An Integrated Biomarker Based Strategy For Ecotoxicological Evaluation of Risk in Environmental Management

Mutation Research 552 (2004) 247268
Review
An integrated biomarker-based strategy for ecotoxicological evaluation of risk in environmental management

Michael N. Moore a, , Michael H. Depledge b , James W. Readman a , D.R. Paul Leonard c
b a Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, UK Science Group, Environment Agency, Rio House, Waterside Drive, Aztec West, Almondsbury, Bristol BS32 4UD, UK c Department for Environment, Food and Rural Affairs (Defra), Marine and Waterways Division, 32-34 Albert Embankment, London SE1 7TL, UK
Received 26 February 2004; received in revised form 14 May 2004; accepted 17 May 2004
Abstract Environmental impacts by both natural events and man-made interventions are a fact of life; and developing the capacity to minimise these impacts and their harmful consequences for biological resources, ecosystems and human health is a daunting task for environmental legislators and regulators. A major challenge in impact and risk assessment, as part of integrated environmental management (IEM), is to link harmful effects of pollution (including toxic chemicals) in individual sentinel animals to their ecological consequences. This obstacle has resulted in a knowledge-gap for those seeking to develop effective policies for sustainable use of resources and environmental protection. Part of the solution to this problem may lie with the use of diagnostic clinical-type laboratory-based ecotoxicological tests or biomarkers, utilising sentinel animals as integrators of pollution, coupled with direct immunochemical tests for contaminants. These rapid and cost-effective ecotoxicological tools can provide information on the health status of individuals and populations based on relatively small samples of individuals. In the context of ecosystem status or health of the environment, biomarkers are also being used to link processes of molecular and cellular damage through to higher levels (i.e., prognostic capability), where they can result in pathology with reduced physiological performance and reproductive success. Complex issues are involved in evaluating environmental risk, such as the effects of the physico-chemical environment on the speciation and uptake of pollutant chemicals and inherent inter-individual and inter-species differences in vulnerability to toxicity; and the toxicity of complex mixtures. Effectively linking the impact of pollutants through the various hierarchical levels of biological organisation to ecosystem and human health requires a pragmatic integrated approach based on existing information that either links or correlates processes of pollutant uptake, detoxication and pathology with each other and higher level effects. It is further proposed here that this process will be facilitated by pursuing a holistic or whole systems approach with the development of computational simulation models of cells, organs and animals in tandem with empirical data (i.e., the middle-out approach). In conclusion, an effective integrated environmental management strategy to secure resource sustainability requires an integrated capability for risk assessment and prediction. Furthermore, if such a strategy is to inuence and help in the formulation of environmental policy decisions, then it is crucial to demonstrate scientic robustness of predictions concerning the long-term
Corresponding author. Tel.: +44-1752-633100; fax: +44-1752-633101. E-mail address: mnm@pml.ac.uk (M.N. Moore).
0027-5107/$ see front matter. Crown Copyright 2004 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.06.028
248
M.N. Moore et al. / Mutation Research 552 (2004) 247268
consequences of pollution to politicians, industrialists and environmental managers; and also increase stakeholder awareness of environmental problems. Crown Copyright 2004 Published by Elsevier B.V. All rights reserved.
Keywords: Biomarkers; Ecotoxicology; Ecosystem health; Governance; Health status; Integrated environmental management; Lysosomal stability; Middle-out approach; Modelling; RAMP; Systems biology; Policy; Risk
1. Introduction Most of the Earths living resources are found in specic geographical locations such as the global coastal environment and the catchment basins of large river systems. Furthermore, more than 3 billion people live in close proximity to these regions and are dependent upon it for either part or much of their food supply and industrial raw materials. The consequence of this situation is that much of the waste, both industrial and domestic, and various other types of ecosystem change and habitat destruction generated by the human population, occur in those areas that are of greatest biological and economic signicance and are likely to impact on quality of life. It is clearly vital that effective internationally accepted procedures for environmental/ecological impact and risk assessment be put in place to manage this problem. Natural and anthropogenic impacts on ecosystem and human health are both an urgent and international issue. The economic scale of the problem is indicated by the ndings of a recent economic study. These placed a value of US$ 12.6 Trillion/year for Coastal Zones & US$ 6.6 Trillion/year on Wetlands, Rivers & Lakes, out of a Global Total of US$ 33.3 Trillion/year [1]. Signicant stressors include toxic industrial chemical contaminants, increased UV-radiation, nutrient enhancement or deprivation, hypoxia, habitat disturbance and pathogen-induced disease. In fact, environmental disturbance will frequently comprise various combinations of such stresses. Furthermore, it is increasingly recognised that assessment of the impact of environmental disturbance on organisms requires understanding of stress effects throughout the hierarchy of biological organisation, from the molecular and cellular to the organismal and population levels, as well as the community and ecosystem levels; and our understanding of the processes of ecosystem recovery are rather limited [2]. In the past, damage to the en-
vironment has largely been identied retrospectively and in response to acute events such as major disasters (e.g., industrial accidents like Seveso and Bhopal; and oil spills (Amoco Cadiz & Exxon Valdez) and chemical pollution of the Great Lakes). Generally, these have been measured in terms of human health impacts and visible changes resulting from the loss of particular populations or communities. However, long term and chronic exposure to environmental stress, including chemical pollutants or other anthropogenic factors, will seldom result in rapid and catastrophic change. Rather, the impact will be gradual, subtle and frequently difcult to disentangle from the process and effects of natural environmental change. Most importantly, the timescale over which the impacts occur are often of the order of decades, rendering them less amenable to study. This latter problem has been a major stumbling block in assessing environmental impact since such investigations began, mainly in the 1960s. Other complicating factors such as diffuse water pollution from agricultural sources may pose major threats in some circumstances [3], which could affect the ecology of rivers and estuaries, reduce drinking water quality and the health of coastal ecosystems. The major issues of concern include: the role of global industrialisation, human population density and haphazard urbanisation as a major source of pollution; the fact that pollution does not respect national boundaries; the loss of living resources and biodiversity; damage to human health; public and political aspirations for mitigation and control; difculties in disentangling natural long term changes from those resulting from human activity and how to achieve support for sustainable nancing and banking in order to support developing economies. The environmental objectives of sustainable industrial development include the sound management of natural resources, effective transfer of environmentally sound technologies in order to reduce, reuse and recycle waste, investment promotion for sustainable industry (see
249
http://www.unido.org), environmental monitoring and control of investments for environmental industry projects. Consequently, we need sound environmental policies underpinned by robust mechanistic science. Environmental components for sustainable industrial development need to include: 1. key environmental health and sustainability indicators; 2. widespread support for the objective of minimising anthropogenic impacts on the environment; 3. an effective environmental policy framework based on an ecosystem approach and the minimisation of environmental impacts; 4. cleaner industrial production and pollution prevention; 5. environmental emission and discharge standards (with the aim of minimising these); 6. enforceable pollution control and waste management; 7. ecotoxicology for assessing environmental impact of pollution and overuse of resources; 8. environmental modelling for policy decisions; 9. risk assessment, reduction and management; 10. the integrating process with socio-economic conditions and governance issues; 11. broad public support for environmental measures. In order to aid governmental and regulatory bodies in protecting the environment through legislation and monitoring, integrated environmental management (IEM) must seek to provide knowledge-based expertise on environmental policy, cleaner production technologies, waste management and pollution control, in order to achieve sustainability [4]. It must also provide expert diagnostic and predictive software to link existing models with physical and chemical information and knowledge of the environment. In addition, IEM needs to support the development and use of indicators, which show effectiveness in moving towards sustainable development, that link environmental, social and economic measures [5]. Environmental stress can be caused by a number of factors including: natural forces such as sea level rise, climate change and soil erosion; poorly planned development, such as haphazard urbanisation and industrialisation; depletion of resources through over-shing, deforestation and poor use of agricultural land; unregulated discharges of municipal sewage and industrial
waste; and illegal practices, such as disposal of dangerous toxic wastes. While it is clearly recognised that stress-induced changes at the population/community/ecosystem/human health levels of biological organisation are the ultimate concern, they are generally too complex and far removed from the causative events to be of much use in developing tools for the early detection and prediction of the consequences of environmental stress (Fig. 1) [6]. Rapid resolution of this link to health is essential if environmental management is to have a sound scientic basis for the regulation of the release of toxic substances, nutrients and habitat disturbance. The basis of such regulation, where it exists at present, is often at best sketchy with a heavy reliance on empirical observations and laboratory-based toxicity tests using organisms that have limited relevance to the real environmental context. In the past there has been an assumption that if man is protected then the ecosystem is protected: this is now somewhat discredited [7]. In fact, Leonard and Hunt [8] showed that it is important to assess human lifetime exposure to radioactive waste and not just annual exposure. Consequently, there needs to be joint support from conservationists, water companies and industry in order to help human societies and ecosystems for both current and future generations. A probable solution to this problem lies in the effective detection of distress signals at the molecular and cellular levels of organisation and linking such signals to the higher level consequences (Fig. 1) [6,915]. This requires the distress signals to be ecologically relevant and to have utility in minimising impact by indicating health status with a prognostic capability. However, the restrictions imposed by our current knowledge, or lack of it, means that within the foreseeable future it is only at the lower levels that we will have the reasonable expectation of developing a basis of mechanistic understanding of how different environmental conditions can modulate organism function, which in turn will ultimately help in linking causality with predictability of response (Fig. 1). This is in part due to our ability to make certain generalisations about biological organisation and function at the molecular and cellular level, which rapidly disappear within the complexity of biological and ecological interactions as we ascend the hierarchical ladder. In principle, distress signals at the molecular, cellular and
250
Fig. 1. Diagrammatic representation of the relationship between environmental distress signal detectability and ecological relevance [9,14,16,17].
physiological levels of organisation should be capable of providing early warning prognostic biomarkers (molecular, cellular, physiological and behavioural) of reduced performance, impending pathology and damage to health [6,18,19]. In fact, there is a direct analogy here with the use of clinical tests (biomarkers) in human and veterinary medicine [14]. Potential prognostic tests are beginning to emerge from a rapidly improving integrated understanding of the cellular, physiological and behavioural processes that inuence uptake, biotransformation, molecular damage and cell injury, impairment of protective systems, degenerative change and impaired reproduction and survival (Fig. 2) [10,13,1517,2022]. At the higher organisational levels, differential sensitivity needs to be assessed according to individual genotype and physiological status/life-history stage, and natural seasonal changes in physiological and/or reproductive status [2325]. This information then needs to be used to develop process-based simulation models of the type increasingly used in quantitative cell biology and cellular
bioengineering [2634]. Process-based simulation models are already being successfully developed for cells, tissues and organs in order to address the complexity of molecular and cellular interactions [17,3133,35,36]. Brenner [37] famously stated that genes can only specify the properties of the proteins they code for, and any integrative properties of the system must be computed by their interactions. This statement implies not only that biological systems themselves compute these proteinprotein interactions, but also, that in order to understand them we need to compute them. Brenner concluded: this provides a framework for analysis by simulation! Therefore, simulation of biological function in cell systems, tissues, organs and individuals is a necessary integrational complement to reductionist molecular and cellular investigation. Simulation models can provide insights into the links between molecular properties and cell and organ behaviour; and indeed, whole animal physiology, while the predictive power of such models can be harnessed to develop tools for risk assessment of toxic
251
toxicity, the toxicity of complex mixtures [39,40]; and linking the impact of pollutants at the various hierarchical levels of biological organisation from the supra-molecular and cellular, through individuals to the population, community and ecosystem health/status (Figs. 1 and 2) [2]. Major longer-term aims need to include the development of conceptual and mathematical frameworks based on an improved mechanistic understanding of contaminant geochemistry and uptake, metabolic biotransformation, toxicity and impact within the biological organisational hierarchy. Large scale tasks include predicting the toxicity of contaminant mixtures [39]; and modelling environmental pollution and impact as a complex adaptive system, which will encompass contaminant geochemistry, biochemical toxicology, cellular pathology, inter-individual variability in response, ecological consequences and human risk (Fig. 2) [17].
2. Environmental risk and integrated environmental management First, we must consider what is at risk in the coastal and riverine zones: clearly these are land use and environmental resources, such as water and sheries; biodiversity and environmental quality; ecosystem status; human health; and nally aesthetics, which can be particularly important in relation to the tourist and ecotourist industries. The acceptability of risk in integrated environmental management (IEM) is not an issue for scientic research alone. It involves economic, social and political issues, which all need to be integrated in order to develop prognostic risk models. There are also important economic components that are directly related to the value of the resources to be protected and the costs of increasing safety margins. We can forecast and reduce environmental risks through the implementation of innovative environmental monitoring and surveillance techniques to understand the extent of the problems (for example, the RAMP (rapid assessment of marine pollution) approach which utilises new rapid low cost immunochemical tests for contaminants, health tests (biomarkers) for animals, plants and humans [41]. These require supporting research into understanding physical, chemical, biological and ecological pro-
Fig. 2. A conceptual framework showing the interconnectedness of environmental pollutant-related processes (including radionuclides and ionising radiation) and their harmful effects as components of a complex interactive system (taken from Moore [17]).
chemicals and other types of environmental perturbation [20]. Developments in this direction are considered to be essential if the question of biocomplexity is to be effectively addressed through the development of computational simulation models, based on multiple interactions at the molecular, cellular and physiological levels [17,38]. These cellular and physiological models must also be capable of experimental validation, which should also include manipulation of key processes. Visualisation of cellular and patho-physiological processes in silico will facilitate the identication of complex strategies for adaptation to altered environmental conditions [20,34]. The key issues pertaining to environmental impact and risk are largely interfacial, and include the effect of physico-chemical speciation on uptake and
252
cesses. We also need to develop decision support (expert) systems to link existing models with our experience and knowledge of the environment; as well as to develop and use indicators of sustainability to show effectiveness in moving towards sustainable development, where there is a need to link environmental, social and economic measures [42]. The objectives of integrated environmental management (IEM) are as follows: prevent, reduce and control deleterious change in the environment, likely to impact on quality of life, thereby maintaining and improving its life support and productivity capacities; develop and increase the potential of living resources to meet human nutritional needs, as well as social, economic and development goals; promote the integrated management and sustainable development of terrestrial, freshwater and coastal marine environments. Conceptually, IEM can holistically assess the changing states of ecosystems based on information from ve operational modules: 1. 2. 3. 4. 5. ecosystem productivity; water, sheries and agricultural resources; pollution and health (ecosystem & human); socio-economic conditions; governance protocols.
biogeochemical and physical processes (e.g., hydrology and sedimentation); bioavailability of toxic chemical pollutants, uptake into plants, sh and animals and the subsequent transfer to humans through the food chain; ecotoxicology and environmental impact of pollution and overuse of resources (e.g., land, water, rivers, forests and sheries); models for integrated environmental management; human health risks; risk assessmenta cross-disciplinary issue; the integrating process for environmental data and predictions, together with appropriate economic and social aspects; appropriate political will and public support to balance minority interests against majority needs.
3. Bioavailability and uptake of chemical contaminants While in no way attempting to comprehensively review this immense eld, we can nevertheless make the general statement that uptake and accumulation of organic micropollutants and metals by aquatic organisms is largely governed by their physico-chemical speciation and by the physiological condition of the organism [43]. Lipophilic pollutants are largely associated with particulates and colloidal organic carbon [44,45] and thermodynamic partition coefcients are traditionally used to model the particulatewater exchange of compounds. There is increasing evidence, however, that these are inaccurate owing to the heterogeneity of sorbants which can alter kinetics of partition to such an extent that equilibrium is never attained (see Fig. 3) [4650]. Quantication of kinetics and mechanisms of sorption of anthropogenic organics to particles is essential to assess transport processes of hydrophobic material through aquatic systems. Research needs to increase awareness and understanding of the heterogeneity of environmental sorbants and to investigate factors affecting linear free energy (non-) equilibrium theories. Physico-chemical speciation is essential in considerations of bioavailability and uptake; and is likely to be inuenced by the local geology and bio-geochemistry. It is probable that pollutant entry into cells is directly related to the extracellular and
These modules link science-based information to socio-economic benets for countries sharing boundaries for international waters; and are used in an integrated interdisciplinary mode to address the consequences of ecosystem change (e.g., GEF-International Waters Programme, Global Environment Facility, http://www.gefweb.org; UNDP, http://www.undp. org; UNEP, http://www.unep.org; UNIDO, http://www. unido.org). The methodology of IEM brings together elements for dealing with the complex interactions of the many demands placed on the environment. The methods can be implemented through training, technology transfer and capacity building, that are rmly grounded on strategic science-based assessments and monitoring and linked to standard internationally agreed QA protocols. Programme components for integrated environmental management should include:
253
Fig. 3. Diagrammatic representation of binding of the ubiquitous polycyclic aromatic hydrocarbon pollutant benzo(a)pyrene to natural particles.
intracellular behaviour of particulates/colloids with adsorbed chemicals and radionuclides (Figs. 3 and 4) [62,70]. It is also important to remember that the physiological status and age of the organism may inuence contaminant uptake. For example, organisms with high lipid reserves may accumulate lipophilic contaminants more readily than those which have not. Also, trace metal accumulation may be inuenced by the activity of ionic pumps involved in osmoregulation [71]. Contaminants are seldom present as a single chemical and usually comprise a complex mixture [39,40]. Uptake of xenobiotics from such mixtures is poorly understood and questions of whether components of the mixture inuence the uptake and biotransformation of other components have not been seriously addressed [72]. Uptake is often viewed as taking place from solution with the contaminant crossing cellular membranes by diffusion [62]. However, as stated above, most contaminant chemicals are bound to particulates
and so, are seldom in true solution [45,46,62]. This is probably of considerable importance in explaining the known compartmentation of many micropollutants and metals within cells and tissues of plants and animals, so it is essential that an appropriate mechanistic understanding of the intracellular transport processes, intracellular chemistry and associated biotransformations is developed in the future [17,62]. This type of knowledge is essential if we are going to be able to attempt to predict the kinds of organisms at risk and, also, whether particular life stages are more vulnerable than others [23,24,73]. There is also a clear need for new rapid cost-effective analytical methods for routine application, with an emphasis on reduced cost when compared to existing techniques. Such methods need to be readily interpretable so that the information is available to policy makers in an understandable form. In the longer term, some of these may be deployed on autonomous sensors, if properly calibrated and maintained. Can-
254
Fig. 4. Diagrammatic representation of the various routes of uptake (both diffusion and endocytotic) of contaminant xenobiotic chemicals into the cell that can result in their accumulation in the lysosomal compartment, based on their physical chemical characteristics [11,12,16,17,5169].
didates here may include immunochemical detection of organic micropollutants [7476]. Clinical chemists have utilised immunoassay (IA) techniques to detect and quantify proteins, hormones, and drugs for decades. The most common version of environmental IA is called ELISA or enzyme linked immunosorbent assay [74]. ELISA is an immunoassay method that uses antibodies and enzyme conjugates to detect and quantify target compounds, otherwise known as compounds of interest (COIs), in eld samples. Therefore, with appropriate calibration and careful comparison with the results obtained by conventional analytical chemistry (e.g., QUASIMEME international intercalibrations, http://www.quasimeme.marlab.ac.uk), IA can be used for rapid low cost pollutant determination in soil, sediment, water and body uids [74,75]. In studies of bioavailability, there has only been very limited use of modelling procedures to simulate intracellular behaviour of pollutants. Here, process simulation modelling will be particularly important in helping to dene the problems and in developing hypotheses in this highly complex area. One such model has been developed that denes
the component processes in endocytosis, lysosomal compartmentation, toxicity and pathology (Fig. 4) [17,39,30,62]. This model focuses on ligand-binding sites associated with endocytosed particles and the role of the endosomallysosomal system in pollutant uptake, toxicity and cell injury (Figs. 4 and 5) [1517,29,45,59,60,7781]. The model has provided a conceptual framework for pollutant uptake and biotransformation, lysosomal accumulation, protein degradation, cellular autophagy and cell injury, as well as excretion of pollutants and bioavailability. It also helps to formulate key hypotheses for experimental testing and validation of the model [29,62].
4. Biomarkers of exposure and effect Research into the molecular mechanisms by which the cells of animals and plants protect themselves against pollutants has produced a wealth of information in the past decade on the membrane protein pumps of the multidrug/multixenobiotic resistance system (MDR/MXR) that directly remove xenobiotics
255
Fig. 5. Distress signals as biomarkers of harmful effect at various levels of animal organisation.
from cells, as well as biotransformation processes by which the enzymes of cells either detoxify pollutants to harmless, excretable products, or activate them to more toxic forms [8284]. However, predicting the impact of pollutant chemicals and other environmental perturbations on bioindicator/sentinel animals and ecosystems requires us to take a different approach, particularly if we are to design effective tests (biomarkers) that can be used to monitor the health of the environment and its biota [6,14,85]. Cells and tissues represent whole biological systems of such immense complexity that it is impossible to envisage the vast number of interactions or biocomplexity from which emerges biological function or physiology [34]. As biologists we are trained to think in terms of the component parts of living systems down to the functional macromolecules (i.e., proteins and nucleic acids), and the success of twentieth century reductionist biology has been the dramatic unravelling of the constituent parts of cells and demonstrating their structures and functional relationships to one another. The challenge here was immense, but the challenge now facing biologists in the new century is how to translate this wealth of detail about cells and tissues into a real understanding of how these systems
function in an integrated way; and are perturbed in disease processes. Likewise, in ecotoxicology, we have to take a systems biology approach if we are to gain insight and understanding of the environmental adaptations and pathologies, in the instances of failure to adapt to physical and chemical stressors. Consequently, we need to develop overlapping process-based models using existing data in order to integrate molecular and cellular processes with physiology and pathology and provide the necessary conceptual and mathematical frameworks that will lead to a predictive capability. Many natural and pollutant organic xenobiotics are detoxied by biotransformation enzymes of phases I and II metabolism [84]. Of central importance in the former is the multi-gene, multi-functional cytochrome P450 (CYP450) family of inducible isoenzymes of which particular forms (e.g., CYP1A) can activate xenobiotics to form covalent adducts with nucleic acid and protein [86]. Interacting with xenobiotic metabolism are pro-oxidant and antioxidant processes involving the production of oxyradicals and their removal by antioxidant defences [69,87,88]. Oxyradical production can be increased by interaction with transition metals (Fe, Cu, Cr, Ni, Co, Va) and redox cy-
256
cling organics (nitroaromatics, quinones), and also by induction of particular components of the biotransformation system, causing oxidative damage to cellular constituents [11,89]. The consequences of xenobiotic activation and enhanced oxyradical production include impaired cellular function, cancer and certain disease processes [21,22,87,90]. Aspects of the defences may be integrated at the gene level, viz. enzymes of the mammalian [Ah]-gene battery, including CYP1A and the antioxidant enzymes DT-diaphorase (DTD) and aldehyde dehydrogenase (ALDH), can be co-induced by organic xenobiotics [91]. The past two decades have seen major advances in genotoxicology in mammals and humans; and some of the methods have been applied to ecotoxicological problems [92]. Fish and invertebrates appear to express similar types of genetic damage to that found in humans and mammals [92]. The consequences of genotoxic damage in natural populations and ecological assemblages are generally believed to constitute a minor threat when considered in the light of the immense wastage that frequently occurs during the early reproductive and life stages [93]. The ecotoxicological signicance of pollutant induced genetic damage, therefore remains an open question. However, given that many species of shellsh and sh are part of the human foodchain, it is very important that we should be concerned about the fate of genotoxins in such animals and whether they accumulate either the parent toxins or more harmful metabolic derivatives. It is here that tests for genotoxicity can be used as warning indicators of possible consumer risk, given the frequent difculties in knowing what contaminants to analyse for or the lack of methods for detecting harmful metabolites [94]. In the meantime, biomarkers of genotoxicity, such as DNA adducts and formation of micronuclei, can provide useful indicators of exposure to mutagens and carcinogens in the environment (Fig. 6A) [21,95,96]. Furthermore, issues such as the genotoxic disease syndrome (GDS) concept developed by Kurelec also raise very important questions concerning the role of contaminant inhibitors of the membrane transporter systems, like MDR/MXR, in evaluating the ecotoxicological signicance of genotoxins [96]. Also, research on the Sea Empress oil spill funded jointly by the UK Ministry of Agriculture, Fisheries & Food (MAFF) and the UK Department for Environment
Transport & the Regions (DETR) on the occurrence of DNAadducts demonstrated that some genetic damage had occurred in sh & mussels during 1996 but a signicant recovery was seen in 1997 [97]. It was therefore not clear that the exposure to Sea Empress oil would result in the eventual generation of tumours in commercial species of sh, as the damage may have been repaired in the interim period [97]. It is now clear that developments in genomics will offer signicant opportunities across all the life sciences, from medicine to ecology; and ecotoxicology will undoubtedly benet from the future application of genomic information in the ecological risk assessment of chemicals. Essentially, all the cautionary issues identied for mammalian toxicogenomics will apply equally to ecotoxicogenomics [98101]. Snape et al. [101] have proposed that such concerns are probably best addressed by research that incorporates genomics, proteomics and metabolomics into well designed in vivo studies, at the system biology level, using environmentally relevant exposures and a range of time points which measure established endpoints of population relevance (e.g., survival, development and reproduction). The phenomenal increase in genomic data for an increasing number of non-mammalian species suggests that such experiments will become more feasible in the near future and ecotoxicology will need to exploit this opportunity to address the issue of whether genotoxicity poses a signicant ecological threat. An important consideration here must also be the fact that many sh and invertebrate species used as environmental sentinels, such as atsh and bivalve molluscs, are broadcast spawners, producing very large numbers of eggs and larvae with a very low survival rate. Pathological cellular reactions to chemical pollutants can provide early-warning distress signals of injurious change in plants and animals [10,1618, 22,58,102105]. Such reactions will be of particular use if they can be shown to be precursors of pathology, since this will relate directly to the risk potential (Figs. 4 and 5). Cellular changes are used as indicators of toxic impact in testing new chemical products in laboratory rodents, and have also been increasingly used in environmental assessment of toxic risk. A further advantage of using cellular reactions is that isolated cells can be exposed to chemicals or complex mixtures of toxicants in vitro, which facilitates rapid
257
Fig. 6. Correlations between lysosomal stability, as a prognostic health status indicator, and (A) genotoxicity (frequency of micronuclei), R = 0.8519, P < 0.01; and (B) total oxyradical scavenging capacity (TOSC), R = 0.8637, P < 0.0001. Data is adapted from the references [21,22].
testing of many samples. Additionally, at a time when the general public is becoming aware and alarmed at animal suffering in the name of science an in vitro approach requires the sacrice of very few animals [16,107,108]. Once disaggregated, a tissue biopsy or sample of body uids (coelomic or blood cells) or eggs can provide sufcient cells to undertake many exposure experiments in the knowledge that genetic heterogeneity has been removed as a confounding factor; which is in sharp contrast to traditional in
vivo exposure studies where it is necessary to treat different animals with a compound. Biomarkers are needed that will accurately indicate the health status of the organism (Fig. 5) [6,12,17,20,109,110]. Promising methods that may offer a simple and rapid way of assessing individual health status include assessments of lysosomal membrane stability [13,105,107]. Increasing use of lysosomal stability methods has demonstrated correlations in blue mussels with oxyradical scavenging
258
Fig. 7. Lysosomal stability as an indicator of whole organism health: lysosomal stability shows a signicant linear relationship with physiological scope for growth in the marine mussel Mytilus edulis (R = 0.8964, P < 0.0001). Data is a composite of several eld and laboratory experiments adapted from the reference [20].
capacity, potential for protein synthesis (translational efciency of ribosomes); genotoxicity (micronucleus formationinverse correlation), physiological scope for growth, tumorigenesis in ounder liver (inverse correlation) and larval viability in oysters (Figs. 6 and 7) [15,2022,105]. The derivation of lysosomal biomarkers was based on the premise that since lysosomal processes are a key component of the cellular economy, they should be indicative of the functional integrity of cells [17]. Specifically, these lysosomal processes include degradative protein and macromolecular turnover, tissue remodelling, rapid mobilisation of reserves (e.g., during gametogenesis in bivalve molluscs) and cellular immune functions. Pathological reactions frequently involve changes in lysosomal function and morphology that can have profound effects on both tissue and whole animal function [13]. Consequently, it is probably not surprising that measures of lysosomal integrity are good indicators of animal well-being [20,105]. Finally, in the context of health status, we believe that an attainable objective is the development of an integrated (holistic) approach to environmental toxicological pathology (Fig. 2). However, this goal requires evaluation of the relative susceptibility of different individuals, populations and species, if accurate
risk assessments are to be achieved [25]. As such, this will require an integration of various tools, including molecular, cellular and physiological toxicology, structure-activity relationships (SARs), modelling the processes of cell injury and linking the models to environmental data [29,30,62,64,65,85,107,108].
5. Modelling Environmental disturbance can result in adaptive responses, stress syndromes, disease and changes in functional biodiversity in living systems (i.e., microbes, plants, animals and functional ecosystems) due to perturbation of the complex interactive functional networks or biocomplexity, that give rise to whole system function. A complex system cannot be understood through a reductionist approach: this would be like trying to understand the global economy by observing a stockbroker in his or her ofce. However, by adopting a middle-out approach, which combines simulation with empirical data, we should be able to describe the behaviour of complex whole systems such as cells, tissues and animals [34,111]. Biocomplexity models will be able to simulate virtual organisms. These models will be based on an in-
259
teomic, metabolomic, cellular and higher level biological processes. Access to such tools will be essential in the future for environmental managers and regulators; where they will be used in integrated environmental evaluation strategies for risk assessment and prediction in order to manage resource sustainability [42].
6. Ecological relevance and risk assessment We need to accept and embrace change, not tacitly accept the need for the quality of the environment to be maintained. Clearly, there will be some areas where environmental quality declines; but, taken at a regional scale, the ecosystem is healthier [115]. A key aim of environmental science is to derive robust, practical and relatively low cost procedures for assessing risk to the health of the biosphere and to use this capability to predict the likely consequences of exposure to potentially harmful toxic pollutants [2]. Until relatively recently, risk assessment procedures have been oriented towards protecting human health. Now, it is widely acknowledged that such procedures must also ensure that complex biotic communities in natural ecosystems are protected if the quality of the environment in which we live is to be maintained. Environmental risk assessments are currently based on a suite of information derived from studies on the physical chemical characteristics of compounds (the QSAR-based approach), and from laboratory-based toxicity tests [6,64,65]. Although these procedures constitute a low cost, pragmatic means of ranking the toxicity of potentially hazardous chemicals, they do not directly evaluate the sublethal toxicity, or other adverse effects (e.g., disturbance of ecological relationships) on organisms exposed to complex mixtures of pollutants in the highly uctuating conditions that prevail in the environment [39,40,72]. There is therefore, a priority requirement to implement the use of robust but simple, easy to learn, cost-effective test systems that can identify early diagnostic changes in biota, which can be linked to ecologically relevant endpoints. The selected endpoints must be capable of facilitating a predictive ranking of the condition of particular ecosystems, thus highlighting environmental situations where a more detailed analysis is justied, as indicated by the use of lysoso-
Fig. 8. Framework for developing new ecotoxicological modelling tools to address future predictive needs for environmental impact and risk assessment showing a schematic of how the disciplines of environmental prognostics relate to each other. Adapted from the reference [20].
tegration of chemical, molecular, cellular, physiological and trophic processes, coupled with generic simulation of virtual whole systems. In respect of the feasibility of the proposal, we have already achieved, and have now moved beyond the proof of concept stage, with the development and publication of generic carbon and carbonnitrogen ux models that have been used to simulate the effects of oil pollution on mussels [20,29,30]. The key to the success of this endeavour lies in the development of a generic series of overlapping computational models for whole systems, which will also encompass their use for individual components such as contaminant behaviour, cells, organs (e.g., digestive gland, liver, cardiac and immune system models) or whole animals (Fig. 8). The development of the virtual heart model by Noble [3133] has demonstrated that such integration and generic transfer from cellular physiology to whole organ electromechanical simulation is a practical reality (Alliance for Cell Signalling Web SiteAFCS.org) [112,113]. Further support for the utility of the generic approach comes from the IUPS International Physiome Project where the methodology for the heart model has been used in the derivation of physiomic models for the lungs and liver [114]. A major objective is to develop prognostic biomarkers and generic simulation models for responses to environmental change in whole systems, that are based on current and developing knowledge of genomic, pro-
260
Fig. 9. Lysosomal stability (NRRneutral red retention times) in mussel haemocytes (blood cells) for the UNESCO-IOC Black Sea Biological Effects Mussel Watch: retention values of less than 60 min indicate severely impaired health. Adapted from the references [116,117].
mal stability (neutral red retention) in the Black Sea (Fig. 9) [6,13,14,106,116,117]. Environmental toxicologists have also to try to anticipate the potential impacts of new or novel products and unwanted by-products of industry [60]. This includes biogenic pollutants and future developments in the chemical and pharmaceutical industries as well as industries using biotechnology and molecular nanotechnology [118121]. Harmful products are likely to include: new targeted drugs and pesticides, particularly those for use in concert with genetically modied crops; natural pesticides resulting from gene transfer into crops; and novel pathogens used for biological control [121123]. The almost exponential increase in published papers in the area of nanotechnology since 1990 is a strong indicator of the rapid development and use in industry and medicine based on engineering molecules for multiple applications [119,120,124]. Nanoparticles are already being used in drug-delivery systems, cosmetics, lters, sprays, coatings, sunscreens,
machine clutches, automobile parts, tennis rackets, scratch-proof eye-glasses, stain-repellent fabrics, self-cleaning windows, CD and DVD pickup heads, computers and many other applications [119,120]. The increasing use of nanoparticles in biomedicine for probing processes and in drug delivery has also opened up considerable possibilities for drug design and therapeutics [125129]. However, there are also risks to be considered, since biological systems did not evolve in the presence of many of the types of nanoparticles now being developed or used in existing nanotechnological applications. The potential toxicity and environmental impacts of such nanoparticles may be related to particle size and biodegradability [60,119]. Ecotoxicological modelling of nanoparticle behaviour in biological systems, coupled with experimental studies of uxes and toxicity will be of immense value in screening candidate nanoparticles for potential pathological effects [17,119]. Without strong international controls on such production there could be serious environmental risks,
261
since it is likely that amongst the practical developments towards nanotechnological application, some will rely on modied biological molecules and supra-molecular assemblages [120,125]. The purpose here is primarily to raise awareness of developments and possible environmental hazards and risks in the eld of nanotechnology, since much of the use of nanoparticles is relatively recent or still in the research and development phase [17]. The overall problem is, in essence: how to develop effective procedures for environmental/ecological impact and risk assessment [2]? The use of biomarkers
and biological effects indices has proven useful in establishing evidence of exposure to pollutant chemicals and damage to the health of sentinel organisms (Figs. 1, 2 and 58) [6,58,102,103,130]. This is obviously of great value in helping to establish causal relationships. However, for impact and risk assessment tools to be effective they must be capable of providing data that relates to ecologically signicant processes [6,16]. This requires a better understanding of particular biomarkers, as they relate to health status (Figs. 2 and 5-9), in order to improve their interpretative value in monitoring (Figs. 5 and 7)
Fig. 10. Conceptual framework for an interactive monitoring programme for the assessment of environmental impact of pollutant chemicals. Adapted from the ICES-WGBEC Report (1997) [132].
262
[14,16,17]. Monitoring, itself, must be made more effective through rational interaction between chemists, ecotoxicologists, ecologists and environmental managers: a framework for this type of interaction is outlined in Fig. 10 (e.g., QUASIMEME; BEQUALM, http://www.bequalm.org; International Council for Exploration of the SeasWorking Group on Biological Effects of Contaminants, ICES-WGBEC, http:// www.ices.dk/iceswork/wgdetail.asp?wg=WGBEC; ICES-OSPAR, http://www.ices.dk/iceswork/wgdetail. asp?wg=WKIMON) [131,132]. Also, rapid and clear interpretation of information for translation to policy will speed legislation on ecosystem management. In order to achieve the above aim, the science must also address the question of linkages between effects at different levels of biological organisation [2,12,17,36,133135]. Establishing these linkages is essential, not only for understanding the current status of the environment, but also to provide a rational basis for prognosis for future improvement or deterioration in environmental quality (Figs. 2 and 10). There is also a need to consider how successful ecosystem change is valued: this is an aspiration of the European Union COST-IMPACT Project (http://www.pml.ac.uk/pml/costimpact.htm).
7. Conclusions and future directions The broad approach developed in this paper to the complex task of assessing the health of the environment will, we believe, facilitate the validation, and further the essential new development of robust and rapid tools for assessment [6,14,16,136]. Future efforts must focus on an integrated approach to the validation of biomarkers that are prognostic for individual health status and relevant to population and community endpoints [6,16]. As with bioavailability and uptake, exposure to physical factors and biological agents as well as pollutant mixtures must also be considered with the possibility of complex synergistic interactions resulting in emergent and novel toxicities and pathologies [62,137]. Other environmental factors such as visible and UV-radiation (Fig. 2) and hypoxia also need to be considered, since these are likely to be important in terms of potentially harmful toxic and genotoxic interactions with contaminants [23,73,88]. This includes xenobiotics acting as photosensitisers and the facili-
tation of cascades of harmful radical production on reoxygenation following a hypoxic interlude, such as that induced by eutrophication (Fig. 2). The development of a predictive capability or environmental prognostics as proposed by Allen and Moore [20], and its use in ecotoxicology, will provide a realistic insight into the limits of reductionism as a very successful universal problem-solving approach (Figs. 2 and 8) [36,138]. Complex biological and ecological processes can generate counter-intuitive and seemingly acausal behaviour that is full of novelty [36,39,40,72,138]. Trying to understand the behaviour of a complex adaptive (or dynamic) system, such as an organism, population, ecosystem or biogeochemical cycle, by a reductionist approach often irretrievably destroys the inherent nature of the problem being addressed [36]. By developing environmental prognostics we will couple our integrated understanding with the conceptual models and then use numerical simulation to derive testable explanatory frameworks [20]. Currently, numerical models are in their infancy and are used for heuristic purposes [29]. However, the great advantage of simulation is that it encourages an understanding of how processes are linked and how system properties emerge in space and time. There must also be a wider recognition that the interface between IEM and ecotoxicology/environmental toxicology is dealing with complex systems that go beyond purely scientic issues; and include economic considerations, societal needs, human values and ecosystem vulnerability (Fig. 2) [36,42]. Consequently, there needs to be a rapid acquisition of the new methods of complexity science and implementation of these into the scientic components of integrated environmental management programmes (Figs. 8 and 9). Therefore, a rational way forward can be conceived, if an integrated multidisciplinary approach to the environmental impact of pollution is adopted as follows: 1. a pragmatic development of realistic whole system (holistic) conceptual frameworks and middle-out simulation models based on an improving mechanistic understanding of processes of contaminant uptake, biotransformation, toxicity and pathological impact within the biological organisational hierarchy (Figs. 2,4,5 and 8) [17,20,2729,3134,62,139,140143];
263
Fig. 11. Proposed IEM solutions for environmental problems related to sustainable development of environmental resources.
2. meet the interdisciplinary challenge of modelling processes in environmental pollution and impact as a complex adaptive system (Figs. 2 and 8) [36], encompassing contaminant geochemistry, mode of uptake and intracellular behaviour, biochemical toxicology (including proteomics), cellular injury and pathology (e.g., using virtual organs and animals; Figs. 4 and 8), ecological consequences and human risk; 3. take a broad view of the current and predicted future problems in environmental management, that incorporates both moderate reductionist and synthesist approaches Figs. 4, 5 and 11); 4. areas of further ecotoxicological integration need to include, among others rapid low-cost biomarker tests and chemical assays [104,144147], remote/satellite surveillance, risk assessment, interpretation of complex information and predictive modelling (Figs. 9 and 11) [138,139]; 5. precautionary anticipation of novel environmental hazards (e.g., from biotechnology & molecular nanotechnology);
6. if such a strategy is to inuence and help form policy decisions, then it is crucial to demonstrate scientic robustness of predictions concerning the long-term consequences of pollution to politicians, industrialists and environmental managers; and also increase stakeholder awareness of environmental problems so that positive pressure is exerted on industry to make its products environmentally sustainable (i.e., eco-labelling).
Acknowledgements M.N. Moore was supported in the preparation of this article by the United Nations Industrial Development Organisation (UNIDO), Vienna, Austria. References
[1] R. Costanza, R. dArge, R. de Groot, S. Farber, M. Grasso, B. Hannon, K. Limburg, S. Naeem, R.V. ONeill, J. Paruelo, R.G. Raskin, P. Sutton, M. van den Belt, The value of the
264
M.N. Moore et al. / Mutation Research 552 (2004) 247268 worlds ecosystem services and natural capital, Nature 387 (1997) 253260. J. Rice, Environmental health indicators, Ocean Coastal Man 46 (2003) 235259. Defra, Diffuse water pollution from agriculture, R&D Newslett. Agric. Environ. 12 (2004) 8. P. Leonard, The role of biological research in supporting policy needs, Mar. Environ. Res. 54 (2002) 209214. R.T. Di Giulio, W.B. Benson (Eds.), Interconnections Between Human Health and Ecological Integrity, Society of Environmental Toxicology and Chemistry (SETAC), Pensacola, FL, 2002, 110 pp. M.H. Depledge, J.J. Amaral-Mendes, B. Daniel, R.S. Halbrook, P. Kloepper-Sams, M.N. Moore, D.P. Peakall, The conceptual basis of the biomarker approach, in: D.G. Peakall, L.R. Shugart (Eds.), BiomarkersResearch and Application in the Assessment of Environmental Health, Springer, Berlin, 1993, pp. 1529. D.R.P. Leonard, K.J. Mondon, M.G. Segal, A systematic approach to control radioactive waste discharges, J. Soc. Radiol. Prot. 13 (1993) 4355. D.R.P. Leonard, G.J. Hunt, A study of sh and shellsh consumers near Sellaeld: assessment of the critical groups including consideration of children, J. Soc. Radiol. Prot. 5 (1985) 129138. B.L. Bayne, D.W. Brown, K. Burns, D.R. Dixon, A. Ivanovici, D.R. Livingstone, D.M. Lowe, M.N. Moore, A.R.D. Stebbing, J. Widdows, The Effects of Stress and Pollution on Marine Animals, Praeger, New York, 1985, 384 pp. A. Khler, E. Wahl, K. Sffker, Functional and morphological changes of lysosomes as prognostic biomarkers of toxic liver injury in a marine atsh (Platichthys esus (L)), Environ. Toxicol. Chem. 21 (2002) 24342444. D.R. Livingstone, Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms, Mar. Pollut. Bull. 42 (2001) 656666. D.R. Livingstone, J.K. Chipman, D.M. Lowe, C. Minier, C.L. Mitchelmore, M.N. Moore, L.D. Peters, R.K. Pipe, Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: recent molecular, genotoxic, cellular and immunological studies on the common mussel (Mytilus edulis L.) and other mytilids, Int. J. Environ. Pollut. 13 (2000) 5691. M.N. Moore, Lysosomal cytochemistry in marine environmental monitoring, Histochem. J. 22 (1990) 187191. M.N. Moore, M.G. Simpson, Molecular and cellular pathology in environmental impact assessment, Aquat. Toxicol. 22 (1992) 313322. A.H. Ringwood, J. Hoguet, C. Keppler, M. Gielazyn, Linkages between cellular biomarker responses and reproductive success in oysters, Crassostrea virginica, Mar Environ. Res. 58 (2004) 151155. M.N. Moore, A. Kohler, D.M. Lowe, M.G. Simpson, An integrated approach to cellular biomarkers in sh, in: M.C. Fossi, C. Leonzio (Eds.), Non-Destructive Biomarkers in Vertebrates, Lewis/CRC, Boca Raton, 1994, pp. 171197. [17] M.N. Moore, Biocomplexity: the post-genome challenge in ecotoxicology, Aquat. Toxicol. 59 (2002) 115. [18] D.E. Hinton, D.J. Lauren, Liver structural alterations accompanying chronic toxicity in shes: potential biomarkers of exposure, in: J.F. McCarthy, L.K. Shugart (Eds.), Biomarkers of Environmental Contamination, Lewis Publishers, Boca Rota, 1990, pp. 1737. [19] J.F. McCarthy, L.R. Shugart (Eds.), Biomarkers of Environmental Contamination, Lewis Publishers, Boca Raton, Ann Arbor, 1990, 457 pp. [20] J.I. Allen, M.N. Moore, Environmental prognostics: is the current use of biomarkers appropriate for environmental risk evaluation, Mar. Environ. Res. 58 (2004) 227232. [21] D.L. Kalpaxis, C. Theos, M.A. Xaplanteri, G.P. Dinos, A.V. Catsiki, M. Leotsinidis, Biomonitoring of Gulf of Patras, N. Peloponnesus, Greece. Application of a biomarker suite including evaluation of translation efciency in Mytilus galloprovincialis cells, Environ. Res. 94 (2004) 211220. [22] F. Regoli, Total; oxyradical scavenging capacity (TOSC) in polluted and translocated mussels: a predictive biomarker of oxidative stress, Aquat. Toxicol. 50 (2000) 351361. [23] I. McFadzen, S. Baynes, J. Hallam, A. Beesley, D. Lowe, Histopathology of the skin of UV-B irradiated sole (Solea solea) and turbot (Scophthalmus maximus) larvae, Mar. Environ. Res. 50 (2000) 273277. [24] I. McFadzen, N. Eufemia, C. Heath, D. Epel, M.N. Moore, D. Lowe, Multidrug resistance in the embryos and larvae of the mussel, Mytilus edulis, Mar. Environ. Res. 50 (2000) 319323. [25] G.W. Winston, S.M. Adams, W.H. Benson, L.E. Gray, H.S. Matthews, M.N. Moore, S. Safe, Biological bases of similarities and differences, in: R.T. Di Giulio, W.B. Benson (Eds.), Interconnections Between Human Health and Ecological Integrity, Society of Environmental Toxicology and Chemistry (SETAC), Pensacola, FL, 2002, pp. 4365. [26] E. Biganzoli, P. Boracchi, M.G. Daidone, E. Marubini, Flexible modelling in survival analysis. Structural biological complexity from the information provided by tumor markers, Int. J. Biol. Markers 13 (1998) 107123. [27] W. Dchting, W. Ulmer, T. Ginsberg, Cancer: a challenge for control theory and computer modelling, Eur. J. Cancer Part A 32 (1996) 12831292. [28] D.A. Lauffenburger, J.L. Linderman, Receptors: Models for Binding, Trafcking and Signaling, Oxford University Press, Oxford, 1993, 365 pp. [29] M.N. Moore, J.I. Allen, A computational model of the digestive gland epithelial cell of the marine mussel and its simulated responses to aromatic hydrocarbons, Mar. Environ. Res. 54 (2002) 579584. [30] A. McVeigh, J.I. Allen, M.N. Moore, P. Dyke, D. Noble, A carbon and nitrogen ux model of mussel digestive gland epithelial cells and their simulated response to pollutants, Mar. Environ. Res. 58 (2004) 821828. [31] D. Noble, Modelling the heart: insights, BioEssays 24 (2002) 11551163. [32] D. Noble, The rise of computational biology, Nature Rev. Mol. Cell Biol. 3 (2002) 460463.
[2] [3] [4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
M.N. Moore et al. / Mutation Research 552 (2004) 247268 [33] D. Noble, Unraveling the genetics and mechanisms of cardiac arrhythmia, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 57555756. [34] D. Noble, J. Levin, W. Scott, Biological simulations in drug discovery, Drug Discovery Today 4 (1999) 1016. [35] M. Chicurel, The bigger picture, New Scientist 164 (2216) (1999) 3842. [36] S.A. Kauffman, The Origins of Order, Oxford University Press, Oxford, 1993, 709 pp. [37] S. Brenner, Biological computation, in: G.R. Bock, J.A. Goode (Eds.), The Limits of Reductionism in Biology, Novartis Foundation Symposium, vol. 213, 1998, pp. 106116. [38] J. Schaff, C. Fink, B. Slepchenko, J. Carson, L. Loew, A general computational framework for modeling cellular structure and function, Biophys. J. 73 (1997) 11351146. [39] C.V. Howard, Synergistic effects of chemical mixtures: can we rely on traditional toxicology? The Ecologist 27 (1997) 192195. [40] F. Kanzawa, K. Nishio, K. Fukuoka, M. Fukuda, T. Kunimoto, N. Saijo, Evaluation of synergism by a novel three-dimensional model for the combined action of cisplatin and etoposide on the growth of a human small-cell lungcancer cell line, SBC-3, Int. J. Cancer 71 (1997) 311319. [41] M.H. Depledge, Rapid assessment of marine pollution (RAMP), in: Proceedings of the VietnamUK Joint Workshop on Marine Pollution Assessment, Hanoi, June 8, 2000, Publication for National Centre for Natural Science and Technology of Vietnam, Hanoi, Vietnam, 2000, pp. 516. [42] M.N. Moore, A strategy for impact and risk assessment in integrated environmental management, Rev. Md. Vt. 153 (2002) 507512. [43] M.H. Depledge, Interactions between heavy metals and physiological processes in estuarine invertebrates, in: P.L. Chambers, C.M., Chambers (Eds.), Estuarine Ecotoxicology, Japaga, Wicklow, Ireland, 1990, pp. 89100. [44] A. Murdoch, K.L.E. Kaiser, M.E. Comba, M. Neilson, Particle-associated PCBs in Lake Ontario, Sci. Total. Environ. 158 (1994) 113125. [45] F. Smedes, Sampling and partition of neutral organic contaminants in surface waters with regard to legislation, Int. J. Environ. Anal. Chem. 57 (1994) 215229. [46] J.W. Readman, R.F.C. Mantoura, M.M. Rhead, The physicochemical speciation of polycyclic aromatic hydrocarbons (PAH) in aquatic systems, Fresenius Z. Anal. Chim. 319 (1984) 126131. [47] M.B. Thomson, J.J. Pignatello, Mechanisms and effects of Resistant Sorption processes of organic compounds in natural particles, in: 214th ACS National Meeting of American Chemical Society, Division of Environmental Chemistry, Preprints of extended abstracts 37 (1997) 159160. [48] D. Thomas, B. Gustafsson, . Gustafsson, Quantication of the sootwater distribution coefcient of PAHs provides mechanistic basis for enhanced sorption observations, Environ. Sci. Technol. 34 (2000) 51445151.
265
[49] J.L. Zhou, T.W. Fileman, S.V. Evans, P. Donkin, C.A. Llewellyn, J.W. Readman, R.F.C. Mantoura, S.J. Rowland, Fluoranthene and pyrene in the suspended particulate matter and surface sediments of the Humber estuary, UK, Mar. Poll. Bull. 36 (1998) 597597. [50] J.L. Zhou, T.W. Fileman, S. Evans, P. Donkin, J.W. Readman, R.F.C. Mantoura, S. Rowland, The partition of ouranthene and pyrene between suspended particles and dissolved phase in the Humber Estuary: a study of the controlling factors, Sci. Total Environ. 244 (1999) 305321. [51] M.P. Cajaraville, I. Abascal, M. Etxeberria, I. Marigmez, Lysosomes as cellular markers of environmental pollution: time- and dose-dependant responses of the digestive lysosomal system of mussels after petroleum hydrocarbon exposure, Environ. Toxicol. Water Qual. 10 (1995) 18. [52] H. Franke, Substructural alterations of liver parenchymal cells induced by xenobiotics, Exp. Pathol. 39 (1990) 139 155. [53] W.H. Halliwell, Cationic amphiphilic drug-induced phospholipidosis, Toxicol. Pathol. 25 (1997) 5360. [54] A.J. Hawkins, A.J. Day, Metabolic interactions underlying the physiological and evolutionary advantages of genetic diversity, Am. Zool. 39 (1999) 401411. [55] L. Hein, R. Lllman-Rauch, K. Mohr, Human accumulation potential of xenobiotics: potential of catamphiphilic drugs to promote their accumulation via inducing lipidosis or mucopolysaccaridosis, Xenobiotica 20 (1990) 12591267. [56] M.U. Hutchins, M. Veenhuis, D. Klionsky, Peroxisome degradation in Saccharomyces cerevisiae is dependant on machinery of macroautophagy and the Cvt pathway, J. Cell Sci. 112 (1999) 40794087. [57] V. Luzikov, NQuality control: from molecules to organelles, FEBS Letts. 448 (1999) 201205. [58] I. Marigmez, L. Baybay-Villacorta, Pollutant-specic and general lysosomal responses in digestive cells of mussels exposed to model organic chemicals, Aquat. Toxicol. 64 (2003) 235257. [59] M.N. Moore, C. Soverchia, M. Thomas, Enhanced lysosomal autophagy of intracellular proteins by xenobiotics in living molluscan blood cells, Acta Histchem. Cytochem. 29 (Suppl.) (1996) 947948. [60] M.N. Moore, D.M. Lowe, C. Soverchia, S.D. Haigh, S.G. Hales, Uptake of a non-caloric, edible sucrose polyester oil and olive oil by marine mussels and their inuence on uptake and effects of anthracene, Aquat. Toxicol. 39 (1997) 307320. [61] M.N. Moore, The Robert Feulgen Lecture 1990. Environmental distress signals: cellular reactions to marine pollution, Prog. Histochem. Cytochem. 23 (1991) 119. [62] M.N. Moore, R.I. Willows, A model for cellular uptake and intracellular behaviour of particulate-bound micropollutants, Mar. Environ. Res. 46 (1998) 509514. [63] G.E. Mortimore, A.R. Poso, Intracellular protein catabolism and its control during nutrient deprivation and supply, Annu. Rev. Nutr. 7 (1987) 539564. [64] F. Rashid, R.W. Horobin, Accumulation of uorescent non-cationic probes in mitochondria of cultured-cells
266
M.N. Moore et al. / Mutation Research 552 (2004) 247268 observations, a proposed mechanism, and some implications, J. Microsc. 163 (1991) 233241. F. Rashid, R.W. Horobin, M.A. Williams, Predicting the behaviour and selectivity of uorescent probes for lysosomes and related structures by means of structure-activity models, Histochem. J. 23 (1991) 450459. P.O. Seglen, DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival, Cell Biol. Toxicol. 13 (1997) 301315. F. Thevenod, J.M. Friedman, Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+ /K+ -ATPase through proteosomal and endo-lysosomal proteolytic pathways, FASEB J. 13 (1999) 17511761. A. Viarengo, L. Canesi, M.N. Moore, M. Orunesu, Effects of Hg2+ and Cu2+ on the cytosolic Ca2+ level in molluscan blood cells evaluated by confocal microscopy and spectrouorimetry, Mar. Biol. 119 (1994) 557564. G.W. Winston, M.N. Moore, M.A. Kirchin, C. Soverchia, Production of reactive oxygen species (ROS) by hemocytes from the marine mussel, Mytilus edulis, Comp. Biochem. Physiol. 113C (1996) 221229. D.R.P. Leonard, R.J. Pentreath, Further 237Pu experiments with the plaice Pleuronectes platessa: sub-cellular distribution of plutonium in the liver, Mar. Biol. 63 (1981) 6771. P. Bjerregaard, M.H. Depledge, Cadmium accumulation in Littorina littorea, Mytilus edulis and Carcinus maenas: the inuence of salinity and calcium ion concentrations, Mar. Biol. 119 (1994) 385395. A. Kortenkamp, R. Altenburger, Synergisms with mixtures of xenoestrogens: a reevaluation using the method of isoboles, Sci. Total Environ. 221 (1998) 5973. B.P. Lyons, C.K. Pascoe, I.R.B. McFadzen, Phototoxicity of pyrene and benzo[a]pyrene to embryo-larval stages of the pacic oyster Crassostrea gigas, Mar. Environ. Res. 54 (2002) 627631. D.S. Aga, E.M. Thurman, Environmental immunoassays: alternative techniques for soil and water analysis, ACS Symp. Ser. 657 (1997) 120. J. Sherry, Environmental immunoassays and other bioanalytical methods: overview and update, Chemosphere 34 (1997) 10111025. P.G. Wells, M.H. Depledge, J.N. Butler, J.J. Manock, A.H. Knap, Rapid toxicity assessment and biomonitoring of marine contaminantsexploiting the potential of rapid biomarker assays and microscale toxicity tests, Mar. Pollut. Bull. 42 (2001) 799804. V.V. Cheung, R.J. Wedderburn, M.H. Depledge, Molluscan lysosomal responses as a diagnostic tool for detection of a pollution gradient in Tolo Harbour, Hong Kong, Mar. Environ. Res. 46 (1998) 237241. C. Hauton, L.E. Hawkins, S. Hutchinson, The use of neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European at oyster Ostrea edulis (L.), Comp. Biochem. Physiol. 199 (1998) 619623. D.M. Lowe, V.U. Fossato, M.H. Depledge, Contaminant induced lysosomal membrane damage in blood cells of mussels M. galloprovincialis from the Venice Lagoon: an in vitro study, Mar. Ecol. Prog. Ser. 129 (1995) 189196. C. Svendseb, J.M. Weeks, The use of a lysosome assay for the rapid assessment of cellular stress from copper to the freshwater snail Viviparus contectus (Millet), Mar. Poll. Bull. 31 (1995) 139142. J. Wedderburn, V. Cheung, S. Bamber, M. Bloxham, M.H. Depledge, Biomarkers of histochemical and cellular stress in Carcinus maenas: an in situ eld study, Mar. Environ. Res. 46 (1998) 321324. B. Kurelec, B. Pivcevic, Evidence for a multixenobiotic resistance mechanism in the mussel Mytilus galloprovincialis, Aquat. Toxicol. 19 (1991) 291302. C. Minier, M.N. Moore, Multixenobiotic resistance in mussel blood cells, Mar. Environ. Res. 42 (1996) 389392. J.J. Stegeman, J.J. Lech, Cytochrome P-450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure, Environ. Health Perspect. 90 (1991) 93100. M.H. Depledge, The rational basis for detection of the early effects of marine pollution using physiological indicators, Ambio (Royal Swedish Academy of Sciences) 18 (1989) 301302. D.R. Livingstone, Organic xenobiotic metabolism in marine invertebrates, Adv. Comp. Environ. Physiol. 7 (1991) 45 185. D.R. Livingstone, Biotechnology and pollution monitoring: use of molecular biomarkers in the aquatic environment, J. Chem. Tech. Biotechnol. 57 (1993) 195211. D.R. Livingstone, P. Garcia Martinez, X. Michel, J.F. Narbonne, S.C.M. OHara, D. Ribera, G.W. Winston, Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs, Funct. Ecol. 4 (1990) 413424. R. Mason, Free radical metabolites of foreign compounds and their toxicological signicance, Rev. Biochem. Toxicol. 87 (1990) 237243. S. Dailianis, G.P. Domouhtsidou, E. Raftopoulou, M. Kaloyianni, V.K. Dimitriadis, Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring, Mar. Environ. Res. 56 (2003) 443470. D.W. Nebert, D.D. Petersen, A.J. Fornace Jr., Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm, Environ. Health Perspect. 88 (1990) 1325. D.R. Dixon, A.M. Pruski, L.R.J. Dixon, A.N. Jha, Marine invertebrate eco-genotoxicology: a methodological overview, Mutagenesis 17 (2002) 495507. A.N. Jha, V.V. Cheung, M.E. Foulkes, H.J. Hill, M.H. Depledge, Detection of genotoxins in the marine environment: adoption and evaluation of an integrated approach using the larval stages of the marine mussel, Mytilus edulis, Mutat. Res. 464 (2000) 213228. R.J. Albertini, J.A. Nicklas, J.P. ONeill, Future research directions for evaluating human genetic and cancer risk from environmental exposures, Environ. Health Perspect. 104 (Suppl. 3) (1996) 503510.
[65]
[80]
[66]
[81]
[67]
[82]
[68]
[83] [84]
[69]
[85]
[70]
[86]
[71]
[87]
[72]
[88]
[73]
[89]
[74]
[90]
[75]
[76]
[91]
[92]
[77]
[93]
[78]
[94]
[79]
M.N. Moore et al. / Mutation Research 552 (2004) 247268 [95] B.P. Lyons, C. Stewart, M.F. Kirby, 32 P-postlabelling analysis of DNA adducts and EROD induction as biomarkers of genotoxin exposure in dab (Limanda limanda) from British coastal waters, Mar. Environ. Res. 50 (2000) 575 579. [96] B. Kurelec, The genotoxic disease syndrome, Mar. Environ. Res. 35 (1993) 341348. [97] D.R.P. Leonard, R.J. Law, C.A. Kelly, Responding to the Sea Empress oil spill, in: Proceedings of the International Symposium on Marine Pollution, IAEA SM-354/86 IAEATECDOC-1094, 1998, pp. 177182. [98] E.F. Nuwaysir, M. Bittner, J. Trent, J.C. Barrett, C.A. Afshari, Microarrays and toxicology: the advent of toxicogenomics, Mol. Carcinogenesis 24 (1999) 153159. [99] W.D. Pennie, J.D. Tugwood, G.J. Oliver, I. Kimber, The principles and practice of toxicogenomics: applications and opportunities, Toxicol. Sci. 54 (2000) 277283. [100] ECETOC, 2001, White Paper on Genomics, Transcript Proling, Proteomics and Metabonomics (GTPM)An Introduction, ECETOC Document No. 42, European Centre for the Ecotoxicology and Toxicology of Chemicals, Brussels, Belgium, 2001, 27 pp. [101] J.R. Snape, S.J. Maund, D.B. Pickford, T.H. Hutchinson, Ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology, Aquat. Toxicol. 67 (2004) 143154. [102] P. Bannasch, H. Enzmann, F. Klimek, E. Weber, H. Zerban, Signicance of sequential cellular changes inside and outside foci of altered hepatocytes during hepatocarcinogenesis, Toxicol. Pathol. 4 (1989) 617628. [103] M.P. Cajaraville, I. Cancio, A. Ibabe, A. Orbea, Peroxisome proliferation as a biomarker in environmental pollution assessment, Microsc. Res. Tech. 61 (2003) 117120. [104] T.S. Galloway, R.C. Sanger, K.L. Smith, G. Fillmann, J.W. Readman, T.E. Ford, M.H. Depledge, Rapid assessment of marine pollution using multiple biomarkers and chemical immunoassays, Environ. Sci. Technol. 36 (2002) 22192226. [105] A. Khler, H. Deisemann, B. Lauritzen, Ultrastructural and cytochemical indices of toxic injury in dab liver, Mar. Ecol. Prog. Ser. 91 (1992) 141153. [106] M.N. Moore, G. Topping, P.-E. Minhea, S. Kiryanov, V. Mikhailov, Design of a monitoring programme for the Black Sea: contaminant levels and biological effects, in: L. Mee, G. Topping (Eds.), Black Sea Pollution Assessment, UN Publications, New York, 1998, pp. 293299. [107] D.M. Lowe, M.N. Moore, B. Evans, Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from Dab (Limanda limanda), Mar. Ecol. Prog. Ser. 91 (1992) 135140. [108] M.N. Moore, Molecular cell pathology of pollutant induced liver injury in atsh: use of uorescent probes, Mar. Ecol. Prog. Ser. 91 (1992) 127133. [109] A.J. Lawrence, A. Arukwe, M.N. Moore, M. Sayer, J. Thain, Molecular/cellular processes and the physiological response to stress, in: A. Lawrence, K. Hemingway (Eds.), Effects of Pollution on FishMolecular Effects and Population Responses, Blackwell Science Ltd., Oxford, 2003, pp. 83133.
267
[110] D.M. Lowe, C. Soverchia, M.N. Moore, Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to ouranthene, Aquat. Toxicol. 33 (1995) 105112. [111] P. Hunter, Putting Humpty Dumpty back together again, The Scientist 17 (2003) 2021. [112] B.H. Smaill, D.C. McGifn, I.J. LeGrice, A.A. Young, P.J. Hunter, A.J. Galbraith, The effect of synthetic patch repair of coarctation on regional deformation of the aortic wall, J. Thorac. Cardiov. Sur. 120 (2000) 10531063. [113] J.J. Rice, M.S. Jafri, Modelling calcium handling in cardiac cells, Philos. Trans. Roy. Soc. 359A (2001) 11431157. [114] P.J. Hunter, P.M.F. Nielsen, D. Bullivant, The IUPS physiome project, in: In Silico Simulation of Biological Processes, Novartis Foundation Symposium 247 (2002) 207221. [115] D.dA. Laffoley, J. Burt, P. Gilland, J. Baxter, D.W. Connor, M. Hill, J. Breen, M. Vincent, E. Maltby, Adopting an ecosystem approach for the improved stewardship of the maritime environment, English Nat. Res. Rep. 538 (2003) 20. [116] M.N. Moore, D.M. Lowe, R.J. Wedderburn, T.L. Wade, G. Balashov, H. Buyukgungor, Y. Daurova, Y. Denga, E. Kostylev, P. Mihnea, S. Moncheva, S. Tabagari, C. Ciocan, H. Ozkoc, M.H. Depledge, International Mussel Watch (UNESCO/IOC) in the Black Sea: a pilot study for biological effects and contaminant residues, in: S. Besiktepe, I. Unluata (Eds.), Environmental Degradation of the Black Sea: Challenges and Remedies, NATO Advanced Research Workshop, Kluwer Academic, New York, Dordrecht, 1999, pp. 273289. [117] M.N. Moore, T.L. Wade, D.M. Lowe, R.J. Wedderburn, G. Balashov, H. Bykgngr, Y. Daurova, Y. Denga, E. Kostylev, P. Mihnea, S. Moncheva, S. Tabagari, C. Ciocan, H. zkoc, M.H. Depledge, The UNESCO/IOC Black Sea Mussel Watch Pilot Study: biological effects and contaminant residues, in: L. Mee, G. Topping (Eds.), Black Sea Pollution Assessment, UN Publications, New York, 1998, pp. 279292. [118] K.E. Drexler, Nanosystems: Molecular Machinery, Manufacturing and Computation, WileyInterscience, New York, 1992, 556 pp. [119] V. Howard, Small particlesbig problems, Int. Lab. News 34 (2) (2004) 2829. [120] M. Gross, Travels to the Nanoworld: Miniature Machinery in Nature and Technology, Plenum Trade, New York, 1999, 254 pp. [121] R.M. Perrin, Crop protection: taking stock for the new millennium, Crop Protect. 16 (1997) 449456. [122] H. Darmency, The impact of hybrids between genetically modied plant crop plants and their related species: introgression and weediness, Mol. Ecol. 3 (1994) 3740. [123] J.M. Dunwell, E.M. Paul, Impact of genetically modied crops in agriculture, Outlook Agric. 19 (1990) 103110. [124] K.E. Drexler, Molecular nanomachines: physical principles and implementation strategies, Ann. Rev. Biophys. Biomol. Struct. 23 (1994) 377405.
268
M.N. Moore et al. / Mutation Research 552 (2004) 247268 (Eds.), Environmental Toxicology & Risk Assessment, ASTM, Philadelphia, 1995, pp. 97110. M.St.J. Warne, D.W. Hawker, The number of components in a mixture determines whether synergistic and antagonistic or additive toxicity predominate: the funnel hypothesis, Ecotox. Environ. Safety 31 (1995) 2328. J.L. Casti, Complexication: Explaining a Paradoxical World Through the Science of Surprise, Abacus, London, 1994, 320 pp. J. Maddox, What Remains to be Discovered, The Free Press, New York, 1998, 434 pp. M.H. Depledge, Genotype toxicity: implications for individuals and populations, Environ. Health Perspect. 102 (1994) 101104. M.H. Depledge, Recovery of ecosystems and their components following exposure to pollution, J. Aquat. Ecosyst. Health 6 (1999) 199206. R.A. Browne, V. Moller, V.E. Forbes, M.H. Depledge, Estimating genetic and environmental components of variance using sexual and clonal Artemia, J. Exp. Mar. Biol. Ecol. 267 (2002) 107119. R.J. Brown, T.S. Galloway, D. Lowe, M.A. Browne, A. Dissanayake, M.B. Jones, M.H. Depledge, Differential sensitivity of three marine invertebrates to copper assessed using multiple biomarkers, Aquat. Toxicol. 66 (2004) 267 278. T.S. Galloway, R.J. Brown, M.A. Browne, A. Dissanayake, D. Lowe, M.B. Jones, M.H. Depledge, A multibiomarker approach to environmental assessment, Environ. Sci. Technol. 38 (2004) 17231731. G. Watson, O.K. Andersen, T.S. Galloway, M.H. Depledge, Rapid assessment of polycyclic aromatic hydrocarbon (PAH) exposure in decapod crustaceans by uorimetric analysis of urine and haemolymph, Aquat. Toxicol. 67 (2004) 127142. G. Fillmann, R.C. Sanger, M.H. Depledge, J.W. Readman, Relative performance of immunochemical (enzyme-linked immunosorbent assay) and gas chromatography-electroncapture detection techniques to quantify polychlorinated biphenyls in mussel tissues, Anal. Chim. Acta 461 (2002) 7584. G. Fillmann, G.M. Watson, M. Howsam, E. Francioni, M.H. Depledge, J.W. Readman, Urinary PAH metabolites as biomarkers of exposure in aquatic environments, Environ. Sci. Technol. 38 (2004) 26492656.
[125] P. Kitov, J.M. Sadowska, G. Mulvey, G.D. Armstrong, H. Ling, N.S. Pannu, R.J. Read, D.R. Bundle, Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands, Nature 403 (2000) 669672. [126] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis, Adv. Drug Delivery Rev. 54 (2002) 631651. [127] A.M. Gatti, F. Rivasi, Biocompatibility of micro- and nanoparticles. Part I. In liver and kidney, Biomaterials 23 (2002) 23812387. [128] P. Gould, Nanoparticles probe biosystems, Mater. Today 7 (2) (2004) 3643. [129] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene delivery to cells and tissues, Adv. Drug Delivery Rev. 55 (2003) 329347. [130] M.P. Cajaraville, M.J. Bebianno, J. Blasco, C. Porte, C. Sarasquete, A. Viarengo, The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian peninsula: a practical aaproach, Sci. Tot. Environ. 247 (2000) 295311. [131] M.N. Moore, Z. Csizer, Integrated coastal zone management as a toolbox for environmentally sustainable industrial development in coastal areas, in: L.F. Cassar (Ed.), Integrated Coastal Zone Management, UNIDO, Vienna, 2001, pp. 161166. [132] ICES International Council for the Exploration of the Seas (ICES), Report of the Working Group on the Biological Effects of Contaminants, ICES, Copenhagen, 1997. [133] C.J. Bayne, M.N. Moore, Non-lymphoid immunologic defenses in aquatic invertebrates and their value as indicators of aquatic pollution, in: J.T. Zelikoff (Ed.), EcoToxicology: Responses, Biomarkers and Risk Assessment, Published for OECD by SOS Publications, Fair Haven, New Jersey, 1998, pp. 243261. [134] M.M. Grundy, M.N. Moore, S.M. Howell, N.A. Ratcliffe, Phagocytic reduction and effects on lysosomal membranes of polycyclic aromatic hydrocarbons, in haemocytes of Mytilus edulis, Aquat. Toxicol. 34 (1996) 273290. [135] M.M. Grundy, N.A. Ratcliffe, M.N. Moore, Immune inhibition in marine mussels by polycyclic aromatic hydrocarbons, Mar. Environ. Res. 42 (1996) 187190. [136] S. Ferson, T.F. Long, Conservative uncertainty propagation in environmental risk assessments, in: J.S. Hughes et al.
[137]
[138]
[139] [140]
[141]
[142]
[143]
[144]
[145]
[146]
[147]

An Integrated Biomarker Based Strategy For Ecotoxicological Evaluation of Risk in Environmental Management

Caricato da

Informazioni sul documento

Descrizione originale:

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

An Integrated Biomarker Based Strategy For Ecotoxicological Evaluation of Risk in Environmental Management

Caricato da

Copyright:

Formati disponibili

Mutation Research 552 (2004) 247268

An integrated biomarker-based strategy for ecotoxicological evaluation of risk in environmental management

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

M.N. Moore et al. / Mutation Research 552 (2004) 247268

[2] [3] [4] [5]

Potrebbero piacerti anche