and are included in this section. The reader is advised to refer to the literature review first before reading the following updates.  1) Microarrays and Toxicology - Review of Recent Developments (S. cerevisiae array): The report (Manuscript I) described the use of Saccharomyces cerevisiae microarray to study shifts in metabolism and amino acid starvation. Interestingly, a similar array has now been used to look at toxic effects of the anionic detergents L A S and SDS (sodium dodecyl sulfate), which is of particular relevance to this thesis (Sirisattha et al., 2004), (Manuscript II). The purpose of the authors' study was to demonstrate whether microarray results correlated with previous findings of anionic detergent toxicity, and to develop microarrays for environmental monitoring. Upon treatment with the, detergents, cells were harvested, R N A extracted and hybridized to a  21 yeast D N A chip, and functional gene groups were analyzed for up- and down-regulation. Highly upregulated genes were often associated with membranes, indicating that L A S and SDS affect membrane structures and proteins. Other genes (PDR15) play a role in detoxification or cellular metabolism, organization and cell cycle control (MET30). Oxidative stress genes related to thioredoxin, glutathione and glutaredoxin were induced as well and indicate that L A S and SDS cause oxidative stress in yeast. Oxidative stress often damages D N A , and some D N A damage repair genes (such as IMP2, PHR1, and HSP12) were induced despite the fact that L A S and SDS are not considered mutagens. Genes related to cell signaling and to the transporters of the pleiotropic drug resistance network were induced as well indicating that L A S and SDS may be pumped out of the cells. A large number of changes in gene expression remain unexplained, either because the ORFs represented on the D N A array code for unclassified proteins or because ORFs within one functional group are both up- and down-regulated and their function is insufficiently known.  2) Microarrays and Toxicology - Review of Recent Developments (Fish D N A arrays): D N A arrays for rainbow trout, sheepshead minnow and largemouth bass were described in the report and are the prototypes for chips intended for aquatic environmental monitoring. This is based on the fact that fish have been used traditionally for environmental monitoring. A local project not mentioned in the report is the Genomic Research on Atlantic Salmon Project (GRASP) ( which is sequencing expressed sequence tags (ESTs) of Atlantic salmon and other salmonid species and constructing cross-species D N A microarrays with approximately 3,700 (Rise et al., 2004) and 16,000 cDNAs with potential application in conservation, health and environmental assessments.  3) A n updated critical evaluation: D N A microarrays have the potential to be powerful tools for high-throughput and complex assessment of alterations in gene expression levels due to an organism's exposures to environmentally relevant concentrations of endocrine disruptors. However, the current choice of D N A microchip for aquatic organisms is very limited (salmonids and other fish, frog and nematode), of which two (salmonids and frog) are based on local expertise based at Environment Canada. At the same time, Vancouver-based medical, university and private institutions have developed the capacity to work with gene chip arrays, mostly in the area of human cancer  22 research. Due to the only very recent development of these arrays, not many results have been presented to link some of the gene expression profiles with effects on the animal-level. Therefore, much validation work remains to be done using the model.animals for which the chips were designed. D N A microarrays do not eliminate the necessity to maintain and develop other test methods currently in use, including the use of exposed and reference animals. What species of animal is to be used (independent of the currently developed gene chips) determines the complexity of the tests that are necessary to link gene expression patterns with effects on the < animal- and population levels.  23  CHAPTER 2 - MANUSCRIPT CHAPTER  24  2.1  MANUSCRIPT I - Genomic Technologies for monitoring of microcontaminants with potential endocrine disrupting effects - a literature review  This literature review was prepared for the purposes of familiarizing with and evaluating current available technologies for using molecular biomarkers for the study and monitoring of endocrine disrupting chemicals. The review was distributed to the thesis advisors and the G V R D Policy and Planning Department. It created a backdrop against which thesis objectives were then formulated.  •  ;  25  GENOMIC TECHNOLOGIES FOR MONITORING OF MICROCONTAMINANTS WITH POTENTIAL ENDOCRINE DISRUPTING E F F E C T S - A L I T E R A T U R E R E V I E W  Submitted by:  Annette Muttray, UBC  Submitted to:  Susan Baldwin, UBC Paul van Poppelen, GVRD, Policy and Planning Department  Date:  10 April, 2003 tn  26  1 INTRODUCTION  During the last few decades thousands of tons of man-made chemicals (xenobiotics) have been produced and subsequently released into the environment. Some of these substances have been found to disrupt the function of the endocrine system and are called endocrine disrupting compounds (EDCs). The list of chemicals with EDC properties is growing, and currently over 500 chemicals are listed as "candidates" by the European Union (Pluygers et al., 2001). They are associated with a variety of adverse health effects in wildlife and humans. Wildlife is especially affected in its reproductive capacity, while the effect in humans has been shown to range from a deterioration of sperm quality and count, congenital malformations, immunosuppression and cancer (Nicolopoulou-Stamati et al., 2001). Rivers and aquatic organisms can be affected by estrogenic substances entering with the effluent from sewage treatment plants. Evidence for estrogenic activity of such effluents comes from the United Kingdom, Germany > Italy, The Netherlands, Sweden, United States, Canada, and Israel (Williams, 2003). Removal rates for natural and synthetic estrogens are relatively high (75-95%) in sewage treatment works, but other compounds, such as nonylphenols, are only thought to biodegrade incompletely. The concentrations of endocrine compounds in the effluent and the receiving waters were found to be highly variable (DeVito et al., 1999).  As the endocrine system regulates a wide variety of functions the hormone disregulation results in a wide array of effects. The effects of xenobiotics are believed to be due to their (1) mimicking the effects of endogenous hormones, (2) antagonizing the effects of normal, endogenous hormones, (3) altering the pattern of synthesis and metabolism of natural hormones, and (4) . . modifying the hormone receptor levels (Colborn et al., 1993). Observable health effects may also be a result of a combination of various endocrine disrupters acting in concert. The observed effects are often a result of an earlier exposure, at a time where the organism and its hormone receptors are more responsive to the hormone or the EDC (i.e. developing embryos). No clear dose-response, relationships exist for most of the EDC candidates or, as discussed below, inverse relationships exist between dose and effect. Some EDCs have been shown to have a large effect  27 at very low concentrations, while higher concentrations result in no effect or an antagonistic effect.  2  OBJECTIVES  The objective of a large part of the current scientific effort is to screen chemicals for their endocrine disrupting effects. To that end, bioassays have been developed which are based on mammalian cell cultures, receptor binding assays, methods measuring cell proliferation and gene expression. These assays have the advantage that they can be performed faster than in vivo assays using life test animals.  The objective of this literature review is to look at some of these assays and also at gene-based assays and evaluate whether they can be developed as tools to determine the effect of environmentally relevant concentrations of (unknown) EDCs in water and wastewater. Potential negative effects of EDCs cannot be averted if long-term in vivo assays are used for environmental risk assessments. Thus, fast and effective techniques are needed for predicting the long-term effects of EDCs. Particularly, Twill focus on the use of gene expression methods to detect sub-lethal concentrations of EDCs in samples. The use of D N A arrays and bioinformatics allows for the screening of a large variety of relevant genes that may be affected by the presence of hormones and EDCs, thus providing a potential for a high-throughput technology for preliminary screening of water samples.  3 BASIC REVIEW OF THE ENDOCRINE  SYSTEM  The endocrine system regulates the body's internal physiology and it's development throughout life and helps it to adapt to nutritional and other external environmental changes. A number of glands produce hormones, which first interact with specific high-affinity receptors in or on cells on target tissues. Receptor activation then initiates a cascade of other biochemical reactions to produce a specific response. The receptor is also the site of EDC binding.  28 The vertebrate endocrine system has three groups of hormones: hormones derived from tyrosine^ steroid hormones which are cholesterol derivatives, and the large and complex group of protein and peptide hormones. j  •  The current discussion of endocrine disruption somewhat implies that EDCs act mainly on steroid hormone system (although the denomination "endocrine system" is most commonly used), because wildlife seems to be affected especially in their reproductive capacity, in highly polluted areas. Similarly, the deterioration in human sperm quality and count, and the higher incidences in prostate and breast cancer in humans, and specific congenital malformations in wildlife can be traced to the disruption of the steroid system. Also, the reproductive capacity is often used as a non-lethal effects endpoint in environmental risk assessment. However, interactions of xenobiotics are not restricted to the steroid hormone group; interaction of xenobiotics resulting in the disregulation of the thyroid-derived hormone functions and of the immune system are also known. There is evidence that thyroid hormones play a significant role in the development of cancer (Pluygers et al., 2001) and are the driving hormones in amphibian development.  Although xenobiotics interact with proteins (for instance serum binding proteins) no reference was found which examined the interactions of EDCs with the protein and peptide hormone group, specifically.  Based on the current assumption that EDCs act mainly on the estrogenic, androgenic and thyroid-derived hormone system, DNA-based detection methods are a potential alternative to in vivo testing. This is because the steroids and the thyroid-derived hormones act on the cellular D N A (via a receptor). Their biochemical reaction cascade shall be described in more detail to help the reader evaluate the aspects relating to the viability and limitations of DNA-based assays.  Steroid hormones are a class of lipids derived from cholesterol. Steroid-secreting cells, unlike protein- and peptide-producing cells, do not store hormones in the cell ready for secretion, but synthesize hormones as required. Therefore, regulation of gene expression and hormone synthesis is crucial to the normal functioning of the steroid system. Examples include Cortisol and aldosterone, which are produced by the adrenal cortex, testosterone, which comes from the  29 testis, and progesterone and estradiol, which come from the ovary. Steroid hormones affect the carbohydrate metabolism, salt and water balance, and the reproductive functions. Small changes in the basic chemical structure cause dramatic changes in the physiological action of this group of hormones (Brook et al., 1996).  .  The hormones derived from tyrosine, such as adrenaline, noradrenaline and dopamine act as neurotransmitters and hormones. This class also includes the thyroid hormones, which are required for growth and development in the young and for basal metabolism. They bind to specific receptors in the nucleus and induce transcription of genes responsive to the thyroid hormones. Their concentration is remarkably constant and is dependent on the regulation by the hypothalamopituitary-thyroid axis, the storage of hormone in the thyroid follicles, the conversion of inactive precursors to the active hormone ("T3") and the binding of hormones to circulating serum proteins. The thyroid hormone receptors are members of the steroid-thyroid receptor superfamily and are acidic proteins associated with chromatin of the cell nucleus. Transcription activation of thyroid-responsive genes is brought about by the activation of a domain of D N A known as thyroid-hormone response element (TRE), which is located close to the promoter of the target gene. (Note: Thyroid receptors, unlike steroid receptors, do not form stable complexes with heat shock proteins and can bind to the TRE without being activated by their ligand.) In this way, the thyroid hormone can activate the transcription of the growth hormone gene in the pituitary, or can repress production of thyroid-stimulating hormone (TSH) (Brook et al., 1996). Because the effect of the thyroid hormone is a general increase in the metabolic rate of the organism, a great many genes may be under the control of the TRE. This in turn would make the study of the effect of the thyroid hormone, or EDCs that mimic the action of the thyroid hormones rather complex.  Both steroid and thyroid hormones are hydrophobic and can easily cross the plasma membrane of target cells and reach the nucleus where they are bound to specific complementary receptor proteins. (Some thyroid hormones require active membrane transport mechanisms.) The intracellular receptors are then activated, which means that they form receptor-hormone dimers and expose a D N A binding site (Figure 5). This binding site permits the dimer to bind-to short  30  Figure 5: Outline of the mechanism of the steroid hormone action. Modified from (Brook et al., 1996). Thyroid hormone action is similar. Free steroid is in equilibrium with that bound to serum binding-proteins (a), and it diffuses across the cell membrane (b). Binding to the steroid receptor protein R may occur in the cell cytoplasm or in the cell nucleus. The receptor looses its inhibitor protein (steroid receptors only) and the hormone-receptor complex forms a homodimer. The hormone-receptor dimer (c) interacts with chromatin, which is attached to the nuclear matrix structures. The hormone-receptor complex binds to a receptor site on the regulatory region of the D N A strand associated with that particular gene (d), the hormone-responsive element (HRE). This interaction influences the promoter region, which then permits D N A dependent RNA-polymerase to start transcription of the gene by separation of the two D N A strands to yield a specific messenger R N A (e). Post-transcriptional modification and exon splicing follows, and mature m-RNA leaves the nucleus (f). Peptides and proteins are formed by translation of the m-RNA by the ribosomes. Post-translational modification occurs to yield the final protein (g).  sequences of D N A inside the nucleus of the cell. In the case of thyroid hormones there may be mitochondrial sites of action as well. These receptor proteins belong to a closely related superfamily of receptors which all consist of hormone-binding, D N A-binding, and transcription-  31 activating domains. The receptor proteins may also contain an inhibitor protein (such as the heat shock protein 90, hsp90), which inhibits the binding of the receptor to the D N A in the absence of the hormone. When the hormone binds to the receptor, the inhibitory protein is released allowing the hormone-receptor complex to bind to the D N A . The complex binds to the D N A in a region upstream from the structural gene and its promoter region. There is a conserved nucleotide sequence which is present in all steroid- and thyroid hormone-regulated genes. These sequences of only a few bases, known as short-sequence-elements or hormone-response-elements (HRE), confer responses to specific hormones and are closely related to each other. The hormonereceptor complex bound to the H R E will suppress or enhance the action at the gene's promoter region. There, DNA-dependent R N A polymerase can bind and begin transcription of the . structural gene into m-RNA. Eventually, this m-RNA will be translated by ribosomes into peptides and proteins. The receptor-hormone dimers are usually homodimers. However, thyroid hormones and their receptors can also form heterodimers with, other receptors, for instance with retinoic acid receptors. This is thought to contribute to the specificity of tissue responses. Also, the promoter control of R N A polymerase may require binding of additional transcription factors (Brook etal., 1996).  The peptide and protein hormones should be mentioned here, although their action might not be affected directly by EDCs. The peptide and protein hormones differ considerably in size and function. Examples are insulin, glycoprotein hormones, gonadotrophins, thyrotrophin, folliclestimulating hormone, luteinizing hormone and others. Pre-hormone is stored in the Golgi complex and the secretory vesicles of a peptide-producing cell. The cell requires some stimulus before the stored pre-hormone is activated and released from the cell. The stimulus may be hormonal (Brook et al., 1996). Thus, while the regulation of gene expression arid synthesis of peptide hormones is independent of the action of hormones, the secretion of peptide hormones may be influenced by hormones and therefore EDCs. However, documented endocrine disruption is only based on the steroid and thyroid hormone systems.  32 4 T E L E O S T ENDOCRINOLOGY  The previous discussion is based on the mammalian endocrine system. However, the affect of EDCs on fish, amphibians, and other aquatic vertebrates and invertebrates is of particular interest. A detailed discussion on comparative endocrinology is beyond the scope of this review, but a short introduction to fish (teleost) reproduction shall be given to exemplify some of the differences.  While the overall teleost endocrine system is very similar to the mammalian endocrine system, it is in the steroid group where major differences are observed. In male fish, 11-ketotestosterone is the predominant hormone for secondary sexual characteristics and behavior, as well as gonadal development, while testosterone (the "male" hormone in mammals) is secreted by both sexes although its function is still far from clear (Klime, 1998). In female fish, testosterone is converted to estradiol. Estradiol, the major "female" hormone in fish is secreted by the developing oocyte and stimulates the liver to produce the yolk protein vitellogenin which is then incorporated into the oocyte and is responsible for the major increase in gonadal weight during recrudescence. It is important to note that male fish also have the estradiol receptor in the liver and are able to produce vitellogenin upon binding an estrogenic compound. Hence, some of the background vitellogenin production observed in male fathead minnows independent of E D C exposure (Cline, 2002). The liver is also a site where steroids are catabolized to metabolites with pheromonal activity important in signaling sexual status.  The embryonic determination of sex is much less clear in fish than it is in mammals.ln mammals, the sex is determined genetically by the X X or X Y combination of chromosomes. Female differentiation does not require any hormone and arises simply from a lack of testicular secretion. Male differentiation is caused by the development of testicular tissue (determined by the presence of the Y chromosome), which secretes an anti-Miillerian factor and testosterone which together destroy the female pattern of development and impose the male pattern. A failure to synthesize or secrete these hormones (or a decreased ability) at critical periods in development will result in either female development or incomplete male development. These effects are irreversible and cannot be changed by hormone administration at a later stage in life.  In fish, the X Y chromosomal pattern is far from universal. In some species, even temperature can determine the proportion of male and female offspring. Sex may also be reversed by administration of hormones during certain critical periods of embryonic development, and phenotypically reversed females can produce sperm which can be used to fertilize normal females and produce eggs which are genetically 100%. female. In some species, natural sex reversal occurs as part of a normal life cycle. Thus, there appears to be no straightforward correlation between feminization of fish and possible EDC effects in aquatic environments. -  5 L I S T S O F E D C S INTERFERING WITH S T E R O I D S OR T H Y R O I D - H O R M O N E A C T I O N  As mentioned previously, the list of candidates for EDCs contains over 500 substances and is growing almost daily. It would be impossible to list all of those substances and, for the majority of these, no information on the mode of action exists. The following list is extracted from Environmental Endocrine Disruptors by L.H.Keith (Keith, 1997). The author based his selection of substances on their inclusion of one or more priority lists (World Wildlife Fund Canada, Center of Disease Control and Prevention in Atlanta, US Environmental Protection Agency, and Colborn et al. "Our stolen future").  Suitability Compound  Class/Use  Action  for  DNA-based  Reference  techniques? Acenaphthene Alachlor  Aldicarb  PAH •  unknown  aromatic  unknown  herbicide carbamate insecticide  USEPA USEPA, CDC, WWF  h i g h l y t o x i c , depresses acetylcholinesterase  USEPA, WWF  activity unknown, c a r c i n o g e n ,  Aldrin  insecticide  acute  intoxication  related to central  EPA  nervous system synthetic Allethrin  pyrethroid, insecticide  unknown  EPA  goitrogenic, interference with triazine  Amitrole  t h y r o i d hormone  herbicide  synthesis causing enhanced TSH l e v e l s and  TSH gene e x p r e s s i o n o r TSHCDC, WWF secretion?  t h y r o i d cancer unknown, b u t Anthracene  r e p r o d u c t i v e , problems,'  PAH  lowered  EPA  fertility,  shortened l i f e s p a n hypothalamic Arsenic  heavy m e t a l  pituitary,  gonadal s t e r o i d biosynthesis supression  steroid biosynthesis  EPA  increased production of triazine  Atrazine  16-a-hydroxyestrone  herbicide  USEPA, CDC,  (estradiol metabolite)  WWF  l e a d i n g t o breast cancer aromatic fungicide Benomyl  and  anthelmintic,oxidizer  unknown  CDC, WWF  in  sewage t r e a t m e n t ! Benz(a)anthrazene  unknown, a n i m a l  PAH  unknown, e s t r o g e n i c ,  B e n z o ( a ) p y r e n e PAH Benzo(b)fluoranthene Benzo(k)fluoranthene  EPA  carcinogen  EPA, WWF  animal carcinogen unknown, a n i m a l  PAH  EPA  carcinogen unknown, a n i m a l  PAH  EPA  carcinogen  paper impregnation, beta-  wood  Hexachloro-  preservative, seed  benzehe  unknown,  USEPA, CDC,  stillbirths  WWF  fungicide,  waste incineration Bisphenol  Butyl  A  binds t o estrogen  plasticizer  benzyl phtalate  phthalate  plasticizer  Cadmium  heavy m e t a l  (receptor?) ^  MCF-7 proliferation assay •  estrogenic, breast  pituitary,  gonadal s t e r o i d biosynthesis supression  WWF USEPA, CDC,  cancer hypothalamic  USEPA/ CDC,  WWF steroid biosynthesis  USEPA, CDC, WWF  35 aromatic  Carbaryl  insecticide, by  used oyster  unknown  CDC, WWF  growers! insecticide,  Chlordane  pesticide,  wood  USEPA, CDC,  unknown, c a r c i n o g e n '  WWF  preservative cholinesterase i n h i b i t o r  Chlorpyrifos  insecticide  (neuroendocrine system  USEPA  toxicity?)  Chrysene Cypermethrin 2,4-D  PAH pyrethroid insecticide herbicide  unknown  USEPA  unknown  WWF USEPA, CDC, •  unknown  WWF  reproductive endocrine  DDD / DDT  insecticide  e f f e c t s , malformations,  USEPA, CDC,  t o x i c e f f e c t on a d r e n a l  WWF  gland  DDE  androgen b l o c k e r (and  insecticide  1,2-Dibromo-3- p e s t i c i d e ,  see DDD) •soil  chloropropane  fumigant  2,4-  disinfection,  Dichlorophenol  2,4-D  yes  USEPA, CDC, WWF  unknown  CDC; WWF  unknown  WWF  seed precursor,  wood p r e s e v e . Lake Apopka s p i l l ;  Dicofol  alcohol  analogue e l e v a t e s e s t r o g e n l e v e l s  of DDT, a c a r i c i d e and reduces t e s t o s t e r o n e  biosynthesis?  CDC, WWF.  levels  Dieldrin  persistent insecticide  unknown  USEPA, CDC, WWF  Di(2ethylhexyl)  plasticizer  unknown  WWF  phthalate Di-n-butyl phthalate  plasticizer insecticide,  Endosulfan  chlorinated hydrocarbon  Endrin Heptachlor  persistent insecticide ' insecticide  estrogenic,  stimulate  b r e a s t cancer c e l l s immunotoxic,  bacterial  mutagen, unknown . unknown unknown  USEPA,  WWF  USEPA, CDC, WWF USEPA, CDC, WWF USEPA, CDC, WWF  Heptachlor  degradation  epoxide  product  Indeno(1,2,3c,d)pyrene  PAH  unknown  CDC,  unknown  USEPA  hypothalamic p i t u i t a r y ,  Lead  heavy'metal  gonadal s t e r o i d biosynthesis  supression  steroid biosynthesis  WWF  USEPA, CDC, WWF  toxic, persistent  Lindane  immunosurpressant,  organochlorine  estrogenic,  insecticide  detrimental  possibly suitable  t o male r e p r o d u c t i v e ,  USEPA, CDC, WWF  system insecticide,  Malathion  unknown  acaricide  Managnese e f f e c t s such  fungicide,  Mancozeb  . as i n l e a d ,  and e t h y l e n e  Maneb  WWF  bisdithiocarbamat es  e t h y l e n e t h i o u r e a has e f f e c t on t h y r o i d •  steroid biosythesis?  CDC,  WWF-  (goiter) hypothalamic p i t u i t a r y , gonadal s t e r o i d biosynthesis  Mercury  supression,  methylmercury  heavy m e t a l  neurotoxin,  isa exact  steroid biosythesis?  USEPA, CDC,. WWF  mechanism f o r b o t h o r g a n i c and i n o r g a n i c Hg unknown  Methorny1  insecticide  unknown  CDC,  WWF  Methoxychlor  insecticide  unknown  CDC,  WWF  CDC,  WWF  fungicide,  Metiram  s w e l l i n g o f t h y r o i d and  ethylene bisdithiocarbamat es  Metolachlor  r a p i d reduction of i o d i n e - uptake s w e l l i n g of t h y r o i d ,  herbicide,  testicular  atrophy  EPA  unknown, adenoma o f  Metribuzin  triazone  l i v e r b i l e duct and  herbicide  p i t u i t a r y gland i n  CDC, WWF•  female r a t s stable  Mi rex  insecticide  and  unknown  CDC,  WWF  CDC,  WWF  CDC,  WWF  flame r e t a r d a n t  Nitrofen  herbicide  Parathion  insecticide  mutagenic, c a r c i n o g e n i c , mechanism unknown unknown  37 estrogenic  PCBs  and t h y r o i d  DSEPA, CDC,  e f f e c t s , b r e a s t cancer  WWF  impact on a d r e n a l ,  Pentachlorophenol  wood  preservative  t h y r o i d and p i t u i t a r y  USEPA, CDC,  functions,  WWF  carciogen,  immuno-srpressant  Pentachloro-  herbicide,  nitro-benzene  fungicide  Phenantharene Pyrene Simazine Styrene  unknown •  USEPA  PAH  unknown  USEPA  PAH  unknown  USEPA  unknown  USEPA  unknown  CDC  unknown  CDC, WWF  algae  control,  herbicide aromatic precursor herbicide,  2,4,5-T  chlorinated aromatic defoliant,  2,3,7,8-TCDD  waste  incineration, Dioxin  a c t s on a r y l hydrocarbon receptor,  isomer  carcinogenic  suitable  WWF  chlorinated terpenes,  Toxaphene  insecticide  and  unknown  CDC, WWF  pesticide, persistent  Trifluralin  herbicide, blocks  chlorinated  Vinclozblin  fungicide  production,  suitable  USEPA, CDC  hermaphroditis and  insecticide,  Zineb  decreases  testosterone  fungicide  WWF  t h e androgen  receptor,  aromatic  USEPA, CDC,  unknown  fluoro-aromatic  ethylene bisdithiocarbamat  sterility, malformations,  CDC, WWF  abortions  es thyroid, reduction i n  Ziram  carbamate pesticide  fertility,  sterility,  retardation of testicular zinc  • suitable  CDC,' WWF-  development, build-up  Table 2: List of suspected endocrine disrupting compounds, based on (Keith, 1997). W W F , World Wildlife Fund Canada; C D C , Center for Disease Control Atlanta; U S E P A , US Environrhental Protection Agency. •  38 The US EPA funded a report by the The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and The National Toxicology Program Interagency Center for the Validation of Alternative Toxicological Methods (NICEATM) in 2002. The study panel released a proposed list of 78 substances for validation of in vitro endocrine disruptor screening assays ( I C C V A M , 2002). The Committee emphasizes that this list is not a complete list of compounds suspected to have endocrine-disrupting properties, but is rather a list of substances used to validate test assays. Currently, studies should be underway for Tier 1 testing using in vivo and in vitro test methods to evaluate which of the 78 substances has the ability to bind to the estrogen (ER) and/or the androgen receptor (AR) or initiates transcription. Part of Tier 1 is to standardize receptor binding assays and a transcription activation assay for in vitro testing. The purpose of the in vitro assays is not to predict relevant biological in vivo reactions, but rather to provide mechanistic information that will be considered in conjunction with in vivo test results in a weight-of-evidence evaluation.  Interestingly, there is only little overlap between the two lists. This may be due to the fact, that L.H.Keith uses many "brand" names for pesticides which are not used by I C C V A M . He also includes most of the heavy metals which are not covered by the I C C V A M list. The I C C V A M list also provides a lot more detail on the current knowledge of E D C action on the molecular levels which is of particular interest here, while L.H.Keith provides descriptions of toxicological effects and results of in vivo studies.  6 W H I C H M O D E L C O M P O U N D TO U S E FOR D N A A R R A Y S T U D I E S ?  Compound Apigenin  Chemical Flavanoid, Phenol  Bisphenol B  Plavone,  ( N a t u r a l Product)  Diphenolalkane, Bisphenol, (Adhesive, intermediate,  Coumestrol  Class  Coumestan,  EPA strong  comments  ER a g o n i s t ,  being  considered for-  t e s t i n g i n . v i v o by US EPA ER a g o n i s t , b e i n g t e s t e d i n vitro by US EPA  Phenol Chemical Coatings)  Benzopyranol,  Coumarin; Ketone  (Natural c  ER  agonist,  being  vitro by US EPA  tested  i n vivo and i n  39 Product) Diethylstilbestrol  Stilbene,  Bezylidene,  Diphenylalkene  ER .agonist, b e i n g c o n s i d e r e d  for testing i n  v i v o by US EPA  (Pharmaceutical) 17a-Estradiol  Steroid, phenolic, Estrene  17(3-Estradiol  (None)  Steroid, phenolic, Estrene  (Pharmaceutical)  ER  agonist,  being  tested  in vivo and i n  yitro by US EPA strong  ER  antagonist, vitro  agonist, being  • AR  tested  agonist  in  and  vivo and  i n  by. US EPA, recommended as p o s i t i v e  c o n t r o l substance f o r t h e ER b i n d i n g and ER 1  TA a s s a y s Steroid, phenolic,  Estrone  Estrene 17a-Ethinyl  (Pharmaceutical)  Steroid,  phenolic  ( P h a r m a c e u t i c a l )•  estradiol meso-Hexestrol  Diphenolalkane, Bisphenol,  Phenol  s t r o n g ER a g o n i s t , AR a g o n i s t , b e i n g t e s t e d in vitro by US EPA s t r o n g ER a g o n i s t , b e i n g t e s t e d i n vivo and in vitro by US EPA and in vivo by OECD  s t r o n g ER a g o n i s t , t e s t e d i n vitro by US EPA  (Pharmaceutical)' n-Nonylphenol  A l k y l p h e n o l ; Phenol (Chemical  intermediate)  ER a g o n i s t  and a n t a g o n i s t ,  AR a n t a g o n i s t ,  b e i n g t e s t e d i n vitro "by US EPA and i n v i v o by OECD  4  -tert-Octylphenol  A l k y l p h e n o l ; Phenol (Chemical  Zearalenone  ER a g o n i s t , b e i n g t e s t e d i n vitro by US EPA  intermediate)  Resorcylic acid lactone, Phenol (Chemical  intermediate  ER  agonist',  belongs  included  i n validation  t o an u n d e r r e p r e s e n t e d  as ' i t  class of  substances  and N a t u r a l p r o d u c t ) Bisphenol  C2  Diphenolalkane, Bisphenol, (Chemical  Equol  Phenol  binds  s t r o n g l y t o t h e ER, but no in vivo  t e s t i n g planned  '•  •  intermediate)  Flavanoid,  Isoflavone,  ER a g o n i s t b u t no i n vivo t e s t i n g p l a n n e d  Benzopyran (Pharmaceutical) Estriol  Steroid, phenolic, (Estrene  Nafoxidine  ER a g o n i s t b u t no i n v i v o t e s t i n g p l a n n e d  pharmaceutical) Stilbene,  Triphenylethylene (Pharmaceutical)  b i n d s to_ER b u t no i n vivo t e s t i n g p l a n n e d  40 Phloretin  (3-Zearalenol  Flavanoid,  Chalcone,  weak  ER a g o n i s t  b u t no in 'vivo t e s t i n g  Phenol ( N a t u r a l product)  planned' • •  Resorcylic acid  ER a g o n i s t in vivo t e s t i n g p l a n n e d  lactone  (Natural product)  Table 3: A selection of compounds from the I C C V A M E D W G proposed substances list, based on their capacity to bind to the estrogen receptor (ER) and activate transcription of genes which are under the control of the ER. .  . The above compounds were picked from the I C C V A M E D W G proposed substances list, based on previous findings as to their ability to a) bind to the estrogen receptor using different assays and to b) be an agonist for transcriptional activation of the ER. As mentioned above there are compounds which bind to the ER, but will either mimic (agonist) or block (antagonist) the activity of endogenous estrogen. To evaluate a detection method it is potentially easier to detect the transcriptional activation of a gene and therefore the elevated presence of a gene product or messenger R N A than a decrease in transcriptional activation or decrease in product. The same selection could have been made for the androgen receptor, however, currently more studies focus on effects of estrogen-mimicking compounds.  7 CURRENT E D C ASSAY TECHNOLOGIES  Assay technologies are focused on the use of biomarkers. Biomarkers are defined as molecules, biochemical pathways, or cellular processes in experimental animals that change in response to contaminated habitats and are indicative of the exposure (Cline, 2002). They are tools that can be used to clarify, the relationship between exposure to a xenobiotic substance and disease. There are a number of biomarkers currently in use and the challenge is to standardize these biomarkers across different laboratories, habitats, species and endocrine disrupting chemicals and effects. The most common biomarkers have been reviewed by (Cline, 2002) for the Water Environment Research Foundation and other in vitro tests by (Zacharewski, 1997), and will only be summarized here briefly.  - 4 1 The gonadosomatic index (GSI) is based on the change of the ratio of the weight of the gonads to the weight of the body throughout the reproductive cycle. It is a simple inexpensive method and can be used to detect endocrine effects in both male and female fish and other organisms. Its limitation is the variability between different batches of fish or between experiments separated in time.  The most commonly used method is to measure* vitellogenin levels in plasma of male and female fish using ELISA (enzyme-linked imunosorbent assay). Estrogens induce the synthesis of. vitellogenin which is detectable for 4-6 weeks after exposure in male fish. Normally, vitellogenin levels are low in males, but ingestion of phytoestrogens, high density of female fish at the height of the season exuding estrogen into the water, and exposure to xenoestrogens can lead to an increased plasma vitellogenin level in males. Lower than expected vitellogenin levels in plasma of female fish can also be used as a biomarker for antagonistic EDCs and may be indicative of more serious adverse effects than enhanced vitellogenin production in males. However, it is essential to determine natural seasonal patterns of fluctuations of vitellogenin plasma levels in females before using the biomarker effectively. Before the protein vitellogenin is produced (approximately 3 days after exposure), its messenger R N A level increases in liver cells. The mRNA is short lived and peaks at about 48 hours after exposure and has been found to be a more sensitive measure than vitellogenin plasma levels. However, this biomarker requires further evaluation, especially the correlation between vitellogenin mRNA levels and vitellogenin protein levels (Cline, 2002).  Sex steroid levels in the plasma are another endpoint that has been used successfully in field and laboratory studies. Easy-to-use kits are available, however interpretation of results is complex, because many factors can contribute to changes in hormone levels, such as alterations in synthesis, secretion, metabolism, excretion and binding to plasma binding proteins (Cline, 2002).  Estrogen does not only induce vitellogenin production in the liver, but also the production of zona radiata proteins. The zona radiata is a membrane situated next the yolk of an ovum. The zona radiata proteins are produced in hepatocytes in response to estradiol (Oppen-Berntsen, 1992) and are transported in the blood plasma to the ovaries and incorporated in the chorion \  layer of the developing egg. The synthesis of protein Zrp-P appears to be more sensitive to low-  42 level estrogen exposure than vitellogenin. This assay had only been used by a limited number of laboratories.  Other proteins who's expression is controlled by the ER-hormone binding and which have been used in studies are: pS2, cathepsin D, prolactin, sex hormone-binding globulin, alkaline phosphatase, ornithine decarboxylase, prostaglandin F2a prostaglandin H synthetase activity, and induction of progesterone receptor levels (Zacharewski, 1992). Measurement usually involves ELIS A (as with the vitellogenin assay) or Western blotting (protein) or Northern blotting (RNA) and require species-specific antibodies for detection. False positives are likely as some of these proteins are also controlled by mechanisms removed from the ER. Several recombinant receptor-reporter gene assays have been developed over the last few years. They usually involve the combination of a reporter gene (firefly luciferase, chloramphenicol acetyltransferase, P-galactosidase, or alkaline phosphatase) with an estrogen-responsive receptor gene (such as progesterone receptor, pS2, cathepsin D, vitellogenin, complement C3, Or collagenase) (Zacharewski, 1992).  The Y E S (yeast estrogen screen) assay is one of the most common and developed in vitro recombinant receptor-reporter gene assays. Native yeast lack known endocrine receptors and can be grown in media devoid of steroids. The yeast Saccharomyces cerevisiae has been used to investigate hormone-receptor interactions. The estradiol receptor (ER) gene has been incorporated in the yeast's genome and has been coupled to a reporter gene. Upon presence and binding of and agonistic EDC to the expressed ER the yeast produces a red dye which can be quantified spectrophotofnetrically. Other constructs utilize p-galactosidase as the reporter. The system is highly sensitive (0.07 pM). If an antagonistic EDC is administered together with estrogen, inhibitory effects can also be measured. As with the measurement of vitellogenin mRNA, this assay does not directly measure effects of proteins that are normally linked to the expression of the E R gene. It does, however, measure the potential of a suspected E D C to bind to and inhibit or activate the ER. The assay was quickly established in two laboratories (Cline, 2002). Note of caution: Interferences were noted in several wastewater and river water samples, requiring additional method development to concentrate and/or process samples. Also, some EDCs (tamoxifen, diethylstibestrol) seem to react very differently in yeast assays compared to  mammalian cell assays. Cell wall permeability, strain-dependent differences, protein lysis mechanisms, and others have been suggested as possible factors to explain these differences.  Another in vitro assay is the cell proliferation assay, for example the E-screen. This screen uses ER-positive, estrogen-responsive MCF-7 or T47-D human breast cancer cell lines. It is one of the most sensitive in vitro assays (10 pg E2/ml) but has a long incubation period (6 days). The E screen compares the number of cells present after this incubation period in the presence or absence of alleged EDCs. It is based on three premises: i) Unidentified factors in the medium (human serum) inhibit proliferation of cells, ii) estrogens induce cell proliferation by negating the inhibitory effect of these unknown factors, and iii) non-estrogenic steroids and growth factors do not neutralize the inhibitory signals present in the medium. Standardization of this assay has been challenging because, the proliferation of human breast cancer cells seems also to depend on differences between cell line clones, culture conditions, receptor level differences, differences in sera used, and cell density. One group of EDCs, phtalate esters, has been reported to stimulate T47-D cells, but not MCF-7 cells.  The strength of in vitro transcriptional activation assays is finding E D C contamination based on ligand binding and protein expression. They are limited by their inability to predict in vivo responses elicited by an exoestrogen. Especially, recombinant in vitro assays (where receptor and reporter genes are transferred to other species such as yeast) cannot predict in vivo effects. Even where non-recombinant assays are used (vitellogenin plasma levels) there is no data indicating that exoestrogens exhibit consistent tissue- and species-specific responses. In vitro assays also do not account for bioaccumulation and interactions involving the induction of binding proteins such as sex hormone binding globulins that may modulate the uptake and metabolism of steroids. Likewise, gene imprinting, where ligand binding exposes the organism to disease in a later stage of life, cannot be resolved by in vitro assays currently.  It is generally understood that no single assay will resolve the potency and environmental effect of a particular EDC. Instead, results from screening-type assays will have to be confirmed with in vivo models at a subsequent study, For instance, once binding and transcriptional activation or blocking has been established using screening assays, effects on the animal- and population-level should be investigated.  44  8 B A S I C PRINCIPLES O F G E N E C H I P A R R A Y S  Among emergent in vitro assays for the detection of EDCs are gene chip arrays. They are influenced by similar limitations as other in vitro assays, but have the potential of offering a more automated and therefore more standardized approach to E D C evaluation.  Gene chip arrays, or microarrays, or D N A arrays, function like biological microprocessors, enabling the rapid and quantitative analysis of gene expression patterns across a large number of genes or gene groups and across various biological models. Small glass chips contain thousands of genes which are used to examine fluorescent samples prepared by labeling messenger R N A (mRNA) from cells, tissues and other biological sources. Molecules in the fluorescent sample react with cognate sequences on the chip causing each spot that contained this sequence to glow with intensity proportional to the activity of the expressed gene. Patterns of gene expression correlate with function, and the capacity of the array allows for the analysis of entire genomes, or parts of genomes across a variety of species. The technology is very versatile which explains the popularity and speed of development in this field.  Microarrays are generated by either printing presynthesized copy-DNAs (cDNA) (500-2000 bases) or synthesizing short oligonucleotides (20-50 bases) onto glass microscope slides or membranes. c D N A is synthetic D N A reverse-transcribed from mRNA which has been extracted from tissues or cells. Thus, c D N A is a copy of genes that have been expressed in a cell. That in turn means that gene chip arrays do not eliminate the necessity of test or sample animals from which the mRNA has to be extracted first. Once a slide has been obtained, fluorescently-labeled c D N A from samples is hybridized to the slide ( Figure 6). Differential gene expression measurements are achieved by competitive simultaneous hybridization using a two-color fluorescent labeling approach, or comparison of two labeled samples hybridized on different chips. One may compare, for example, two different tissues, treated versus untreated. The slides are then scanned and the images are processed. Scanned images representing the two samples are then overlaid using specialized image-processing software that assigns a color intensity to. the corresponding amount of fluorescence. The ratio of intensity of one sample compared to the other is used as a measurement of whether a gene is  45 significantly different (i.e. induced or repressed) in one sample from the other (Lobenhofer et al., 2001).  As a result, researchers are able to pinpoint which gene or potentially groups of genes are either upregulated or downregulated due to the effect of the treatment with an EDC. Detailed doseresponse studies are necessary, has been shown that EDCs can do both, up- of downregulate genes, depending on their concentration and time of exposure. Again, the changes in pattern of gene expression subsequently have to be related to actual effects on the animal- and population level before microarrays can be used as a monitoring tool.  Treated Population  Control Population  T Cy3 dye o o ° OG o  m-RNA isolation  Reverse Transcription  r  1  Cy5 dye El  Mix'c-DNAs and apply to array  I  Data Processing  Figure 6: Simplified overview of the method for sample preparation and hybridization to c D N A chips.  46 Microarrays are a tool for the analysis of large numbers of genes. Where a smaller number of target genes are of interest, other methods may be more appropriate. Instead of reversetranscribing the entire mRNA into cDNA for hybridization to a microarray, targeted species of mRNA can be transcribed using sequence-specific primers. This reverse transcription can be carried out in a quantitative manner using "real-time" RT-PCR which allows for the quantification of the original mRNA present in the tissue. Thus, mRNA of known indicator proteins can be analyzed in a less complex manner and perhaps more easily correlated to known effects on the animal level.  9 G E N E CHIP A R R A Y S AND TOXICOLOGY - REVIEW OF R E C E N T DEVELOPMENTS  Gene chip arrays have been used in the areas of oncology, molecular basis of infectious disease, cellular biology and development, drug discovery and, last but not least, toxicology. We have seen the advent of a new discipline, called toxicogenomics (Nuwaysir et al., 1999), a combination of the fields of toxicology and genomics, which is concerned with the identification of potential human and environmental toxicants, and their putative mechanism of action, through the use of genomics resources. The principal hypothesis underlying toxicogenomic studies is that chemical-specific patterns of altered gene expression can be revealed using high-density microarray analysis on the tissues from treated organisms (Lobenhofer et al., 2001).  The assessment of risk associated with different chemical exposures is limited partially by difficulties relating to cross-species extrapolation and dose response as well as estimation of exposure levels (Lobenhofer et al., 2001). Current in vitro models have to be sufficiently close to humans (i.e. rodents, canines, monkeys) to be able to extrapolate findings to humans. Environmental risk studies have to be tailored toward key species, either aquatic or terrestrial depending on the objectives of the study. This makes screening studies very expensive and timeconsuming. Using, for instance, human genome microarrays may circumvent problems associated with cross-species extrapolation. Using a chip with a number of responsive genes from key fish or other aquatic species may aid a comprehensive risk assessment. Currently, there is only a limited inventory of expression profiles reflecting the response to chemical treatment in biological models. Also, as seen in Table 2, there is limited information about the molecular  47 effects of xenobiotics. Thus, one application of whole-genome microarrays is the discovery of novel toxicant-induced gene expression alterations. Simpler arrays can be made for simpler target organisms such as recombinant yeasts which contain genes for endocrine receptors. They can easily be grown in the lab in media containing the sample water, have a fast generation time and routine R N A extraction protocols.  So, what kinds of arrays are out there?  High-density microarrays for human, mouse and rat have been used in drug testing (Grier et a l , 2001) and work is expanding to include environmental toxicants. Laboratories, such as the National Institute of Environmental Health Sciences in Research Triangle Park, N C (focusing on human toxicology), Oak Ridge National Laboratory, T N , and the US Army Engineer Research and Development Center in Vicksburg, M S , (focusing on stress response gene expression) monitor pollutant impacts using microarrays. The National Center for Biotechnology Information maintains many databases and resources for use by scientists working in biomedical research including toxicology (  The lab of E.F. Nuwaysir at Triangle Park has developed the ToxChip vl.O which contains 2090 human genes selected for their well-documented involvement in basic cellular processes as well as their responses to different types of toxicants, such as genes responsive to D N A replication and repair, apoptosis genes, genes responsive to PAHs and dioxin-like compounds (12 genes), estrogenic compounds (63 genes), oxidant stress genes, some oncogenes, tumor suppressor genes, and house-keeping genes (for signal normalization) (Nuwaysir et al., 1999). When a toxicant signature of a known toxicant is determined it can later be compared to signatures from unknown toxicants. The lab is also working on a ToxChip v2.0 and chips for other model systems, such as rat, mouse, Xenopus and yeast.  Affymetrix (Santa Clara, CA) offers custom-made and routine chips for yeast, the worm Caenorhabditis elegans, rat (toxicology), mouse and human. Andrew Watson et al. described in detail the process of array creation and printing, sample processing, hybridization, detection and software applications (Watson et a l , 1998). (However,  48 the design of a new array is time- and labor intense and involves a number of interacting disciplines such as bioinformatics, genetics, toxicology and others.) The nematode worm C. elegans has been used for a long time in developmental and toxicology studies. It is therefore not surprising that after the genome had been completely sequenced, a D N A microarray was created for C. elegans and used for environmental monitoring and identification of possible endocrine disrupting chemicals (Custodia et al., 2001). Notable is that C. elegans has 228 nuclear receptor genes (sites were EDCs bind), while only 48 are known in humans. C. elegans also has a fast life cycle and its embryonic development has been well studied. In addition to D N A array studies, the researchers also looked at the production of vitellogenin when exposing the worms to estrogen, progesterone and testosterone. Unfortunately, regulation of vitellogenin production in invertebrates is not as well understood yet as it is in vertebrates. However, results suggest that 10" M estrogen decreases vitellogenin, and 10" M and 10" M increases vitellogenin production, but the microarray only showed an increase in vitro 5  gene expression at 10" M estrogen. Other genes examined in this preliminary study were 5  cytochrome P450, glutathione s-transferase, metallothionein, and heatshock proteins. The authors conclude that C. elegans microarrays have a good potential for environmental monitoring but concede that their work is very preliminary.  A number of D N A microarrays containing genes from the yeast Saccharomyces cerevisiae have been used for toxicological studies, with compounds such as the pesticide thiuram, heavy metals (cadmium), the alkylating agent methyl methanesulfonate (MM), the herbicide sulfometuron methyl (SM), hydrogen peroxide, menadione, DDT, and diamide. Results will be reviewed here briefly. E. Kitagawa and co-workers (Kitagawa et al., 2002) used a chip with 6000 open reading frames from D N A Chip Research, Inc. (Yokohama, Japan) to look at the effects of the pesticide thiuram [bis(dimethyldithiocarbamoyl)disulfide] (75uM) on gene expression. They found that i) the number of genes induced or repressed by thiuram increases with the time of exposure, and ii) the affected genes belonged to a wide variety of functional groups (such as D N A repair, cell growth, detoxification, stress response, a.o.). Four genes were selected based on their expression ratios on the arrays and used to make a promoter-GFP construct for use as a bioindicator for thiuram. A group at DuPont Central Research (Jia et al., 2000) monitored the expression of 1,529 yeast genes after treatment with S M . S M is not characterized as an endocrine disruptor, but an  49 inhibitor of acetolacetate synthase, a branched chain amino acid biosynthetic enzyme. Thus, results are not directly applicable, but they show the complexity of responses to chemical stressors on the genetic level. After a 15 min exposure, lower and higher concentrations of S M resulted in similar expression profiles (induction of 125 and repression of 80 genes). At the lower S M concentration, the level of induction and repression lessened as a function of time.Tn contrast, at high concentrations, no such relaxation was observed and the change in expression profile became more pronounced. This indicates, that at lower concentrations, yeast cells adapt to the new conditions and adjust the initial gene expression profile accordingly. At higher concentrations, effects caused by the depletion of amino acids and the accumulation of ketoacids in the cells are observed, such as induction of D N A damage repair- and oxidative stress response genes.  1  The heavy metal cadmium is a strongly suspected EDC (EPA listed) and has been used as a model compound to evaluate a yeast microarray bioassay (Momose et al., 2001). A concentration of 0.03 m M inhibits growth of the yeast, while cell death was observed at 1 m M . A n exposure of 0.3 m M of cadmium chloride for 2 hours was chosen by the group for the array studies. A wide variety of different genes related to cell death, stress response, sulfur metabolism, transport were either upregulated (310 genes) or downregulated (322 genes). Because the yeast used was not engineered to have steroid hormone receptors, the effect of cadmium as an E D C could not be observed. A global array of the S. cerevisiae genome has also been used to investigate the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration (DeRisi et al., 1997) and during amino acid starvation (Natarajan-et al., 2001). Similarly, several different stress factors, some of chemical nature, have been investigated using a yeast array (Gasch et al., 2000). It was found that some sets of genes (stress response) respond in a stereotypical manner to many different environmental changes, whereas the response of other sets of genes is unique to specific conditions. It was also found that (at a cell viability of 80% upon exposure) most changes in gene expression are of transient nature. Thus, there is a quite large knowledge base for the use of yeast arrays in baseline and in toxicology studies.  Lobenhofer and co-workers (Lobenhofer et al., 2001) used a microarray containing 1700 rat genes and mRNA extracts from the liver of exposed rats to successfully look for chemicalspecific patterns of gene expression. However, only two classes were tested: peroxisome  50 proliferators and barbiturates. In his paper, Lobenhofer describes in more detail the use of bioinformatics for image analysis, data acquisition, data processing and multivariate analysis. Another mammalian gene chip (mouse) was used by Bartosiewicz et al. (Bartosiewicz et al., 2000) to look at the expression patterns of a subset of 148 genes relevant to toxicology (xenobiotic metabolism, D N A repair, stress proteins, cytokines, and housekeeping genes) upon exposure to beta-naphthoflavone (flavones are currently being tested as EDCs by the USEPA). The study demonstrates that less complex arrays with spot replication are a more useful approach to toxicological studies than global gene expression arrays, and that animal-to-animal variability is generally greater than variability associated with spotting, hybridization and data acquisition associated with the gene chips. The test animal was a mouse model. Mice were fed with various doses of beta-naphthoflavone and mRNA was extracted from the liver at time intervals up to 29 hours after exposure. The spot-to-spot variability was 10-20%, and replicate slides yield data with an overall coefficient of variance of 14%. Dust on slides increased the variability dramatically underlining the need for a clean working environment. The study also illustrates that dose-response relationships have to be established first before data can be interpreted in a meaningful way. For instance, metallothioneins were induced at some doses, but seem to be repressed at other doses. The study also showed that the labeling efficiency of the Cy5 dye is . about 40% less than that of the Cy3 dye, and that controls have to be labeled with both dyes to establish a baseline before determining induction or repression in treated samples. A key question remains of whether the induction or repression of genes is sufficiently sensitive so that changes at environmentally realistic exposures can be detected. The same array was used in a subsequent dose-response study using cadmium chloride, Benzo(a)pyrene (BaP) and trichloroethylene (TCE), of which the first two are suspected EDCs (Bartosiewicz et al., 2001). However, the compounds were chosen based on their differing classification as heavy metal, polycyclic hydrocarbon and oxidant stressor, and to examine whether a toxicant-focused array can provide distinct patterns of gene expression for separate chemical classes. The data supported a proof of concept that the three different contaminants elicited unique patterns of gene expression over the doses tested. Cautionary notes are again presented, because i) increasing doses of cadmium elicited a change in expression patterns and ii) although the effect of benz(a)pyrene is facilitated via the aryl hydrocarbon (Ah) receptor, a number of genes which are under control of the A h receptor were not upregulated as was  51 expected. Currently, the group is testing the chip with additional chemicals from the same classes of chemicals as a basis for studying the potential additive or subtractive effects of mixtures.  There are not too many D N A arrays for aquatic studies available at this point. The Center for Animal Functional Genomics at Michigan State University makes D N A chips available for general use by scientists. According to Paul Coussens, Associate Professor and Director of the Center for Animal Functional Genomics, a chip for rainbow trout may be available by summer/fall of 2003. (So far, the focus of the center has been bovine and swine gene chips.) The Pacific Northwest National Laboratory, under the lead of Dr. D.P. Chandler is proposing to use a random nonamer (9 bases oligomers as probes) microarray to fingerprint different species of salmonids . Locally, the Symposium on Endocrine Disruptors: Mechanisms and Impacts in Vancouver (July 2002) unearthed different gene chips specifically designed to detect genetic effects of EDCs in fish. For instance, chips for sheapshead minnow and largemouth bass were designed with 30 or 132 EDC-responsive and constitutive genes, respectively (Denslow et al., 2002).'Booth and co-workers (Booth et al., 2002) presented work on the use of a 150 gene-chip for rainbow trout to investigate expression profiles after treatment of juvenile trout with 50, 100 and 250 p.g/1 of nonylphenol. To reduce the fish-to-fish variability the chip was also used to hybridize R N A extracted from hepatocytes in primary culture exposed to estradiol, nonylphenol and beta-sistosterol. However, no results were presented in the abstract (Wiseman et al., 2002). The macroarray with 132 largemouth bass genes (see above) was also used to investigate the up- and down-regulation of various genes after injecting adult male bass with 50 mg/kg of nonylphenol (NP) or a combination of the estrogen-mimic NP and the antiestrogen ICI-182-780. The steroidal ICI-182-780 reduced the up-regulation of the alpha estrogen . receptor due to NP, but did not affect the expression of the vitellogenin gene. The question remains, of course, if the chip is sensitive enough for environmentally relevant doses and exposure pathways.  Another gene chip applicable to aquatic environments is a chip designed to address the effects of thyroid hormone mimicking compounds on the frog Xenopus laevis (Crump et al., 2002). Binding of thyroid hormone to the T H receptor is an absolute requirement for the induction and repression of genes involved in tadpole metamorphosis. The widely-used herbicide acetochlor 2  .52 accelerates the T3-dependent metamorphosis and it was proposed to use the gene chip " M A G E X array" 3 containing 420 known frog genes to determine the effects of acetochlor. It is crucial to use tadpoles at a stage where they are competent to respond to thyroid hormone, but are functionally devoid of the hormone (athyroid) themselves. This will ensure that possible compounding effects of natural thyroid hormone is eliminated from the test. Tadpoles were exposed to 10 n M (concentration detectable in some US streams) of acetochlor in the presence and absence of the hormone T3. Based on the results (Table 4) the following was concluded: Acetochlor generally accelerates frog metamorphosis. Shorter exposure times may not result in morphological alterations, but can result in an increase in T H receptor levels as measured by quantitative PCR. Despite a clear association between T H responsiveness and acetochlor action for many genes, not all T3-regulated genes are targets of acetochlor. Reduction in the rate of T3 inactivation by a deionidase enzyme due to acetochlor exposure may also manifest itself as an acceleration of metamorphosis. Although not all of the gene expression profile can be explained  Premetamorphic  Precotious  (premature) metamorphosis (T  (no T )  added)  3  Treatment  Acetochlor alone  TRa mRNA  not  affected  TR(3 mRNA  not  affected  Gene Expression  Morphology  3  T not  3  alone  T  3  affected  + Acetochlor increased  increased  increased  11 down-, 1 up-regulated gene a f t e r 48 hours o f exposure  10 T - r e s p o n s i v e genes u p r e g u l a t e d 8 T - r e s p o n s i v e genes n o t a f f e c t e d 6 genes where T d o w n r e g u l a t i o n i s a t t e n u a t e d 9 genes n o t r e g u l a t e d b y T a r e u p r e g u l a t e d  modest decrease' i n body a r e a a f t e r 72 h o u r s of exposure  reduced body area and s c u l p t u r i n g o f head  3  3  3  3  much r e d u c e d b o d y a r e a and a c c e l e r a t i o n o f metamorphosis  Table 4: Summarized results for the effects seen after exposure of Xenopus laevis to 10 n M acetochlor. Results are based on (Crump, 2002). Levels of T R mRNA were measured by quantitative PCR, while gene expression profiles were based on the M A G E X gene array.  3  Viagen X Biotech Inc. homepage at (accessed May 5, 2003)  .53  currently studies are underway to further elucidate the TH-dependent signaling in X. laevis (Veldhoen et a l , 2002). Acetochlor was also reported to inhibit binding of 17 beta-estradiol to rat uterine estrogen receptors, indicating that the herbicide can target multiple endocrine pathways.  There are several sites in the synthesis, transport and metabolism of THs that can be altered by xenobiotics. In addition, it is possible that xenobiotics can alter T H signaling through the T H receptor (TR) either by directly binding to the TR or indirectly by altering phosphorylation of TR or through interaction with accessory proteins. Unlike the estrogen receptors, there is little evidence of environmental chemicals binding to the TR, but the hypothesis has not been adequately tested. Because of the complexity of T H function and regulation, it is unlikely that a single test will be available to detect chemicals that act on any or all of the pathways in all or some species. Thyroid hormone receptors have only been cloned in one species of fish, two species of frogs, in chickens and in mammals. A variety of screening methods for thyroid hormone disrupters exist for mammalian species, but no validated methods are available for aquatic species. The tadpole metamorphosis assay, is an indirect assay that can be influenced by other environmental or endogenous factors, and is in need of validation (DeVito et al., 1999). Because many differences exist between the mammalian and teleost or amphibian thyroid hormone system, tests that are available for mammalian species cannot easily be adapted to nonmammalian species. For instance, PCB's which are efficacious in decreasing plasma or serum T4 levels in rodents have little effects on plasma T H in fish.  10 F A C T O R S  WHICH  INFLUENCE  HORMONE/EDC  ACTION  BEYOND  THE  TRANSCRIPTIONAL L E V E L  Assays based on receptor binding and transcriptional regulation cannot predict directly, how an E D C affects the entire animal. This is due to other factors beyond the receptor/transcriptional level that influence EDC action and may limit genomic technology application. Some of those factors are synergistic between different EDCs or other non-EDC contaminants, protein factors in the serum of the animal, additional transcription factors, supply of ligands due to other enzymatic activities, concurrent "natural" expression and action of hormones, timing of exposure, latent effects in development, threshold levels, and others. Assays such as yeast-based  54  receptor-reporter expression, are wholly inadequate to address these factors. Assays based on aquatic model organisms (with subsequent hybridization to microarrays) indirectly address these factors because the organism itself is part of the assay and possesses the serum binding proteins, uptake mechanisms, transcriptional regulation of target genes etc. which, all influence the final measurable outcome in the form of mRNA. However, D N A microarrays cannot present information about translational activities, i.e. whether the differentially increased (or decreased) concentrations of mRNA are actually translated into functional proteins within the cells. Therefore, microarrays must be complemented with proteomics techniques to become more meaningful on the cellular and animal level.  55  REFERENCES  . Bartosiewicz, M . , Trounstine, M . , Barker, D., Johnston, R., Buckpitt, A., 2000. Development of a toxicological gene array and quantitative assessment of this technology. Archives of Biochemistry & Biophysics 376, 66-73. 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Technol. 37, 1744-1750. Wiseman, S., Vijayan, M . , 2002. Endocrine disruption in rainbow trout hepatocytes: A c D N A microarray study. In: Vijayan, M . , Hontela, A., MacKinlay, D. (Eds.), International Congress on the Biology of Fish, vol. American Fisheries Society, Vancouver, B C , 29-39. Zacharewski, T., 1997. In vitro bioassay for assessing estrogenic substances. Environ. Sci. Technol. 31,613-623.  58  2.2  II - Fate and effects of linear alkylbenzene sulfonates in the aquatic environment MANUSCRIPT  This report, with minor channges, was submitted to the thesis supervisors and the Environmental Monitoring Committee of the G V R D in Novermber 2003.  59  F A T E AND EFFECTS O F LINEAR A L K Y L B E N Z E N E S U L F O N A T E S IN T H E A Q U A T I C E N V I R O N M E N T  Annette Muttray,  PhD, RPBio  Department of Civil Engineering, University of British Columbia, 2324 Main Mall, Vancouver, British Columbia, V6T 1Z4  Abstract: Linear alkylbenzene sulfonates (LAS) are commonly used surfactants in detergents and household cleaners. They have been shown to have acute and chronic toxic effects on aquatic species. A wide range of literature covers the environmental occurrence, toxicity effects, natural (bio)degradation and wastewater treatment aspects of L A S . Some of the literature is a very comprehensive review in itself aimed largely at policy and risk assessment, and the reader is advised to refer to these reviews for a more in-depth coverage of all available data on L A S . This review introduces the reader to basic aspects of L A S pollution and frames the L A S discussion as it pertains to. current local (Vancouver, British Columbia, Canada) municipal wastewater issues. Therefore, special emphasis is placed on the behaviour of L A S in the marine environment and how the blue mussel, a genus used extensively for environmental.effects monitoring, and other bivalves are affected by the presence of L A S in the environment. Keywords: Linear alkylbenzene sulfonates, Mytilus, bivalves, sediment, marine environment  1. Introduction  The Greater Vancouver Regional District (GVRD) operates several primary wastewater treatment plants. The primary effluents have been shown to be acutely toxic from time to time using standard test methodology. EVS Environment Consultants Inc. produced reports for the G V R D in 2001 and 2003 (EVS Consultants, 2001; EVS Consultants, 2003) in which they conducted Toxicity Identification and Evaluation (TIE) (Ford, 1992) studies and concluded that a large part of the toxicity is due to anionic "methylene blue-active" substances (MBAS). The M B AS analysis effectively measures the concentration-of anionic surfactants in the water. The MBAS-associated toxicity was highest in C18 column fractions eluted with 85-90% methanol which represent the less polar fractions of M B A S compounds. The most common M B A S associated compounds are linear alkylbenzene sulfonates (LAS). Dodecyl benzene sulfonate is the most commonly used surfactant in industrial detergents and cleaners, although commercial detergents are often a mix of different L A S molecules. Currently, the G V R D is developing a receiving environment effects monitoring program using caged bivalves as well as reasonable interim treatment options for the most toxic components of the primary effluent. It is of importance to be able to evaluate the effects L A S may have in the marine environment and in particular on the test species. Therefore, the objective of this report is to review available literature on the behaviour in the environment and toxic effects on some marine species of linear alkylbenzene sulfonates.  2. What are linear alkylbenzene sulfonates?  Linear alkylbenzene sulfonates (LAS) are, by volume, the most important group of synthetic anionic surfactants in use today. It has been estimated that developed countries use 5.5 g per person per day of L A S (Rubio et al., 1996) resulting in an annual production of 2,400,000 tons globally (Gonzales-Mazo et al., 1997). They consist of an alkyl chain attached to a benzene ring in the para position to the sulfonate group. Sometimes, toluene, naphthalene and xylene are used instead of the benzene. The homologue distribution in commercial products covers alkyl chain length from Cio to C n . The benzene ring may be attached to any of the C atoms from C2 to C6 (referred isoforms). A summary of the chemical L A S data, as well as L A S occurrence and  61 effects in the environment can be found in Madsen (Madsen et al., 2001). This comprehensive technical review was issued by the Danish Environmental Protection Agency and some parts of this literature review will be based on its analysis. However, because wastewater treatment plants in Europe are mostly secondary, the focus of the European literature is largely on L A S degradation products attached to sludge and applies to the Vancouver scenario only to some extent. For instance, while Madsen et al. report concentrations of L A S in the effluent of seven representative municipal sewage treatment plants in the Netherlands between 0.019 and 0.071 mg/1, Vancouver data show a M B A S concentration of 1.6 to 4.0 mg/1 in the tested effluents.  3. Environmental fate of LAS  Generally, the fate of a substance in the environment depends on its chemical structure and the prevailing environmental conditions. Substances can accumulate in the environment (either in their original form or in some partially degraded or altered form), they can bioaccumulate or bioconcentrate in organisms, they can be completely degraded by physical, chemical or biological pathways, or they can be transferred from the receiving water column to sediments by attachment and settling, or volatilized to the atmosphere. L A S are considered non-volatile organics (Vejrup et al., 2002) and therefore loss of L A S from the aquatic environment due to volatilization can be disregarded. However, it may be conceivable that the exposure of L A S laden sediments to air (draw-down zones of reservoirs, tidal estuaries) and subsequent mobilization of dust by wind action will contribute to the dispersal of L A S . No study was found investigating this proposition and it is likely that this route of dispersal is of only very minor importance. The common pathways by which L A S are transported and transformed in the aquatic environment will be reviewed here.  3.1. Sorption Although quite water-soluble, L A S consistently attach to particles due to their surfactant nature and their stable ionized SO3" functional group. Therefore, some of the L A S in municipal wastewater will attach to primary sludge and thus be removed from the primary effluent. However, enough L A S remain in the primary effluent to contribute to a large part of the toxicity (EVS Consultants, 2001; E V S Consultants, 2003). Hand and Williams (Hand et al., 1987) systematically investigated the relationship between chain length and benzene ring position and  62 sorption to river sediments and found that sorption increases with increasing alkyl chain length and as the benzene position approaches the end of the chain. Sorption and desorption were rapid and reversible. This is important because although initial sorption will render L A S unavailable to some organisms it will be released over longer periods of time from sediments as the dissolved fraction decreases in the water column due to degradation. Also, the longer more toxic chains will persist in the environment much longer due to their preferred attachment to particulate matter. The sorption partition coefficient varied between 3 to 26,000 1/kg, as river sediment types, chain lengths and benzene ring positions were changed. A similar study was conducted with marine sediments and similar L A S concentrations in 1996 by Rubio and co-workers (Rubio et al., 1996), however, methodology and results were quite different when compared to the 1987 study. For instance, Rubio concluded that L A S sorption to marine sediments was irreversible. O f importance also is the conclusion that the sorption coefficient increased with increasing salinities (partially explaining why L A S sorption may be irreversible in marine sediments). Vives-Rego et al. (Vives-Rego et al., 1987) have reported that the half-life of L A S in seawater is two to three times as long as in fresh water, up to 10 days. This was attributed to the association of L A S to Mg  2 +  and C a  2+  ions in the seawater which reduces L A S bioavailability due to precipitation.  3.2. Particle dispersion Because L A S tend to attach to suspended particles, particle transportation mechanisms are of importance in the dispersal of L A S . Gonzales-Mazo (Gonzales-Mazo et al., 1998) evaluated L A S dispersion and removal in the Bay of Cadiz (Southwest of Spain), a shallow estuarine area subject to strong tidal currents and receiving untreated wastewater of about 100,000 inhabitants. A vertical concentration gradient was found close to the outfall that showed a strong sorption of L A S at the water-atmosphere interphase. No such gradient was observed at more distant sampling stations where the flow regime of the narrow bay is more turbulent. The percentage of L A S associated with particulate matter increases in line with the distance of the sampling stations from the point of discharge, reaching 60% at the farthest station (17 km). The evolution of L A S concentrations in the sediments is very similar to the L A S concentrations in the water column. Although C M chains are of small quantities in the original commercial L A S mixture, they were found at elevated concentrations in the sediments, confirming their slower biodegradation (and higher toxicity) potential.  63 Pollutants can also be supplied to coastal environments via riverine input. The flux of detergentderived pollutants to the Tokyo Bay was studied in detail by Takada and co-workers (Takada et al., 1992). The authors describe that L A S mixtures also contain small amounts of non-sulfonated benzene alkyl derivatives (LAB) which are more toxic, less biodegradable, and more hydrophobic than L A S . As a result, riverine input of the two structurally similar substance groups is quite different and shall be described here briefly. Untreated wastewater is the source of L A S and L A B to the Tamagawa River. Their concentration and flux were measured at a dam approximately 15 km before the river enters into the Bay of Tokyo. It was found that when the river flow increased during/after rainfall, the suspended L A B concentration significantly increased due to resuspension of sediments containing L A B . The dissolved L A B concentration at low flows were 2-3 times higher than the suspended concentration, whereas at high flow the dissolved L A B concentration was insignificant compared to the suspended L A B concentration. Flush events contributed about 74% of the annual L A B flux. In contrary, dissolved L A S concentration represented up to 99% of the total L A S at low flow, and flush events did not dramatically increase the flux (only 4% of the annual flux was carried during flush events). L A S concentrations are lower in the summer months than in the winter months due to higher biodegradation rates in warmer water. L A B concentrations, however, did not change with the seasons because L A B is not readily biodegraded. It was also found that the contribution of L A B by the Tamagawa River to the Bay of Tokyo accounts for what could be expected from the population living in this watershed. L A S concentrations in the bay, on the other hand, were much lower than what would have been expected from riverine inputs. This confirms that L A S is degraded during transport in the river. Another interesting finding showed that higher concentrations of L A B were present in the dissolved phase than predicted from the octanol-water partition coefficient ( K ) . This was attributed to colloid-LAB association (such as humic and ow  fulvic acids) and/or solubilization of L A B by surfactants, such as L A S present in the river water.  3.3 Biodegradation in the environment Of the degradation mechanisms, biodegradation plays a predominant role in affecting persistence of L A S in the environment (WHO, 1996). Basic principles of L A S biodegradation were described in detail by Schoberl (Schoberl, 1989). The initial biodegradation step is a co-oxidation of the terminal methyl group of the alkyl chain to form a carboxylic acid. The alkyl chain is then step-wise shortened by p-oxidations. The resulting short-chain sulphophenyl carboxylic acid is  64 further degraded by aromatic ring oxidation and desulfonation leading to assimilation of L A S into bacterial biomass, C 0 , water and sulfate, to an extend of 45-84% (Schoberl, 1989). Both 2  initial and ring oxidation require molecular oxygen. Anaerobic L A S mineralization (degradation to C O 2 ) is therefore not possible, or occurs only at very slow rates, and has not been shown to occur in the environment to date (Madsen et al., 2001). Anaerobic primary degradation ( M B A S reduction) may occur in the environment as it has been shown to occur in continuous stirred tank reactors with anaerobically digested sludge (Angelidaki et al., 2000; Madsen et al., 2001). Aerobic biodegradation involves a variety of enzymatic mechanisms and it is thought that different species or genera of bacteria are involved in the catabolism of the L A S molecule. Since commercial L A S is a mixture of different isoforms, one has to bear in mind that not all isoforms are degraded with equal efficiencies and rates. The "distance principle" (Swisher, 1978) applies, in other words, L A S isoforms that are degraded most rapidly are those where one methyl group is farthest from the sulfobenzene configuration. One can therefore expect an enrichment of isoforms in sediments which contain the benzene ring at a position larger than C2. Terzic et al. (Tefzic et al., 1992) also noted that commercial "linear" alkylbenzene sulfonate mixes can contain branched benzene sulfonates which have a strong negative effect on biodegradation due to steric hindrance of the formation of the substrate-enzyme complex. As L A S isoforms degrade to various intermediary products, depending on their initial chemical properties, the potential toxicity of these intermediaries has to be considered to properly evaluate environmental effects of L A S on the environment. While the longer chain length and more terminal phenyl isomers are the more toxic components of L A S , they are also biodegraded at faster rates than short-chain and less-terminal phenyl isomers. LC50 values (lethal concentration at which 50% of the exposed population dies) for fish for C12 and C14 isoforms increased from 3 and 0.6 mg/1, respectively, to greater than 100 mg/1 after biodegradation in laboratory activated sludge units (Swisher et al., 1964). The initial decarboxylation of the terminal methyl group reduces most of L A S toxicity and surfactancy (Kimerle et al., 1976). There is a clear correlation between acute toxicity to Daphnia magna and the extent of biodegradation of L A S (Kimerle et al., 1976). A considerable volume of literature in the 1960's and 1970's is devoted to hazardous effects of L A S to marine organisms, but will not be reviewed here further (see Table 5 for a list of toxicity data). When surfactants are discharged into the sea, the high ionic strength of the medium causes a fall in their critical micelle concentration and consequently their solubilities are greatly  65 reduced (Quiroga et al., 1989). As a result, L A S accumulate close to outfalls and present a potential threat to benthic communities, which are of great importance in marine food chains. In general, sediments contain more bacteria than the water column which, as shown above, will lead to an enhanced degradation of L A S if those sediments are aerobic. Biodegradation was shown to be dependent on temperature (Quiroga et al., 1989): There was hardly any biodegradation at 5-10°C, while at 25°C 90% of the surfactant disappeared 15 days after the start of the sediment incubations. Terzic et al. showed that the biodegradation rates were two times lower at 14°C (0.106-0.192 day ) when compared to 23°C (0.180 day to 0.303 h" ). 1  1  1  4. Toxicity and Bioconcentration in Bivalve Mollusks  One of the most comprehensive reviews of chemical data of L A S and other commonly-used chemicals has been published by the International Programme of Chemical Safety (IPCS) (WHO, 1996) and the Canadian Centre for Occupational Health and Safety (CCOHS). IPCS Inchem is a "means of rapid access to internationally peer reviewed information on chemicals commonly used throughout the world, which may also occur as contaminants in the environment and food. It consolidates information from a number of intergovernmental organizations whose goal it is to assist in the sound management of chemicals." Extensive toxicological data can be found for fish and daphnia, and a range of other invertebrates (Table 5). I will focus this review on the effects of L A S exposure observed in bivalve mollusks. Bivalves have been used in environmental effects monitoring for effluent from pulp and paper mills, and are now introduced as a monitoring tool for municipal effluent of some wastewater treatment plants in the Greater Vancouver Regional District. Because L A S have been shown to be a major component of the toxicity of the primary effluent in the G V R D area, the effects of L A S on bivalves is of special interest. %  .  Versteeg and Rawling (Versteeg et al., 2003) caged mollusks, fish, and crustaceans in the  stream of a C 1 2 L A S containing model ecosystem and assessed bioconcentration and chronic toxicity. Test concentrations were 0.15 to 3.0 mg/1 of C 1 2 L A S under flow-through conditions. Total L A S bioconcentration factor (BCF) for the Asiatic clam (Corbicula fluminea) ranged from 9 to 33 and was affected by exposure concentration and phenyl position. B C F values increased with decreasing exposure concentration arid in the more external phenyl isomers. This is  66 Species/Group  Ranges o f t o x i c i t y  data  Microorganisms  0.5: - >1000 mg/1 .test systems)  Freshwater a l g a e  1 - 100 mg/1 EC50 ( v a r i o u s p e r i o d s and species)  Freshwater green algae  .10-235 mg/1  Freshwater b l u e algae  5-56 mg/1  Freshwater  1.4-50 mg/1  diatoms  EC50 ' ( v a r i e t y  Secondary Reference of  exposure  ( C - C ) EC50 10  (WHO, 1996)  ( C . - C ) EC50 6  (Madsen, 2 0 0 1 ) (WHO, 1996)  14  ( C n ^ - C ^ ) EC50  n  (WHO, 1996)  (WHO, 1996)  1 3  Freshwater macrophyte s  2.7-4.9 mg/1  Marine a l g a e  1 - 1 0 periods  mg/1 EC50 . ( v a r i o u s and species)  exposure  (Madsen, 2 0 0 1 )  Daphnla magna  1 - 1 0 mg/1 LC50 ( v a r i o u s p e r i o d s , d e p e n d i n g on c h a i n  exposure length)  (Madsen, 2 0 0 1 )  Daphnia magna  3-6 mg/1 LC50 ' ( C . ) 1.2-3.2 mg/1 NOEC ( C . )  (C  1 L 8  )  u  EC50  worms insects Marine  (WHO, 1996)  8  u  Freshwater invertebrates: . molluscs crustaceans  (WHO, 1996)  8  (WHO, 1996)' 4.6-200 mg/1 (C13) a c u t e LC50 0.12-27 mg/1 (C11.2-C18) a c u t e LC50 0.2-10' mg/1 NOEC (C11.8) 1.7-16 mg/1 (C11.8) a c u t e LC50 1.4-270 mg/1 ( C i - C ) a c u t e LC50 0  invertebrate  15  1 .- >100 mg/1 LC50 (C12) f o r 13 species' 0.025-0.4 mg/1 NOEC ( C . )  (WHO, 1996)  Acartia tonsa (marine c r u s t a c e a n )  0.5 - 2  (Madsen, 2 0 0 1 )  Fathead minnow  a p p r . 1 mg/1 LC50 (48/96 h, 21/30 d, depending on.chain length  (Madsen, 2 0 0 1 )  Freshwater  2-15 mg/1 LC50 (c'n.s) 0.48-0.9 mg/1 NOEC ( C . )  (WHO, 1996)  1-6.7 mg/1 LC50  (WHO, 1996)  n  fish  mg/1 LC50  (8 d, 48 h)  n  Marine  fish  • Chironomus  riparius  Bivalvia Unio elongatulus Anodonata cygnea  8  8  (C . ) n  8  319 mg/kg s e d i m e n t NOEC 993 mg/kg' s e d i m e n t LOEC 2.4 mg/1 NOEC f o r s o l u b l e LAS  (Madsen, 2 0 0 1 )  (Madsen,_ 2001) 182.5 mg/kg s e d i m e n t 96h-LC50 200 mg/kg s e d i m e n t 96h-LC50  67 Table 5: Range of toxocity values for a variety of species or groups of organisms. Sensitivity to L A S is generally higher in marine organisms, but depends largely on carbon chain length and test system used.  consistent with the direct relationship between L A S B C F values and hydrophobicity. Acute (4 day) LC50 were >3.0 mg/1 associated with a >0.078 mmoles/kg acute LBB50 (body burden at which 50% of the population dies, here, L A S tissue concentration). Chronic toxicity (body length, 32 days) was at 0.27 (0.204-0.352) mg/1 EC o (effective concentration in water that 2  reduces the biological endpoint 20% compared to control levels) and 0.035 (0.0310 - 0.0405) mmoles/kg EBB2o(effective body burden concentration at which the biological endpoint is reduced 20%). The chronic EC20 value for Corbicula is among the lowest chronic toxicity value available for C 1 2 L A S . It may be due to the ability of bivalves to detect the presence of surfactants at low concentrations and reduce their siphoning. This behavioral response reduces feeding resulting in slower growth. Bressan and co-workers (Bressan et al., 1989) investigated the effect of dissolved L A S and L A S associated with sediments on the European mussel Mytilus galloprovincialis and other invertebrates. Chronic toxicity was assayed by growth and it was found that growth, expressed as mean length of the major axis of the shell was not significantly different at 0, 0.25, and 0.5 mg/1. However, when growth was calculated as increment of the major axis, the effect of the individual variability is suppressed and a significant reduction (P<0.001) in growth is observed in treated animals. Oxygen uptake and retention of neutral red (NRRR, a measurement of the filtration rate) showed a significant decrease, while no effect was detected on the nitrogen (ammonia) excretion rate. When L A S was added attached to sediment (40-45 mg/kg suspended solids with approximately 280 mg/kg of L A S , 7-day treatment) no significant effects on oxygen uptake and nitrogen excretion rate were detected, but a slight increase in N R R R was observed. The authors concluded that when compared with other test animals, M. galloprovincialis was more sensitive to L A S than freshwater clams. However, the effects of saltwater on L A S solubility, hydrophobicity and bioavailability were not discussed. In general, L A S have a higher effect on bivalve eggs and larvae than on adult animals. Granmo (Granmo, 1972) found that the fertilization and growth of eggs were affected at L A S concentrations as low as 0.05 mg/1. The length attained by the larvae was affected by concentrations of 0.1 mg/1. Hansen et al. (Hansen et al., 1997) explored other chronic effects such as swimming, grazing and growth of larvae in the laboratory and settling and population  68 growth of larvae in mesocosm experiments. Swimming behavior was altered at 0.8 mg/1 L A S , grazing was reduced 50% at 1.4 mg/1, and the specific growth rate was reduced by 50% at 0.82 mg/1 over 9 days. During mesocosm experiments, the larval population showed a dramatic decrease in abundance within 2 d at concentrations as low as 0.08 mg/1 due to reduced settling. Larvae also showed^delayed metamorphosis and reduced shell growth. The authors hypothesized that the observable effects were due to the damage by L A S of the larval ciliary apparatus (see below). Bivalves filter particles from the water and use digestible particles, such as microalgae, as a food source. To more fully understand the chronic effects of a pollutant on an organism it is of importance to consider the effect this pollutant has on the organisms primary food supply. Consequently, what is the effect of L A S on microalgae in the marine environment? Generally, microalgae have been shown to be more sensitive to L A S homologues in toxicity tests than other organisms such as mollusks, crustaceans or fishes. Moreno-Garrido (Moreno-Garrido et al., 2001) investigated several microalgae and found a wide range of EC50 and N O E C 5 0 values in growth inhibition tests depending on the species of microalgae used for the tests. The range of EC50  was between 0.3 to >2 mg/1 for commercial L A S . C13 L A S homologues were usually an  order of magnitude more toxic than C | 1 homologues. The authors noted that it is known that euryhaline microalgae (can live in a wide range of salinities) are more resistant to toxicants than stenohaline microalgae (exist only within a narrow range, of salinity). It is reasonable to assume that L A S will have an effect on (the more sensitive) microalgae-food. In order to take this effect into account for receiving environment monitoring studies, one has to consider the species composition at the monitoring sites as well as the standing (algal) biomass crop to determine whether food limitation may have an influence on bivalve growth, development, and reproduction. L A S do not occur in a vacuum in the environment, but co-occur with a range of other pollutants or naturally occurring substances. The physiological effects of L A S on a tissue and cellular level can be interpreted either as a direct effect of L A S alone or an effect of L A S plus other substances. This is partially due to the fact that L A S , as a lipophilic surfactant, will damage cellular membranes by intercalation into the membrane matrix and solubilization and substitution of the component lipids and proteins (Hansen et al., 1997), thereby allowing other substance to cross the membrane boundary into the cell more easily. Other physiological effects of L A S are  69 changes in enzyme activities and tissue structures in organisms (Blasco et al., 1997). In particular the inhibition of esterase activity has been a useful endpoint in assessing L A S toxicity. In fish, damage to the gills has been observed. In the presence of L A S and heavy metals, an increase in cadmium transfer through perfused gills of Salmo gairdneri has been noted (Part, 1985). Significant difference in the thinning of the epithelium in the digestive tubule of mussels exposed to cadmium and L A S , in comparison with those exposed to L A S alone, has been reported (Da Ros et al., 1995). On the other hand, the antioxidant enzymes SOD (superoxide dismutase), catalase, NAD(P)H-DT-diaphorase and G P X (glutathione peroxidase) did not show any significant differences between exposed and non-exposed mussels (Gupta et al., 1989; Da Ros et al., 1995). Blasco et al. (Blasco et al., 1999) showed that copper and lead were bioaccumulated in smaller quantities in the clam Ruditapes philipinarum exposed simultaneously to L A S and metal than when the clams were exposed to the metal alone. This is opposite to behaviour seen in fish gills. The authors hypothesize that the observed 6-time increase in mucus production by the clam digestive tract and gill when exposed to. copper and the subsequent loss of mucus to the water assists in regulating and maintaining levels in the tissue. Also, L A S can form chelates with the metals, thus reducing the effective concentrations. Similarly, there is an interaction of L A S with calcium of the seawater forming insoluble calcium sulfonates. It was hypothesized by Bressan et al. (Bressan et al., 1991) that the inhibition of growth and skeletal development observed in sea urchin embryos exposed to L A S and varying calcium concentrations were a result of the reduced bioavailability of calcium ions. Especially noteworthy was the. observation that an increase in the environmental calcium concentration, did not mitigate the toxic effects of L A S ; on the contrary it had a significantly worse effect when 2+  compared to the natural Ca  concentration. Therefore, the toxic effects of L A S cannot be  explained solely on the basis of its capacity to sequester calcium. It may also affect the different enzyme systems involved in the production and transport of HCO3" ions which are required for CaC03 deposition and spicule calcification in sea urchin embryos (Bressan et al., 1991). Municipal effluents have been found to contain estrogen-like compounds which may lead to endocrine disruption in receiving environments. It has not been shown that L A S have endocrine-mimicking activities. However, a recent study by Miyamoto et al. (Miyamoto et al., 2002) found that estrogen-like activities detected by the recombinant D N A yeast.assay (YES assay) overestimated the concentration of nonylphenol and estrogen detected by chemical analysis (LC/MS/MS) expressed as the theoretical estrogenicity (which is the detection  70 concentration multiplied by the relative estrogenic potential of the chemical). It was found that the fractionated samples which contained most of the estrogenicity as detected by the Y E S assay also contained high concentrations of L A S , and that the presence of L A S in samples may lead up to 2.5 times larger estimation of estrogen-like activities in the YES assay. Therefore, chemical analysis alone may underestimates the synergistic endocrine effects of a mixture of compounds. It remains to be studied whether those synergistic effects between L A S and endocrine disrupting compounds seen in recombinant yeast cells are also applicable for other eukaryotic cells and organisms.  5. Conclusions  Linear alkylbenzene sulfonates are of considerable concern in untreated or primary treated effluents at their point of discharge in the receiving environment. Due to extensive aerobic degradation, LAS.are removed from the environment. L A S dispersal is determined by the proportion of L A S that is dissolved in the water or attached to particles. L A S may accumulate in anaerobic sediments. Organisms in the affected receiving environment may experience chronic toxic effects either by L A S alone or by a cumulative effect from L A S and other interacting pollutants or naturally-occurring substances. Treating municipal effluents aerobically and thus achieving removal or-reduction of L A S may indirectly reduce toxic or endocrine-disrupting effects from other pollutants.  6. Acknowledgement The author would like to thank Paul van Poppelen from the Greater Vancouver Regional District for his helpful comments on the manuscript.  7. 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Gonzales-Mazo, E., Forja, J.M., Gomez-Parra, A., 1998. Fate and distribution of linear alkylbenzene sulfonates in the littoral environment. Environ. Sci. Technol. 32, 1636-1641. Gonzales-Mazo, E., Honing, M . , Barcelo, D., Gomez-Parra, A . , 1997. Monitoring long-chain intermediate products from the degradation of linear alkylbenzene sulfonates in the marine environment by solid-phase extraction followed by liquid chromatography/ionspray mass spectrometry. Environ. Sci. Technol. 31, 504-510. Granmo, A . , 1972. Development and growth of eggs and larvae of Mytilus edulis exposed to a linear dodecylbenzenesulphonate, L A S . Mar. Biol. 15, 356-358. Gupta, B.N., Mathur, A . K . , Agarwal, C , Singh, A., 1989. In vitro effect of linear alkylbenzene sulphonate (LAS) on some enzymes in liver and gills of the teleost Channa punctatus. Bull. Environ. Cont. Toxicol. 42, 375-381. Hand, V.C., Williams, G.K., 1987. 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Fractionation method for estimating the cause of estrogen-like activity in sewage. W E F T E C 2002, vol. 14. Water Environment Federation, Chicago, Illinois Moreno-Garrido, I., Hampel, M . , Lubian, L . M . , Blasco, J., 2001. Marine microalgae toxicity test , for linear alkylbenzene sulfonate (LAS) and alkylphenol ethoxylate (APEO). Fresenius J. Anal. Chem. 371, 474-478. Part, P., 1985. The influence of surfactants on gill physiology and cadmium uptake in perfused rainbow trout gills. Ecotoxicol. Environ. Safety 9, 135-144. Quiroga, J.M., Sales, D., Gomez-Parra, A., 1989. Experimental evaluation of pollution potential of anionic surfactants in the marine environment. Water Res. 23, 801-807.. Rubio, J.A., Gonzhlez-Mazo, E., Gomez-Parra, A., 1996. Sorption of linear alkylbenzene sulfonates (LAS) on marine sediments. Marine Chemistry 54, 171-177. Schoberl, P., 1989. Basic principles of L A S biodegradation. Tens. Surf. Deter. 26, 86-94. Swisher, R., O'Rourke, J., Tomlinson, H., 1964. 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Vives-Rego, J., Vaque, M.D., Sanchez Leal, J., Parra, J., 1987. Surfactants biodegradation in sea water. Tens. Deter. 24, 20-22. WHO -International Programme on Chemical Safety (1996) Linear alkylbenzene sulfonates and related compounds (Environmental health criteria 169), available online at http://www.inchem.Org/documents/ehc/ehc/ehcl69.htm#SectionNumber:l.6, last accessed on October 1,2004.  73  2.3  MANUSCRIPT III - Identification and phylogenetic comparison of p53 in two distinct mussel species {Mytilus)  This manuscript was submitted to the Journal of Comparative Biochemistry and Physiology, Part B, September 22, 2004. A. Muttray started the experimental work in the laboratory of C. Reinisch (Woods Hole Marine Biological Laboratory, M B L ) under the guidance of R. Cox. Data obtained by A . Muttray in the laboratory of S. Baldwin (Chemical and Biological Engineering, UBC) was subsequently analysed by R. Cox and A. Muttray at Woods Hole M B L and at U B C . S. St-Jean and P. van Poppelen provided much assistance in the sampling, processing and observation of the species. A l l authors participated in the design of research objectives and tasks and provided assistance, each in their own area of expertiese, at various stages of the project.  TITLE: IDENTIFICATION AND PHYLOGENETIC COMPARISON OF P53 IN TWO DISTINCT MUSSEL SPECIES (MYTIL US)  Annette F. Muttray '*, Rachel L. Cox '*, Sylvie St-Jean , Paul van Poppelen , Carol L . 1,5  Reinisch , Susan A . Baldwin 2  2  3  4  5  1 Department of Civil Engineering, University of British Columbia, 5250 Applied Science Lane, Vancouver, B C , V6T 1Z4, Canada; 2 Laboratory of Aquatic Biomedicine, Marine Biological Laboratory, L7 M B L Street, Woods Hole, M A 02543, United States; 3 National Water Research Institute, Environment Canada, 867 Lakeshore Rd., Burlington, O N , L7R 4A6, Canada; 4 Greater Vancouver Regional District, 4330 Kingsway, Burnaby, B.C. V 5 H 4G8, Canada; 5 Department of Chemical and Biological Engineering, University of British Columbia, 2216 Main Mall, Vancouver, B . C , V6T 1Z4 Canada; * both authors have contributed equally to this publication  Running title: Mussel p53  Corresponding author: Annette Muttray, Department of Chemical and Biological Engineering, University of British Columbia, 2216 Main Mall, Vancouver, B. C , V6T 1Z4 Canada. Tel: (604) 827-5273; Fax: (604) 822-6901; E-mail:  75 ABSTRACT The extent to which humans and wildlife are exposed to anthropogenic challenges is an important focus of environmental research. This work examines gene expression as a molecular, indicator for a leukemia that occurs in mussels (Mytilus spp). Potential use ofp53 gene family marker(s) for aquatic environmental effects monitoring is the longterm goal of this research. The p53 gene is a tumor suppressor gene that is fundamental in cell cycle control and apoptosis. It is mutated or differentially expressed in about 50 % of all human cancers and has been implicated in leukemia development in clams. Here we report the identification and characterization of the p53 gene in two species of Mytilus, M. edulis and M. trossulus, using RT-PCR with degenerate and specific primers to conserved regions of the gene. The Mytilus p53 proteins are 99.8 % identical and closely related to clam (Mya) p53. In particular, the 3' untranslated regions were examined to gain understanding of potential post-transcriptional regulatory pathways of p53 expression. We found nuclear and cytoplasmic polyadenylation elements, adenylate/iiridylaterich elements, and a K-box motif previously identified in other, unrelated genes. We also identified a new motif in the p53 3'UTR which is highly conserved across vertebrate and invertebrate species. Differences between the p53 genes of the two Mytilus species may be part of genetic determinants underlying variation in leukemia prevalence and/or development, but require further investigation. Protein expression analysis with p53 antibodies revealed that the m R N A may not be translated into a 53 kDa protein in mussel tissues, but that there may be a post-translationally modified 120 kDa protein in leukemic (but not in normal) haemocytes in Mytilus edulis. This finding further illustrates the importance of including post-translational regulation in considering gene expression as a potential biomarker for genotoxins in the environment. K E Y W O R D S : environmental effects monitoring, leukemia, mussel, Mytilus, p53 gene family, phylogenetic footprint analysis, toxicogenomics, 3' untranslated region  INTRODUCTION Environmental effects monitoring often includes in-situ biological information provided by fish and benthic invertebrate species to assess the health status of their environment (Canada, 2003). The marine bivalve mollusc, mussel Mytilus sp. is widely used by. Mussel Watch Programs (O'Connor et al., 1995), for monitoring pulp and paper mill effluents (Salazar et al.,  76 1997; St-Jean et al., 2003) and more recently, in municipal effluents effects monitoring (St-Jean et al., 2004). The sessile nature of this species facilitates the establishment of cause and effect relationships in time and space (Widdows et al., 1995) and minimises the possible confounding factor associated with the use of migratory species and those using larger areas for foraging. Our research is directed toward the development ofp53 gene family expression in sessile bivalves as molecular biomarker for environmental assessment. One sublethal monitoring endpoint currently under investigation by our laboratory is haemic neoplasia (leukemia), an ultimately fatal condition well documented in clams and mussels. Leukemia is characterized by continuously dividing malignant cells in the haemolymph of shellfish and is thought to be caused by anthropogenic substances (pesticides, PCBs), abnormal temperatures, viral transmission (McGladdery et al., 2001) and/or genetic background and seasonality (Elston et al., 1992). We and others have demonstrated that the p53 gene and its family member, p73, are implicated in the onset of molluscan leukemia (Barker ei al., 1997; Kelley et al., 2001; Stephens et al., 2001). The p53s, a well charcterized family of transcription regulators, act to promote expression of genes controlling D N A editing and repair, apoptosis and carcinogenesis. The p53 tumor suppressor gene was first discovered as a suspected oncogene by three independent research groups in 1979 (DeLeo et al., 1979; Lane et al., 1979; Linzer et al., 1979). Many studies illustrate the importance of the p53 gene, as it is either mutated or lost in over 50% of human cancers (Hollstein et al., 1991). p53 has been termed the "gatekeeper of the genome" as well as a "network hub"(Vogelstein et al., 2000) because of its central role in the molecular networks that decide the fate of cellular life and death. As a transcriptional activator, p53 is normally turned "off, but becomes activated upon damage to D N A by radiation or chemical treatments, hypoxia or activation of oncogenes. Thus, it will prevent cells from passing on the wrong D N A message potentially turning these cells into malignant tumors. . The first molluscan p53 to be identified originated from the squid Loligo forbesi (Ishioka et al., 1995) but the sequence is more similar to the close homologues of p53, p63/73. Kelley et al. isolated p53 and p73 homologues from the bivalve mollusc Mya arenaria (Kelley et al., 2001), and a partial sequence for a p53 homologue is available for the oyster Crassostrea rhizophorae (AY442309). Bhaskaran and co-workers (Bhaskaran et al., 1999) described p53 from various fish species and suggested the use of p53 to study mutagenesis in fish and genotoxins in the aquatic environment.  77 The p53 protein is comprised of several conserved regions. The N-terminal transactivation domain provides the generic transactivation function and the binding site for M D M 2 , the main regulator of p53 stability. A proline-rich region has complex roles as a protein-binding site and a specific regulator of apoptosis (Courtois et al., 2004) (and references therein). The C-terminus includes several domains involved in oligomerization and regulation of the specific D N A binding activity. It also possesses a non-specific D N A binding activity thought to be involved in nonspecific p53-mediated D N A repair and in D N A / R N A reannealing (Wolkowicz et al., 1997). The regulation of p53 function is tightly controlled through several mechanisms including p53 transcription and translation, protein stability and post-translational modification, as well as p53 location in the cell nucleus or cytoplasm (O'Brate et al., 2003)(and references therein). Several authors have used various monoclonal and polyclonal antibodies raised against human and clams (Spisula, Mya) p53 family members to study the expression of the p53 gene family and to distinguish leukemic cells from normal cells (Kelley et al., 2001; Stephens et al., 2001; JessenEller et al., 2002; Cox et a l , 2003). We showed that in leukemic haemocytes of the softshell clam Mya arenaria, the p53 transcript is consistently mutated (Barker et al., 1997). In addition, Kelley et al (2001) showed that for thep53 gene family memberp73, transcription is up-regulated in leukemic haemocytes of adult M. arenaria and that p53 family member protein expression is altered in the transition of normal hemocytes to leukemia cells (Stephens et al., 2001). These observations suggested that p53 gene expression in Mytilus spp. could be used as a potential early-warning biomarker for evaluation of anthropogenic impacts affecting D N A structure or function. The goal of the research presented here was to identify and characterize the p53 gene in two species of Mytilus, M. edulis and M. trossulus, both of which are currently used for bio-monitoring. We isolated haemocytes from two species of bivalve mollusc, Mytilus spp. and used these as a source for identification of p53 homologues. Here we report the identification, phylogenetic characterization and footprint analysis of distinct p53 homologues from Mytilus edulis and Mytilus trossulus. M. edulis is prevalent on the North Atlantic coast, while M. trossulus is prevalent on the North Pacific coast. These two species, albeit similar morphologically, differ significantly at the physiological level, such as gamete incompatibility (Rawson et al., 2003), temporal separation and duration of spawning in Atlantic mussels, and total egg production and size (Toro et al., 2002). In addition, species isolation in the Mytilus complex is partially ensured by the doubly uniparental inheritance (DUI) of the mitochondrial D N A (Saavedra et al., 1996).  78 Supporting these physiological differences are also their respective growth patterns, where M. edulis exhibits a faster growth rate than M. trossulus on the East coast of Canada (Penny et al., 2002). Work carried out on the Pacific coast (BC) by our research team on caged mussels has confirmed differences in growth and reproductive cycles between the species. In addition, there is evidence of differing responses in haemic neoplasia prevalence to the same environmental challenges between the species (unpublished data). Here we report distinct variability in the primary gene structure of the p53 paralogues from these two species, a difference, which may help understand their individual susceptibilities to environmental challenges (Pesch et al., 2004). Phylogenetic comparisons as well as footprint analyses revealed that unique and highly conserved sequence sites occur in the 3' untranslated regions (3' UTRs). Our previous reports suggest that the occurrence of m-acting signaling sites in the 3' UTRs of the p53 gene family members in clams may control gene expression (Cox et al., 2003). Further investigations are required to determine the potential role of these sites in post-transcriptional signaling or regulation of gene expression. This work makes use of comparative data to identify potential regulatory mechanisms controllingp53 gene expression. This represents an important first step toward the use ofp53 gene family expression as a marker for leukemia and a valid environmental assessment monitor.  M A T E R I A L S A N D METHODS  Organisms Certified M. edulis were obtained from Island Scallops, Qualicum, British Columbia and deployed in cages in March 2003 at various locations in the Vancouver Harbour, B C . M.. edulis for this study were collected in October 2003 from one of the cages. M. trossulus was collected in March 2004 at Jericho Beach, Vancouver Harbour, British Columbia. In a previous report, Jericho Beach (Locarno) mussels were found to be 98% M. trossulus based on shell morphometry measurements (Mallet, 2003). Mussels used for the isolation of p53 were subsequently identified and confirmed by the same method by Mallet Research Services, NS (McDonald et al., 1991; Mallet et a l , 1995).  Total RNA extraction Haemolymph was withdrawn from the posterior adductor muscle area using a dry syringe with 22V2  gauge needle. A drop of haemolymph was deposited on a microscope slide and haemocytes  79 were allowed to adhere to the glass surface for five minutes at room temperature. Samples were then examined for potential parasite and mantle fluid contamination by microbial source or by tissue such as gamete, and moribund individuals were discarded. Estimate of percentage of neoplastic versus normal haemocytes was determined using phase contrast microscopy (McGladdery et al., 2001). mRNA was extracted from haemocytes of an M. edulis animal in. transitional phase (Farley et al., 1991) and an M. trossulus animal in normal phase using the Trizol reagent and protocol (Invitrogen Life Technologies, Mississauga, ON). Haemolymph was 3000 rpm using a microfuge and carefully resuspended in 0.5 ml chilled Trizol. • The suspension was snap-frozen in a dry-ice/ethanol bath and stored at -80°C until further extraction.  RT-PCR First strand cDNA synthesis was carried out on approximately 5 \ig of total R N A extract with oligo-dT primers following the guidelines for PowerScript™ Reverse Transcriptase (BD Biosciences Clontech, Mississauga,. ON). Initial sequences were obtained for M. edulis by degenerate PCR using primer design that was based on Mya p53 protein D N A binding region (forward primer DegF2, reverse primer DegR2K, Table 1, kindly provided by Charles Walker), This resulted in the amplification of an initial 320 bp product which was further extended to the 5' and 3' region by R A C E PCR (BD Biosciences Clontech). Final full length clones for both species for the coding region were obtained with forward primer pMe-28F and reverse primer pMel323R (Table 1), and for the 3' untranslated region (3'UTR) by 3 ' R A C E PCR with forward primer 3'RACE829. Step-down PCR cycling conditions for all reactions were as follows: Initial melting at 95 °C for 1 min, 5 cycles of 94 °C for 30 s, 68 °C for 45 s, 72 °C for 3 min; 5 cycles of 94 °C for 30 s, 66 °C for 45 s, 72 °C for 3 min; and so on to 5 cycles of 94 °C for 30 s, 62 °C for 45 s, 72 °C for 3 min; and finally 20 cycles of 94 °C for 30 s, 60 °C for 45 s, 72 °C for 3 min; followed by a final extension at 72 °C for 10 min. Clones were obtained by T A cloning into plasmid vector pCR2.1 and transformation of E.coli I N V a F ' or TOPI OF' (Invitrogen Life Technologies). PCR products for the coding regions were cloned directly, while PCR products from the 3 ' R A C E PCR were gel-purified from 0.7 - 1.0 % agarose T A E gels using the Roche High Pure PCR Product Purification kit (Roche Applied Science, Laval, QC) or the QiaQuick Gel Extraction kit (Qiagen Inc., Mississauga, ON) using manufacturers instructions. Plasmids with the correct length insert based on PCR with universal forward and reverse M l 3 primers and  80 gel electrophoresis were then submitted for sequencing to the Nucleic Acid and Protein Sequencing facility, University of British Columbia. A l l clones were sequenced in both directions using the universal M13 primers. The number of clones sequenced for the final sequences were as follows: Three clones each for M. edulis and M. trossulus coding region, 10 clones for M. edulis 3'UTR, and one clone for M. trossulus 3'UTR. •  Amino acid sequence analysis A l l Mytilus sequences were submitted to Discontiguous Mega B L A S T searches. We performed pairwise amino acid alignment of the deduced Mytilus p53 proteins with selected species using AlignX (residue substitution matrix Blosurh) to illustrate conserved protein domains. Gap opening penalty was set to 10; gap extension penalty at 0.05; gap separation penalty at 8. The alignment was edited in order to match highly conserved regions of the protein. A second pairwise multiple alignment with 32 other known p53 proteins was performed using Clustal X 1.83 (residue substitution matrix Gonnet) (Jeanmougin et al., 1998). The species and assession numbers are listed in Table 2. Gap opening penalty was set to 10; gap extention penalty at 0.1. The phylogenetic tree was produced in Clustal X , bootstrapped 200 times and displayed using TreeView (Page, 1996). Protein sequence identities were obtained from Clustal X alignment.  Nucleic acid sequence analyses 3'UTR sequences were submitted to UTRdb (Pesole et a l , 2002) for analysis. 3'UTR sequences were aligned in ClustalX 1.83 and known regulatory elements were edited by hand. We also submitted a wide range of p53 3'UTRs to a phylogenetic footprint analysis using the FootPrinter 2.1 Webserver available at Parameters were set to default, except for motif size and maximum number of mutations, which were set according to the diversity of the species examined. For instance, for a broad diversity search we allowed for two mutations in a small motif of 6 to 10, while for a narrow diversity search we allowed no mutation and,a longer motif size of 8 to TO.  Microscopic analysis of leukemic cells Five hundred microlitres of haemolymph obtained from an animal with leukemia was fixed (1:1) in modified Barker's formol calcium that contained 4% v/v formaldehyde, 2% w/v sodium chloride and 1% w/v calcium acetate diluted in high salt tris buffered saline (high salt). Samples  81 were subsequently spun 3 times for 5 minutes in high salt at 3000 rpm using an Eppendorf Model 5415C microfuge. Either high salt solution, murine monoclonal antibody 1E10 that recognises M. arenaria leukemia cells, or normal mouse IgG was added to pelleted cells as previously described (Reinisch et al, 1983). The samples were then incubated on a rocker at 10°C overnight, spun 3 times in high salt and a 1:100 dilution of tetramethylrhodamine conjugated (RITC) rabbit anti mouse IgG (Molecular Probes, Eugene, Oregon), added to each sample. A l l antibodies were spun at 10,000 rpm for 20 minutes prior to use to remove aggregates. The cells were then incubated an additional two hours at 10°C, washed 3 times, and evaluated using an Olympus BH2 microscope at 500x magnification using a rhodamine filter. Any positive samples were then visualized at an excitation wavelength of 488 nm using a Zeiss L S M 510 laser scanning confocal microscope. In each analysis, normal IgG as well as high salt solution was used to control for nonspecific binding of the secondary antibody or background signal.  RESULTS A N D DISCUSSION  Identification and characterization of Mytilus p53 cDNA The nucleic acid sequence of the cDNA for p53 was determined from two different mussel species, Mytilus edulis and Mytilus trossulus, and deposited in Genbank as AY579472 and AY611471, respectively. The total length of the cDNAs are 2288 nucleotides (nt) for both species, with an open reading frame (ORF) of 1302 nt, predicting a protein of 434 amino acids in length with a calculated molecular mass of 50 kDa, based on an average amino acid weight of 115 Da. This is within the range of previously reported p53 proteins. The nucleic acid sequences of the ORFs of M. edulis and M. trossulus are 96.5 % identical. However, predicted amino acid (aa) sequences for the two species are 99.8 % identical, indicating slightly different codon usage by the two species.  Comparative analysis of p53 amino acid homologues We performed a comparative analysis between the two Mytilus p53 sequences and representatives of other major lineages: Mya arenaria , a representative for bivalve molluscs and likely a close relative of Mytilus, and Drosphila melanogaster a second representative for invertebrates, and four species of vertebrates, Xenopus laevis, two fish, Danio rerio and Barbus  82 barbus, and one well-characterized mammalian representative, Homo sapiens (Table 2 and Figure 1). Overall amino acid sequence identities between M. edulis p53 and the other aligned species, based on Clustal X indentity tables, were as follows: M. arenaria Map53, 69 %; D. melanogaster Dmp53, 18 %; X. laevis Xlp53, 36 %; D. rerio Drp53, 38 %; B. barbus Bbp53, 38 %; and H sapiens Hsp53, 31 %. Drosophila p53 has the least identity with Mytilus p53. As predicted, Mya p53 is the closest known relative to Mytilus p53. However, no N-terminal extension was detected in Mytilus when compared to Mya. In M. arenaria, a N-terminal extension was detected 8 aa distance upstream from the conventional start site which leads to a potential second start site for protein translation (Kelley et al., 2001).  Mytilus p53 DNA Binding Domains Mytilus p53s have five D N A binding domains (DBDs, including the transcriptional activation domain, TAD), which are also found in Mya and other species. D B D II to V are located between amino acid residues 150 and 319 in both Mytilidae. These domains are highly conserved between species. Overall predicted protein identities of these four DBDs of Mytilus p53 are 88% when compared to Mya p53 but only .63% when compared to Homo p53. The transcriptional activation domain (TAD) provides the binding site for proteins that regulate p53 expression, D N A editing and repair, and apoptosis. The T A D is highly conserved across species (except D. melongaster). The Mep53 T A D is 100% identical with Map53 and 75% identical with human p53. Negative feedback regulation of p53 activity is driven by a protein called M D M 2 , and positive regulation of p53-dependent tumor suppression is driven by the transcriptional coactivator p300 (Shimizu et al., 2003) (and papers therein). M D M 2 functions as an ubiquitin ligase in the nucleus and, upon binding to p53 T A D , destines p53 for export to the cytoplasm and subsequent degradation. It is involved in an autoregulatory feedback loop that maintains p53 at basal level in heaithy non-stressed cells. Defects in the pathways of M D M 2 regulation of p53 are common in tumors that retain wildtype p53 (Vousden et al., 2002). The crucial M D M 2 contact sites in human p53 are F19, W23 and L26, (shown in Figure 1 with # signs) which are also found in Mep53 and Mtp53. Key p300 contact sites in human p53 are overlapping with the M D M 2 contact sites and are less conserved between species. Only three out of the six identified p300 binding sites are conserved between human and Mytilus p53. Different kinases, such as A T M  83 (ataxia telangiectasia mutated) or C H K 2 (checkpoint kinase 2) can modify p53 at specific amino acids within the T A D region. Serl5, Thrl8 and Ser20 have been identified as important phosphorylation sites (Shimizu et al., 2003) (and papers therein). As shown in Figure 1 (with P) Serl5 and Thrl8 are highly conserved between species, including Mep53 and Mtp53, while the residue at position 20 is less conserved (fish, Mya and Mytilus display a glutamate at this position). It has been found that a number of human tumors are associated with cytoplasmic localization of p53. As mentioned above, M D M 2 regulates p53 activity by ubiquifation, followed by nuclear export and degradation. M D M 2 overexpression was frequently found in advanced leukemic cells of human patients which have a p53-null phenotype due to rapid p53 degradation (Konikova et al., 2003), but also in relation with low p53 levels or conformational mutants of p53 as measured with antibody PAb240. Similarly, leukemic clam haemocytes show localization of Map53 and Map73 in the cytoplasm and not in the nucleus (Kelley et al., 2001). Since these M D M 2 binding sites are also conserved in Mytilus this particular mechanism of leukemia may also be conserved in this species.  The region between the T A D and D B D II is the most divergent region in p53 and contains a number of proline residues. It appears that the molluscs (including Loligo forbesie p53, sequence not shown) have a much longer region just before the proline-rich region than other organisms (Figure 1), the function of which is not known. The proline-rich region of the mussel p53 contains two P X X P motifs at positions 116 and 128. This is very similar to Map53, which also has only two P X X P motifs, while human p53 contains three such motifs and additional proline residues. These motifs are entirely absent from Drosophila and Xenopus p53, and are present at non-homologous sequence positions in other organisms. This proline-rich region is involved in apoptosis and may bind SH3-containing kinases involved in signal transduction (Kelley et al., 2001). This region also contains the only amino acid position which is different between the two mussel species: Ser86 and Thr86 for M. edulis and M. trossulus, respectively. It is unlikely that this variation will have an effect on p53 function and regulation as it is located in a less-conserved region of the protein. Mussel p53 D B D II is 92% idential with Mya p53, but only 69% identical with human p53. D B D III shares 92 % identity with Mya and 81% identity with human p53. It contains two zinc binding sites which are fully conserved in all presented species. Similarly, two mutational hot-spots (Val 173 and Cys 175 in Hsp53) are 100% conserved (May et a l , 1999). Mep53 DBD  IV is 91% identical with Mya and 73 % identical with human p53. The three mutational hotspots contained in that region are highly conserved, with the exception of Drosophila p53. Zinc and D N A binding sites are 100% conserved between all presented species. Known residues for mutational hot-spots and D N A binding sites for neighbouring D B D V are slightly less conserved between species. Overall, identities for this region between Mytilus and Mya are 100 %, and between Mytilus and Homo 93 %.. Mutational hot-spots are based on the frequency of point mutations found in human cancers. Point mutations with the highest frequencies have been termed hot-spots. Analysis of distribution of mutations in human p53 shows that they are essentially clustered in the central region of the protein, and in particular within the four D N A binding domains (Soussi et al.,T996). Conservation of human mutational hot-spots in other species not only points to the essential nature of these amino acids for p53 function but also raises the possibility that these residues may be involved in cancers in other species.  Mytilus p53 has a tripartite nuclear localization signal (NLS I, II and III)) which is similar to human p53. In our alignment, Myap53 also shows a tripartite N L S , although Kelley et al: (Kelley et a l , 2001) indicated that Mya may only have two N L S . A l l NLS are rich in lysine residues. It is known that lysine K320 in Hsp53 N L S I, which is highly conserved between species, is acetylated to enhance p53 stability and p53- specific D N A binding with p53-regulated proteins (Liu et al., 2000). Nuclear import of p53 is enabled by its N L S while nuclear export is enabled by its nuclear export signal (NES) which is located within the tetramerization domain (see below). In human, when D N A is damaged, p53 gets imported into the nucleus via its N L S and undergoes tetramerization, binds and activates DNA-damage response genes (O'Brate et al., 2003) (and papers therein). The tetramer state of p53 masks the nuclear export signal thereby . preventing export to the cytoplasm. As mentioned above, p53 functions as a tetrameric protein. The tetramerization domain is 59 % identical with Mya p53 and 53 % identical with human p53 tetramerization domain. Glycine 334 (in humans) is crucial for stability of p53 and conserved throughout all presented lineages. Leucines 348 and 350 (in humans) are crucial for NES. They are conserved in Mytilus, but interestingly not in Mya where the second leucine is substituted by isoleucine. Positions 341 and 344 (in humans) are critical for oligomerization and are conserved non-polar residues, mostly leucines. The ability to form tetramers allows p53 to behave in a dominant-negative fashion (Hofseth et al., 2004). Our identification of highly conserved regions within the functional domain of Mytilus p53 suggests that its overall function is conserved.  85 These observations, taken with previous data (Kelley et al., 2001) implicating p53 family members in the onset of leukemia further suggest the utility of Mytilus p53 gene expression as an indicator for early onset leukemia and an important molecular target of genotoxins in the environment.  Phylogenetic analysis The recent finding of a p53 homologue in Entamoeba histolytica (Mendosa et al., 2003) suggests that the p53 gene family is of ancient origin. We performed a phylogenetic analysis of many to-date available p53 amino acid sequences to a) confirm Mytilus p53 sequence relationships with other invertebrates, and b) clarify the phylogenetic relationship of the D. melanogaster p53 homologue in relation to other invertebrates and vertebrates (Figure 2, Table 2). We set entamoeba Ehp53 as the outgroup as it is likely the most distantly related p53 known thus far. We found that Mytilus p53s cluster with other known mollusc invertebrate p53s, especially with the other known bivalve p53s, as would be expected. The family Mytilidae dates back to the Jurassic or perhaps even Devonian times (Soot-Ryen, 1969) (ca. 400 million years ago). The tree topology supports the now accepted view that the molluscan lineage is not part of a common lineage with the annelids as was suspected previously (Wilmer, 1990). It is remarkable that p53 of D. melanogaster (Dmp53) is monophyletic and does not cluster with the other insect p53, and suggests that p53 of D. melanogaster may have undergone more recent mutations, for instance loss of the M D M 2 binding region. Because of its role as guardian of the genome as well as its recent identification in entamoeba it may be possible to use p53 and its relatives p63 andp73 (Yang et al., 2002) as central regulatory genes for phylogenetic studies as more sequences become available.  Analysis of the 3'untranslated region of Mytilus p53 cDNA Untranslated regions at the 3' end of the mRNA contain signals, for mRNA translation, polyadenylation, stability and subcellular localization, and play therefore an important role in gene regulation and expression at the post-transcriptional level (Pesole et al., 2002). We obtained the 3'UTR by 3 ' R A C E PCR starting at the conserved D B D V . We sequenced 10 clones in M. edulis and subsequently one clone in M. trossulus. The total length, starting after the stop codon T G A and ending at the start of the polyA tail, is 917 nt in M. edulis, and 874 nt in M.  86 trossulus, and fall well within <the range of 3'UTR lengths (maximum 9142 nt, minimum 15 nt, average 444.5 nt) reported for invertebrate species (Pesole et al., 2001) (Figure 3). Myap53 3'UTR contains 2027 nt and is therefore comparatively longer than the Mytilus p53 3'UTRs. The sequences vary widely between the mussels and the clam 3'UTRs, probably because these regions are under less evolutionary constrains than the coding regions (Conne et al., 2000; Grzybowska et al., 2001). Sequence alignment of the three consensus sequences (Mep53 v l , v2, v3) obtained by manual alignment from 10 M. edulis clones revealed various deletions in the 3'UTR ofp53 for this species (Figure 3). One clone had a deletion of 114 nt starting at position 40 (termed version 3), while three other clones had a deletion of 42 nt starting at position 551 (termed version 2) of the full-length 3'UTR. Six of the ten clones were fulllength (termed version 1). M. trossulusp53 3'UTR was most similar to the full-length version of the M. edulisp53 3'UTR (vl) with a minor deletion of 7nt at 128, and a deletion of 31 nt at position 817. Regions bordering these deletions were not found to be reverse complement to each other and it is therefore unlikely that these deletions are artifacts of polymerase slippage. It is currently unknown whether these deleted (or inserted) regions affect expression of the transcript or function of the protein. While post-translational regulation and cellular localization of p53 has received considerable attention (for a review see (O'Brate et al., 2003)), post-transcriptional regulation of p53 activity has perhaps been undervalued in recent years. We identified the following regulatory regions based on current literature in bivalvep53 3'UTR: 1) Tandem nuclear polyadenylation sites " A A T A A A " are located 20 nt upstream of the polyA tail in M. edulis. A single polyadenylation site is located 14 nt upstream of the polyA tail in M. trossulus. This sequence is required for proper poly(A) tail addition and mRNA stabilization in the nucleus and binds to a complex of four polypeptides, the polyadenylation specificity factor (CPSF) (Verrotti et al., 1996). mRNAs initially receive their poly(A) tails of approximately 250 nucleotides in length in the nucleus of the cells. Upon entering the cytoplasm, poly(A) is removed in most cells. 2) We located three cytoplasmic polyadenylation elements (CPEs) in a region starting at 482 nt after the stop codon ' (Figure 3). CPEs are AU-rich regions and are generally defined as  "T4.6AT"  (Pesole et al., 1999),  (see also UTRsite at*l). Located near the nuclear polyadenylation element, the CPEs are evolutionarily conserved sites, known to regulate translational activation by elongation of the poly(A) tail in the cytoplasm of the cell. Generally, poly(A) elongation confers translational activation while deadenylation promotes  87 translational silencing (Richter, 1999). CPEs have mostly been studied in mouse, Xenopus and Drosophila oocyte maturation and early development. Detailed mutagenesis experiments established that minimal perturbations of Xenopus CPEs can abolish their function (Verrotti et al., 1996) (and references therein). Sequence comparison across species is difficult because of the AT-richness of the region and because of a "substantial context and position effect on CPE function" (Verrotti et a l , 1996), which is illustrated by the alignment of the Mytilus and Mya 3'UTRs (Figure 3). Whether the deletion of regions of the Mep53_v2 and the Mtp53 3'UTR located within the CPE region have an effect on cytoplasmic polyadenylation and translational activation is entirely speculative at this point. In Xenopus, CPEs are recognized by C ? E binding proteins which, together with the CPSF, may form a core cytoplasmic polyadenylation apparatus that is conserved across species (Verrotti et al., 1996). Recent evidence suggests that cytoplasmic polyadenylation not only plays a role in mRNA activation in early development, but also in synaptic memory (or plasticity) after stimulation in the brain (Richter, 2001) and in cell cycle control upon recognition of D N A damage in yeasts (Read et al., 2002). CPE-dependent polyadenylation has also been demonstrated in human M C F 7 breast cancer cell line, in a CPEcontaining 3'UTR fragment of cyclin B mRNA (Groisman et al., 2002). 3) The 3'UTR ofp53 also contains six adenylate/uridylate-rich elements (AREs) which are usually defined by the pentamer A U U U A or the nonamer UUAUUUA(U/A)(U/A), and have previously been found in labile mRNAs for regulatory proteins such as proto-oncoproteins, growth factors and their receptors, inflammatory mediators and cytokines (Grzybowska et al., 2001). The AREs primary function is to target mRNAs for selective degradation. However, A R E mediated decay is itself regulated: under stress conditions, cell stimulation, or during oncogenic transformation A R E containing mRNAs are stabilized. Their regulatory functions are expressed through the specific binding of proteins which can modify transcript stability. The mussel sequences contain 5 consecutive A R E sequences in comparison to the clam p53 sequence which contains only one (Figure 3). As already concluded by Read and Norbury (Read et al., 2002), the further characterization of cytoplasmic polyadenylation implicate this mechanism of translational control in the regulation of increasingly diverse cellular processes, but seemingly most often in cellular responses to stress, D N A damage, replication block, and normal cell cycle events. It may therefore be of no surprize to find CPEs and AREs in p53 as well as in its relatives, p63/73 (Cox et al., 2003). One of the main advantages of translational control is that it enables rapid changes in gene expression without requiring gene transcription or mRNA transport (Read et al., 2002).  88 In an attempt to identify novel signaling elements in the 3'UTR of Mytilus p53, we submitted the sequences to the UTRscan program f (Pesole et al., 1999) and identified a K-box motif as indicated in Figure 3. The K-Box motif ( A T G T G A T A ) occurs 179 nt upstream of the poly-A tail in all Mytilus p53 and potentially in Mya p53. The more conserved " T G T G A T " motif ocurrs in Mya p53, 380 nt upstream of the poly A tail and may be a second K-box in this species. We also searched other aligned species for the conserved " T G T G A T " K-box motif and found it in Danio reriop53 3'UTR, at 327 nt upstream from the poly-A tail. We did not find it in Drosophila p53 or in Loligo p53. This is the first identification of the K-box motif in any species as a potential site of 3'UTR transcriptional regulation in the p53 gene family. The K-box was originally identified in Drosophila Notch signalling proteins E(spl)-C (Lai et al., 1998) where it is loosely associated with a C A A C motif, not present in Mytilus, Mya and Danio p53 3'UTR. Lai and co-workers (Lai et al., 1998) found that the presence of the K-box resulted in a decreased level of mRNA as well as protein in developing Drosophila embryos. Mutation or deletion of the K-box motif resulted in over-expression of the reporter construct in all developing tissues. These K-box motifs may be recognized by micro-RNAs (Lai, 2004), which mediate translational inhibition in Drosophila and C. elegans (Lai et al., 2004) (and references therein). We hypothesize that the K-box motif found in Mytilus and Myap53 3'UTR may play a role in regulation of p53 levels in addition to, or in concert with other 3'UTR translational elements as well as the M D M 2 positive regulatory feedback loop. Phylogenetic conservation of K-boxes in the p53 gene family remains to be investigated. The p53 3'UTRs of the two Mytilus species examined are 93 % identical based mostly on point mutations and two regions missing from M. trossulus when compared to the full-length M. edulis 3'UTR. The point mutation at position 857 eliminates one of the two nuclear polyadenylation sites. However, this may be due to polymerase error and needs to be confirmed in subsequent sequences. The deleted regions do not affect any of the conserved functional regions, but would affect spacing between the regions. Whether any of the observed differences result in differential expression of the p53 protein in the two species is unclear, and may also be due to other factors up- and downstream of p53 transcription/translation within the regulatory pathways.  89  Phylogenetic Footprint Analysis of the 3'UTR  >•  If the p53 family is indeed of ancient origin it is possible that functional elements have . been conserved in the untranslated regions. Selective pressure would cause functional elements to evolve at a slower rate than that of nonfunctional sequences. Phylogenetic footprinting deduces novel regulatory elements by considering orthologous regions of a single gene from several species (Blanchette et al., 2002). We ran a series of phylogenetic footprint analyses using FootPrinter 2.1 (Blanchette et al., 2003) for a very diverse group of p53 sequences to potentially identify novel regulatory elements conserved across species. The analysis includedp53 3'UTR sequences from Mytilus spp., clam M. arenaria, flour beetle T. castaneum, puffer fish T. miurus, trout O. mykiss, chicken G. gallus, frog X. laevis, and H. sapiens (see Table 2 for accession numbers). The 3'UTR for Cep-1 and Ehp53 were not included. We searched for conserved elements of lengths 6 to 10 in consecutive runs with a parsimony score (= number of mutations) of 2. Only the smallest motif length, 6, returned two conserved motifs across this wide range of species. Figure 4A gives the location and sequence of the two motifs: tg(t/c)(g/t)tt (green) and tattta (red). The first motif may be a new previously unidentified regulatory element in the 3'UTR ofp53, and is also indicated as green background in Figure 3. Of all the sequences analysed, only T. miurus has a duplication of this motif. The second motif identified by FootPrinter overlaps with the C P E / A R E region identified in Figure 3 for the Mytilus sp. (484, Mep53_y\). FootPrinter identified a similar region in Map53 at position 386 which was not previously identified. The ClustalX alignment (Figure 3) failed to align the CPEs found in Mytilus with potential CPEs in M. arenaria, likely due to low sequence conservation and A T richness of the region. We used the FootPrinter program on the Mytilidae and M. arenaria to look for highly conserved potential regulatory elements within the mollusc bivalve family. For this, we increased the motif length to 9 and allowed no mutations, because the evolutionary distance is short between Mytilus and Mya. Two tandem motifs were identified (Figure 4B): Motif #1 (pink) is an unidentified conserved motif adjacent to the highly conserved motif identified in the previous footprint analysis (and is also indicated with a pink background in Figure 3), motif #2 (green) coincides with the CPE region identified for Mytilus sp. in Figure 3 (722, Mep53_y\), motif #3 coincides with the highly conserved motif identified in the previous footprint analysis (and indicated with a green background in Figure 3), and motif #4 (blue) is an unidentified conserved motif. Motif #2 can be interpreted as a potential CPE in M. arenaria at location 833 (green frame in Figure 3). Our analysis found previously known regulatory  90 elements as well as highly conserved motifs with unknown function. We found that the FootPrinter results are highly sensitive to the input criteria such as species selection, motif length and parsimony score. During the analysis we chose a low parsimony score to ensure that the motifs reported were well conserved. Therefore, we may have missed potential functional motifs with a higher degree of variability. The data presented here contribute to a rapidly emerging story that describes potential transcriptional control of expression in the p53 gene family. Further analysis of phylogenetically diverse sequences will contribute to this model. Variability in the 3'UTR of molluscan p53 family members was previously identified by Kelley and co-workers (Kelley et al., 2001) who found that unlike in mammals, the molluscan p73 is a 3' gene variant of the p53 gene, suggesting that a divergence in gene function occurred early in evolution. In addition, Cox et al. (Cox et al., 2003), found that two unique polyadenylation site variants may control expression of the p73 gene in another molluscan species, Spisula solidissima. Taken together, these observations and the data presented here serve to demonstrate the complexities of p53 gene family regulation and the insights gained by analysis of non-mammalian species  Detection of p53 protein in Mytilus edulis tissues At equivalent protein loading on Western blots, a p5 3-specific polyclonal antibody (Ab1) detected expression of a 53 kDa protein in gill and mantle of Mytilus edulis but not in haemocytes, from which we isolated the mRNA (R. Stephens, personal communication). Interestingly, Ab-1 did cross-react with a single 120 kDa protein predominantly expressed in leukemia haemocytes (St-Jean et al., 2004). The Ab-1 antibody recognizes an epitope near the C-terminal of the protein that is variable between species (aa 371-380). Several possibilities may explain the discrepancy: 1) In Mytilus tissues, the antibody cross-reacts with an unrelated protein having a similar size and epitope as p53, an unlikely yet plausible coincidence. 2) In Mytilus haemocytes, the antibody detects a 120 kDa protein which is a post-translationally modified member of the p53 family, as was previously detected in Spisula solidissima embryos (JessenEller et al., 2002). 3) The antibody can recognize a true 53 kDa p53 Mytilus homologue, but it is not sensitive enough to detect low levels of p53 protein in haemocytes (St-Jean et al., 2004). 4) p53 mRNA transcripts in Mytilus haemocytes may not be translated into protein. We feel that the most likely conclusions are that "native" p53 protein in haemocytes may be below the detection limits for Ab-1 and that the,p 120 protein that is detected by Ab-1 may be a post-  91 translationally modified p53 or p53 family member. In fact, an antibody to a peptide that includes much of the D B D V region of Mya arenaria p53 (Kelley et al. 2001) also does not detect a 53 kDa p53 in Mytilus haemocytes, yet it readily cross-reacts with p73 and p63 while detecting higher molecular weight forms as well (St-Jean et al., 2004). p53 may be transcribed into pre-mRNA and spliced into mature mRNA at levels high enough to be detected by RT-PCR, but is either not translated into protein or the protein may be degraded or modified at such a rate that it cannot be detected by Western blotting. Interestingly, it was shown that there is an inverse relationship between mRNA levels and protein levels of a pi20 (now believed to be a post-translationally modified p63/73 homologue) during embryonic development of the surf clam S. solidissima (Jessen-Eller et al., 2002). Also, Conne and coworkers (Conne et al., 2000) found that p53 protein is often undetectable in acute myelogenous leukemia (AML) cells in human, and raised the possibility that its half-life is altered in A M L cells and/or that p53 gene expression is translationally regulated. The identification of translational regulatory elements in the 3'UTR ofp53 corroborate this hypothesis, but would require further detailed investigations to confirm cellular localization, stability and translation of p53 mRNA.  Comparison between M. edulis and M. trossulus The question has been for a long time, whether differences between M. edulis Linnaeus, 1785, and M. trossulus Gould, 1850, are sufficiently large to warrant full species status for each, or should be considered a semispecies, ecotype of variaty of M. edulis (Gosling, 1992). Morphologically they are almost identical, with M. trossulus initially growing faster in width than in length when compared to M. edulis. The minor differences in shell morphology have been exploited in the multivariate analysis of adults for species identification (McDonald et al., 1991), although it is generally accepted that morphology is affected by environmental conditions. Allozyme analysis has been very useful at distinguishing species from different geographic locales, but is not powerful enough when comparing species from a single locale, likely due to hybridization between the two species and enzyme loci selection pressure in one locale (Kenchington et al., 1995) (and references therein). The 18S rRNA sequences are also very similar within the genus, and are not reliable at distinguishing between M. edulis and M. trossulus collected from different geographic regions (Kenchington et al., 1995). We found that the p53 protein is also almost identical between the two species. However, p53 from a greater  .  92 number of animals should be sequenced to confirm the single amino acid change that we have observed. Despite all the similarities, M. edulis and M. trossulus only form unstable hybrids and are reproductively and physiologically different (Toro et al., 2002). It was also shown that general prevalence of haemic neoplasia is higher in M. trossulus (0.0-45%) than in M. edulis (0.0-4.3%) (Elston et al., 1992) (and references therein), although the examples listed were from different geographic regions. In M. trossulus, highest prevalences are found from January to March followed by decreased prevalence during summer and early autumn (Elston et al., 1992); (Bower, 1989). Consequently, induction and occurrence outside these cycles provide strong evidence for the influence of anthropogenic influences and factors affecting D N A structure and function. Therefore, this may serve as a useful biomarker for environmental effect monitoring in the marine environment. A study is currently underway in the Burrard Inlet and Howe Sound (BC, Canada) to assess the differences in natural leukemia prevalence between the two mussel species caged at various locations. Preliminary results confirmed that susceptibility to leukemia is greater in M. trossulus (unpublished data). Susceptibility to leukemia is different somewhat between the two species, as is the mechanism. This difference is also reflected in the aggregation of a protein that is recognized by the antibody 1E10, which is a leukemia-specific antibody. Antibody 1E10 recognizes a glycosylated cell surface protein on the non-adhesive leukemia cells. It has previously been used to stain leukemic cells of Mya arenaria and Mytilus edulis (St-Jean et al., 2004). Interestingly, while this protein appears to be quite patchy on the cell surface of M. edulis leukemic haemocytes, it is rather equally distributed on the surface of M. trossulus leukemic haemocytes (Fig. 5). Its function is currently unknown, but one may speculate that the secretion of the glycosylated protein on the cell surface is more advanced in M. trossulus and that M. edulis'' leukemic haemocytes are "caught in the act" of translocating the protein to the cell surface. It provides a further indication that the two mussel species are different not just physiologically but also in leukemia-related characters. This has to be taken into serious consideration when mussels and mussel leukemia is used for environmental monitoring in different geographic regions, and should be further investigated. Based on our sequence analysis, it is unlikely that the differences in leukemia progression and prevalence observed on a macro-level can be explained solely on the basis of p53 regulation via its 3' untranslated region. Other regulatory mechanisms up- and  93 downstream in the p53 network, as well as other factors, are likely involved and require further investigation.  CONCLUSIONS As two species of Mytilus, M. edulis and M. trossulus, are often used for coastal or marine environmental effects monitoring, their responses to similar challenges must be quantified. In addition, differences in the natural prevalence, cyclical nature, and progression of the disease between the two species must also be determined to enable differientiation between natural, phenomena and anthropogenic effects, in order to be an effective bioindicator. To achieve these objectives it was required to identify and compare the p53 sequences in both species as a first step in the development of a rapid and repeatable screening techniques for H N development in Mytilidae. The p53 cDNA sequences, designated Mep53 and Mtp53, were identified in Mytilus edulis and Mytilus trossulus, respectively, and found to be 96.5 % similar to each other. The coding regions of the p53 c D N A contain highly conserved regions similar to most identified p53. Putative p53 proteins of the two Mytilus species are 99.8 % similar to each other. The 3' noncoding region contains a number of known regulatory regions: adenylate/uridylate-rich elements, cytoplasmic polyadenylation sites, nuclear polyadenylation sites and a K-box motif, which has not been identified previously in ap53. Western blot analysis showed that p53 may be posttranslationally modified or degraded in haemocytes, from which the c D N A was obtained. Further studies on p53 mRNA presence and/or regulation will ascertain whether it is differentially expressed or mutated in leukemic haemocytes of Mytilus.  ACKNOWLEDGEMENTS  The authors would like to acknowledge Melissa Kelley and Raymond Stephens for their expertise and review of the manuscript. A . F . M . was supported by a grant from the Greater Vancouver Regional District, B C , Canada, and R.L.C. was supported by STAR grant R82935901 from the Environmental Protection Agency (USA). 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Scope for growth and contaminant levels in North Sea mussels Mytilus edulis. Mar. Ecol. Prog. Ser. 127, 131-148. Wilmer, P., 1990. Invertebrate relationships: patterns in animal evolution. Cambridge University Press, Cambridge. Wolkowicz, R., Rotter, V., 1997. The D N A binding regulatory domain of p53: see the C. Pathol. Biol. (Paris) 45, 785-796. Yang, A., Kaghad, M . , Caput, D., McKeon, F., 2002. On the shoulders of giants: p63, p73 and the rise ofp53. Trends Genet. 18, 90-95.  99  TABLES AND FIGURES  Table 6 (Table 1 of the submitted manuscript): Primers used in the identification of p53 in Mytilus sp. Capital letters indicate degenerate residues: I, inosine; R, A/G; M, A/C; Y, C/T; K, G/T; N, A/T/G/C; D, A/G/T.  Name  Sequence 5' -> 3'  DegF2  gtlaaRMgltgYccIaaYcaK  DegR2K  NggRcaNgcRcaDatNcKNacYtc  pMe-28F.  tggaaagttcactcatcatcacc  pMel323R  atatatcctcaatgttcctgaacc  3'RACE829  catgtgtaggaggaccaaacagaaggcc  100 Table 7 (Table 2 of the submitted manuscript): List of species, accession numbers, and abbreviations used for the phylogenetic analysis of p53 proteins in Figure 2. Common name Entamoeba Blue mussel Bay mussel Softshell clam Squid Fruit fly Potato beetle Flour beetle Nematode Swordtail Medaka Flounder Congo puffer Barbel Zebrafish Channel catfish Rainbow trout Clawed frog Chicken Human Dog Cattle Vervet monkey Chinese hamster Guinea pig Cat Natal rat Sheep European rabbit Rat Mouse Pig Beluga whale  Scientific name Entamoeba histolytica Mytilus edulis Mytilus trossulus Mya arenaria Loligo forbesie Drosophila melanogasier Leptinotarsa decemlineata Tribolium castaneum Caenorhabditis elegans Xiphophorus helleri Oryzias latipes Platichthys flesus Tetraodon miurus Barbus barbus Danio rerio Ictalurus punctatus Oncorhynchus mykiss Xenopus laevis ' Gallus gallus Homo sapiens Canis familiaris Bos taurus Chlorocebus aethiops Cricetulus griseus Cavia porcellus Felis catus Mastomys natalensis Ovis aries Oryctolagus cuniculus Rattus norwegicus Mus musculus Sus scrofa Delphinapterus leucas  Abbreviation  Accession #  Phylogenetic taxa  Ehp53 Mep53 Mtp53 Map53 Lfp73 Dmp53 Ldp53 Tcp53 Cep-1 Xhp53. 01p53 Pfp53 Tmp53 Bbp53 Drp53 Ipp53 Omp53 Xlp53 Ggp53 Hsp53 Cfp53 Btp53 Cap53 Cgp53 Cpp53 Fcp53 Mnp53part Oap53 Ocp53 Rnp53 Mmp53 Ssp53 Dlp53  AJ489250 AY579472 AY611471 AF253323 U43595 AF263722 BD250011 BD250012 AF440800 AF043946 U57306 Y08919 AF071571 AF071570 AF365873 AF074967 M75145 X77546 NM_205264 AB082923 AB020761 NM_174201 X16384 U50395 AJ009673 D26608 U48617 X81705 X90592 NM_030989 X00741 AF124298 AF475081  Eukaryota, Entamoeba Invertebrate, Mollusca Invertebrate, Mollusca Invertebrate, Mollusca Invertebrate, Mollusca Invertebrate, Arthropoda Invertebrate, Arthropoda Invertebrate, Arthropoda Invertebrate, Nematoda Vertebrate, Neoteleostei Vertebrate, Neoteleostei Vertebrate, Neoteleostei Vertebrate, Neoteleostei Vertebrate, Ostariophysi Vertebrate, Ostariophysi Vertebrate, Ostariophysi Vertebrate, Ostariophysi Vertebrate, Amphibian Vertebrate, Aves Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal Vertebrate, Mammal  101 Figure 7 (Figure 1 of the submitted manuscript): Multiple pairwise alignment of amino acid sequences for p53 homologues from Mytilus edulis (Mep53), Mytilus trossulus (Mtp53), Mya arenaria (Map53), Drosophila melanogaster (Dmp53), Xenopus laevis (Xlp53), Danio rerio (Drp53), Barbus barbus (Bbp53), and Homo sapiens (Hsp53). Color coding: Black on white, non-homologous residues; green on white, weakly similar residues; blue on white, block of similar residues; black on grey, conserved residues; red on grey background, identical residues. Abbreviations: T A D , Transcriptional activation domain (or D N A binding domain I); D B D II-V, D N A binding domains II-V; N L S I and II, nuclear localization domain I and II; NES, nuclear export domain; PxxP, proline-rich domains in shellfish species; ® , putative conserved phosphorylation sites; #, key M D M 2 binding sites; §, residues where Hsp53 binds to D N A ; *, Hsp53 mutational hot-spots; Z, residues involved in zinc binding. The open box indicates the single amino acid change between Mep53 and Mtp53.  Figure 8 (Figure 2 of the submitted manuscript): Phylogenetic relationship between p53 proteins of diverse species indicating separate lineages for mollusca and annelida. See table 2 for species list and abbreviations. The neighbour-joined consensus tree was based on a pairwise Clustal X alignment, boostrapped 200 times and rooted with entamoeba p53 as an outgroup. Numbers at the nodes indicate bootstrap values. The bottom scale measures genetic distances in substitutions per nucleotide. The clustering of p53s in phylogenetic groups (Mammals, Ostariophysi, Neoteleosti, Mollusca and Insecta) is indicated with brackets.  Figure 9 (Figure 3 of the submitted manuscript): Clustal X alignment of the 3'UTR of the p53 gene of three variants of M. edulis, Mep53_vl, Mep53jv2 (AY735341), Mep53_v3 (AY735340), M. trossulus (Mtp53) and M. arenaria (Map53). Colour coding: Red, identical residues; blue, similar residues; black, non-similar residues. Abbreviations: CPE, cytoplasmic polyadenylation signal; A R E , adenylate/uridylate-rich elements. Conserved known regulatory regions are indicated by open and closed boxes.  102 Figure 10 (Figure 4 of the submitted manuscript): Output for the phylogenetic footprint analysis. The graphic panel shows the phylogenetic tree relating the sequences. Each horizontal line is labeled with the name of the gene (see Table 2 for species identification) and represents the entire 3'UTR sequence. The colored bars above the lines indicate the position of discovered motifs. The bar colors correspond to the font colors in the table below. This table shows the exact sequences and positions of each motif. A,p53 3'UTRs from a diverse range of species. B, p53 3 'UTRs from the bivalve species.  Figure 11 (Figure 5 of the submitted manuscript): Leukemic haemocytes from mussels Mytilus spp treated with murine monoclonal antibody 1E10 and analysed with laser scanning confocal microscopy. The images were obtained using Carl Zeiss L S M 510 software. Scale bars = 5 um. A) Mytilus edulis; B) Mytilus trossulus. Arrows in A indicate typical lElO-negative cells.  103  Figure 1: TAD (i) (i) (i) (i) (i) (i) (i) (i)  MSQASVSTTCTPSGPPMSQET--FEYLWNTLGEVTQEGGYTNITSKESIDYAFSEAEDET|I MSQASVSTTCTPSGPPMSQET--FEYLWNTLGEVTQEGGYTNITSKESIDYAFSEAEDETfI MSHEALHKMSQVAIHGTLP--NQPMSQET--FEYLWHTLEEVTDNVDYTHINTRE-LDYSYDDSEDGTSL MYISQPMSWHKESTDSEDDSTEVDIKEDIPKTVEVSG SEL PLQ -ME-PSSETGMDP---PLSQET- -FEDLWSLLP-D PPG MAQ---NDSQE-- -FAELWEFNLIIQ MAE SQE- - -FAELWERNLIST QEA - F . DLWKLLPENNVLS PI SC. -gEEPQSDPSVEP PLS  ®  ® #® #  #  ® PxxP  (61) (61) (66) (41) (31) (24! (22) (39)  (128) (128) (134) (78) (68) (63) (59) (95)  SVEKYRITSNDS-ISDLLNPIIGQ-TTfT|A.SSMSPDSQTNIIGSSASSPYNDT-lTSPPPYSPHTSMQSPI SVEKYRITSNDS-ISDLLNPIIGQ-TlsksSMSPDSQTNUGSSASSPYNDT-ITSPPPYSPHTSMQSPI ..VEKFRINQHHTDVSDLLNPIIG--TTSSSSMSPDSQTNISGSTASSPYQEMALTSPPPYSPHTNLTSPI TT|PMAFLQGLN SGNLMQFSQQSVX REMMLQDIQIQ A TVTCRLDNLS EFP. YPLAADMSVLQEGLMGNAVfT VT GGSCWDlflNDE E-YLPGSFDPK'FF- XVLEEQPQPS- - - T - -LPPT G-TCWELIND E - Y L P SFDP JIFDNVLTEQPQPS T--SPPT AMDDLMLSPDDI EQWFTEDPGHBEAPRMPEAAPRVAPAP -APTPAAPAPAPS - - WPLS PxxP PxxP PxxP D B D II PxxP SVPSNTDY-PGDYGFTIS FSQPS KE:KS!TWTYSESLKKLYVRMATTCPIRFKCLR-QPPQG VIRAMP : SVPSNTDY-PGDYGFTISFSQPSKETKSXTWTYSESLXKLYVRMA TCPIRFKCLR-QPPQG VIRAMP : TVPSNTXY-PGDYGFEISFATPSKE : KS : TWTYSDIliKjaYVRMATTCPVRFKTLR-QPPPGrVIRSMP XTLPKLENHNIQOYCFSMV: D E P - - - PICS - LWMYSIPLNKLYIRMNK'-FNVDV ; FKSKMPIQPLNLRVFL S 7 PS :DDY-AGKYGXQLDFQQ- :,GTAKSVTCTYSPELNKLFC. LAKTCPLLVRVES - PPPXGSILRATA STVPE TSDY- PGDHGFRLRFPQ-SGTAKSVTCTYSPDLNKLFCLAKTCPVQMWDV- APPQGSWRATA ASVPVATDY-PGEHGFKLGFPQ-SGTAKSVTCTYSSDliNKLFC; LAKTCPVQ>WVNV- APPQGSVIRATA SSVPSQ TY-QG YGFRLGFL. -SGTAKSVTCTYSPALNKMFC.LAKTCPVQLWVDS-TPPPGTRVRAMA  D B D III (196) (196) (202) (144) (135) (130) (126) (162)  IFMK EHV,.EPVKRCPNHATSKEHNENHPAP-THLCRCEHKL-AKFVEDP YTSR;SVLIPHEI - - IFMK EHV.E PVKRCPNHATS KEHNENH PAP-THLCRCEH XL-AKFVEDP YTSR.}SVLIPHEI- - IFMK EHV,E/WKRCPNHATSKEFNENHPAP-NHLVRCEHKV-SKYVEDP YTNRQSVLIPQE - - CFSND--VSAPWRCQNHLSVEPLTANNAKMRESLLRSE1CPN-SVYCGNAQGKGISERFSVWPL;XMSRS VYKKSEHVAEWKRCP:iHERSVEP-Gt.DAAPPSHLMRVEGNLQAYYMEDV- - - - NSGRHSVCVPYEG - - ITLRHSVFVPYEA--IYKKSEHVAEWRRCPHHERTPD- -GDNLAPAGHIt IRVEGNQRANYREDN VNSRHSVWPYEV- - IYKKSEHVAEWRRCP; ;HERTPD- -G GLAPAAHLIRVEGNSRALYREDD NTFRHSWVPYEP- - IYK.S.HM:EWRRCP:;HER SD-- GLAPPQHLIRVEGNLR EYLDDR  * *z z  DBD V  D B D IV  .GSEW .  (257)  -PQ  (257)  -PQ  (263)  -PQ GSEW TNLFQFMCL SCVGGPNRRPLQIV; TLE-KDXQVLGRR VEVRICACPGRDRK-XDER--XS v: XSGLTRQTLAF FvfQNB|i|- - -RKETSLVFCM^CJDIV|QHVIH|KI|TH^ -PQVGTECTTVLYNYMCNSSCMGGMNRRPILTIITLETP XGLLLGRR FEVRVCACPGRDRRTEE - -NY -PQLGAEWTTVL NYMCNSSCMGGMNRRPILTIITLETQEGQLLGRRSFEVRVCACPGRDRRTEE -NF -PQLGSE FTTVLYNFMCNSSCMGGMNRRPILT11SLETHDGQLLGRRS FEVRVCACPGRDRKTEE -NF -P VGSDCTTIHYNYMCNSSCMGGMNRRPILTiITLEDS GNLLGR SFEVXVCACPGRDRRTEE -NL  (211) (197) (191) (187) (223)  TNLFQFMCL ;SCVGGPNRRPIQIVLTLE-KD::QVLGRRAVEVRICACPGRDRK-VDEK--AA SEW TNLFQFMCL-1SCVGGPNRRPIQIVLTLE-KDXQVLIGRRAVEVRICACPGRTJRK*\DEK- AA  Z  §* z  (323) (323) (329) (278) (264) (258) (254) (290)  *  ** §  §* § § NLSI  §* § Tetramerization  LPPCKQS-PKKGQ KVNIINEITTVTP---GGKKRK-- -AEDEP- -FTLSVRGRE . YEILCRL.-DSL LPPCKQS-PKfGQ KVNIINEITTVTP---GGKKRK---AEDEP--FTLSVRGRE YEILCRL DSL LPPMVSGGVKKSQMP--KFSMGTEITTVS GKKRK FEDDEQTFTLTVRGREXYDMLCKI DSL KfRKSVPEAAEEDEPSKVRRCIAIKT: DTE XNDSR-DCDXSAAEWNVSRTPiXG'XYRLAITCP-NKEW KK RGLKPSG- -KRELAHPPSSEi PLP- - -KKR-LWXDDEE- IFTLRIKGRXRYEMIKKLNDAL KKDQETKTMAKTTTGT-KRSLVKESSSATLRPEGSKKAKGS-S DEE-IFTL.VRGRERYEILKKLNDSL RKDQETKTLDKIPSAN-KRSL'. KDSTSS PRPEGSKKAKLSGS: DEE - IYTL'.VRGKERYEMLKKINDSL RKKG--EPHHXLPPGSTKRALSNXTSSS. - Q P - - - K K K : LDGE-YFTL.IRGRERFEM,RXLNEAL NES-  104 domain Mep53 Mtp53 Map53 Dmp53 Xlp53 Drp53 Bbp53 Hsp53  (380) (380) (390) (343) (322) (325) (322) (349)  N L S II  N L S III  ELS."MVPQNQIDVYKQKQLDTNRQWLSMILAR:-.K ::KLMKKVKRPQHRP G- - IKS TELSMVPQNQIDVYKQKQLDTNRQWLSMILARE> K > KLMKKVKRPQHRPG--IKS TEIAALLPQNQLQSLKQKQVEVQRQWLTNVLAKEGK SRLIKKK HRPGKIIRHPLK L L . JIEGMIKE AAAEVLENPNQENLRRHA .MCLLSLKK RAY ELP- E L . SLDQ QKV IKCR • CR. EIK- KKG KKLLVKDE QPD SE- ELSiJVVPASDAEKYRQKFMTKNKKENRESSEPKQG KKLMVKDEG RSD SD- ELS:;WPPSEMDRYRQKLLTK K KD Q P PKRG KKLMVKDE K. D SD - ELK. AQAG KEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTE GPD SD - -  ®  ® ®  105 Figure 2: r Oap5  A  '200  1  200 Ssp5 Btp5  191  Dlp5  ril60  —  199  H  Cfp5 Fcp5  141  100  -| 200 92  Hsp5  Mammal  Cap5  Ocp5 Cpp5 200  Mmp5  H172  - Rnp5  1199 200  H  194  Mnp53pa Cgp5 J  H 119  Ggp5 — Xlp5  181  200 193  Drp5 Bbp5 Ipp5  H200  Ostariophy  — Omp5  200  — Pfp5  duo  — Tmp5  195  —  200 200  Olp5  Neoteleost  Xhp5 Mep5 200  200  186 200  Mtp5  Mollus  Map5 — Lfp5  153 200  - Tcp5 Ldp5  Insect  Dmp5 Cep1 Ehp5 0.1  106 Figure 3 Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  -AGA-AGA-AGA-AGA-  -GAGAA- -GTTTCCA -GAGAA- -GTTTCCA - GAGAA- -GTTTCCA - GAGAA- -GTTTCCA  ATTGGT- -CCAG- -GAATTGGT- -CCAG- -GAATTGGT- -CCAG- -GAATTGGT- -TCAG- -GA-  AAATACCAACTCAAATCGTTGTGAAAATGGTTATTATGAGGTCGATGGCCAGTCGATGTT  38 38 38 38  - T ATATAAAGACCTTGCTTTA - TATATAAAGACCTTGCTTTA-TA TATATAAGGACCTTGCTTTA  - ATGTGGTGCATTCATTTTGGA - ATGTGGTGCATTCATTTTGGA  1  ACATTGA - GGA ACATTGA-GGA ACATTGA-GGA-ACATTGA-GGATACATTGACGGAACTGACGA  CAATTATAGA CAATTATAGA  CAT CAT  ATGTGGTGCATTCATTT - GGA- - -CTATAATAGA  TAT  81 GTGCTCTACTGAAAAACTTTACTCCGTTTTTTGAGATCTGATGAAAT - ATCCAAGATGGCAGTTGTAAAAACACTAACAT  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map 5 3  TTATCAAA-ATCTTTTGGTATACAGTGAGATCCAGATGACTCCATGCTCCTGAAAGCAAGGATAATTGATATTTTATAGA TTATCAAA-ATCTTTTGGTATACAGTGAGATCCAGATGACTCCATGCTCCTGAAAGCAAGGATAATTGATATTTTATAGA ATTGATATTTTAT AGA 91 TTATCAAA-ATCTTTTGGTATACAGTAAGATCCAGATG CGTCCTGAAAGCAAGGATAATTGATATTTTAACGA 160 TAGTCCAACAAAACTGGATATGTAACGAGCCAAATCTGAGATAATATTACCCAAACCACCACAAATAAACAAGGAAAGGC  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  171 171 56 163 240  ACTTATCACGTCACCTCCTGCCATTTCACTGATGACCAAGGCAGCAAC AT* ACTTATCACGTCACCTCCTGCCATTTCACTGATGACCAAGGCAGCAAC AT* ACTTATCACGTCACCTCCTGCCATTTCACTGATGACCAAGGCAGCAAC AM ACTTATCACGTCACCTCCTGCCATTTCACTGATGACCAAGGCAGCAAC TJ ACTTGTCCAGCGACCTT CCATTCTCCAAGAGTTCCAAACAGCAGCCAATGA  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  243 243 128 235 317  TCATTGTTGGT GGTGGTGGG- -TCTCAT ATTG GTTT-GTTTGTTAGTTA-TCATTGTTGGT GGTGGTGGG- - TCTCAT ATTG GTTT - GTTTGTTAGTTA-TCATTGTTGGT GGTGGTGGG- -TCTCAT ATTG GTTT-GTTTGTTAGTTA-TCATTGGTTGT TGGTAGG- -TCTCAT ATTG GTTT-GGTTGTTAGTTA--GCTGTTAACATACCGTCAATAAACATCTCATTCAATCATAAACATTATCCCTTCATTTCAAAGAGTTCATT^TTTAbG p o t e n t i a l ARE  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  289 289 174 279 395  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  345 345 230 336 475  ATTTT-A GA GGATAATTTTATATTTTT- -TTCTTGTTTTTGAATGACATGGATTGTCTTTTAGTGAAAAGC ATTTT-A GA GGATAATTTTATATTTTT- -TTCTTGTTTTTGAATGACATGGATTGTCTTTTAGTGAAAAGC ATTTT-A GA GGATAATTTTATATTTTT- -TTCTTGTTTTTGAATGACATGGATTGTCTTTTAGTGAAAAGC ATTTTTA-- - -GA GGATAAATATATATATTTGTTTCTTGTTTTTGAATGACATTGATTGTCTTTTAGTGAAAAGC AgTTTTA^CACAATTATTAGATAAAAAGATATGCCTAGATCCTATT AATTAGAAGAATCTTGTTTTAGTTTAAA-T CPE  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  413 413 298 407  ATTTGAATCTGTTTCTGTATGAAAGAAATTTATTTGAATCTGTTTCTGTATGAAAGAAATTTATTTGAATCTGTTTCTGTATGAAAGAAATTTATTTGA-TCTGCTTCTATATGAAAGAAATTT-  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  480 480 365 471  92 92  4  0  -TAAGAGTTT-GAGTA- -CATAG- - ATATTT ATT - TTAT - TAAGAGTTT - GAGTA- - CATAG- - ATATTTATT - TTAT - TAAGAGTTT-GAGTA- -CATAG- - ATATTTATT - TTAT - TAAGAGTTTTGAGGA--CATAG- - ATATTTATT - TTAT  :AAT—TCATTATOQTT |CAAT---TCATTATCGTT CAAT- --TCATTATCGTT |CAAA---TCATTATCGTT JCAGTCGCTCATTATCGAT  AGGGCATGGTTTT AGAAAT - CAAGA AGGGCATGGTTTTAGAAAT - CAAGA AGGGCATGGTTTTAGAAAT - CAAGA AGGGCATGGTTTTAGAAAT - CAAGA  TCACTTTTAAAGTTCAGAATCATTATAACATATATTCACTCTTATCATGCTCAAAAGTGCCTGAAATTTAAAATATAAGC  -AAGACGAGAGTT- - -CTGAATC-TCATGTTG -AAGACGAGAGTT- - -CTGAATC-TCATGTTG -AAGACGAGAGTT- - -CTGAATC-TCATGTTG -AQGGCAAGAGTT- - -NTGAATC-TCAAGT  TTTT- -GTTTA TTTT- -GTTTA TTTT- -GTTTA TTTT- -TTTTG  550 ATAGAAATCACTTTTATTGTTAAAGGATTTTTTAGTCTGGTAGTTTTTCTTACTTATTATATAGAAACATTTTAAGTTTA CPE-1  630  GATGATGATTTT-  ARE-1  TTTTTTA1 TTTTTTJAI TTTTTTA1 TTTTTTJAI  TTA^ATTGAATC TTARATTGAATC TTA^ATTGAATC TTAAATATAAGC  CPE-2 AAAGTCTATCGGChTTTT AAAGTCTATCGGC TTTTT AAAGTCTATCGGC TTTTT AAAGTCTATCAGC TTTTT  ATJCTTTTGCCTAACAATTTGAAT CTTTTGCCTAACAATTTGAAT CTTTTGCCTAACAATTTGAAT CTTTTGCCTAACAATTTGA-  AGTCATATTGTCCGTCCAAGTAGGATTGTTTTTAGAATCTTCTCACACATTCACAGAGATCTGATTCCTAAGGATCTGAA  Figure 3 continued  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  ARE-2 ARE-3 542 - -^ATVT^TGAMIAfTT^GGTGTTATAGACTTCCCTCCCAA- - - -AATTAGATATGGA- - -ACTAGATGGTTTTAC- 542 --T.ATTTATTG---| , GA---ACTAGATGGTTTTAC 427 - -BATTTAJTTGAAJIATTTAjrGGTGTTATAGACTTCCCTCCCAA- - - -AATTAGATATGGA- - - ACTAGATGGTTTTAC - 533 - - tATTTjffTGAA^JATTTAgGGTGTTATAGACTTACATCNCAA- - - -AATTTGATATGAA- - -ACAAGGTGGTTTTAC- 710 TTTTTCTCTCTCTTTTTTGTAGTGCTATGTACATAGAGTG^TpTCAATTAAATCTGATTTTACTTGGCAGTCTGfl K-Box  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  611 568 496 602 790  -AAAA-AAAA-AGATCTTTTGAA GAAATTTT-TGATATTTTTGAAGAATCTCTTTT- --CTTCCTAGTT--ATTG-AAAA-AAAA-AGATCTTTTGAA GAAATTTT-TGATATTTTTGAAGAATCTCTTTT- - -CTTCCTAGTT- -ATTG- AAAA-AAAA-AGATCTTTTGAA GAAATTTT-TGATATTTTTGAAGAATCTCTTTT- - -CTTCCTAGTT- -ATTG- CCTACAAAA-AAAACTTTTTAA GAAATTTT - TGATATATTTGAAGTATCTCTTTT - - - CTTCATAGTT - -ATTGVTAAATAGTGGGAAATCTGAAGTTCCATTATATTQ^T^TlATfTCTTlrTTAGCTTCTTAGCTCCAATGT p o t e n t i a l CPE  CPE-3 -GCT- -TACATT TGTAAATA- -ATTTTCTATATAATATTTT TTTJTTTTT -GCT- -TACATT TGTAAATA- -ATTTTCTATATAATATTTT TTT TTTT1 - GCT - - TACATT TGTAAATA- - ATTTTCTATATAATATTTT TTT TTTTT -GCT- -TACATT TGTAAATA-ATTTTCTATATAATATTAA TTT TTTTT IUIMAIA--ATTTTCTATATAATATTAA TTTfTTTTT) TCATAATCTCATACACCGCTATTCCATTGTCATGGAAACAGAATTCACCA-AGAATATGCCGAGCTTGCATTTAAAGGAA  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  677 -AATAGT634 -AATAGT562 -AATAGT669 •CATAGT-  Mep53_vl Mep53_v2 Mep53_v3 Mtp53 Map53  727 684 612 719  865  K-Box CPE-3 ARE-4 ARE-5 -TA1TTCTTTA ATGTGATA TG 3TGCTATTTGTGTAATATATAATAATT - ATTGGATAWAT MTCTTTFTAC - TAT TTCTTTA ATGTGATA TG 3TGCTATTTGTGTAATATATAATAATT-ATTGGATA]TATT^ATTT]AAC - TAT TTCTTTA ATGTGATA TG' GTGCTATTTGTGTAATATATAATAATT-ATTGGATATTATTTHATTTRAC - - AT TTCTTTC ATGTGATA TG I——I ''"^"^"^""'"^"^^•^•^•^UCTATTTGTGTAATATATAATAATT-ATTTGATATATTtfATTTA* GTGCTATTTGTGTAATATATAATAATT-ATTTGATA.'TATTJT^TTTJ^AC 944 CTATACTGCCA|ATGT^A^ATGATTGATTGCGTTTCATATGTTTTACCTGTGAAATTTTATTGTAAOTATTmTTTR*-IrpIIIIIi/-I » m* mitrrymmm n i-irtrnitms— a m < m — .  Mep53_vl 804 TGATGTAAATATTTT--AAGGGTTATTTGTGAAGCATTGCAATAAGATGTTTCTGTTTATTAACCTATAGTTGGTAGCAT Mep53_v2 761 TGATGTAAATATTTT - -AAGGGTTATTTGTGAAGCATTGCAATAAGATGTTTCTGTTTATTAACCTATAGTTGGTAGCAT Mep53_v3 689 TGATGTAAATATTTT-AAGGGTTATTTGTGAAGCATTGCAATAAGATGTTTCTGTTTATTAACCTATAGTTGGTAGCAT Mtp53 795 TGATGTAAATATTTT--AAG CATTTTTAACTTATAGTTGTCAGCAT Map53 1021 TTGTATATTTATTTTCAAAATGTTACCTGT ATATTTGTAATATTTTACTTACATTTGTATGAAA Nuclear polyadenylation sites  Mep53_vl 882 Mep53_v2 839 Mep53_v3 767 Mtp53 839 Map53 1085  TTGTATAATGCCATTC AATAAAAI AA - T AAAfciTA - ATG TTGTATAATGCCATTC AATAAAAIAA-TAAA 3TA-ATG TTGTATAATGCCATTC AATAAAA t AA-TAAA 3TA-ATG TTGTATAATGCCNTTCAAGAAAAT AA-TAAA 3TG-ATG CTGCAAAAAGCAGGATGAAAGAGC AAATAAA TTGTATAAAACG  108 oo o  o o CM  as i. 0  u  w  G  o  gH  G  o m -rl M JJ it) •H 0) a o  ft  N tt  to  fl)  H •H •H o 0  (0 4J JJ JJ (0 JJ  00  t*  o ro  to  4J  <N  VO  rro  CM O ro  (fl  00  tfl  10 JJ 4J 4J 10 4J  JJ 4J 4J  4J 4J JJ  tfl JJ  (fl  to  JJ JJ JJ (0 JJ  JJ JJ JJ  to  (fl  JJ JJ JJ  JJ JJ JJ  JJ  JJ  JJ  <T\ 0 0 oi CM  10 00  ro in  00 00  tfl  tfl  m JJ JJ JJ  (fl J(flJ  ro  H  H  ro  ro  CM CM  co m  co m  ro m  ro in  ro in  ro in  ro in  ro in  4-1 4J  JJ JJ  JJ JJ  JJ JJ JJ JJ 01 01 JJ JJ 01 01 4J J J  JJ JJ 01 JJ 01 JJ  JJ JJ 01 JJ  JJ JJ JJ JJ 01 J J JJ 01 01 JJ JJ  o •tf  > > > > > >  ro in  ft ft ft ft ft ft ft ft o H H W H W W fn E-t ft 10 g g 9 g S g g h 0)  g  10 4J JJ JJ 10 JJ  o o CN  as  u  0  u  ta  en 01  a 0  g  G 0  JJ 0) 4J  JJ 0) JJ  Dl  JJ 01 4J  Oi  JJ  O u  -H •H a J J H vo H ro CM ro CM m in <N o tN H cn ro ro ro ro n(0 •H CO (N H CM CM CM CM H CM ro PH  H  4J  0  g  H  ro  >|  >  1  ro in  in co  < g  2  g  3  L  ft <U  > i  ft <U  ro in  ft d)  g  ro in  ft  4J  g  n in  ro m  (fl  U H  ft  2  ft  ro m ft  ro  m  61  co  m ft  (0  S  L I  ro in ft  ro m ft  01 JJ  a ro  0  ft  to <D  -H  JJ JJ JJ  H  ro  1 •H ro ro O in in a> £3 W  ft  (0  CM  > l • i i >1 ro in  ro in  ro in  ro in  ro in  ro in  ro in  ro in  ft ft ft ft ft ft ft ft PH ft U *2 ft 0 g  w s  S  £5  s  H  0  109 ON  o  JJ 4J 4J  JJ  (fl  JJ  «  4J 4J  <o nt (0  4J  t>) JJ (fl  0  4J  4J 4J  JJ JJ JJ JJ (fl J J JJ JJ JJ JJ  n) JJ JJ JJ JJ JJ  a) JJ JJ J J JJ J J  id id  JJ J J  mHcNiocNVocNrno  rororororococororo ininininiAinuiifliA Pi P< b Pi a O C9 CO J  O U  JJ J J JJ J J Cn Cn u JJ Cn Cn JJ J J O  M  H  ro  ro ro m in  5  g  X O  g  Jl  X o  110  o o  <U M O O w  ID  id  id  id  Xi  •U  Xi 4J  Xi 4J  4J  4J  U  O  O  id V Xi 4J  id V Xi 4J  cn cn cn tr> cn  I oa  •rl  in  rl Id ft  JJ  4J  4J  Oi Oi Oi Oi Oi  « 4J  •rl 4J  id 4J  4J  •d  id  n o o w  I i  'H  ca  u  in C N C N in CN! CH id 0 CN CN o CN H CN ft  •H  O  O  ft  H  CN ro  a > > >  J-l  4J  4J  id id id <d id id id id id id id id id id id 0» Oi •U  Xi  -U  4J  01 01 01 4J 4J 4J 4J 4J 4J 4J  U  U  u o o o CN 00 VO r-s ^1ro ro 00 o \o r> u  H  CN  ro  n > > > • I I I  • I I I ro <H - H ro ro co ro ro •H ro ro ro ro - H o in in in in in u in in in in m ^ m ft ft A A ft « H ft ft ft ft  (I § 3 &a s n n£ g Oi 0i 01 01 o o u o 4J  to  id Xi Xi id  G O g •H  id  •U  (0  o o  4J  10 u  (d ft  >  ro in  ro in  i  ft <D  CQ  SB  S3  ft  1  0) 53  > n in  ft  Me  CN  >|  Co  CO  U  4J  in in ft ft  Xi  Xi  o o 4J  •U  4J 4J  id Xi Xi  m CN O 10 ro CN 00 H H ro r- 10 vo r- oo  H  H  CN ro  I  I  I  •H ro ro ro ro ro U in in in in in a) ft ft ft ft ft  H  U  4J Xi Xi id xi Xi  H v> ro ro ro H CN CN CN H CN  a > > > V  4J  CN  ro  > >>  ro | ro ro ro ro in in in in in  ftft g  MTP MAP  0  O  MEP  4) M  B  3  Ill  Figure 5:  112  tvi-fenliun  cJtt.  fl  LhnL  113  C H A P T E R  3 - A D D I T I O N A L U N P U B L I S H E D  M E T H O D S ,  R E S U L T S  A N D  O B S E R V A T I O N S  In the previous chapter, Manuscript III describes methods used and results obtained during the course of this project. However, I feel that there are more observations that should be included in this thesis for the benefit of future investigations in this area, which would either have exceeded the publication limits of journals or make an "incomplete" story. Also, methods, which are commonplace in one discipline, can place insurmountable hurdles when applied in another discipline where they are new or less common. As part of this thesis is to try and bridge different disciplines I will describe here some methods in more depth and list unpublished results and observations.  /  1 M I C R O S C O P I C OBSERVATIONS A N D S A M P L E SELECTION FOR P C R  The microscopic examination of haemocytes was performed according to (McGladdery et al., 2001a). Normal (healthy), transitional (Elston et al., 1988b) and leukemic haemocytes display clear morphological differences. Generally, a healthy haemocyte, which is spread on a surface of the glass slide, is about 10 to 15 pm in diameter and has spike-like pseudopods (see (Cheng, 1981) for illustrations). They can occur as single cells or in clumps, especially when in the process of pathogen defense. Leukemic haemocytes, on the other hand, have lost their ability to spread and adhere to a surface, are round and of much more regular shape and smaller diameter, and have a much higher cell density due to active cell division. More frequently, haemocytes are not fully leukemic, but are in a transitional state. This state is harder to define and to detect, but commonly the.cells are larger and have to some extent lost the ability to form pseudopods. Often, their cell, shape is somewhat "mine"-like (round and with short spikes protruding from the cell surface), but cells are still able to stick to surfaces in most cases. Their nucleus to cytoplasm ratio is higher than in normal cells and additional nucleoli may be observable.  Haemolymph samples were generally taken from all three stages (normal, transitional and leukemic), although a heavy-leukemic animal was sought for the isolation of a p73 m R N A  114 transcript. This was based on the observation that thep73 protein was expressed at a higher level in leukemic haemocytes in M. edulis (C. Reinisch, R. Stephens, R. Cox, personal communication) and that therefore the likelihood of isolating a p73 above a p53 would increase. When choosing samples for R N A extraction care was also taken to take clean samples, which were not contaminated, by either parasites or bacteria, or mussel sperm cells. When the gonads are fully developed (spring-summer), sperm cells will very easily contaminate the haemolymph during the sampling procedure.  2 RNA EXTRACTION AND OBSERVATIONS R N A is very sensitive to enzymatic degradation and requires special handling. A l l bench and . other surfaces arid pipettes used in the extraction were decontaminated for elimination of RNases and D N A with "RNase Away" reagent (Invitrogen, Burlington, ON). Pipette tips used were certified RNase/DNase-free.. Water for make-up of solutions was DEPC-treated (diethyl pyrocarbonate) and gloves were worn at all times. Quality of total R N A extracts was confirmed by agarose gel electrophoresis (1% agarose). Commonly, the presence of two distinct bands at 1.5-2.0 kb and 4.5-5 kb for 18S rRNA and 28S rRNA, respectively, indicates that the R N A extraction procedure has yielded good-quality undegraded R N A , including mRNA (which is not visible on these gels without further labeling). However, repeated extractions only yielded one band, at 1.2 kb, and it was feared that by inference the R N A extracts did not have quality mRNA. However, it was found that, for instance in arthropods, 28S rRNA is nicked easily which makes the molecule migrate at the same speed as 18S rRNA in the gel (Ambion Technical Support, personal communication). In Spisula, R N A extracts appear as a smear despite being of good quality (Jill Kreiling, personal communication). In this case, the total R N A extracts from Mytilus yielded enough mRNA for c D N A synthesis and subsequent amplification of the p53 sequence.  115 3 IN-DEPTH DESCRIPTION O F M O L E C U L A R METHODS A N D MATERIALS  3.1 cDNA synthesis First strand cDNA (complementary D N A ) synthesis from R N A was carried out on approximately 5 pg of total R N A extract with oligo-dT primers following the guidelines for PowerScript™ Reverse Transcriptase (BD Biosciences Clontech, Mississauga, ON). Oligo-dT primers (1 pi, 0.5 pg/ul), total R N A and DEPC-treated ddFfiO were added to a total volume of 11 pi. After incubation for 10 minutes at 70°C, the mixture was transferred onto ice, and 4 pi of first-strand-buffer, 2 pi of dNTP mix (10 m M each dNTP), 2 pi DTT and 1 pi reverse transcriptase were added, mixed and incubated for 80 minutes at 42°C. The reaction was stopped by incubation at 70°C for 10 minutes and transferred to ice. 3.2 Degenerate PCR Two microliters of the resulting cDNA was then PCR-amplified using Taq polymerase (Invitrogen, Burlington, ON) and a touch-down PCR protocol: Initial melting at 95 °C for 1 min, 3 cycles of 94 °C for 30 s, 62 °C for 45 s, 72 °C for 45 s; 3 cycles of 94 °C for 30 s, 60 °C for 45 s, 72 °C for 45 s; and so on to 3 cycles of 94 °C for 30 s, 48 °C for 45 s, 72 °C for 45 s; and finally 16 cycles of 94 °C for 30 s, 46 °C for 45 s, 72 °C for 45 s; followed by a final extension at 72 °C for 10 min. The reaction mix was as follows: 0.25 pi Taq polymerase, 5 pi PCR buffer, 1 pi dNTP (10 m M each dNTP), 4 pi 50 m M M g C l , 2.5 pi or 1% (final) formamide, 36.75 pi of 2  dl-bO and 1 pi of each primer. Primers used for initial c D N A amplification were DegF2 and DegR2K (Table 8, designed based on known p53 sequences from a protein sequence alignment of various p53s, and based on the soft-shell clam Mya arenaria (Kelley et al., 2001). P C R reactions were analyzed on an agarose gel and resulting strong and weak bands bands were stabbed with a pipette tip and reamplified using the same primers and cycling conditions. P C R products were then gel-purified, cloned and sequenced as described below.  116 Name  Sequence 5' -> 3'  SsplF  ggcttccaggaaaaatg  Ssp73Ralpha  ttcacaaaatcaaatacatgaatccaa  Deg  F  Deg  R  ggRcalgcRcaKatlcKWacYtc  Deg  F2  gtlaaRMgltgYccIaaYcaK  Deg  R2  RtcNcKNccNggRcaNgcRc  Deg  R2 K ( e l l e y )  NggRcaNgcRcaDatNcKNacYtc  • catgcIgglWcWgaRtgg  5'RACE1  ggtgcccgaatcatgccacatctaaagagc  5'RACE2  tgaaaatcatccagctccaacacattatg  5'RACE3  tgtcgatgtgagcacaaacttgc  5'RACE4  ggacttgatgcagagctaccgatg  5'RACE5  cagggctcatagagctagctgtagtcg  3'RACE1R  gtacacatcctcctggtttgtcttccgg  3'RACE2R  cccagtggttaaacaaggtcaagtacacgg  3'RACE3R  cggcggtctcacaagattagggtgtactc  3'RACE829- 857  catgtgtaggaggaccaaacagaaggcc  3'RACE7 93- 822  ,  gggtcaccaatttgttccagttcatgtgcc  3'RACE742- no  gccgccagagtgttctaattccacatgag  3'RACE917- 937  ggtccgtatttgcgcctgccc  pMelF  atgtcacaagcttcagtttcaac  pMe-28F  tggaaagttcactcatcatcacc  Deg  NatKttcatYttNgcNaRRtcRtc  p73R  pMel323R  atatatcctcaatgttcctgaacc  Deg p73F2  gaYYtNgcNaaRatgaa  Deg  p73R2  KttcatYttNgcNaRRtc  Deg  p73R3  KttNaRNgcNccNaRRtc  5'RACE6  agtagttggctggcacatggcaatg  pF832seq  gtgtagcaggaccaaac  Table 8: Catalog of primers used during the course of this project. Note: Not all primers are referred to in the text; only primers with relevant results are described. Acronyms:. Ssp, S. solidissima primer; Deg, degenerate primer, pMe primer for M. edulis; R A C E , gene-specific primers for rapid amplification of cDNA ends; F, forward; R, reverse; seq, primer used for sequencing. Bases in capital letters are degenerate, using Y = C or T, R = G or A , K= G or T, W = A or T, N = G, A , T or C, I = inosine.  117 3.3 Gel purification of PCR products PCR products were run on a 0.7 - 1.0 % agarose T A E gel (40 m M Tris acetate, 1 m M EDTA) and bands were excised using an U V light table or a hand-held U V lamp taking care not to expose the D N A for too long to damaging U V . The gel slices were weighed and the D N A was purified from the slices using the Roche High Pure PCR Product Purification kit (Cat #1732668, Roche Applied Science, Laval, QC) or the QiaQuick Gel Extraction kit (Cat# 28704, Qiagen Inc., Mississauga, ON) and the manufacturers instructions. Concentration and quality of D N A was assessed spectrophotometrically at 260 and 280 nm and/or by gel electrophoresis. Gels were visualized using the Alphalmager system and the band intensity of the 1636 bp band of the 1 kb D N A ladder (Invitrogen) was used to estimate sample D N A concentration.  3.4 3' A-tailing Repeated gel purification steps and exposure to U V light can degrade the 3'A-overhangs on the PCR amplicon which are automatically created by Taq polymerase and which are required for sticky-end cloning using vector pCR 2.1 (see below). In cases where it was suspected that 3'Aoverhangs had been degraded the following procedure was used to add 3'A-overhangs to the gelpurified amplicons: 2 pi of amplicon, and 1 pi of 1 Ox PCR buffer, 1 pi of 25 m M M g C b , dATP to a final concentration of 0.2 m M , 5 U of Taq D N A polymerase and water to 10 pi were added. This reaction mix was incubated at 72°C for 15-45 minutes and then transferred to ice. 1 to 2 pi of this reaction was used for the ligation reaction. 3.5 Ligation and cloning The purified PCR products were ligated into the vector pCR2.1 (Invitrogen Life Technologies, Mississauga, ON; complete vector sequence is available at overnight at 14°C according to the manufacturers instructions for the Original T A Cloning kit. The amount of PCR product was determined by absorption at 260 nm and the amount used for ligation was determined as instructed by the manufacturer. E. coli PNVaF' or TOPI OF' cells were transformed with the ligation reaction according to the manufacturer. Recombinant cells were grown on selective L B plates containing 100 pg/ml (final concentration) ampicillin, 40 pi of 100 m M IPTG and 40 pi of 40 mg/ml X-Gal, overnight at 37°C. Colonies were picked and  118 transferred to 5 ml L B broth. The liquid cultures were grown overnight at 37°C and used to isolate the plasmid (Qiagen Mini Plasmid Purification kit, Cat # 12123, Qiagen, Mississauga, ON). Presence of insert was confirmed by either EcoRI restriction enzyme digest of the resulting plasmid or by PCR amplification of colony material with M l 3 forwardand reverse primers. Colonies were transferred to PCR reaction tubes containing PCR reaction components and cycled at 55°C annealing temperature. Plasmids with the correct length insert were then submitted for sequencing to the U B C Nucleic Acid and Protein Sequencing facility.  3.6 3' and 5' RACE PCR R A C E PCR (Rapid Amplification of cDNA Ends) is used when priming sequence of only one end of an amplicon is known and only one gene-specific primer can therefore be designed for PCR. The S M A R T R A C E c D N A amplification kit (Clontech, Palo Alto, CA) was used to reverse transcribe and amplify sequences to either end of the initial sequence obtained by Degenerate PCR. Gene specific PCR primers were designed (Table 8) such that they could be used for semi-nested PCR, and the following step-down amplification, protocol was used: Initial melting at 95 °C for 1 min, 5 cycles of 94 °C for 30 s, 68 °C for 45 s, 72 °C for 3 min; 5 cycles of 94 °C for 30 s, 66 °C for 45 s, 72 °C for 3 min; and so on to 5 cycles of 94 °C for 30 s, 62 °C for 45 s, 72 °C for 3 min; and finally 20 cycles of 94 °C for 30 s, 60 °C for 45 s, 72 °C for 3 min; followed by a final extension at 72 °C for 10 min (Eppendorf Mastercycler® gradient, Brinkmann Instruments, Inc.). Initial amplification products were usually confirmed by a nested or seminested R A C E - P C R . PCR products were gel purified, ligated and cloned, checked either by EcoRI digest or PCR with M l 3 primers, and submitted for sequencing to the Nucleic Acid and Protein Sequencing laboratory at U B C .  4P63/73 The following paragraphs will describe the rationales and approaches used to try and elucidate a sequence for a putative p63/73 transcript in M. edulis. Results were negative and no p63/73 sequence can be presented at this time.  119  Several approaches have been used to isolate p63/73 sequences. Previous literature has shown that p53 and p63/73 have very similar core sequences. Therefore, one approach has been to R A C E - P C R outward from the 3' end of the core region of the p53 and sequence clones of the expected size range (Bhaskaran et al., 2000; Kelley et al., 2001; Cox et al., 2003). A second approach is to use highly conserved domains from available p63/73 sequences from a closelyrelated species, to design degenerate primers (Pan et al., 2003). The M. arenariap53 andp73gene sequences are the most closely related known sequences to M. edulis. In M. arenaria, the lengths of the two sequences are very similar, 2537 and 2451 bp for p53 andp73, respectively (Kelley et al., 2001). In Danio rerio, thep53 is 2166 bp andp73 is 2335 bp long; in Barbus barbus, thep53 is 1766 bp andp73 is 2193 bp long. During the course of 3 ' R A C E - P C R experiments with primers for,the end of the D N A binding region for the elucidation of a p53 in M. edulis, it was observed that commonly several amplification bands were shown on the gels. The strongest band was identified first to be ap53. "N  Because of the narrow size range observed in other species, 10 clones of slightly varying sizes originating from this strong band were sequenced in the hopes that ap63/73 was among those. Several variants of ap53 were identified (see Manuscript III for a detailed analysis),.but none were similar to ap63/73.  '  Degenerate reverse primers to the most highly conserved region (about 560 bp Mya numbering, S A M domain) of the p73 were designed based on the p73 protein sequence found in Mya and Spisula, and paired with a Mytilusp53 forward primer (-28F or IF). Various concentrations of PCR additives such as D M S O and formamide, which destabilize secondary structures and lower melting temperatures, as well as varying MgCl2 concentrations were used in step-down PCR reactions designed to amplify a Mytilus p73. However, no reaction condition yielded a product. It may be that a) the concentration ofp73 mRNA transcripts was too low to obtain enough c D N A for amplification (Forney et al., 2004), or b) that the S A M domain is not highly enough conserved in Mytilus to enable degenerate primers to bind, or c) secondary structure in that domain hinders primer and polymerase accessibility.  Next, I examined other weaker bands that were co-amplified with the p53 band during 3 ' R A C E PCR. For that, I used R N A extracts from highly leukemic haemocytes from M. trossulus, collected during the sampling for the G V R D caged bivalve study in May 2004 off Hopkins  120 Landing, B C . It was assumed that ap73 transcript would be at higher concentration in highly leukemic animals because it had previously been shown that a p63-73 like protein was more highly expressed in leukemic haemocytes in M. edulis than in normal haemocytes (Raymond Stevens, personal communication). Unfortunately, no highly leukemic M. edulis was obtained, but presumably, similar gene expression patterns could be expected in highly leukemic M. trossulus. Figure 12 shows the results of the 3 ' R A C E - P C R amplification using several nested primers for transitional M. edulis and highly leukemic M. trossulus haemocytes. A strong p53 band is obtained iii both species at approximately 1.5 kb, but M. trossulus leukemic haemocytes also show a second band at approximately 1.9 kb. This band was subsequently excised and gelpurified. Gel-purification was repeated two to three times to exclude contamination from the strong p53 band. Cloning proofed to be difficult, despite. A-tailing the PCR product after repeated gel purification. Cox et al. (Cox et al., 2003) also found that one of the splice variants of thep73 in S. solidissima was not clonable. Therefore, direct sequencing using the 3 ' R A C E primers was attempted but did not yield any results for unknown reasons. Lastly, direct sequencing from the 3'end using the universal primer was attempted, but the sequence was unreadable due to polymerase slippage on the 3' poly-A tail.  121  2  3  5  6  7  H B  mmm  ^ ^ ^ ^  M eJw/w transitional haemocytes  4  SHB  ii^Ms  ? p53  M trossulus leukemic haemocytes  Figure 12: Ethidium bromide-stained agarose gels showing results of 3'Smart™ R A C E P C R reactions: Lanes 1, no target control; 2, Universal Primer M i x only; 3, primer Fp742-77fj- 4 nested primer Fp793-822; 5, nested primer Fp829-857; 6, nested primer Fp917-937 (all M. edulis p53 sequence numbering); 7, lkb ladder (0.5 mg).  5  Mytilus trossulus M Y T I L I N  C  Surprisingly, one of the M. trossulus clones amplified with primer Deg p73F2 yielded a sequence that was found to be closely related (77 % similarity) to the mRNA of Mytilus galloprovincialis mytilin B antimicrobial peptide precursor (Figure 13 and Figure 14) when blasted against GenBank. The clone sequence was submitted to GenBank, accession number AY730626. The clone has 525 nucleotides that translate into a putative protein of 103 residues in length.  Mytilins are small cystein-rich peptides with antimicrobial activity found in the haemolymph and haemocytes in Mytilus. They were first discovered and characterized by Charlet et al. in 1996 in the blood supernatant of M. edulis collected at the Zoological Outstation of the St. Petersburg  122 >NA_209-13 IGACCTGGCGA GTCCAAGACG TGTAGATATT TCCAGCGAGC CAGATGAATG ACTGGAATGT AAATTCGAAC TTTCTGTAAT AAAAAAAAAA  AAATGAAIGTT CAGATGCAAG ACGTCTCAGT ACACCATGAA ACCATGAGCA AAATAGACGG TGTGTTTATA TGTTATTCTA NGAAAAAAAA  AGCAGTTATC CTGTGCTTCC CAGATATGGA ATTCTCACCA ATTCCAGGAC CCTGATAAAG GACTTTTCTC AATAAAATTG AAAAAAAAAA  CTGGCTATTG AGATGTAAAT TGGTTCTGCT GAAAGTGAAG ATGCAGAAAG TGACATTGAT TTTCGTTTCA TGTTTCAACT AAAAAAAAAA  CTCTTGCAGT CTCGTTGTAG ACTGCAAATG GACCAGCTGA GAGAAACCGA ACACATTCTG CTTGTATAAA TAAAAAAAAA AAAAA  ACTTCTTATA AAGCAGACGC TCTCAGATGT GATGCCAGCA ACAAGGTGAA TTTTGGAGTG ATGTCTTAGA AAAAAAAAAA  >AA_209-13 DLAfaMKLAVILAIALAVLLIVQDADASCASRCKSRCRSRRCRYYVSVRyGWFCYCKCLRC SSEHTMKFSPESEGPAEMPAQMNDHEQFQDMQKGETEQGETGM 103  60 120 180 240 300 360 420 480  60  Figure 13: M. galloprovincialis mytilin C antimicrobial peptide precursor - like sequence in M. trossulus. Nucleic acid and putative protein sequence for M. trossulus clone 209-13, direct entry to GenBank, accession number AY730626. Red font indicates the ORF translated below, the box indicates the primer binding site for primer Deg p73F2, underlined bases are the stop codon for the ORF, and bold face indicates the nuclear polyadenylation signal. Scanning of the 3'UTR using UTRScan did not yield any further known 3'UTR signaling sites. The gray font in the protein sequence indicates amino acid residues prior to the start methionine, which are unlikely part of the protein, the blue font indicates the mature mytilin B sequence following cleavage by peptidases.  University at the White Sea (Charlet et al., 1996). They found two peptides, termed mytilin A and B, both 34 AA in length, 3.7 kDa mass, and rich in cystein residues. Mytilin A was rapidly active against gram positive and gram negative bacteria, but displayed specificity when challenged with different strains of E. coli. Mitta and co-workers (Mitta et al., 2000) examined a mytilin B precursor in Mytilus galloprovincialis (AF162336), as well as other isoforms which were termed mytilin C, D, and Gl. They found that the mytilins are stored only in some haemocytes, which exhibit large granules and infiltrate adductor muscle, gill, digestive gland, and intestine. Mytilin B positive haemocytes accumulated around sites where solutions containing bacteria were injected into the mussel. Only mytilin B positive haemocytes seemed to be able to phagocytose these bacteria.  123 > g i | 5 5 3 3 0 8 8 | g b | A F 1 6 2 3 3 6 . 1 | A F 1 6 2 3 3 6 - M y t i l u s g a l l o p r o v i n c i a l i s m y t i l i n ,B a n t i m i c r o b i a l p e p t i d e p r e c u r s o r , mRNA, c o m p l e t e e d s L e n g t h = 504 S c o r e = 162 b i t s ( 8 4 ) , E x p e c t = 8e-37 I d e n t i t i e s = 2 4 8 / 3 2 0 ( 7 7 % ) , Gaps = 18/320 (5%) Strand = Plus / Plus  Q u e r y : 13  atgaagttagcagttatcctggctattgctcttgcagtacttcttatagtccaagacgca  72  Sbjct:  atgaaggcagcagttattctggctatcgctcttgtagcaattcttgcagtccatgaggca  85  gatgcaagctgtgcttccagatgtaaatctcgttgtagaagcagacgctgtagatattac  132  26  Q u e r y : 73 Sbjct:  86  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 II I I M 1 1 • 1 |l 1 1 1' II  Ill-Ill.  II  '1 1 1 1 1  1 1 1 1 1 1 1 1 1  1 1 1 II  1  1 1 1 1  1 1 1 1 1 1 1  1 1 1 1 1 1  11 1  1 1 1 1 II  1  gaggcaagttgtgcttccagatgtaaaggccattgtagagcaagacgctgtggatattat  145  Q u e r y : 133 g t c t c a g t c a g a t a t g g a t g g t t c t g c t a c t g c a a a t g t c t c a g a t g t t c c a g c g a g c a c  II  Sbjct:  1 1 1 1 1 1  IN  II II  II 1 II  1 1 1 1 1 1 1  1 II  1 1 1 1 1 1 1 1  206 t c c a t g a a a t t c  Q u e r y : 247 a a t g a c c a t g a g 1 1 1  Sbjct:  1 1 1  1  1 1 1 1 1 1 1 1  1  1  1  I I  146 g t a t c a g t c c t a t a c a g a g g g c g t t g c t a c t g c a a a t g t c t t c g t t g t t c c a g t g a g c a t  Q u e r y : 193 a c c a t g a a a t t c t c a c c a g a a a g t g a a g g a Sbjct:  11  II  1 1 1 1 1  1 1  1 1 1 1  192 205  c c a g c t g a g a t g c c a g c a c a g a t g 246  Ml MM IN  1 1 1 1 1 1 1  II II II II  c c t g a a a a t g a a g g a t c a t c t c c a t c t g a c a t g a t g c c a c a g a t g 262  caattc  II  1 1. 1  c a g g a c a t g c a g a a a g g a g a a a c c g a a c a a g g t 297  II II  1 1 1 1 1 1  1  1 1 1,1  1 1  II  1 1 1 1 1  II II II  263 a a t g a a a a t g a g a a c a c t g a a t t c g g t c a g g a c a t g c c c a c a g g a g a a a c c g a a c a a g g t 322  Figure 14: Results for the Blast search (Discontiguous rhegablast) conducted May 9, 2004 at, indicating an overall 77 % similarity with the mRNA of Mytilus galloprovincialis mytilin B antimicrobial peptide precursor.  The mytilin isolated in this study was originally thought to be closely related to a mytilin B in M galloprovincialis, based on Blast searches (Figure 14). However, subsequent alignment with sequences not represented in GenBank but described in references (Charlet et al., 1996; Mitta et a l , 2000) indicate that the new mytilin (designated MtJVlytC in Figure 15) is probably more closely related to the mytilin C of M galloprovincialis (Mg_MytC), based on only three amino acid differences in the core region between these sequences, compared to eight aa differences between the new Mt_MytC and Mg_MytB (Figure 15). The new mytilin is therefore termed M. trossulus mytilin C precursor protein. The precursor protein has a 22-residue signal peptide which is likely cleaved by signal peptidase between the alanine and the serine residue in position 1 of the mature peptide (Figure 15) (Mitta et al., 2000).  124  Mg_MytB Me_MytB Mg_MytC Mt_MytC Mg_MytGl Mg_MytD Me_MytA consensus  Mg_MytB Me_MytB Mg_MytC MtJMytC Mg_MytGl Mg_MytD Me_MytA consensus  MKAAVILAIALVAILAVHEAEA  YYVSVI1' YYVSVI SCASRCKHCRIRRClYYVSV  |R0G|F|  MKLAVILAIALAVLLIVQDADA  asm  m um n  :  :FM.  ]R§GWF  I  C A S R C I AK  * * **  57  *  * *  \s  0 *** *  SSEHSMKFPENEGSSPSDMMPQMKENENTEFGQDMPTGETEQGETGI  35  61 SSEHTMKFSP-ESEGPAEMPAQMNDHEQ—F-QDMQKGETEQGETGM  37  35 35  61  Figure 15: ClustalW alignment of available mytilin sequences. Me ( M edulis) MytA and MytB (mytilin A and B) sequences were obtained from (Charlet et al., 1996). M g (M. galloprovincialis) MytB, C, D, and G l were obtained from (Mitta et al., 2000). M t M y t C was obtained during this study. The alignment is presented using Boxshade 3.2 available at Identical amino acid residues are highlighted in black, similar residues are highlighted in gray. The mature protein of M t M y t C is more similar to mytilin C then to mytilin B from M. gal I oprovincialis.  There is also a 43-residue C-terminal extension not found in mature peptides. Previously, peptide sequences were obtained by extraction from blood and purification by solid phase extraction and HPLC, and subjected to Edman degradation (Charlet et al., 1996). The ClustalW alignment also shows that the mature mytilin B from M. galloprovincialis and from M. edulis are 100 % identical, and that the mytilin D from M. galloprovincialis only differs in one aa residue from the mytilin A of M. edulis. Therefore, the mytilin D would have better been termed mytilin A in M. galloprovincialis. The 100 % sequence identity is curious, and can perhaps be explained by a failure to correctly identify the species from which they were isolated. The White Sea is predominately M. edulis based on evidence from morphological and/or genetic (allozyme) data  (Gosling, 1992). The French coast has both populations in the wild, but M. galloprovincialis used for the study was obtained from a farm in the Gulf of Lyon. However, both species can be intermixed, can have very similar morphometry and can hybridize with each other. Canonical variate analysis based on 18 morphological shell characters (McDonald et al., 1991), which has so far been the most reliable method for species distinction, was apparently not applied. It can v therefore not be excluded that all available mytilin sequences to date are from M. edulis only.  126  CHAPTER 4 - CONCLUDING CHAPTER  1 Discussion and recommendations for future research  1.1 p53 expression and environmental monitoring  Health and function o f individual  Population effects  Figure 16: Primary interpretation permutations of response reading of one gene in an array. (This figure was based on communications with P.v.Poppelen and S.A. Baldwin.)  The objectives of this thesis were not to only elucidate p53 family member in mussels chosen as bioindicators for environmental monitoring, but also to critically evaluate the usefulness of gene expression, and in particular expression of p53 for environmental monitoring. While the timeframe was too short for an experimental evaluation, the detailed analysis of the new p53 sequences in Mytilus (Manuscript III) as well as literature reviews make it quite clear that there is unlikely going to be a simple relationship between p53 up- or downregulation and environmental  127 exposure to contaminants. This, by no means, is to say that p53 should be discarded as a candidate for early-warning indicators of biological effects due to environmental degradation, but it has to be shown first that p53 up- or downregulation is correlated with detrimental effects for the animals, such as seen in haemic neoplasia. This should be the objective of a future study. p53 is a gateway, a 'hub' in the highly-connected regulatory network that leads to apoptosis or cell cycle arrest upon DNA-damaging signal recognition. Removal of the hub is the most effective way of destroying a network. Monitoring a hub's activity should be the most effective . way of monitoring effects that lead to cancer (Vogelstein et al., 2000). Its central position, however, also requires that the different converging factors that affect p53's regulation and activity need to be thoroughly understood before conclusions about p53 expression and environmental genotoxins as causative agents can be drawn.  A simple correlation of upregulated p53 expression with negative environmental effects cannot hold (Figure 16). There are several potential permutations that can occur just at the p53 gene expression level as well as at the functional protein level (i.e. health and function of the individual animal) upon induction by DNA-damaging events. Although there may be p53 inducers present in the environment, highly expressed and active p53 can mitigate potential effects and the animal remains healthy. Mutation of p53 itself may result in non-functional p53, in which case the p53, even so it is highly expressed, may not be able to mitigate detrimental effects on the animal level. Alternatively, other parallel pathways, independent of p53, may fill in the gap and effects on the animal-level are mitigated. In case of up-stream interferences with the p53 signaling pathway, p53 expression levels may not be changed despite the presence of DNA-damaging contaminants, and effects may be seen on the animal level, but not at the p53 gene expression level. And, lastly, negative environmental effects that do not damage D N A may not be detectable with this biomarker at all. To conclude, upon exposure to environmental contaminants, p53 expression has to be evaluated very carefully, both at the mRNA as well as the protein level before p53 can be applied as a biomarker. This, and the linkage between.p53 failure and haemic neoplasia in mussels requires further investigation.  Other genetic biomarkers have been used in the past to link environmental effects to diseases in fish, such as hepatocellular carcinomas, and their brief description here will serve to highlight  128 some of the issues that need to be addressed before interpreting results from an environmental monitoring program using gene expression data. The expression of the C Y P 1 A gene in Atlantic Tomcod and other fish was examined in laboratory experiments, feral and caged fish at pristine and polluted sites on the East Coast (Hudson River, Miramichi River, and other sites with varying levels of anthropogenic influence) (Wirgin et al., 1994). C Y P 1 A is part of the cytochrome P450 system which encodes for proteins responsible for metabolism of both endogenous and exogenous substrates. Phase I enzymes encoded for by this system oxidize xenobiotics to an inactive form in which they may be excreted from the body by Phase II enzymes. Alternatively, classes of environmental procarcinogens are activated to their carcinogenic state by C Y P 1 A enzymes. Activated carcinogens form D N A adducts responsible for the neoplastic process. Thus, induction of C Y P 1 A gene expression may not only be indicative of xenobiotic exposure but also of detrimental biological effect. Field studies showed that levels of C Y P 1 A mRNA expression correlated well with environmental levels of aromatic hydrocarbon contamination, and concordance was seen between relative levels of C Y P 1 A expression and other biological markers, such as aromatic compounds in the bile and levels of hepatic D N A adducts. Laboratory studies demonstrated that expression of C Y P 1 A mRNA was sensitive to very low levels of exposure and a dose response was observed. The following precautions were raised: Genetic differences in the inducebility and activity of the C Y P 1 A mRNAs should be quantified and evaluated before instituting environmental monitoring programs. Variability exists at varying levels of taxonomy, including between individuals within a population, among populations, and among species. Because of large interspecies variability, selection of the appropriate sentinel species is critical. Observed variation in the C Y P 1 A mRNA and protein structure suggests that the biological consequences of induction may vary widely even among individuals. Also, prior exposure history had an effect on levels of C Y P 1 A gene expression in exposed populations of feral fish. One may add, that it is also critical to select an appropriate biomarker depending on the xenobiotics that one wishes to examine. While C Y P 1 A is a very good biomarker for contamination by polyaromatic hydrocarbons, PCBs, dibenzofurans and dioxins, it may not be suitable for other aquatic contaminants. 1.2 p53 and the endocrine system The field of research studying connections between p53 and the endocrine system is still in its infancy (starting in the early nineteen nineties). This section will discuss recent developments in  129 this field based on a review by Sengupta and Wasylyk (Sengupta et al., 2004) and references therein. It is important to keep in mind that studies have focused on the glucocorticoid, androgen and estrogen receptors in humans (and to some extent in rodents) only, and that interaction between p53 and these receptors are based on receptor binding of their native ligand hormones, and not at all of EDCs. However, I feel that the potential connections between E D C exposure, p53 mRNA expression and regulation and p53 protein function is one avenue that needs to be explored in the future, because the rising age- and country-specific incidence rates of human diseases such as prostate cancer and breast cancer suggest that tumor risks are in part determined by lifestyle choices and environmental factors (Henderson, 1993), such as potentially EDCs.  The glucocorticoids (GCs) in humans maintain homeostasis in response to internal and external stresses via the regulation of the function of the glucocorticoid receptor (GR). GRs remain in the cytoplasm in large complexes with chaperons, such as the heatshock factor hsp90. When GRs are activated by binding GCs, they release the chaperons and translocate to the nucleus where they modulate various genes' expression levels. p53 cross talks with GR both as an antagonist as well as a complement. GCs can induce cell death upon stress, and p53 activation causes an enhancement of the resistance to GC-induced cell death. Reciprocally, it was demonstrated that GCs protect against apoptosis induced by p53. It has been shown that the core domain and the nuclear localization signal of the p53 protein interact with the GR. The physical interaction of p53 and G R in the presence of its ligand GC can result in cytoplasmic sequestration of p53, a condition observed in neuroblastoma and other diseases (O'Brate et al., 2003). (Other cytoplasmic "anchors" for p53 are hsp70, vimentin, tubulin, F-actin, and Pare, an ubiquitin ligase.) Much like p53, the G R is also a substrate for M D M 2 , the ubiquitin ligase destining p53 for cytoplasmic degradation. Apart from cytoplasmic sequestration, p53 and G R can also regulate each other's activity at the transcriptional level by affecting each others target genes, for instance via the cyclin-dependent kinase inhibitor p21 leading to growth arrest, and others. The question is, of course, what determines whether these two transcription factors act in positive or negative loops? While far from clear, the overall primary response to a particular stress seems to depend on the cell type, which overrides and modulates the response in individual gene products.  Androgens are critical for the development, growth, and maintenance of the male reproductive system. They bind to the androgen receptor (AR) and regulate its activity as a transcription  130 factor. A R is also important in pathological situations, such as prostate cancers, where it regulates genes involved in proliferation and differentiation. Primary prostate tumors usually have wildtype p53 and respond to anti-androgen therapy, whereas advanced prostate tumors often express mutant p53 and are hormone resistant. M D M 2 overexpression is associated with cancer progression and aggressive behavior. Just as with GR, M D M 2 can bind to A R and lead to ubiquitination-mediated degradation of A R . Molecular and functional evidence provide a link between p53 and A R : Agonist activation of A R in rats led to a decrease in nuclear p53 by posttranscription mechanism, suggesting that A R negatively regulates p53 nuclear localization and stability.  Estrogens, responsible for proliferation and differentiation of the female reproductive organs, bind to estrogen receptors (ERa and ERP). E R a expression is a useful predictor of prognosis and therapeutic response in breast cancer. Several studies have linked p53 with estrogens and E R a . Estrogens have'been shown to increase p53 protein levels through an indirect mechanism that involves increased transcription of the c-myc gene followed by c-Myc activation of the p53 promoter. Estrogens have also been shown to stabilize wild-type p53 protein in the absence of increased p53 transcription via M D M 2 . Curiously, mutant p53 protein turnover was found to be insensitive to estrogens. E R a has been shown to form a triple complex with p53 and M D M 2 , and protect p53 from MDM2-mediated degradation. Estrogens have also been shown to induce cytoplasmic accumulation and functional inactivation of wild-type p53 in MCF-7 cancer cells, which could mirror the cytoplasmic accumulation observed in breast cancer tissue. A common feature of all these studies is that estrogen activation^of E R a leads to increased expression and cytoplasmic accumulation of p53 and M D M 2 . In a number of tumor types that retain wild-type p53, loss of p53 activity is associated with a cytoplasmic "zip code", where p53 is excluded from the nucleus (O'Brate et al., 2003).  Lastly, one of p53's target genes is 14-3-3sigma, which sequesters the cyclin/cyclin-dependentkinase complex and is responsible for a block at the G2 stage. Estrogen binding to the E R can induce the transcription of a gene for another ubiquitin ligase (Efp), which, upon specific binding to 14-3-3sigma, will tag it with an ubiquitin chain and destine 14-3-3sigma for degradation (Figure 17). That will release the cyclin/CDK complex, annulling p53's protective measures, and the cell will enter into mitosis. Because this pathway is downstream and therefore independent of  131 p53 expression itself, it may, by extrapolation, explain two observations from bivalve neoplasia: 1) haemic neoplasia is found at higher prevalence in areas contaminated with EDCs (such as some herbicides and PCBs) (Farley et a l , 1991,Van Beneden, 1994 #155; Gardner, 1994; Harper et al., 1994), and 2) p53 protein was not found to be at higher or lower concentrations in neoplasia compared to healthy animals (Jessen-Eller et al., 2002), (Kelley et al., 2001), (Stephens et al., 2001). If this pathway is a significant mechanism for leukemia development under E D C influence then using p53 itself (concentration of protein or p53 somatic gene mutations) as a biomarker may not be a suitable approach.  Common to all three hormonal networks is the connection between p53, M D M 2 and their respective hormone receptors (Table 9). In all three cases, p53 is inactivated by the receptors and/or is sequestered in the cytoplasm. G R and A R downregulate p53 stability, while E R upregulates p53 stability. However stable, cytoplasmic p53 cannot function as a transcriptional regulator in the cell nucleus. It was shown that p53 in leukemic haemocytes in M. arenaria were exclusively present in the cytoplasm and not in the nucleus (Kelley et al., 2001), while normal haemocytes retained p53 in the nucleus. It emerges, that assaying for protein p53 cellular localization may be a useful approach for studying endocrine effects on p53 activity and function. Pathological and physiological consequences of the receptor-p53 interactions are still unknown, especially for the A R and E R (Sengupta et al., 2004).  132  Figure 17: Estrogen-responsive protein Efp controls cell cycle and breast tumor growth. Adopted from the BioCarta collection of the Cancer Genome Anatomy Project at Pathway contributed by Kosi Gramatikoff, based on several published contributions. Abbreviations: C D K , cyclin dependent kinase; Ub, ubiquitin. See text for explanation.  133  GR  AR  ER  down  down  up  down  down  up  MDM2 s t a b i l i t y  7  7  up  MDM2 gene t r a n s c r i p t i o n  7  7  up  Cytoplasmic accumulation o f receptor  yes  yes  yes  Receptor i s t a r g e t f o r MDM2 E3 l i g a s e  yes  yes  yes  yes  7  yes  7  yes  no  I n h i b i t i o n o f r e c e p t o r DNA b i n d i n g  yes  yes  yes  p53  i n a c t i v a t i o n by r e c e p t o r  yes  yes  yes  p53  reactivation  yes  7  yes  P a t h o l o g i c a l consequences o f r e c e p t o r - p 5 3 i n t e r a c t i o n  yes  7  7  Physiological  yes  7  7  Receptor p53  stability  stability  T r i p l e complex: receptor-p53-MDM2 Inhibition o f receptor dimerization  b y downregulation  o f t h er e c e p t o r  consequences o f r e c e p t o r - p 5 3 i n t e r a c t i o n  Table 9: Effects of the interactions of p53 and M D M 2 with GR, A R and ER. Adopted with permission from the authors from (Sengupta et al., 2004).  1.3 Chemical agents for haemic neoplasia in bivalves and p53 expression While a true causative chemical agent or combination of agents for haemic neoplasia have yet to be identified, there is abundant evidence in-situ as well as in laboratory exposures that toxicants and possibly other stresses can increase the prevalence of haemic neoplasia (see (Elston et al., 1992) for review). Toxicants may be just an additional stress, rather than the causative agent. It is therefore of importance that caged mussel studies use animals which have originated from the same population, i.e. have the same genetic background and background prevalence of the disease. Controls for handling and background prevalence should be placed in "clean" environments unaffected by potential toxicants and resembling, as much as possible, sampling sites in salinity, temperature, and other environmental factors. It is also important to fully understand the natural background and temporal cycle of the disease in the local mussel population. Any disease prevalence above the background and outside the natural cycle will indicate compounding negative effects in the environment possibly induced by anthropogenic impacts.  '  134  Somatic mutations in the p53 gene in humans, more so than protein expression, have been shown as a result of chemical exposures. The p53 mutation spectrum (location and type of mutations) differs in different cancer types associated with different exposures. Mutation spectra are fairly well characterized for human cancers (see (Hofseth et al., 2004) for a collection of p53-related websites and databases). Hot spots for mutations (sites of frequent mutations, see also Manuscript III) seem to occur in codons encoding amino acids in direct contact with p53regulated D N A . One determinant in the type of mutation found in tumors is the selection for growth advantage they give the cell while destroying normal p53 protein function. A second determinant is the etiology of the tumor, i.e. tumors in different organs have different p53 mutation spectra. And finally, in some cases, evidence now exists that exogenous carcinogenic exposures contribute to different mutation spectra (Olivier et al., 2001; Vahakangas, 2003; Pesch et al., 2004). P A H compounds in cigarette smoke as well as radon and mustard gas exposure induce certain codon changes in p53 in lung cancer, vinyl chloride seems to induce A:T to T: A mutations in.p53 in liver cancer, and chromate and nickel was shown to induce G:C to A:T changes in p53, again in lung cancers (Vahakangas, 2003), and references therein). In Mya arenaria, a. single nucleotide change from C to G (changing the aa residue from proline to' alanine) was detected in the p53 sequence from leukemic clams from New Bedford Harbor, an area highly polluted with PCBs (Barker et al., 1997).  Numerous studies have been published on the immunohistochemistry of p53 protein in human tumors. However, the status of the protein (wildtype, mutant) being detected needs consideration and cannot easily be addressed with antibodies. Generally, mutant p53 protein has an increased half-life in the cell and can therefore be more easily detected than wild-type protein. But similarly, the binding of other (such as viral) proteins to p53 can affect its half-life, and induction of p53 upon induction by DNA-damaging agents can increase its detectability. Therefore, detection of the p53 protein alone is not sufficient to link p53 protein expression with exposure to environmental carcinogens. The increase in protein level is generally attributed to an increase in p53 stability which can be achieved through post-translational modifications (e.g. phosphorylation) and reduced interaction with the M D M 2 protein, but, to my knowledge, there is currently no significant literature attempting to link exposure to environmental carcinogens to effects on post-translational modification of p53 directly. However, new projects within the  135 Environmental Genome Project at the US National Institute for Environmental Health Sciences (NIEHS) ( are attempting just that.  To conclude, there is evidence that p53 (mostly human) as well as cancers (in humans) and haemic neoplasia (in bivalves) are affected by environmental carcinogens. Previous studies have linked haemic neoplasia to p53 family members in Mya arenaria. It is therefore highly probable that a connection can be established between p53 family members, haemic neoplasia, and . environmental effects in Mytilus. The exact nature of that link, such as p53 mutations, or differential p53 regulation (post-translational, cellular localization, phosphorylation), or indeed an involvement of other p53 family member (such as p63 and p73) or p53 regulators (such as M D M 2 or myc proteins), remains to be elucidated in future studies.  1.4 Other causative agents for haemic neoplasia in bivalves and p53 expression Other potential causes of increased prevalence of haemic neoplasia in bivalves have been studied (see (Elston et al., 1992) for review). Are there any agents that negatively affect p53 gene family function which are also implicated in the development of haemic neoplasia? The following is a brief list of such coincidal agents, other than environmental contamination, with the purpose of raising awareness, rather than to draw any conclusions about their validity as agents for simultaneous induction of haemic neoplasia and p5 3 expression.  Viral oncogenes: Evidence for infectional triggers by retroviruses are weak, but have not been excluded from the list of causative agents for haemic neoplasia (in M. arenaria) (Elston et al., 1992). In humans, some D N A viruses (not retroviruses) such as simian virus 40, human papilloma virus and adenoviruses, encourage the cells they infect to become cancerous. They . produce proteins that bind to and inhibit tumor suppressors such as p53 (Vogelstein et al., 2000).  General stress conditions (including high temperatures) may enhance the likelihood of haemic neoplasia. Chaperons, or heatshock proteins, such as Hsp70 are induced under stress and generally stabilize other proteins necessary for cell survival, growth and development. Hsp 70 and other heatshock proteins have been shown to be able to bind to wildtype p53 and conformationally mutated p53. It has been proposed (Zylicz et al., 2001) that wild-type p53 may  136 be .escorted to the nucleus by chaperones, such as Hsp90, where it becomes activated by phosphorylation. However, another hsp (Mot-2 protein), in the absence of an appropriate hsp, may induce the aggregation of wild-type p53 in cytoplasm, which results in the inhibition of apoptosis in cancer cells.  Hypoxia: It was shown that haemolymph with highly leukemic haemocytes in M. arenaria is hypoxic (Sunila, 1991). This is likely not a cause for, but rather and effect of haemic. neoplasia, because partial oxygen pressure in the haemolymph of early cases of sarcoma did not differ from control animals. Interestingly, p53 can also be induced by hypoxia (Vousden et al., 2002). In the case of hypoxia, it is known that the physiological response is oppositely regulated by p53 (leading to apoptosis) and by the glucocorticoid receptor (GR) (leading to cell survival) (Sengupta et al., 2004). As shown above, the G R linked to its ligand can have an antagonistic effect on p53 activity.  1.5 LAS treatability studies and effects on EDC removal Treatability studies showed that anionic surfactants and surfactant-associated toxicity can be removed from the Lions Gate WWTP sewage by several treatment options (Bradley, 2004). Biological treatment was most effective at removing M B AS activity, followed by ozonation, air flotation, partitioning to solids and alum coagulation/flocculation. IC20 Microtox™ toxicity was reduced by approximately 45 % in the total sample using air flotation. Air flotation and subsequent biological treatment of the resulting froth, which likely contains most of the M B A S related toxicity, is currently considered as a potentially viable interim treatment option at the Lions Gate plant.  Based on the study by Miyamoto and coworkers (Miyamoto et al., 2002), the effects of E D C likely present in the primary treated sewage may also be reduced in the final effluent. However, this requires analysis of EDC activity of the influent, the primary effluent, and the effluent resulting from the biotreatment of the froth generated by air flotation. It may be of interest to analyze the partitioning of EDC activity into the effluent and the froth fraction after air flotation.  137  2 Major conclusions and recommendations The p53 mRNA sequences were elucidated in two mussel species, Mytilus edulis and Mytilus trossulus, both currently used for environmental monitoring. The sequences share 99 % amino acid homology. They differ in one residue and in the length of the 3' untranslated region of the mRNA.  Based on the previously-shown involvement of p53 gene family members in haemic neoplasia, and based on the fact that haemic neoplasia is, at least in part, affected by anthropogenic contamination, it is suggested that p53 expression can be used as an early warning indicator for biological effects of anthropogenic influences i f it is applied judiciously and understood to a ' reasonable extent. Based on detailed analysis of the mRNA sequences and especially the 3' untranslated region, I suggest that there is unlikely a simple positive or negative dose-responselike relationship between anthropogenic contamination and p53 expression. Future experiments need to address the presence/absence of other p53 family members in mussels, which have been implicated in haemic neoplasia in clams, such as p63 or p73-like mRNAs. Further, expression levels of these and other translational activators and regulators involved in the p53 network should be quantified in leukemic, transitional and healthy mussels at the mRNA and also at the protein level. The effect of endocrine disruptors other than PCBs or PAHs on the development of haemic neoplasia and p53 gene family expression is of future interest. Other contaminants, such as linear alkylbenzene sulfonates can increase the biological effects of endocrine disrupting compounds, but EDCs are likely to be present even after (partial) elimination of the surfactant by physical and biological wastewater treatment.  138  REFERENCES  Atkin, N . , 2000. M Y C . In: (Ed.), Atlas of Genetics and Cytogenetics in Oncology and Haematology, vol. 2004. Barker, C M . , Calvert, R.J., Walker, C.W., Reinisch, C.L., 1997. Detection of mutant p53 in clam leukemia cells. Exp. Cell Res. 232, 240-245. Benard, J., Douc-Rasy, S., Ahomadegbe, J . - C , 2003. 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