Mutagenesis vol. 19 no. 3 pp. 165-168,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Biomarkers of genotoxicity and other end-points in an integrated approach to environmental risk assessment
1Unitat de Toxicologia Experimental i Ecotoxicologia, Parc Científic de Barcelona, Josep Samitier 1–5, 08028 Barcelona, Spain and 2Departament de Biología Animal-Vertebrats, Facultat de Biología, Universitat de Barcelona, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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Risk is defined as the probability of a given toxicological hazard resulting in actual biological harm. This involves some form of mathematical relationship between exposure and toxic effects. Simplified models based on laboratory testing in surrogate species neglect potentially important factors in real life situations. Our own approach to the study of atmospheric and edaphic pollution, focused on realism, includes the use of sentinel species (animals as prospectors and integrators of information, along both the spatial and the temporal axes) and selected biomarkers. We aim to: (i) consider pollution as a complex mixture; (ii) take into account homeostasis of the environment and of living organisms; (iii) be realistic (all data obtained in the field; calculations based on actual effects; exposure measured as internal dose). The proposed test battery divides toxicological information into four blocks: systemic effects (serum biochemistry and histopathology in wild wood mice), reproduction (epididymis cell count in mice, malformations in amphibian larvae), genotoxicity (Comet test in mice and earthworms) and population effects (abundance and diversity in arthropods). Each block is represented by the sum of the results of the tests performed within the block (presented as a severity score from 0 to 3). A final value is obtained to represent the integrated toxicological harm (ITH) occurring at a given location. To assess exposure, taking into account bioavailability, we propose (i) for soil contamination studies, measuring EROD activity in liver; (ii) for atmospheric pollution, the gaseous fraction is taken from immission gases analysis, while the solid fraction is assessed through levels of metals in sentinel organisms, the values of both fractions then being combined. Finally, a regression line is established for exposure versus ITH in four to five locations with decreasing exposure levels, ranging from the immediate neighbourhood of the pollution focus to controls, following the main dissemination line. In this model we may interpolate new exposure data to find the corresponding predicted ITH. Such a prediction may be directly interpreted as a form of risk assessment or, alternatively, these pairs (toxicological harm/exposure) could then be related to a conventional scale of ecotoxicological risk.
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Introduction |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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Research in the life sciences is committed, due to the built-in complexity of the subject under study, to the use of models. We want our models to be comprehensive, meaningful and able to afford some insight into the mechanisms involved, but, at the same time, they ought to be quick, cheap, simple and practical. We seek for realistic models yet we want to keep full control of the experimental parameters. In practice, these may be incompatible. Rapidity, economy and technical ease frequently imply a drastic simplification of the real world, sometimes to the extreme of becoming meaningless; realism and control of the experimental variables are also often contrary factors of a dialectic pair.
In the field of environmental toxicity assessment, the need for in-time risk management decisions requires setting up a battery of standardized and relatively easy to perform tests, allowing quick answers to pressing questions. The use of substitute species, subjected in the laboratory to conventional routes and patterns of exposure to xenobiotics, is therefore unavoidable.
Risk is defined as the probability of a given toxicological hazard producing actual biological harm. This idea involves some form of mathematical relationship between exposure and toxicology. Present regulatory issues on quantitative ecotoxicological risk assessment are mostly based on the ratio PEC/PNEC, where PEC is the predicted exposure concentration (derived from the analysis of chemicals in the environment) and PNEC is the predicted no-effect concentration, estimated from regulated laboratory tests in which surrogate species are exposed to known amounts of single pollutants. However, such simplified models fail to take into account some potentially very important factors, such as cross-reactions among pollutants within a complex mixture, the action of climatic or physico-chemical conditions, the homeostatic capacity of the environment itself and species-specific detoxifying/activating metabolism in living things.
On the other hand, a realistic approach often means a long-term field study, producing overwhelming amounts of disparate results which have to be integrated.
Midway between the two extremes of realism and control there may exist some intermediate solutions, going from mesocosms to keeping laboratory animals under field conditions. Of course, integration of studies based on multiple, independent approaches is also advisable, and probably most scientifically relevant.
We here present a model of field study, based on the use of sentinel species, that we are currently applying to risk assessment in the area around a focus of pollution.
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Previous work |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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Our own approach to the study of atmospheric and soil pollution over the past 10 years has been a bid for realism.
We have studied the effects of a coal-fired power plant in Cercs (Catalonia, northeast Spain), of urban air pollution in the area of México DF, of a dumping site for domestic and general industry waste in the metropolitan area of Barcelona and of another landfill in the Catalan countryside (La Seu d’Urgell).
Haematological, biochemical, histopathological, ultrastructural and genotoxicological analysis, as well as density and biodiversity studies, have been carried out on animals captured in the wild in the contaminated areas and in matched, clean ones. These animals include rodents (Llacuna et al., 1993
; Górriz et al., 1994, 1996), passerine birds (Llacuna et al., 1995, 1996; Brotons et al., 1998) and arthropods (Salgado et al., 1996, 1997). Some still unpublished data cover effects on insectivores, metal deposition and mobilization and genotoxicity in rodents and in earthworms (Delgado et al., 2000). Biogeographic and nutritional studies in rodents are presently being carried out in one of the above-mentioned landfill areas.
This is a realistic approach, but it does not allow any control over the sex, age, health status or genetic characteristics of the study subjects nor of the real duration or pattern of exposure nor the possibility of selective predation of the most severely affected animals. In this context we considered an interesting alternative, of undertaking an intermediate approach (in the case of air pollution), placing homogeneous, caged laboratory rodents under field conditions in both the polluted (near the power plant) and the control zones. Weight gain, food and water intake, serum biochemistry and haematological parameters were controlled in a 6 month study (Borràs et al., 1998) and, eventually, a histopathological evaluation was also performed (Borràs et al., 1999).
In the atmospheric pollution studies (Cercs), data from 8–10 wild Apodemus sylvaticus and 20 caged Mus musculus per location showed alterations in the individuals in the polluted zone, including an increase in serum transaminases (during the first month in Mus, returning to normal by the end of the study; persistent as a background in Apodemus), ciliar pathologies in the trachea (changes in the ciliated area and ciliar orientation, malformations), severe inflammatory processes in the respiratory tract, liver inflammation and lipofuscin storage in Kupffer cells (indicative of enhanced lipid peroxidation), renal glomerular changes and interstitial nephritis. No differences were observed between control and polluted zones in the micronucleus test. There were also behavioural effects, such as a marked decrease in activity and also in water consumption in the caged Mus or an increase in the time spent by birds (in partially defoliated trees) watching for the presence of possible predators, resulting in a noticeable decrease in their feeding efficacy. At the population level, reduced diversity indices in birds were reported, as well as a demographic boom in the springtime (compensated by lower numbers during the rest of the year) and an increase in numbers of carnivorous arthropods.
Air pollution studies in México were performed on more than 500 individuals belonging to several species of small wild mammals (Peromyscus maniculatus, Peromyscus melanotis, Peromyscus dificilis, Microtus mexicanus and Neotomodon alstoni). Results include tracheal mucous cell hyperplasia, ciliar malformations, pneumoconiosis, increased Toxoplasma gondii parasitism and mild renal pathologies related to pollution exposure. No genotoxicity assessment was carried out.
The studies of soil pollution generated by landfills (Barcelona and La Seu d’Urgell) were carried out in groups of 
20 wild Apodemus per location (histopathology, serum biochemistry and genotoxicity) and 7–9 Allollobophora sp. earthworms per soil sample for the Comet test in coelomocytes (Barcelona). Significant increases in transaminases were detected in both polluted zones, as well as varying degrees of hepatic chronic inflammation and interstitial nephritis. Hepatic apoptosis was seen in Barcelona and splenic pathologies (esplenomegalia, lymphocitary autophagocitosis and megakaryocyte proliferation) in about one third of the animals from La Seu d’Urgell, 25% of which, in addition, showed heavily parasitized livers. In both landfill areas we obtained significant positive results (compared with controls) in the micronucleus and Comet tests. Moreover, in the Barcelona landfill the Comet test proved to be more sensitive than the micronucleus test, allowing (in mice) a significant distinction between a zone of maximum effect, near the leachates pool, and a second one some 4 km downstream of the torrent. These results stress the importance of genotoxicity tests as an independent (and relevant) part of integrated ecotoxicological assessment.
Such a polynomial, mid-term type of study, with wild as well as laboratory reared animals, has enabled us to gain some understanding of the chain of events that are taking place in contaminated terrestrial environments. Moreover, it has helped us identify species and biomarkers that may better contribute to establishing the quality and extent of the toxicological impact.
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Model features |
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Living things, especially animals, because of their capacity for movement, are prospectors and integrators of information, along both the spatial and temporal axes. It is in that sense that such species are defined as sentinel, because their characteristics of ubiquity, abundance, sedentarism, relative longevity, capacity for bioaccumulation and a certain degree of tolerance to toxicants are able to provide some information either on the exposure to xenobiotics or on their biological effects, acting as an alarm signal. Biomarkers are the biological end-points through which such information is gathered.
Our draft proposal for a realistic model of ecotoxicological risk assessment in terrestrial environments should include:
• characterization of the appropriate sentinel species and biomarkers to assess every relevant aspect of toxicological harm;
• integration of the different aspects of toxicity in a polynomial expression;
• measurement of exposure as internal dose (i.e. level of pollutants found within the organism);
• development of a predictive mathematical model of risk, relating effect to exposure.
Such a procedure aims to:
• consider pollution as a complex mixture;
>• take into account the homeostasis of the environment and of the living organisms;
• be realistic (all data obtained in the field; calculations based on actual effects, rather than on hazard).
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Toxicological harm |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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The selection of species and biomarkers should be made according to practical criteria, combining maximum information with affordability. Our previous experiences of atmospheric and soil pollution have allowed us to evaluate the relevancy of the various assays performed. On that basis, we divide toxicological assessment into four blocks (systemic effects, reproduction, genotoxicity and population effects), and several biomarkers are combined to gain information within each one.
The wood mouse, A.sylvaticus, is taken as the mammalian reference species, while amphibians and earthworms are used to obtain complementary information on teratogeny and genotoxicity. The effects at the population level, for the sake of simplicity, are assessed in arthropods. The test battery is summarized in Table I. Each one of the above-mentioned blocks is represented by the sum of the results of the tests performed within the block. In order for all values to be expressed in compatible magnitudes, results for each biomarker are presented as a severity score from 0 to 3 (compared with controls). Subsequently, a final value is obtained by adding the blocks considered, to represent the integrated toxicological harm (ITH) occurring in a given location.
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Genotoxicity |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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The extraordinary boom in genotoxicity assays in the 1970s and 1980s, due to high priority placed by the pharmaceutical industry on finding alternatives to expensive life-term carcinogenesis bioassays, led eventually to a certain feeling of disappointment in the 1990s. This loss of momentum was due in part to the identification of an increasing number of epigenetic carcinogens, to the difficulty of interpretation and extrapolation of certain data and to the awareness that no one single test may be really meaningful by itself. However, a battery of soundly established genotoxicity tests nowadays constitutes an essential part of drug development.
The most creative aspects of genotoxicity assessment have experienced a renaissance in the last decade with their application to environmental toxicology (Schoen, 1998). A broad range of techniques and species (ranging from bacteria to humans) have been proposed as experimental models, as recently reviewed (Godet et al., 1993; Depledge, 1996; Tawn, 1999; Sato and Aoki, 2002).
For our battery we have chosen the Comet test because, in the context of environmental studies:
• we believe clastogenicity to be a biologically relevant end-point;
• its sensibility affords a higher resolution power than other clastogenicity tests (such as the micronucleus test) in situations of low exposure level; and
• its versatility allows for special applications, particularly suited for the study of soil pollution.
Circulating lymphocytes from wood mice provide an adequate mammalian model. In turn, earthworms are extremely efficient prospectors of soil. The test devised by Verschaeve et al. (1993) and Verschaeve and Gilles (1995), based on a Comet assay performed on the coelomocytes obtained (after Eyambe et al., 1991) from worms maintained for several days in contaminated soil, has proved to be very useful in our studies of landfills.
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Exposure |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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The other main factor in risk prediction is exposure, and in environmental toxicology this is often the decisive one. To assess it, taking into account bioavailability, we propose the following:
• In the case of atmospheric pollution, the gaseous fraction, fully available, may be directly taken from immission gases analysis, while the solid fraction can be assessed through metal levels in animals, the values of both fractions being combined.
• In soil contamination studies, metals have proved to be excessively sensitive to factors such as soil pH, vegetation, leaching, etc., giving erratic results. As a more suitable marker, we are presently assessing EROD activity in mouse liver. Probably, a specially adapted model of exposure assessment will eventually be necessary for each type of pollution system (meaning the ensemble formed by pollution focus, dissemination/transport mechanisms and bioavailability modulators).
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Risk |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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As a first approach for risk assessment, we propose the establishment of a regression line for exposure versus ITH in four or five locations with decreasing exposure levels, ranging from the immediate neighbourhood of the pollution focus to controls (such different levels of exposure may be diversely distributed, according to climatic or edaphic factors or to the dissemination mechanisms acting in the zone; in a landfill located in a valley, for example, the maximum exposure is usually found in the area around the leachates pool and the main dissemination line follows the drainage of the valley). We can interpolate new exposure data into that regression line to find the corresponding predicted ITH. Such a prediction may be directly interpreted as a form of risk assessment or, alternatively, these pairs (toxicological harm/exposure) could then be related to a conventional scale of ecotoxicological risk, for example from 1 to 10.
This kind of very simple model accepts the assumption of a linear dose–effect relationship. This should probably be true for low exposure levels. For higher doses of toxicants we may find a saturable model, in which a hyperbolic-rectangular correlation curve may perhaps give a better fit.
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Final remarks |
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TopAbstractIntroductionPrevious workModel featuresToxicological harmGenotoxicityExposureRiskFinal remarksReferences |
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This model constitutes our working methodology at the present time. We do not intend to define a closed conceptual system, but just to try a tool that must be trimmed in practice.
When acquiring more experience we may find that it is still not realistic enough to achieve a holistic evaluation of risk and that each of the blocks into which we have divided the toxicological information should in turn be subdivided, to include several sentinel species representative of the different ecological niches. Or, on the contrary, we may perhaps conclude that things are much more simple and that a single measurement, let’s say of the microbiological status of soil, is eventually sufficient.
Nevertheless, we think that our method represents an effort for realism (pollution considered as a complex mixture with complex effects) and for accuracy (covering different aspects of risk) and that, at the present state of knowledge, it may be helpful in getting a more solid basis for environmental risk management.
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Notes |
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3To whom correspondence should be addressed. Email: mborras{at}pcb.ub.es
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References |
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