Biochemical ecotoxicology : principles and methods PDF
Preview Biochemical ecotoxicology : principles and methods
Biochemical ECOTOXICOLOGY Principles and Methods Biochemical ECOTOXICOLOGY Principles and Methods FRANÇOIS GAGNÉ Senior Research Scientist Biochemical Ecotoxicology at Environment Canada Québec, Canada With AdditionalContributions From CHANTALE ANDRÉ JOËLLE AUCLAIR ÉMILIE LACAZE BRIAN QUINN AMSTERDAM(cid:1)BOSTON(cid:1)HEIDELBERG(cid:1)LONDON NEWYORK(cid:1)OXFORD(cid:1)PARIS(cid:1)SANDIEGO SANFRANCISCO(cid:1)SINGAPORE(cid:1)SYDNEY(cid:1)TOKYO AcademicPressisanimprintofElsevier AcademicPressisanimprintofElsevier 32JamestownRoad,LondonNW17BY,UK 225WymanStreet,Waltham,MA02451,USA 525BStreet,Suite1800,SanDiego,CA92101-4495,USA Copyrightr2014ElsevierInc.Allrightsreserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinanyformor byanymeanselectronic,mechanical,photocopying,recordingorotherwisewithoutthepriorwritten permissionofthepublisher. 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BritishLibraryCataloguing-in-PublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress ISBN:978-0-12-411604-7 ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com PrintedandboundinUnitedStatesofAmerica 14 15 16 17 10 9 8 7 6 5 4 3 2 1 LIST OF CONTRIBUTORS ChantaleAndre´ Emerging methods, Aquatic ContaminantsResearchDivision,EnvironmentCanada, Montre´al,Que´bec, Canada Joe¨lle Auclair Emerging methods, Aquatic ContaminantsResearchDivision,EnvironmentCanada, Montre´al,Que´bec, Canada Sylvie Bony Ecole Nationale des Travaux Publicsde l’Etat France Alain Devaux Ecole Nationale des Travaux Publicsde l’Etat France Franc¸ois Gagne´ Emerging methods, Aquatic ContaminantsResearchDivision,EnvironmentCanada, Montre´al,Que´bec, Canada E´milie Lacaze Institut Armand-Frappier-INRS, Laval,Que´bec, Canada Brian Quinn Universityof WestScotland,Scotland, UK xiii DEDICATION I dedicate this book to Lucie, my life-long companion, for her unconditional support in the many projects I undertake. I also dedicate this book to my daughters Genevie`ve, Catherine and Vale´rie for providing me with reasons to fight for the protection of the environment and to my parents for their support during my studies in biochemistry and thereafter.IalsodedicatethisbooktomymentorsProfFrancineDenizeau(inmemoriam) and Dr Christian Blaise for their guidance, foresights and for opening my eyes to the scienceofecotoxicology. Franc¸ois Gagne´ January 2014 xv PREFACE The science of ecotoxicology is the study of contaminant exposure and its effects on all organisms in the biosphere, including humans. This discipline started in the late 1960s and was defined as the study of the contamination of the biosphere and the resulting toxic effects to all life forms. The scope of ecotoxicology is quite broad because it deals with the studies of the fate and effects of contaminants from the molecular level to the ecosystem. To address this complexity, ecotoxicology was subdi- vided into three phases or pillars: exposure and bioavailabilityof contaminants, toxicity evaluation of substances with their mixtures, and biomarkers to determine the long- term risk of pollution in organisms and corresponding populations. This relatively new science was further complicated by the observation that not only chemical agents are at play but the presence of physical (radiation, temperature variations) and biological agents (infectious diseases) are also involved. Hence, there is a need to understand the cumulative contribution of these agents to organism health and the maintenance of populations. In addition, the advent of new exotic products such as nanotechnology, climate change, and landscape transformation will bring new types of toxic interactions that could overwhelm the ability of organisms to cope with existing environmental stressors such as prey(cid:1)predation changes, eutrophication, food availability, water levels (and quality), and seasonal temperature changes. Biomarkers are broadly defined as a measured variable that results in or follows the interactions of (toxic) agents with a molecular or physiological target. It is generally recognized that fundamental interactions occur at the biochemical level in cells pro- vided that this interaction forms the basis of initiating toxic events in organisms. Some biomarkers are more specific (more closely related) to the toxic agents so they can be used in testing schemes to identify the contribution or the cumulative effects of vari- ous toxic agents. These types of biomarkers are particularly valuable since organisms are seldom exposed to one compound or toxic agent at a time. Indeed, organisms are usually exposed to low concentrations of hundreds and perhaps thousands of chemicals in their lifetime. Biochemical markers could also determine some biochemical changes in organisms that could lead to deleterious effects on organism health in the long term. To provide a clear representation for the reader, the occurrence of high lipids (cholesterol) and oxidative stress (inflammation) in the plasma is associated with the degeneration of blood vessels and related diseases in humans. Hence, the measure of plasma lipids or cholesterol represents a biomarker of risk in developing a vascular pathology in time. In other words, a biomarker could represent a biochemical xvii xviii Preface condition that could lead to damage at a higher level of biological organization. In order to predict effects at the level of organism’s health and maintenance of popula- tion, we need to understand how a biochemical change resonates at a higher level of biological organization. Biochemical markers could contribute to this understanding and find their use and place in ecotoxicology. When I started as an experimental scientist, many techniques were not always read- ily available in the laboratory. We (my colleagues and myself) often had to develop and adapt methods from those reported in the primary literature. We also had to examine hypotheses on issues related to a particular biochemical effect of given pollutants to a physiological target in organisms under budget and time constraints. As a preliminary approach we sometimes first used generic and cost-effective biomarkers to determine whether a type of effect occurred in exposed organisms before using more sophisti- cated and expensive techniques or approaches. We quickly came to realize that these preliminary testing tools were of great interest to laboratories with tight budgets (which is often the case for most laboratories) or located in remote areas. We discov- ered that not only highly sophisticated techniques are valuable in ecotoxicology but generic ones as well. Another advantage of generic assays, which are based on a partic- ular and fundamental property of the biomarker, is their potential adaptability to be used in new species. These experiences made me decide to produce a book of meth- ods in biochemical ecotoxicology that illustrates both generic and highly sophisticated and species-specific biomarkers that are accessible to many laboratories with budget and other constraints. We also realized that biochemical markers should stand the test of time, be cost-effective, simple to use, and accessible by science laboratory students without major investment. In this respect, this book will assist the experimenter in ecotoxicological sciences to quickly start biochemical-based assays on various organ- isms. Our laboratory receives many requests by graduate students, young scientists beginning in this field, and other researchers getting acquainted with a new area of research to learn these techniques. Each chapter of this book could be considered comprehensive on its own and follows a common structure to enable the reader to quickly learn and try the assay. Each chapter has a brief introduction showing the con- text and principle of the assays, a section on reagents and solutions required for the procedure, data handling, and references. In some instances, case studies or examples are provided to demonstrate how the methods can be used. I am confident that this book will be well appreciated by both researchers and students who need quick and direct guidance in performing biochemical assays in ecotoxicology. Franc¸ois Gagne´ Montre´al, January 2014 ACKNOWLEDGMENTS The principal author is very grateful to the contributions of Dr. Brian Quinn for the preparation and use of cell cultures in toxicity investigations in Chapter 3, Dr. E´milie Lacaze, Sylvie Bony and Alain Devaux for the contribution on the COMETassay in Chapter 10, Chantale Andre´ for her contribution on gene expression assays using quantitative reverse-transcriptase polymerase chain reaction in Chapter 4, and to Joe¨lle Auclair for her contribution on protein transfer and detection techniques (Western blots) in Chapter 5 for the detection of protein-based markers. Franc¸ois Gagne´ xix INTRODUCTION Biochemical ecotoxicology is the study of the effects of contaminants in ecosystems using biochemical methods. The scope of this discipline is vast and complex because it implicates exposure to low concentrations of mixtures in the long term (chronic), encompassing most of the life stages of organisms. Although the toxicity of chemicals mostly depends on the physicochemical properties of chemicals, the resulting effects will depend on the lifestyle of the organisms, such as nutritional status, reproduction activity, prey(cid:1)predation pressures, and habitat characteristics. The advent of climate change adds another level of complexity to predicting the toxic outcomes of pollutants, which is a challenge for the riskevaluation community. The biochemical approaches in ecotoxicologyhavetheadvantageoffindingearlywarningsignalsatthemolecularlevel, which can lead to effects at higher levels of biological organization. The interactions of chemicals with biochemical targets in cells or tissues that could lead to altered function represent a special type of “biomarker” in toxicology. There are many definitions pro- posed in the literature for biomarkers, but we think this one best reflects the biochemi- cal markers: the term biomarker is used in a broad sense to include almost any measurement reflecting an interaction between a biological system and an environmen- talagent,whichmaybeofchemical,physical,orbiologicalorigin[1]. Thisbookpresentspracticalmethodsandapproachesusedinthefieldofbiochemical ecotoxicology. It is destined for laboratory investigators involved in environmental toxi- cologyinvestigationsfor theprotectionofwildlifeandaquaticecosystemsagainstpollu- tionandother stressorssuchasclimatechanges.Asexplainedpreviously,biomarkersare definedasmeasureshighlightinganinteractionbetweenaxenobioticandotherstressors (e.g., temperature) and a biological target in the physiological sense [2]. Biomarkers are also determined at different levels of biological organization starting at the molecular, subcellular (organites), cellular, organ/tissues, organism, and population levels (Figure 1). Often called the ecotoxicology continuum, biochemical ecotoxicology is mainly focused at the molecular and subcellular levels and the conditions or criteria by which a molecular toxic interaction resonates at a higher level of biological organiza- tion. For example, the expression of genes involved in the biotransformation of xeno- biotics that leads to increased activity of a given enzyme, which in turn leads to potentially toxic protein or DNA adducts. Changes at the molecular level are dynamic and occur rapidly in low timescales (seconds to minutes and up to many days), while effects at the tissues/individual level are manifested generally at later times (hours and days to years). In some cases, biochemical alterations take place at the early stages of xxi xxii Introduction Individual/ population y cit Systemic xi effects/health e pl Cells m o (altered cellular C function) Organites/ Subcellular function (altered function) Molecular (primary interaction) Minutes Hours Days Weeks Months Year Time scale Figure1 Levelsofbiologicalorganizationinthemanifestationoftoxicity. pathogenesis, but the observed effects are not always irreversible, hence their usefulness in prevention. In other cases, responses observed at the biochemical level (i.e., gene expression changes) do not imply an impact on the organism’s health status and perfor- mance, hence their lower “physiological or ecological” relevance. Endless arguments about which levels are more physiologically or ecologically relevant for ecotoxicology studiesareconsidereduselessandoutdated.Itdependsonthequestionsbeingaskedina givenstudyandthecontextofpollutionstudies.Thescienceofecotoxicologyishierar- chized,i.e.,onecompartmentisapartof anothercompartmentatahigherlevelofbio- logical organization. It is generally accepted that an impact at any level is likely to produce changes at a higher level provided the intensity of the responses and/or dura- tion of effects are sufficient. It is a question of knowing when an alteration at one level (e.g., enzyme inhibition) will translate to an effect at the next level of complexity (e.g., altered metabolic pathway from an inhibited enzyme). From the risk assessment per- spective, biomarkers that provide information on health status toward survival, growth, andreproductionareparticularlyrelevantfortheprotectionofpopulationtowardpollu- tionandotherstressorsinvolvedinthesetimesofclimatechange. Investigators in ecotoxicology are often confronted to examine or work with all types of species from which no genetic information or specific antibodies or other probes are (yet) available. They have to fall back on generic tests, which focus on spe- cial biochemical properties of the biological target. Indeed, modern and state-of-the- art techniques are highly dependent on the information of the test species under
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