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Fish as biomarkers in immunotoxicology
TOXiCOlOGY E L S F V I I R S( [[ N T I [ I(
Toxicology 86 (1994) 213-232
Fish as biomarkers in immunotoxicology P.W. Wester
~a
, A . D . Vethaak b, W.B. van Muiswinkel c
aLaboratory for Pathology, National Institute ~/" Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, Netherlands hMinistry of Transport, Public Works and Water Management, Directorate-General./br Public Works and Water Management, Tidal Waters Division, Eeotoxicology Section, P.O. Box 207, 9750 AE tlaren. Netherlands 'Department of Experimental Animal Morphology and Cell Biology, Agricultural Universit)' Wageningen, P.O. Box 338, 6700 AH Wageningen, Netherlands
(Received 23 July 1993; accepted 17 October 1993)
Abstract
This paper presents an overview on the state of the art in the development and application of biomarkers for immunotoxicology in fish. There are several reasons for developing this field: many fish diseases are related to environmental quality, various environmental pollutants have immunotoxic potential and many fish diseases have an immunological component. As in immunotoxicology in general, this aspect, in fish, has received ample attention in the recent past. Much benefit has been obtained from progress in related fields of science, such as fish immunology and rodent immunotoxicology. Meanwhile there is a broad spectrum of potential biomarkers for immunotoxicology in fish, from which macrophage parameters seem to be most widely used. The application of others, such as lymphoid cell parameters is still limited, probably due to practical problems such as lack of experience with conduct, validation and interpretation. Specific problems include the paucity of background data in the case of epidemiological field studies and the important role of other (non-chemical) stress factors in the immune response, and hence the lack of specificity of potential biomarkers. Key words. Environmental pollution; Macrophages; Toxicology; Immunology; Pathology
1. Introduction Immunotoxicology has become of major interest in the last decade. This was stimulated by increasing knowledge of immunology and the importance * Corresponding author. 0300-483X/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02735-Y
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of the immune response in maintaining the integrity of the organism. To date several mechanisms of immunotoxicity are established, various tests are developed and validated, and a large array of chemicals are known to have immune effects, being either inhibiting or stimulating. Several of these chemicals are common environmental pollutants, such as (organo)metals, halogenated hydrocarbons, heterocyclic compounds, carbamates and organophosphates (Wong et al., 1992). Also the aquatic environment is known to be polluted with these chemicals, either as water soluble, sediment or organically bound. Therefore these chemicals have been the main target compounds in aquatic {fish) immunotoxicity studies (Zeeman and Brindley, 1981). Fish are probably the best studied class of aquatic animals for immunology and toxicology for two reasons; there is concern about the health status of the aquatic ecosystems in relation to pollution; fish will be useful target species when developing biomarkers: they are easy to obtain and there is a extensive body of knowledge and economic interest (aquaculture) that facilitates research resources. This paper will review the currently available tools and possibilities in the study of fish immunotoxicology, with particular reference to its use as a biomarker for environmental pollution. Biomarkers are given increasing attention as tools for the monitoring of various types of exposure or effect. This emphasises the importance of interaction between laboratory research and practical applications. Several applications, characteristics and criteria have been formulated and discussed (Hugget et al., 1992; Mayer et al., 1992; NRC, 1992; Peakall and Shugart, 1993). Some general characteristics important for fish as biomarkers for immunotoxicological stress are: - - a biomarker should be rather sensitive: it should reflect the changes in the underlying causes, and within the order of magnitude relevant for the actual field situation; It must be relevant and informative: - - It must be specific: a cause-and-effect relationship should be established; - - It must be easy, reproducible and validated: - - It should be measured preferably by non-invasive methods. -
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When these prerequisites are met, the monitoring has the potential to result in management decisions (e.g. pollution control). Such influence should be reflected in the biomarkers, in other words, the biomarker should be a potential regulatory tool. By all means, it is clear that for the practical application of biomarkers, despite their widespread popularity, a thorough background knowledge on the validity, variability and relevance is still needed but often lacking. In the present paper special attention will be given to
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the latest aspects of the use of fish as immunotoxicological biomarkers for environmental pollution. It has become evident that mammalian immunotoxicology has developed towards a widely accepted scientific discipline within toxicology. It has reached a certain level of organisation in that its implementation in testing regimens with mice and rats is presently under consideration and, in particular, the tiered approach is endorsed by various investigators (e.g. Vos and van Loveren, 1987; Luster et al., 1988; Schuurman et al., 1991). This implies that at first some overall functional parameters of the whole organism, including the immune system, are studied (screening phase, Tier I), and when applicable, further detailed immune function studies (Tier II) are carried out before any conclusion is drawn as to the immunotoxic effect of the toxicant under study. These detailed tier II studies are particularly aimed at covering the whole range of immune pathways to identify the particular target in the immune response, using test protocols that are currently being validated by interlaboratory testing. Tier II studies include challenge tests and host resistance studies (occasionally called Tier III) to measure the ultimate effect on the cascade of the immune response which is important for risk assessment. In fish toxicology, effect studies are not as established as is the case in (man-oriented) mammalian toxicology. Classical ecotoxicology studies were mainly focussed at quantitative ecological parameters as target instead of 'quality of life'. This means that in fish toxicology more emphasis has been given to parameters for population dynamics (mortality, growth, reproduction), and less to effects on the organism or lower levels of organisation. More recently, when disease problems in wild fish populations were suspected to be related to environmental deterioration, special attention was given to toxicological pathology and other toxicological parameters at the cellular or tissue level.
2. Immunology in fish Fish immunology - - in this context in particular teleosts (bony fish) are referred to - - has undergone obvious progress in the seventies and eighties. This science was stimulated by immunology in general, the growth of aquaculture and the concern for environmental and wildlife quality. The importance of aquaculture provides a economic basis for fundamental and applied research on immunology and infectious diseases. This has resulted in development of vaccination strategies and the discovery of interference of certain drugs, in particular antibiotics, with immunization (Anderson et al., 1984; van der Heijden et al., 1992). The observation of fish epidemics has offered another opportunity for research development. Handbooks have
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been published, including techniques in fish immunology (Stolen et al., 1990, 1992). The immune system of fish appears to be fairly well developed and is in various aspects comparable to the mammalian immune system. Both nonspecific and specific immune pathways can be recognised, the latter including T- and B-cell mediated immunity, both of the cellular and humoral type. In contrast to mammalian immunology where cellular immunity is used for specific T-cell mediated immunity, in fish immunology this term may also be used for non-specific cellular defence. The most striking differences with higher vertebrates are the absence of lymph nodes, tonsils and (avian) bursa, and the presence of a head kidney, an analog of the (non-existing) bone marrow. The head kidney (pronephros), in some species an integrated part of the interrenal haemopoietic tissue, and the thymus are paired organs, while the thymus is intimately connected to the gill cavity (Fig. 1). 3. lmmunotoxicology in fish The coincidence of the phenomena described above, namely the increasing interest in toxic effects at lower levels of biological organisation and development in fish immunology resulted in the development of immunotoxicology in fish. In classical mammalian (immuno)toxicology the basic rule is to start at the beginning, i.e. the Tier I study in which target organs are identified followed by functional assays (Tier II). This approach is important as the immune system can be dramatically (secondarily) affected by other (primary) pathologies which may escape attention when not searched for. Unfortunately, in many studies with fish described in the literature, this systematic approach was not always followed. It is often unclear whether the level of exposure investigated in the laboratory has other major effects such as body weight loss or significant mortality during an extended period, or is within the range of actual exposure in the field. Therefore spurious conclusions as to a specific immunotoxic effect can be drawn. On the other hand, in contrast to mammals in which toxicology starts from the safety aspects of a compound, in fish immunotoxicology the incentive is mainly an observation of a disease problem. At present, the level of sophistication in fish immunotoxicology clearly lies behind that in mammals. Screening and function tests are currently under development in the laboratory, but there is still a long way to go before they can be applied in the field. In mammalian immunotoxicology a variety of effects is considered, not only immunosuppression, but also imnmnostimulation, allergy and autoimmunity (Wong et al., 1992). In fish, the reduction of immune reactivity seems mainly to be the target of the study. This reflects the interest in economically important infectious disease epidemics rather than in the individual's discomfort from an immunopathologic condition.
:ig. 1. A. Histological picture (H&E, x 225) of a control guppy, showing the complex localization of the thymus (arrowheads). Obviously, ,xcision of the whole thymus is hardly possible in such a species, and therefore histopathology is the method of choice to study effect on this mportant immunological organ, This is demonstrated in (B), where severe atrophy has occurred after exposure to tributyltinoxide, a well known immunosuppressant. From Wester and Canton (1987), with permission.
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For field and laboratory studies, a wide range of species is being used (see overview in Zeeman and Brindley, 1981). Apparently the choice depends on the author's experience and the laboratory's historical background. The large array of species studied (in contrast to the limited number of species used in mammalian immunotoxicology) makes this research field rather diffuse and extended and may thus generate limited progress. On the other hand, either a variable or a consistent effect over a variety of species certainly is a valuable observation. Of the species used, however, some seem to be preferred, such as trout, salmon and carp. These animals are more manageable due to their size, which enables the sampling of blood and tissues for laboratory studies. Moreover, homozygous inbred strains of these fish are available (Komen et al., 1991) which will improve standardisation of tests. Smaller species such as guppies and medaka have secured a niche in aquatic toxicology owing to easy husbandry and relatively low cost; moreover, because of their small size, whole animals can be used for histopathological examination (Wester and Canton, 1991). However, their application in immunotoxicology may be limited due to problems in obtaining adequate blood and tissue samples due to their small size. In saltwater studies, bottom dwelling flatfish are more commonly used in field and, to a certain extent, mesocosm and laboratory studies. In Europe flounder (Platichthys flesus) and dab (Limanda limanda) are popular target species since they are susceptible to certain recognisable diseases and are commonly available. 4. Field observations
In the European situation, fish diseases are being monitored in the North Sea on a routine basis. Most of these programmes are carried out under the auspices of the International Council for Exploration of the Sea (ICES). More local studies focus on suspected marine or brackish water such as in the vicinity of industrial areas or after major oil spills. The more common diseases that are discussed in relation to pollution are skin diseases such as lymphocystis, papillomas, fin rot and skin ulcers (Vethaak and ap Rheinallt, 1992; Vethaak, 1993). They are easily identified at the gross level and therefore potentially useful for biomonitoring. Moreover, - - most have a viral or bacterial etiology - - the immunological component is rather obvious. Unfortunately, for these specific conditions a causal relation with pollution has not been established yet, and indeed for many diseases in free living animals, the pathogenesis is not fully understood. A condition more promising as a biomonitor for environmental pollution in flatfish to date is liver neoplasia and precursor lesions (Malins et al., 1988; Vethaak et al., 1992; Vethaak and ap Rheinallt, 1992; Vethaak, 1993). However, the role of the immune system in this disease is less evident, the observation needs kill-
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ing of the animals and subsequent histopathological examination, and the long lag time in the development of this chronic disease implies only longterm studies and management decisions. Unlike laboratory studies where single-chemical-exposure is studied (mainly pesticides, heavy metals and chlorinated hydrocarbons), the effects observed in field studies are generally related to unspecified pollution (e.g., harbour sediment, sewage sludge) or to a major (group of) suspected pollutant(s) (e.g. polycyclic aromatic hydrocarbons, pulp mill effluent). Moreover, these effects are often modified or confounded by numerous factors, in particular for feral fish with a deficient case history, migratory patterns and limited knowledge on biology (Vethaak et al., 1992). Extensive epidemiological surveys are required, with specific parameters that have been validated, for instance, by experiments under (more) controlled conditions (mesocosms or laboratory studies). Changes in disease patterns may be suggestive for immune alterations, but this should be further demonstrated. Since most diseases have a complex etiology, it will be difficult to establish the role of immunotoxic effect as such under field conditions. Circumstantial evidence can be obtained in these instances, although for feral animals a mesocosm or laboratory experiment needs to be carried out to yield a final conclusion (Secombes et al., 1992; Vethaak et al., 1993). In using immunological parameters as biomarkers, it should be emphasised that these parameters can be influenced by a large variety of factors (stressors). This implies that immunological biomarkers can be useful and sensitive, but often nonspecific. This has been demonstrated in laboratory studies which showed, for instance, that various immune parameters were suppressed in endrin-exposed rainbow trout (Bennet and Wolke, 1987), as was the case in cortisol-treated animals while treatment with metapyrone, a steroid suppressive drug, compensated, at least partly, this suppressive effect. Another example, demonstrating a neuroendocrine-immune regulation, showed that several immunological parameters were depressed in subordinate specimens of aggressive fish species (Faisal et al., 1989). Moreover, this suppression could be transferred to normal fish by serum from 'suppressed' animals. Another example of natural variation in immune function of lower animals mediated by neuroendocrine mechanisms (sex- and cortocosteroids) is the sex difference and seasonal variation (Fletcher, 1986~ Zapata et al., 1992, Slater and Schreck, 1993). Also temperature variation plays an important role (Rijkers et al., 1980; Pickering and Pottinger, 1985). Various stressors, including pollution-related factors~ have been reviewed (Snieszko, 1974). The non-specific response to general stressors should be taken into consideration when reviewing literature data claiming immunosuppression. Thus inhibiting effects have been induced by corticosteroids on leukocyte migration (Fletcher, 1986), cellularity of lymphoid organs (Chylmonczyk,
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1982; G h o n e u m et al., 1986), antibody production (Wechsler et al., 1986; T r i p p e t al., 1987) and disease susceptibility (Picketing and Porringer, 1985). In particular, epidemiological field studies often lack specificity and sensitivity to allow conclusions as to immunosuppresive effects~ while stress related to handling and temperature changes may cause major fluctuations. In practice, field studies should be confirmed, wherever possible, under more controlled conditions. On the other hand, when immune suppression, specific or non-specific, occurs under practical circumstances at a level where resistance or immune response is affected, it should be interpreted as a serious signal.
5. Laboratory studies For many diseases the etiological components and their role in the pathogenesis, and consequently the biomarkers to be developed, are poorly understood. Hence laboratory experiments are required for background knowledge on the biology of the candidate biomarker. Once these scientific deficiencies are solved, laboratory studies are needed, since function tests under controlled conditions yield the most reliable and sensitive methods of assessing immunological stress, and often need to accompany field studies. However, findings from laboratory situations are not necessarily valid to evaluate effects for the field. In particular when results from the laboratory are extrapolated to field situations, there are often discrepancies between the levels of exposure. It has been c o m m o n in toxicology to extrapolate in a linear fashion, but the existence of non-linear dose-response curves need to be considered (Davis and Svendsgaard, 1990). Thus it has been demonstrated in various cases, including fish, that at lower levels of exposure contradictory, and even favourable effects ('hormesis') (Stebbing, 1982; Calabrese et al., 1987), could be induced. This might, at least in part, explain equivocal effects reported in literature.
6. Immune parameters in fish and their application in toxicology When considering the candidate immune parameters in fish, these can be subdivided in order of complexity (Zeeman and Brindley, 1981): ~structural' parameters such as cell counts, tissue weight and morphology, and serum proteins; functional response of the immune system (specific and nonspecific) and immune-regulated functions such as susceptibility to disease. Comprehensive lists of possible biomarkers, and their applicability have been discussed (see reviews by Anderson (1990a) and Weeks et al. (1992)). It appears that for fish many assays may be applicable, though it should be mentioned that not all are applied or validated yet in immunotoxicology as will be pointed out below. In relation to this, it will be evident that not all
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criteria for usefulness as biomarkers are met. A limited list of candidate biomarkers for immunotoxicity screening for tier I (screening) and tier lI (functional assays) is given in Table 1, and explained in more detail below.
6.1. Blood cell counts and differential counts White blood cells (reviewed in Ellis, 1977) play a major role in specific and non-specific, humoral and cellular immune response. Therefore it is attractive to use this parameter as a measure for the status of the defence system, in particular in the tier I phase of the study. It is a rather easy test to carry out in a blood sample drawn from a live animal. However, many other environmental factors not related to defence may modify the white blood cell status, and therefore its indicative function is limited (Anderson, 1990a). It can be expected that the use of monoclonal antibodies will improve the differential leukocyte counts in the future (Bly et al., 1990; van Diepen et al., 1991). Another parameter proposed is haematocrit; this seems, however, rather optimistic since it has no known specific indicative function for any immune function, but may be considered as a general stress indicator.
6.2. Non-specific defence In various papers other parameters for non-specific defence are studied and proposed as indicators for immunological stress. Examples are acute phase proteins (Fletcher, 1986), the levels of which appeared to be stress hormone dependent; lysozyme and ceruloplasmin activity, reduced in carp after in vivo trichlorphon exposure (Siwicki et al., 1990).
6.3. Spleen weight~morphology The spleen is easy to excise and weigh in animals of adequate size, and thus this parameter could well serve as a biomarker. This is, however, not commonly reported in the literature. A reason could be the fact that a major and variable portion of the spleen consists of storage blood or erythropoietic tissue (Fringe and Nilsson, 1985); the lymphoid tissue is poorly developed and mainly associated with melanomacrophage centers (Zapata, 1982; 1983; F/inge and Nilsson, 1985), and after immunization, only a small proportion of the plaque-forming cells is found in the spleen in contrast to the (head) kidney (Muiswinkel et al., 1991 ). Therefore the role of the spleen in immunity in most fish species seems to be limited, although melanomacrophage centers are abundant.
6.4. Thymus weight~morphology Experimental immunotoxicology in mammals has demonstrated the weight of the thymus, a primary and exclusive immunological organ, to be a sensitive parameter in the case of thymus effects. However, in fish this
Agglutination, ELISA: Allograft rejection (scale/skin, eyes): Phagocytosis, bacterial killing, migration, chemiluminescence; Bacterial infections:
Humoral immune response: Cellular immune response: Macrophage functions:
Host resistance:
Tier H: Functional assays
Serum Ig-concentrations: Lymphoid organ weights: Histopathology:
Total and differential white blood cell count, surface markers flow cytometry, Macrophage density/morphology: Naive (unstimulated): Mainly spleen, occasion, thymus: Thymus, spleen, kidney;
Conventional haematology:
Tier I: Screening assays:
Table 1 Parameters in fish as candidate biomarkers for immunotoxicity
possible, can be specific, relevant
possible, specific possible, specific limited specificity
easy, nonspecific easy, limited specificity impractible possible, can be specific; relevant.
t,a
4~
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parameter is not commonly used. One reason might be the fact that the thymus has a complex localisation in some species which makes clean dissection nearly impossible. An alternative is thymus morphology, studied by histopathological methods or morphometry (Ghoneum et al., 1986; Wester and Canton, 1987). The latter paper describes toxicity studies with tributyltinoxide (TBTO) in guppies where a dose-dependent atrophy of the thymus was demonstrated as was also the case in rats (Krajnc et al., 1984), resulting in immune suppression (Vos et al., 1984). Interestingly, there was a concomitant increase in 'neutrophils' (in guppies and rats) which suggests a functional compensation. Surprisingly, this could not be reproduced in medaka (Wester et al., 1990), and also experiments in sticklebacks and flounder were unsuccessful in this respect (unpublished results), which illustrate the possibility of species-specificity. Another approach towards the thymus is the in vitro testing of thymic lymphocyte functions as described below. 6.5. Melanornacrophage centers Melanomacrophage centers (MMC), or alternatively also termed macrophage aggregates, are found widely distributed throughout the fish body, in particular in spleen, liver and kidney. They are composed of clusters of swollen rounded cells (macrophages) that stain pale tan to black. As a matter of course, this parameter requires a histopathological approach. Numerous papers have been published describing their occurrence and morphology (reviewed in Agius, 1985; Wolke, 1992), but their function is not yet fully understood. The main functions are considered as follows (Wolke, 1992): the presence of pigments (haemosiderin, lipofuscin and ceroid) indicate a storage of effete biological material (erythrocytes, biomembranes). The presence of melanin can be supposed to act as a generator for the bactericidal hydrogen peroxide (Roberts, 1975), and the presence of antigens indicate a role in immune reactions (e.g. Ag-presentation). An increase can be found with age and after stress (Blazer et al., 1987). In our material from field studies this could be confirmed (Vethaak and Wester, 1993). Moreover, a peculiar finding in that study was the high number of relatively small and pale MMCs in animals caught in late winter, the period with more stressful conditions, such as spawning with associated migration and starvation (Vethaak and Wester, 1993). In our view, this small size and pale appearance may be an indicator for recent development. This is supported by the observation that in liver tumours, composed of relatively young and fast growing tissue the MMCs are usually absent or definitely smaller (Fig. 2). As a consequence, when MMCs are being used as (general) stress parameters, age matching of the study groups is a prerequisite. Because of their characteristic nature for fish and the multiplicity of functions, these structures deserve special attention in the context of potential
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Fig. 2. Liver of a flounder bearing a tumour (hepatocellular adenoma, indicated by arrowheads). Staining is by PAS, exhibiting brightly stained melanomacrophage centers, due to their ceroid/lipofuchsin content. Within the turnout, apparently of younger age, the MMCs are nearly absent and at least smaller and more pale-staining in H&E. PAS, x 135.
biomarkers for immunotoxicity. In addition, they are easy to monitor since they do not require specific preparation techniques other than routine histological procedures, including morphometry. Since MMCs may be considered as primitive analogs of the mammalian lymph follicle (Payne and Fancey, 1989), it has been suggested that these centers are an indicator of immune capacity or function, although their role in this context has not yet been established. This indicates that the implications of a change in this parameter for the integrity of the defence systems remains unclear. In various studies the density of MMCs in liver or spleen has been successfully correlated with environmental pollution. Some of these studies showed a decrease in MMCs after exposure to contaminated sediment (Payne and Fancey, 1989) or along a pollution gradient in the North Sea (Bucke et al., 1992), which can be interpreted as an indicator for immune suppression. By contrast other studies have reported an increase in M M C density after contact with chemical contaminants (Blazer et al., 1975, Secombes et al., 1991), which may indicate accumulation of cytotoxic waste (or immune stimulation).
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Fig. 3. Skin grafts in carp at 5 days after grafting (19°C). The autograft (left) is accepted (normal melanophores) while the allograft (right) is rejected (haemorrhagic tissue).
100000
~Ag ~Ag
dose 10 / z g / m l dose 100 /zg/ml
10000 Z
0
1000
100
10
0
5
I/ 30
exposure time (min.) Fig. 4. Numbers of sptenic plaque forming cells (PFC) from rainbow trout given no treatment (0 min), 5 or 10 min. of 10 ppm phenol before immunisation with Yersinia ruckeri O-antigen by bath. The fish were sampled for PFC at 14 days alter immunisation (12°C). The phenolexposed animals show a significant reduction in the humoral immune response. Error bars represent S.E.M. (n---5). From (Anderson et al., 1990), with permission.
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6.6. Macrophage Junction tests Macrophages constitute an important cell population for both specific (antigen processing and presentation) and non-specific (phagocytosis and destruction) defence. They are considered to provide a relatively primitive defence mechanism and are therefore of major importance in lower animals (Ratcliffe and Rowly, 1981). Much effort has been given to macrophage parameters as biomarker for immune effects in fish; a possible reason for this preference is the fact that these cells are fairly easy to obtain, e,g. by peritoneal washing or removal of head kidney, and many function tests do not require sophisticated techniques or species-specific reagents or markers. Several tests with these cells in various estuarine species and their applicability as bioindicators has been discussed (Mathews el al., 1990). Such tests are chemotaxis, phagocytosis, pinocytosis and chemiluminescence. The applicability of trout peritoneal macrophages in immunotoxicology has been studied stressing the need for systematic baseline information. In addition to the tests mentioned before, the morphology and spreading of resident and stimulated peritoneal macrophages have been studied. The conclusion was that these cells share many morphological and functional properties with their mammalian counterparts, and that this cell may be a useful indicator in immunotoxicology (Zelikoff et al., 1991). Several case studies in various fish species have been published, demonstrating the sensitivity of one or more of the above mentioned parameters for chemical stress in vivo (contaminated waters, including polycyclic aromatic hydrocarbons (Weeks and Warinner, 1986; Weeks et al., 1988)) and in vitro (Pentachlorophenol (Anderson and Brubacker, 1993)). At present macrophage function tests and melanomacrophage centers seem to be most widely used as a promising parameter for effects of environmental stress (Blazer et al., 1987). It will be clear, however, that its relation to other components of the immune system needs to be clarified using positive immunotoxicants. 6.7. Humoral immune response Determination of circulating immunoglobulin levels in serum is a useful function test of the net result of an immunological pathway in vivo. It can be measured in 'naive' animals (total lg) or after exposure to a specified antigen, such as to verify vaccination efficacy in veterinary fish immunology (aquaculture). As a standard antigen, sheep red blood cells can be used, the immune response to which can be measured by agglutination tests. Also ELISA tests are applied, which can be considered a sensitive and specific assay (Arkoosh and Kaattari, 1990), A related test is the haemolytic plaque assay which identifies antibody producing cells (splenic lymphocytes) (Anderson, 1990b: Anderson et al., 1990). To date this test has been applied only to a limited extent in fish immunotoxicology.
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6.8. Specific lymphocyte stimulation tests Functional tests are widely used parameters in mammalian immuno(toxico)logy, in which lymphocytes are stimulated in vitro by exposure to mitogens such as LPS, PHA and Concanavalin A. Proliferation is monitored by measuring the incorporation of radioactive thymidine in DNA. The test is not antigen-specific, but provides information on the capacity of the whole B (LPS) or T cell population (PHA, ConA). This test is already used in a limited scale in fish immunotoxicity, and suppression for this parameter under field conditions in Spot (Leiostomus xanthurus) could be elegantly demonstrated (Faisal and Hugget, 1993), a result which was further confirmed to be site- and pollution-related after controlled laboratory experiments.
6.9. Speciji'c cellular immune response Tests described in the literature to measure the cellular immune response are the scale/skin allograft rejection, a relatively simple test (Zeeman and Brindley, 1981 ), or eye allograft rejection (Khangarot and Tripathi, 1991 ), by whom a delayed rejection in carp after copper-exposure was demonstrated. These tests are yet applied to a limited extent. The tests described above are mainly classified as 'Tier 1I' tests. The most integrated test in immunotoxicology, however, is the host resistance test (challenge by infections or tumours). Such tests are hardly reported in the literature and as a matter of course, hardly validated. For this ultimate proof of immunotoxicity, all phases (maintaining, exposure and infection) need to be conducted under strictly controlled laboratory conditions. When suitable (often species-specific) pathogens are standardised, such a test is a valuable and necessary tool in estimating the practical consequences of suspected immunotoxicity. Although the incentive for immunotoxicological studies in fish is usually an epidemiological observation with a suspected toxic component, there remains a need for ultimate challenge experiments before a final conclusion, as to the immunotoxic mechanism, can be drawn. 7. Conclusions
As in many possible applications ofbiomarkers, also in fish, more emphasis has been given to the development rather than the application in the field. This has several causes, such as the lack of specificity and the lack of linkage between effect at the level of the biomarker and the population effect (Mayer et al., 1992). Some other gaps and needs in this field tire as follows: - - There is a great potential of immunological biomarkers in fish. Many of them have not yet been fully explored, probably due to practical limitations or lack of specificity/predictability. - - It can be advantageous to limit the number of animal species in order to
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concentrate the research which requires often species-specific knowledge and reagents. Standardisation could be achieved choosing well-defined inbred strains of fish (e.g. carp or trout). A tiered approach is highly recommended in order to contribute to the knowledge on and the specificity of the biomarker. More knowledge is desired on epidemiology and mechanism/etiology of disease, in particular the predictive value of immune parameters and the influence of hormesis. In terms of relevance for the organism, a test that monitors the net result from a cascade of reactions (e.g. specific antibody production; host resistance) is more predictive than a single non-specific cell parameter (e.g. in vitro macrophage activity). In identifying potential biomarkers for immunotoxicity there should be evidence that the levels tested in the laboratory are relevant for field conditions and that the effect is directly related to the immune system. Acknowledgement
The authors wish to thank Dr. J.G. Vos for critical comments on the manuscript. 8.
References
Agius, C. (1985) The melanomacrophage centers of fish; a review. In: M.J. Manning and M.F. Tatner (Eds), Fish Immunology, Academic press, London, p. 85, Anderson, D.P. (1990a) Immunological indicators: effects of environmental stress on immune protection and disease outbreaks. Am. Fisheries Soc. Syrup. 8, 38. Anderson, D.P. (1990b) Passive hemolytic plaque assay for detecting antibody producing cells in fish. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Robertson and W.B. van Muiswinkel (Eds), Techniques in Fish Immunology I, SOS Publications, Fair Haven, N J, p. 9. Anderson, D.P., Dixon, O.W. and van Muiswinkel, W.B. (1990) Reduction of the numbers of antibody-producing cells in rainbow trout, Onkorhynchus mykiss, exposed to sublethal doses of phenol before bath immunization. In: W.G. Landis and W.H. van der Schalie (Eds), Aquatic Toxicology and Risk Assessment, 13th Vol., ASTM STP 1096. Am. Soc. Testing and Materials, Philadelphia, p. 331. Anderson, D.P., Van Muiswinkel, W.B, and Robertson, B.S. (1984) Effects of chemically induced immune modulation on infectious diseases of fish. In: Chemical Regulation of Immunity in Veterinary Medicine, Alan R. Liss, New York, p. 187. Anderson, R.S. and Brubacher, L.L. (1993) Inhibition by pentachlorophenol of production of reactive-oxygen intermediates by medaka phagocytic blood cells. Mar. Environ. Res. 35, 125. Arkoosh, M.R. and Kaanari, S.L. (1990) Quantitation of fish antibody to a specific antigen by an enzyme-linked immunosorbent assay (ELISA). In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S, Robertson and W.B. van Muiswinkel (Eds), Techniques in Fish Immunology I, SOS Publications, Fair Haven, N J, p. 15.
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Mathews, E.S., Warinner, J.E. and Weeks, B.A. (1990) Assays of immune function in fish macrophages. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Robertson and W.B. van Muiswinkel (Eds), Techniques in Fish Immunology I, SOS Publications, Fair Haven, N J, p. 155. Mayer, F.L., Versteeg, D.J., McKee, M.J., Folmar, L.C., Graney, R.L., McCume, D.C. and Rattner, B.A. (1992) Physiological and Nonspecific Biomarkers. In: R.J. Hugger et al. (Eds), Biomarkers, Biochemical, Physiological and Histological Markers of Anthropogenic Stress, SETAC Publication, Lewis Publishers, London, p. 5. National Research Council Biologic Markers in lmmunotoxicology, (1992) NRC, National Academy Press, Washington DC, 206 pp. Payne, J.F. and Fancey, L.F. (1989) Effect of polycyclic aromatic hydrocarbons on immune responses in fish: Change in melanomacrophage centers in flounder (Pseudopleuronectes americanus) exposed to hydrocarbon-contaminated sediments. Mar. Environ. Res. 28, 431. Peakall, D.B. and Shugart, L.R. (Eds) (1993) Biomarkers: Research and Application in the Assessment of Environmental Health, NATO - - ASI series, Springer Verlag, Berlin, 119 PP. Picketing, A.D. and Pottinger, T.G. (1985) Cortisol can increase the susceptibility of brown trout, Salmo trutta L., to disease without reducing the white blood cell count. J. Fish Biol. 27, 611. Ratcliffe, N.A. and Rowly, A.F. (1981) Invertebrate Blood Cells, Academic Press, London. Rijkers, G.T., Frederix-Wolters, E.M.H. and van Muiswinkel, W.B. (1980) The immune system of cyprinid fish. Kinetics and temperature dependence of antibody-producing cells in carp (Cyprinus carpio). Immunology 41, 291. Roberts, R.J. (1975) Melanin-containing cells of the teleost fish and their relation to disease. In: W.E. Ribelin and G. Migaki (Eds), The Pathology of Fishes, Univ. of Wisconsin Press, Madison, p. 399. Schuurman, H.-J., Krajnc-Franken, M.A.M., Kuper, C.F., van Loveren H. and Vos, J.G. (1991) "Immune System" In: Handbook of Toxicologic Pathology, Academic Press, p. 421. Secombes, C.J., Fletcher, T.C., O'Flynn, J.A., Costello, M.J., Stagg, R. and Houlihan, D.F. (1991) Immunocompetence as a measure of the biological effects of sewage sludge pollution in fish. Comp. Biochem. Physiol. C: Comp. Pharmacol. Toxicol. 100, 133. Secombes, C.J., Fletcher, T.C., White, A., Costello, M.T., Stagg, R. and Houlihan, D.F. (1992) Effects of sewage sludge on immune response in the dab, Limonda limanda. Aquatic Toxicol. 23, 217. Siwicki, A.K., Cossarini-Dunier, M., Studnicka, M. and Demael, A, (1990) In vivo effect of the organophosphorus insecticide trichlorphon on immune response of carp (Cvprinus carpio). Ecotox. Environ. Safety 19, 99. Slater, C.H. and Schreck, C.B. (1993) Testosterone alters the immune response of chinook salmon, Oncorhynchus tshwytscha. Gen. Com. Endocrinol. 89, 291. Snieszko, S.F. (1974) The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6, 197, Stebbing, A.R.D. (1982) Hormesis - - T h e stimulation of growth by low levels of inhibitors. Sci. Total Environ. 2, 213 Stolen, J.S., Fletcher, T.C., Anderson, D.P., Kaattari, S.L. and Rowley, A.F. (Eds) (1992) Techniques in Fish immunology II, SOS Publications, Fair Haven, NJ. Stolen, J.S., Fletcher, T.C., Anderson, D.P., Robertson, B.S. and van Muiswinkel, W.B. (Eds) (1990) Techniques in Fish Immunology 1, SOS Publications, Fair Haven, NJ. Tripp, R.A., Maule, A.G., Schreck, C.B. and Kaattari, S.L. (1987) Cortisol mediated suppression of salmonid lymphocyte responses in vitro. Dev. Comp. Immunol. I1, 565.
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van der Heijden, M.H,T., van Muiswinkel, W.B., Grondel J.L. and Boon, J.H. (1992) lmmunomodulating effects of antibiotics. In: C. Michel and D.J. Alderman, (Eds), Chemotherapy in aquaculture: from theory to reality. Office Int. des Epizooties, Paris, p. 219. van Diepen, J.C.E., Wagenaar, J.T.M. and Rombout, J.H.W.M. (1991) Immunocytochemical detection of membrane antigens of carp leukocytes using light and electronmicroscopical microscopy. Fish Shellfish Immunol. 1, 47. van Muiswinkel, W.B., Lamers, C.H.J. and Rombout, J.H.W.M. (1991) Structural and functional aspects of the spleen in bony fish. Res. lmmunol. 142, 362. Vethaak, A.D. (1993) Diseases of flounder (PlatichtthysJlesus) in Dutch coastal waters, with particular reference to environmental stress factors. In: Fish disease and marine pollution, Acad-thesis, University of Amsterdam, pp. 36-59. Vethaak, A.D. and ap Rheinallt, T. (1992) Fish disease as a monitor for marine pollution: The case of the North Sea. Rev. Fish Biol. Fisheries 2, 1-32. Vethaak, A.D. and Wester, P.W, (1993) Diseases of flounder (Platichtthysfiesus) in Dutch coastal waters, with particular reference to environmental stress factors. Part II. Liver histopathology. In: Fish Disease and Marine Pollution, Acad-thesis, University of Amsterdam, p. 60. Vethaak, A.D,, Bucke, D., Lang, T., Wester, P.W., Jol, J. and Carr, M. (1992) Spatial trends of gross disorders and hepatic lesions in dab (Limanda limanda L.) sampled along a pollution gradient in the German Bight. Mar. Ecol. Prog. Series 91, 173. Vethaak, A.D., Jol, J., Wester, P.W., Zande, T., Bergman, A., Eggens, M.L., Ariese, F., Meijboom, A., Dankers, N., ap Rheinalt, T., Marquenie, J.M., Everts, J.M. and Opperhuizen, A. (1993) A large scale mesocosm study of the effects of marine pollution on disease development in flounder (Platichtthysflesus) In: Fish Disease and Marine Pollution. Acadthesis, University of Amsterdam, pp. 145-168. Vos, J.G. and van Loveren, H. (1987) Immunotoxicity testing in the rat. In: E.J. Burger, R.G. Tardiff and J.A. Bellanti (Eds), Environmental Chemical Exposures and Immune System Integrity. Advances in Modern Environmental Toxicology, Series Vol. XII1, Princeton Science Publishing Co, Inc., N J, p. 167, Vos, J.G., de Klerk, A., Krajnc, E.I., Kruizinga, W., van Ommen B, and Rozing, J. (1984) Toxicity of bis(tri-n-butyltin)oxide in the rat. II. Suppression of thymus-dependent immune responses and of parameters of nonspecific resistance after short-term exposure. Toxicol. Appl. Pharmacol. 75, 87. Wechsler, S.J., McAllister, P.E., Hetrick, F.M. and Anderson, D.P. (1986) Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus. Vet. lmmunol. Immunopathol. 12, 305. Weeks, B.A. and Warinner, J.E. (1986) Functional evaluation of macrophages in fish from a polluted estuary. Vet. Immunol. Immunopathol. 12, 313. Weeks, B.A., Anderson, D.P., DuFour, A.P., Fairbrother, A., Goven, A.J., Lahvis, G.P. and Peters, G. (1992) Immunological biomarkers to assess environmental stress. In: R.J. Hugget et al. (Eds), Biomarkers, Biochemical, Physiological and Histological Markers of Anthropogenic Stress, SETAC Publication, Lewis Publishers, London, p. 5. Weeks, B.A., Warinner, J.E. and Mathews, E.S. (1988) Influence of toxicants on phagocytosis, pinocytosis and melanin accumulation by fish macrophages. Aquat. Toxicol. 11,424. Wester, P.W. and Canton, J.H. (1987) Histopathological study of Poecilia reticulata (guppy) after long-term exposure to bis(tri-n-butyltin)oxide (TBTO) and di-n-butyl-tinchloride (DBTC). Aquat. Toxicol. 10, 143. Wester, P.W. and Canton, J.H. (1991) The usefulness of histopathology in aquatic toxicity studies. Comp. Biochem. Physiol. 100C, 115.
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Toxicology 86 (1994) 213-232
Fish as biomarkers in immunotoxicology P.W. Wester
~a
, A . D . Vethaak b, W.B. van Muiswinkel c
aLaboratory for Pathology, National Institute ~/" Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, Netherlands hMinistry of Transport, Public Works and Water Management, Directorate-General./br Public Works and Water Management, Tidal Waters Division, Eeotoxicology Section, P.O. Box 207, 9750 AE tlaren. Netherlands 'Department of Experimental Animal Morphology and Cell Biology, Agricultural Universit)' Wageningen, P.O. Box 338, 6700 AH Wageningen, Netherlands
(Received 23 July 1993; accepted 17 October 1993)
Abstract
This paper presents an overview on the state of the art in the development and application of biomarkers for immunotoxicology in fish. There are several reasons for developing this field: many fish diseases are related to environmental quality, various environmental pollutants have immunotoxic potential and many fish diseases have an immunological component. As in immunotoxicology in general, this aspect, in fish, has received ample attention in the recent past. Much benefit has been obtained from progress in related fields of science, such as fish immunology and rodent immunotoxicology. Meanwhile there is a broad spectrum of potential biomarkers for immunotoxicology in fish, from which macrophage parameters seem to be most widely used. The application of others, such as lymphoid cell parameters is still limited, probably due to practical problems such as lack of experience with conduct, validation and interpretation. Specific problems include the paucity of background data in the case of epidemiological field studies and the important role of other (non-chemical) stress factors in the immune response, and hence the lack of specificity of potential biomarkers. Key words. Environmental pollution; Macrophages; Toxicology; Immunology; Pathology
1. Introduction Immunotoxicology has become of major interest in the last decade. This was stimulated by increasing knowledge of immunology and the importance * Corresponding author. 0300-483X/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02735-Y
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of the immune response in maintaining the integrity of the organism. To date several mechanisms of immunotoxicity are established, various tests are developed and validated, and a large array of chemicals are known to have immune effects, being either inhibiting or stimulating. Several of these chemicals are common environmental pollutants, such as (organo)metals, halogenated hydrocarbons, heterocyclic compounds, carbamates and organophosphates (Wong et al., 1992). Also the aquatic environment is known to be polluted with these chemicals, either as water soluble, sediment or organically bound. Therefore these chemicals have been the main target compounds in aquatic {fish) immunotoxicity studies (Zeeman and Brindley, 1981). Fish are probably the best studied class of aquatic animals for immunology and toxicology for two reasons; there is concern about the health status of the aquatic ecosystems in relation to pollution; fish will be useful target species when developing biomarkers: they are easy to obtain and there is a extensive body of knowledge and economic interest (aquaculture) that facilitates research resources. This paper will review the currently available tools and possibilities in the study of fish immunotoxicology, with particular reference to its use as a biomarker for environmental pollution. Biomarkers are given increasing attention as tools for the monitoring of various types of exposure or effect. This emphasises the importance of interaction between laboratory research and practical applications. Several applications, characteristics and criteria have been formulated and discussed (Hugget et al., 1992; Mayer et al., 1992; NRC, 1992; Peakall and Shugart, 1993). Some general characteristics important for fish as biomarkers for immunotoxicological stress are: - - a biomarker should be rather sensitive: it should reflect the changes in the underlying causes, and within the order of magnitude relevant for the actual field situation; It must be relevant and informative: - - It must be specific: a cause-and-effect relationship should be established; - - It must be easy, reproducible and validated: - - It should be measured preferably by non-invasive methods. -
-
When these prerequisites are met, the monitoring has the potential to result in management decisions (e.g. pollution control). Such influence should be reflected in the biomarkers, in other words, the biomarker should be a potential regulatory tool. By all means, it is clear that for the practical application of biomarkers, despite their widespread popularity, a thorough background knowledge on the validity, variability and relevance is still needed but often lacking. In the present paper special attention will be given to
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the latest aspects of the use of fish as immunotoxicological biomarkers for environmental pollution. It has become evident that mammalian immunotoxicology has developed towards a widely accepted scientific discipline within toxicology. It has reached a certain level of organisation in that its implementation in testing regimens with mice and rats is presently under consideration and, in particular, the tiered approach is endorsed by various investigators (e.g. Vos and van Loveren, 1987; Luster et al., 1988; Schuurman et al., 1991). This implies that at first some overall functional parameters of the whole organism, including the immune system, are studied (screening phase, Tier I), and when applicable, further detailed immune function studies (Tier II) are carried out before any conclusion is drawn as to the immunotoxic effect of the toxicant under study. These detailed tier II studies are particularly aimed at covering the whole range of immune pathways to identify the particular target in the immune response, using test protocols that are currently being validated by interlaboratory testing. Tier II studies include challenge tests and host resistance studies (occasionally called Tier III) to measure the ultimate effect on the cascade of the immune response which is important for risk assessment. In fish toxicology, effect studies are not as established as is the case in (man-oriented) mammalian toxicology. Classical ecotoxicology studies were mainly focussed at quantitative ecological parameters as target instead of 'quality of life'. This means that in fish toxicology more emphasis has been given to parameters for population dynamics (mortality, growth, reproduction), and less to effects on the organism or lower levels of organisation. More recently, when disease problems in wild fish populations were suspected to be related to environmental deterioration, special attention was given to toxicological pathology and other toxicological parameters at the cellular or tissue level.
2. Immunology in fish Fish immunology - - in this context in particular teleosts (bony fish) are referred to - - has undergone obvious progress in the seventies and eighties. This science was stimulated by immunology in general, the growth of aquaculture and the concern for environmental and wildlife quality. The importance of aquaculture provides a economic basis for fundamental and applied research on immunology and infectious diseases. This has resulted in development of vaccination strategies and the discovery of interference of certain drugs, in particular antibiotics, with immunization (Anderson et al., 1984; van der Heijden et al., 1992). The observation of fish epidemics has offered another opportunity for research development. Handbooks have
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been published, including techniques in fish immunology (Stolen et al., 1990, 1992). The immune system of fish appears to be fairly well developed and is in various aspects comparable to the mammalian immune system. Both nonspecific and specific immune pathways can be recognised, the latter including T- and B-cell mediated immunity, both of the cellular and humoral type. In contrast to mammalian immunology where cellular immunity is used for specific T-cell mediated immunity, in fish immunology this term may also be used for non-specific cellular defence. The most striking differences with higher vertebrates are the absence of lymph nodes, tonsils and (avian) bursa, and the presence of a head kidney, an analog of the (non-existing) bone marrow. The head kidney (pronephros), in some species an integrated part of the interrenal haemopoietic tissue, and the thymus are paired organs, while the thymus is intimately connected to the gill cavity (Fig. 1). 3. lmmunotoxicology in fish The coincidence of the phenomena described above, namely the increasing interest in toxic effects at lower levels of biological organisation and development in fish immunology resulted in the development of immunotoxicology in fish. In classical mammalian (immuno)toxicology the basic rule is to start at the beginning, i.e. the Tier I study in which target organs are identified followed by functional assays (Tier II). This approach is important as the immune system can be dramatically (secondarily) affected by other (primary) pathologies which may escape attention when not searched for. Unfortunately, in many studies with fish described in the literature, this systematic approach was not always followed. It is often unclear whether the level of exposure investigated in the laboratory has other major effects such as body weight loss or significant mortality during an extended period, or is within the range of actual exposure in the field. Therefore spurious conclusions as to a specific immunotoxic effect can be drawn. On the other hand, in contrast to mammals in which toxicology starts from the safety aspects of a compound, in fish immunotoxicology the incentive is mainly an observation of a disease problem. At present, the level of sophistication in fish immunotoxicology clearly lies behind that in mammals. Screening and function tests are currently under development in the laboratory, but there is still a long way to go before they can be applied in the field. In mammalian immunotoxicology a variety of effects is considered, not only immunosuppression, but also imnmnostimulation, allergy and autoimmunity (Wong et al., 1992). In fish, the reduction of immune reactivity seems mainly to be the target of the study. This reflects the interest in economically important infectious disease epidemics rather than in the individual's discomfort from an immunopathologic condition.
:ig. 1. A. Histological picture (H&E, x 225) of a control guppy, showing the complex localization of the thymus (arrowheads). Obviously, ,xcision of the whole thymus is hardly possible in such a species, and therefore histopathology is the method of choice to study effect on this mportant immunological organ, This is demonstrated in (B), where severe atrophy has occurred after exposure to tributyltinoxide, a well known immunosuppressant. From Wester and Canton (1987), with permission.
,,a .,o -.a
I
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2.
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For field and laboratory studies, a wide range of species is being used (see overview in Zeeman and Brindley, 1981). Apparently the choice depends on the author's experience and the laboratory's historical background. The large array of species studied (in contrast to the limited number of species used in mammalian immunotoxicology) makes this research field rather diffuse and extended and may thus generate limited progress. On the other hand, either a variable or a consistent effect over a variety of species certainly is a valuable observation. Of the species used, however, some seem to be preferred, such as trout, salmon and carp. These animals are more manageable due to their size, which enables the sampling of blood and tissues for laboratory studies. Moreover, homozygous inbred strains of these fish are available (Komen et al., 1991) which will improve standardisation of tests. Smaller species such as guppies and medaka have secured a niche in aquatic toxicology owing to easy husbandry and relatively low cost; moreover, because of their small size, whole animals can be used for histopathological examination (Wester and Canton, 1991). However, their application in immunotoxicology may be limited due to problems in obtaining adequate blood and tissue samples due to their small size. In saltwater studies, bottom dwelling flatfish are more commonly used in field and, to a certain extent, mesocosm and laboratory studies. In Europe flounder (Platichthys flesus) and dab (Limanda limanda) are popular target species since they are susceptible to certain recognisable diseases and are commonly available. 4. Field observations
In the European situation, fish diseases are being monitored in the North Sea on a routine basis. Most of these programmes are carried out under the auspices of the International Council for Exploration of the Sea (ICES). More local studies focus on suspected marine or brackish water such as in the vicinity of industrial areas or after major oil spills. The more common diseases that are discussed in relation to pollution are skin diseases such as lymphocystis, papillomas, fin rot and skin ulcers (Vethaak and ap Rheinallt, 1992; Vethaak, 1993). They are easily identified at the gross level and therefore potentially useful for biomonitoring. Moreover, - - most have a viral or bacterial etiology - - the immunological component is rather obvious. Unfortunately, for these specific conditions a causal relation with pollution has not been established yet, and indeed for many diseases in free living animals, the pathogenesis is not fully understood. A condition more promising as a biomonitor for environmental pollution in flatfish to date is liver neoplasia and precursor lesions (Malins et al., 1988; Vethaak et al., 1992; Vethaak and ap Rheinallt, 1992; Vethaak, 1993). However, the role of the immune system in this disease is less evident, the observation needs kill-
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ing of the animals and subsequent histopathological examination, and the long lag time in the development of this chronic disease implies only longterm studies and management decisions. Unlike laboratory studies where single-chemical-exposure is studied (mainly pesticides, heavy metals and chlorinated hydrocarbons), the effects observed in field studies are generally related to unspecified pollution (e.g., harbour sediment, sewage sludge) or to a major (group of) suspected pollutant(s) (e.g. polycyclic aromatic hydrocarbons, pulp mill effluent). Moreover, these effects are often modified or confounded by numerous factors, in particular for feral fish with a deficient case history, migratory patterns and limited knowledge on biology (Vethaak et al., 1992). Extensive epidemiological surveys are required, with specific parameters that have been validated, for instance, by experiments under (more) controlled conditions (mesocosms or laboratory studies). Changes in disease patterns may be suggestive for immune alterations, but this should be further demonstrated. Since most diseases have a complex etiology, it will be difficult to establish the role of immunotoxic effect as such under field conditions. Circumstantial evidence can be obtained in these instances, although for feral animals a mesocosm or laboratory experiment needs to be carried out to yield a final conclusion (Secombes et al., 1992; Vethaak et al., 1993). In using immunological parameters as biomarkers, it should be emphasised that these parameters can be influenced by a large variety of factors (stressors). This implies that immunological biomarkers can be useful and sensitive, but often nonspecific. This has been demonstrated in laboratory studies which showed, for instance, that various immune parameters were suppressed in endrin-exposed rainbow trout (Bennet and Wolke, 1987), as was the case in cortisol-treated animals while treatment with metapyrone, a steroid suppressive drug, compensated, at least partly, this suppressive effect. Another example, demonstrating a neuroendocrine-immune regulation, showed that several immunological parameters were depressed in subordinate specimens of aggressive fish species (Faisal et al., 1989). Moreover, this suppression could be transferred to normal fish by serum from 'suppressed' animals. Another example of natural variation in immune function of lower animals mediated by neuroendocrine mechanisms (sex- and cortocosteroids) is the sex difference and seasonal variation (Fletcher, 1986~ Zapata et al., 1992, Slater and Schreck, 1993). Also temperature variation plays an important role (Rijkers et al., 1980; Pickering and Pottinger, 1985). Various stressors, including pollution-related factors~ have been reviewed (Snieszko, 1974). The non-specific response to general stressors should be taken into consideration when reviewing literature data claiming immunosuppression. Thus inhibiting effects have been induced by corticosteroids on leukocyte migration (Fletcher, 1986), cellularity of lymphoid organs (Chylmonczyk,
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1982; G h o n e u m et al., 1986), antibody production (Wechsler et al., 1986; T r i p p e t al., 1987) and disease susceptibility (Picketing and Porringer, 1985). In particular, epidemiological field studies often lack specificity and sensitivity to allow conclusions as to immunosuppresive effects~ while stress related to handling and temperature changes may cause major fluctuations. In practice, field studies should be confirmed, wherever possible, under more controlled conditions. On the other hand, when immune suppression, specific or non-specific, occurs under practical circumstances at a level where resistance or immune response is affected, it should be interpreted as a serious signal.
5. Laboratory studies For many diseases the etiological components and their role in the pathogenesis, and consequently the biomarkers to be developed, are poorly understood. Hence laboratory experiments are required for background knowledge on the biology of the candidate biomarker. Once these scientific deficiencies are solved, laboratory studies are needed, since function tests under controlled conditions yield the most reliable and sensitive methods of assessing immunological stress, and often need to accompany field studies. However, findings from laboratory situations are not necessarily valid to evaluate effects for the field. In particular when results from the laboratory are extrapolated to field situations, there are often discrepancies between the levels of exposure. It has been c o m m o n in toxicology to extrapolate in a linear fashion, but the existence of non-linear dose-response curves need to be considered (Davis and Svendsgaard, 1990). Thus it has been demonstrated in various cases, including fish, that at lower levels of exposure contradictory, and even favourable effects ('hormesis') (Stebbing, 1982; Calabrese et al., 1987), could be induced. This might, at least in part, explain equivocal effects reported in literature.
6. Immune parameters in fish and their application in toxicology When considering the candidate immune parameters in fish, these can be subdivided in order of complexity (Zeeman and Brindley, 1981): ~structural' parameters such as cell counts, tissue weight and morphology, and serum proteins; functional response of the immune system (specific and nonspecific) and immune-regulated functions such as susceptibility to disease. Comprehensive lists of possible biomarkers, and their applicability have been discussed (see reviews by Anderson (1990a) and Weeks et al. (1992)). It appears that for fish many assays may be applicable, though it should be mentioned that not all are applied or validated yet in immunotoxicology as will be pointed out below. In relation to this, it will be evident that not all
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criteria for usefulness as biomarkers are met. A limited list of candidate biomarkers for immunotoxicity screening for tier I (screening) and tier lI (functional assays) is given in Table 1, and explained in more detail below.
6.1. Blood cell counts and differential counts White blood cells (reviewed in Ellis, 1977) play a major role in specific and non-specific, humoral and cellular immune response. Therefore it is attractive to use this parameter as a measure for the status of the defence system, in particular in the tier I phase of the study. It is a rather easy test to carry out in a blood sample drawn from a live animal. However, many other environmental factors not related to defence may modify the white blood cell status, and therefore its indicative function is limited (Anderson, 1990a). It can be expected that the use of monoclonal antibodies will improve the differential leukocyte counts in the future (Bly et al., 1990; van Diepen et al., 1991). Another parameter proposed is haematocrit; this seems, however, rather optimistic since it has no known specific indicative function for any immune function, but may be considered as a general stress indicator.
6.2. Non-specific defence In various papers other parameters for non-specific defence are studied and proposed as indicators for immunological stress. Examples are acute phase proteins (Fletcher, 1986), the levels of which appeared to be stress hormone dependent; lysozyme and ceruloplasmin activity, reduced in carp after in vivo trichlorphon exposure (Siwicki et al., 1990).
6.3. Spleen weight~morphology The spleen is easy to excise and weigh in animals of adequate size, and thus this parameter could well serve as a biomarker. This is, however, not commonly reported in the literature. A reason could be the fact that a major and variable portion of the spleen consists of storage blood or erythropoietic tissue (Fringe and Nilsson, 1985); the lymphoid tissue is poorly developed and mainly associated with melanomacrophage centers (Zapata, 1982; 1983; F/inge and Nilsson, 1985), and after immunization, only a small proportion of the plaque-forming cells is found in the spleen in contrast to the (head) kidney (Muiswinkel et al., 1991 ). Therefore the role of the spleen in immunity in most fish species seems to be limited, although melanomacrophage centers are abundant.
6.4. Thymus weight~morphology Experimental immunotoxicology in mammals has demonstrated the weight of the thymus, a primary and exclusive immunological organ, to be a sensitive parameter in the case of thymus effects. However, in fish this
Agglutination, ELISA: Allograft rejection (scale/skin, eyes): Phagocytosis, bacterial killing, migration, chemiluminescence; Bacterial infections:
Humoral immune response: Cellular immune response: Macrophage functions:
Host resistance:
Tier H: Functional assays
Serum Ig-concentrations: Lymphoid organ weights: Histopathology:
Total and differential white blood cell count, surface markers flow cytometry, Macrophage density/morphology: Naive (unstimulated): Mainly spleen, occasion, thymus: Thymus, spleen, kidney;
Conventional haematology:
Tier I: Screening assays:
Table 1 Parameters in fish as candidate biomarkers for immunotoxicity
possible, can be specific, relevant
possible, specific possible, specific limited specificity
easy, nonspecific easy, limited specificity impractible possible, can be specific; relevant.
t,a
4~
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parameter is not commonly used. One reason might be the fact that the thymus has a complex localisation in some species which makes clean dissection nearly impossible. An alternative is thymus morphology, studied by histopathological methods or morphometry (Ghoneum et al., 1986; Wester and Canton, 1987). The latter paper describes toxicity studies with tributyltinoxide (TBTO) in guppies where a dose-dependent atrophy of the thymus was demonstrated as was also the case in rats (Krajnc et al., 1984), resulting in immune suppression (Vos et al., 1984). Interestingly, there was a concomitant increase in 'neutrophils' (in guppies and rats) which suggests a functional compensation. Surprisingly, this could not be reproduced in medaka (Wester et al., 1990), and also experiments in sticklebacks and flounder were unsuccessful in this respect (unpublished results), which illustrate the possibility of species-specificity. Another approach towards the thymus is the in vitro testing of thymic lymphocyte functions as described below. 6.5. Melanornacrophage centers Melanomacrophage centers (MMC), or alternatively also termed macrophage aggregates, are found widely distributed throughout the fish body, in particular in spleen, liver and kidney. They are composed of clusters of swollen rounded cells (macrophages) that stain pale tan to black. As a matter of course, this parameter requires a histopathological approach. Numerous papers have been published describing their occurrence and morphology (reviewed in Agius, 1985; Wolke, 1992), but their function is not yet fully understood. The main functions are considered as follows (Wolke, 1992): the presence of pigments (haemosiderin, lipofuscin and ceroid) indicate a storage of effete biological material (erythrocytes, biomembranes). The presence of melanin can be supposed to act as a generator for the bactericidal hydrogen peroxide (Roberts, 1975), and the presence of antigens indicate a role in immune reactions (e.g. Ag-presentation). An increase can be found with age and after stress (Blazer et al., 1987). In our material from field studies this could be confirmed (Vethaak and Wester, 1993). Moreover, a peculiar finding in that study was the high number of relatively small and pale MMCs in animals caught in late winter, the period with more stressful conditions, such as spawning with associated migration and starvation (Vethaak and Wester, 1993). In our view, this small size and pale appearance may be an indicator for recent development. This is supported by the observation that in liver tumours, composed of relatively young and fast growing tissue the MMCs are usually absent or definitely smaller (Fig. 2). As a consequence, when MMCs are being used as (general) stress parameters, age matching of the study groups is a prerequisite. Because of their characteristic nature for fish and the multiplicity of functions, these structures deserve special attention in the context of potential
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Fig. 2. Liver of a flounder bearing a tumour (hepatocellular adenoma, indicated by arrowheads). Staining is by PAS, exhibiting brightly stained melanomacrophage centers, due to their ceroid/lipofuchsin content. Within the turnout, apparently of younger age, the MMCs are nearly absent and at least smaller and more pale-staining in H&E. PAS, x 135.
biomarkers for immunotoxicity. In addition, they are easy to monitor since they do not require specific preparation techniques other than routine histological procedures, including morphometry. Since MMCs may be considered as primitive analogs of the mammalian lymph follicle (Payne and Fancey, 1989), it has been suggested that these centers are an indicator of immune capacity or function, although their role in this context has not yet been established. This indicates that the implications of a change in this parameter for the integrity of the defence systems remains unclear. In various studies the density of MMCs in liver or spleen has been successfully correlated with environmental pollution. Some of these studies showed a decrease in MMCs after exposure to contaminated sediment (Payne and Fancey, 1989) or along a pollution gradient in the North Sea (Bucke et al., 1992), which can be interpreted as an indicator for immune suppression. By contrast other studies have reported an increase in M M C density after contact with chemical contaminants (Blazer et al., 1975, Secombes et al., 1991), which may indicate accumulation of cytotoxic waste (or immune stimulation).
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Toxicology 86 (1994) 213-232
Fig. 3. Skin grafts in carp at 5 days after grafting (19°C). The autograft (left) is accepted (normal melanophores) while the allograft (right) is rejected (haemorrhagic tissue).
100000
~Ag ~Ag
dose 10 / z g / m l dose 100 /zg/ml
10000 Z
0
1000
100
10
0
5
I/ 30
exposure time (min.) Fig. 4. Numbers of sptenic plaque forming cells (PFC) from rainbow trout given no treatment (0 min), 5 or 10 min. of 10 ppm phenol before immunisation with Yersinia ruckeri O-antigen by bath. The fish were sampled for PFC at 14 days alter immunisation (12°C). The phenolexposed animals show a significant reduction in the humoral immune response. Error bars represent S.E.M. (n---5). From (Anderson et al., 1990), with permission.
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6.6. Macrophage Junction tests Macrophages constitute an important cell population for both specific (antigen processing and presentation) and non-specific (phagocytosis and destruction) defence. They are considered to provide a relatively primitive defence mechanism and are therefore of major importance in lower animals (Ratcliffe and Rowly, 1981). Much effort has been given to macrophage parameters as biomarker for immune effects in fish; a possible reason for this preference is the fact that these cells are fairly easy to obtain, e,g. by peritoneal washing or removal of head kidney, and many function tests do not require sophisticated techniques or species-specific reagents or markers. Several tests with these cells in various estuarine species and their applicability as bioindicators has been discussed (Mathews el al., 1990). Such tests are chemotaxis, phagocytosis, pinocytosis and chemiluminescence. The applicability of trout peritoneal macrophages in immunotoxicology has been studied stressing the need for systematic baseline information. In addition to the tests mentioned before, the morphology and spreading of resident and stimulated peritoneal macrophages have been studied. The conclusion was that these cells share many morphological and functional properties with their mammalian counterparts, and that this cell may be a useful indicator in immunotoxicology (Zelikoff et al., 1991). Several case studies in various fish species have been published, demonstrating the sensitivity of one or more of the above mentioned parameters for chemical stress in vivo (contaminated waters, including polycyclic aromatic hydrocarbons (Weeks and Warinner, 1986; Weeks et al., 1988)) and in vitro (Pentachlorophenol (Anderson and Brubacker, 1993)). At present macrophage function tests and melanomacrophage centers seem to be most widely used as a promising parameter for effects of environmental stress (Blazer et al., 1987). It will be clear, however, that its relation to other components of the immune system needs to be clarified using positive immunotoxicants. 6.7. Humoral immune response Determination of circulating immunoglobulin levels in serum is a useful function test of the net result of an immunological pathway in vivo. It can be measured in 'naive' animals (total lg) or after exposure to a specified antigen, such as to verify vaccination efficacy in veterinary fish immunology (aquaculture). As a standard antigen, sheep red blood cells can be used, the immune response to which can be measured by agglutination tests. Also ELISA tests are applied, which can be considered a sensitive and specific assay (Arkoosh and Kaattari, 1990), A related test is the haemolytic plaque assay which identifies antibody producing cells (splenic lymphocytes) (Anderson, 1990b: Anderson et al., 1990). To date this test has been applied only to a limited extent in fish immunotoxicology.
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6.8. Specific lymphocyte stimulation tests Functional tests are widely used parameters in mammalian immuno(toxico)logy, in which lymphocytes are stimulated in vitro by exposure to mitogens such as LPS, PHA and Concanavalin A. Proliferation is monitored by measuring the incorporation of radioactive thymidine in DNA. The test is not antigen-specific, but provides information on the capacity of the whole B (LPS) or T cell population (PHA, ConA). This test is already used in a limited scale in fish immunotoxicity, and suppression for this parameter under field conditions in Spot (Leiostomus xanthurus) could be elegantly demonstrated (Faisal and Hugget, 1993), a result which was further confirmed to be site- and pollution-related after controlled laboratory experiments.
6.9. Speciji'c cellular immune response Tests described in the literature to measure the cellular immune response are the scale/skin allograft rejection, a relatively simple test (Zeeman and Brindley, 1981 ), or eye allograft rejection (Khangarot and Tripathi, 1991 ), by whom a delayed rejection in carp after copper-exposure was demonstrated. These tests are yet applied to a limited extent. The tests described above are mainly classified as 'Tier 1I' tests. The most integrated test in immunotoxicology, however, is the host resistance test (challenge by infections or tumours). Such tests are hardly reported in the literature and as a matter of course, hardly validated. For this ultimate proof of immunotoxicity, all phases (maintaining, exposure and infection) need to be conducted under strictly controlled laboratory conditions. When suitable (often species-specific) pathogens are standardised, such a test is a valuable and necessary tool in estimating the practical consequences of suspected immunotoxicity. Although the incentive for immunotoxicological studies in fish is usually an epidemiological observation with a suspected toxic component, there remains a need for ultimate challenge experiments before a final conclusion, as to the immunotoxic mechanism, can be drawn. 7. Conclusions
As in many possible applications ofbiomarkers, also in fish, more emphasis has been given to the development rather than the application in the field. This has several causes, such as the lack of specificity and the lack of linkage between effect at the level of the biomarker and the population effect (Mayer et al., 1992). Some other gaps and needs in this field tire as follows: - - There is a great potential of immunological biomarkers in fish. Many of them have not yet been fully explored, probably due to practical limitations or lack of specificity/predictability. - - It can be advantageous to limit the number of animal species in order to
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concentrate the research which requires often species-specific knowledge and reagents. Standardisation could be achieved choosing well-defined inbred strains of fish (e.g. carp or trout). A tiered approach is highly recommended in order to contribute to the knowledge on and the specificity of the biomarker. More knowledge is desired on epidemiology and mechanism/etiology of disease, in particular the predictive value of immune parameters and the influence of hormesis. In terms of relevance for the organism, a test that monitors the net result from a cascade of reactions (e.g. specific antibody production; host resistance) is more predictive than a single non-specific cell parameter (e.g. in vitro macrophage activity). In identifying potential biomarkers for immunotoxicity there should be evidence that the levels tested in the laboratory are relevant for field conditions and that the effect is directly related to the immune system. Acknowledgement
The authors wish to thank Dr. J.G. Vos for critical comments on the manuscript. 8.
References
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