CHAPTER 3 GUT PHYSIOLOGY, CHEMISTRY AND NUTRITION

Transcription

1 CHAPTER 3 GUT PHYSIOLOGY, CHEMISTRY AND NUTRITION Peter G.C. Campbell, Susan J. Clearwater, Paul B. Brown, Nicholas S. Fisher, Christer Hogstrand, Glenn R. Lopez, Lawrence M. Mayer, Joseph S. Meyer v

2 CHAPTER 3: TABLE OF CONTENTS 1. Introduction Variability of gut morphology, physiology and function among aquatic vertebrates and invertebrates Influence of digestive processes on the bioavailability of dietborne metals: the gut as a biochemical reactor Dissolution of metals from ingested matrices The matrix The gut fluid The reaction Possible modeling approaches Metal uptake pathways in the intestine Potential effects of dietborne metals on the digestive tract before they are assimilated across the gut wall Digestive enzymes and intestinal microorganisms Mucus secretion Gut motility, hormone secretion and nutrient absorption Inhibition of metal uptake Influence of exposure pathway on the internal distribution of metals and their toxicity Invertebrates Fish Waterborne and dietborne metal exposures: practical considerations Concurrent waterborne and dietborne exposures Diet preparation Nutritional considerations Type of diet... 32

3 Chapter 3 p.ii 7.2 Feed Acceptance Nutritional Adequacy Comparison of Diets Nutritional Deficiency vs. Toxicological Effect Combined approaches Summary, conclusions and recommended future research Summary and Conclusions Recommended future research: References... 48

4 1. Introduction To evaluate the potential toxicity of dietborne metals to aquatic animals, one should consider what happens to ingested metals when they reach the gut environment. In this chapter, the term "gut" refers to the entire gastrointestinal (digestive) tract from the mouth to the anus of an animal. The term "ingested metals" refers to metals that are sorbed to or incorporated into solids (e.g., food or abiotic sediment particles) consumed by an organism. Ingested metals do not include dissolved forms that enter the digestive tract via drinking (e.g., in saltwater fish) or along with ingested solids. Ingested metals constitute a Trojan Horse of potential harm to organisms, the impacts of which can be unlocked by the digestive processes of the gut. Ingested forms are largely harmless in their particulate state, but their release by the powerful solubilizing forces of digestive chemical milieux may allow them to run amok in gut or somatic tissue. Both the chemistry and the biology (i.e., gut structure, function and physiology) of digestion are important. The digestive process normally involves an initial solubilization of particulate food material followed by various hydrolytic processes, all of which might expose the previously bound metal. The extent of this breakdown varies among aquatic animals, as a function of the nature of the ingested particle, the chemical conditions in the gut (e.g., ph, E H ) and the gut passage time. All of these factors will affect the biological availability of the metals that originally entered the gut with the ingested food.

5 Chapter 3 p.2 In this chapter we first consider the gut as a biochemical reactor, then discuss metal speciation in the gut environment, and finally review the mechanisms by which metals can be assimilated. The gut epithelium differs from other epithelial barriers in that it offers a wider variety of uptake mechanisms for metals, which can pass across the gut wall both as dissolved inorganic ions and as dissolved metal complexed with assimilable organic ligands such as histidine or cysteine, and as metal sorbed to or incorporated into particles that are internalized via phagocytosis. These piggyback uptake mechanisms represent an extension of the Trojan Horse metaphor to the absorption step, wherein the animal must, for example, accept a dose of metal in order to assimilate an essential amino acid. In the second part of the chapter we consider how ingested metals can affect the organism. After reviewing the evidence that metals can influence the digestive process, even before assimilation occurs, we turn our attention to assimilated metals and analyze the growing body of data that shows how the internal fate of a metal assimilated from the gut can differ from that of a metal assimilated from water. In some cases, these differences in pharmacokinetics lead to different effects of metals in the consumer organism. In the third section of this chapter, we address some practical concerns regarding the design and conduct of dietborne-metal experiments, notably with respect to the need for concurrent exposure to both waterborne and dietborne metals. Given that some researchers have hypothesized that the mechanism for some adverse effects of metal contamination might involve metal-induced changes in the nutritional quality of the prey organisms rather than direct toxicity from the ingested metal, we also consider how nutritional (in)adequacy can be identified.

6 Chapter 3 p.3 During the preparation of this chapter, we compiled a number of data gaps and identified key research needs. These are summarized in the final section of the chapter. 2. Variability of gut morphology, physiology and function among aquatic vertebrates and invertebrates Gut morphology, function and physiology vary widely among aquatic vertebrates and invertebrates. This subject is reviewed in Appendices A and B. Additional useful reviews of the digestive physiology of fish are Fange and Grove (1979), Smith (1989), Horn (1992, 1998). The guts of omnivorous and carnivorous teleosts tend to be shorter than those of herbivores. Carnivorous and omnivorous teleosts tend to have stomachs, some of which include a gizzardlike region important for mechanical breakdown of food. Herbivorous fish often lack a stomach, and thus lack the "acid digestion" phase that may be important in increasing the bioavailability of metals for absorption, by solubilizing them from the food. Many fish without stomachs possess an intestinal bulb that functions in food storage, but for acid digestion. Some fish species possess a single large caecum that may be the site of fermentative digestion promoted by symbiotic microorganisms, or that may simply increase the absorptive area of the intestine; other fish species have anywhere from two to hundreds of caecae. If present, the caecae are pouches that branch off the digestive tract near the junction of the stomach with the "anterior portion of the intestine". Fermentative digestion occurs in the posterior intestine of some species. The anterior intestine often has numerous fine villi that increase the absorptive area of the gut. The posterior intestine is often characterized by lower numbers of villi and the presence of muscular

7 Chapter 3 p.4 annular rings. Biliary and "pancreatic" secretions enter the gut directly after the pyloric sphincter, where the stomach ends and opens to the pyloric caecae or anterior intestine. These secretions neutralize the acidic stomach contents and solubilize fats present in the digesta. Because marine fish also must constantly drink seawater in order to osmoregulate, they are exposed to waterborne metals via the digestive tract as well as via external surfaces such as the gill and epidermis. Teleost fish display a large variety of digestive specializations that are intimately linked to their diets. Some species show ontogenetic changes in digestive physiology that are linked to changes in diet, e.g., development from carnivorous juveniles to herbivorous adults. 3. Influence of digestive processes on the bioavailability of dietborne metals: the gut as a biochemical reactor 3.1 Dissolution of metals from ingested matrices The dissolution of metals from ingested substrates may be regarded as a chemical reaction between two phases: the ingested matrix holding the metal and the gut fluid, which has unique properties. In a thermodynamic sense, the chemical nature of each phase controls the possible extent of this dissolution, whereas biotic factors such as gut residence time and food grinding affect the kinetics and hence the actual extent of the reaction.

8 Chapter 3 p The matrix Ingested metals are contained in a variety of chemical matrices. Digestive availability can be limited by the chemical nature of these matrices, which consist of both live and dead materials. A classic example of this partitioning within live tissues comes from studies examining metal assimilation by herbivorous invertebrates that consume live algae. Assimilation of metals in marine herbivores is directly related to the site of localization of the metals in the algal cells that comprise the diet. As a general rule, the assimilation efficiencies of ingested metals in marine copepods (Reinfelder and Fisher 1991; Hutchins et al. 1995; Stewart and Fisher 2003) and bivalve larvae (Reinfelder and Fisher 1994a) are directly related to the cytoplasmic distribution of the metals in the algal diet (Fig. 3.1). This partitioning has been demonstrated for a variety of essential and non-essential metals and for a variety of algal species used as diet. No exceptions have been noted to this trend for invertebrates with rapid gut transit times and relatively simple guts. Thus, metals bound to cell walls and membranes (which are less readily digested) are rapidly packaged into fecal pellets and defecated, whereas metals in the cytoplasm of the ingested cells are assimilated by the animals. For example, when a diatom containing about 20% of its total cellular Ag in the cytosol was ingested by a copepod, about 20% of the total ingested Ag was assimilated in the animal; all other metals (and non-metallic elements as well, including C, S, and P) also displayed this 1:1 relationship between assimilation efficiency and cytoplasmic distribution (Reinfelder and Fisher 1991). A less robust but still suggestive relationship between metal assimilation efficiencies and cytoplasmic distribution in algal cells was observed for adult bivalve mollusks, which have more complex gut physiology and much longer gut residence times for ingested food particles (Wang and Fisher 1996; Wang et al. 1996; Reinfelder et al.

9 Chapter 3 p ). However, it is noteworthy that assimilation efficiencies of ingested metals in adult bivalves can also correlate with gut passage times and the extent of intracellular digestion to which the ingested food particle is subjected (Wang and Fisher 1996; Roditi and Fisher 1999). The research on live algae relied on determination of the cellular distribution of metals in the prey items by lysing the cells and analyzing different cellular components with a differential centrifugation scheme, which produces operationally defined categories of cellular components. We recommend that these results be validated by comparison with independent assessments of sub-cellular metal distributions. Such independent assessments are difficult, but recent work in one of our laboratories (N. Fisher) with synchrotron-based x-ray fluorescence to determine the metal contents of different cellular components shows great promise for identifying the cellular distributions of some common metals of concern, including Fe, Ni and Zn, in phytoplankton and other aquatic protists (Twining et al. 2003). This relationship between cytoplasmic distribution of metals in algal prey cells and assimilation efficiencies in herbivores derives from studies in which algae were exposed to low metal concentrations. If cells are exposed to much higher metal concentrations, such as might exist in contaminated waters, the resulting distribution of the metal in the cell might differ. Different distributions of metals might be due to saturating "normal" pools of metal within the cell or to biochemical responses of the cells such as phytochelatin or metallothionein induction. The extent to which some exposures would lead to different cytological distributions in cells and its relationship to metal assimilation in herbivores remains unexplored.

10 Chapter 3 p.7 Extending this caveat, metal detoxification by many prey organisms can strongly alter metal absorption and toxicity in their predators. Live organisms in areas that have been severely contaminated can have elevated metal body burdens in forms that are less available during digestion by predators (e.g., Bryan and Gibbs 1983). Metal-enriched granules often found in polychaete worms and molluscs (Brown 1982) are an example. Such prey organisms could attenuate the "food-chain effect", i.e. so-called "secondary poisoning" or the transfer of metals from prey to predator (Dallinger et al. 1987). Alternatively, elevated metal burdens can be stored as soluble metallothionein complexes or as other metal-thiol complexes, in which case metal assimilation efficiency in the predator might increase (i.e., an enhancement of the "food-chain effect"; Wallace and Lopez 1997). Other insoluble body parts might also affect digestive availability. For example, in another study designed to measure the trophic transfer of metals from zooplankton to fish, Reinfelder and Fisher (1994b) found that metals bound to the chitinous exoskeleton of marine copepods were largely unassimilated by Atlantic silversides, Menidia menidia, whereas 80% of the metal in the soft parts was assimilated in the fish. If they can be extrapolated to other predator-prey relationships, these results suggest that in diets composed of animal prey, metals bound to the soluble organic fraction of the diet will be assimilable, in contrast to the metals bound to mineral fractions or indigestible, relatively inert components of the diet. These results are analogous to those obtained in experiments with herbivores fed diets composed of phytoplankton, as discussed above. In general, the extent to which particulate metals are solubilized in the gut of an animal can vary with the ph, E H, and surfactancy characteristics of the digestive tract, as illustrated in

11 Chapter 3 p.8 contrasting assimilation efficiencies of Ag, Cd, and Co in clams and mussels from the same sediment diets (Griscom et al. 2002). Metal speciation in nonliving detritus is much less understood than in live organisms. Detritus probably contains a mixture of original metal-containing biomolecule fragments (e.g., metals in pigments or proteins), metals involved in cross-linking among detrital organic molecules, and metals complexed or adsorbed on external functional groups. Many metals are associated with inorganic matrices, including inside mineral lattices or adsorbed on mineral surfaces. Highly insoluble inorganic forms such as sulfides are often thought to restrict digestive availability (Chen and Mayer 1999b), though some metals bound to acid volatile sulfides appear to be assimilated in some benthic animals (Lee et al. 2000). Although contaminant metals can also be found in a wide variety of persistent anthropogenic matrices, such as paint chips or brake lining dust (Davis et al. 2001), very little systematic work has been performed on these matrices The gut fluid Gut fluids in the extracellular lumen of the gut are intended to dissolve food biochemicals from particles and to decrease their molecular weight to enable transport across cell membranes. Organisms secrete a large variety of digestive enzymes to effect this dissolution and molecular weight decrease, and these proteins often dominate the dissolved material in gut fluids. In some animals, especially those ingesting large amounts of indigestible detritus e.g., deposit-feeding invertebrates and vertebrates (Vonk, 1969; Mayer et al. 1997; Smoot and Findlay 2000) surfactants are added to this mixture. Gut fluid also contains a wide variety of lower molecular weight biochemicals dissolved from food, awaiting absorptive uptake. Their concentrations can

12 Chapter 3 p.9 be of similar magnitude as that of the digestive agents. The combination of all of these biochemicals can lead to solutions that have remarkably high concentrations of dissolved organic matter. For example, concentrations of dissolved organic carbon in the gut fluid can reach 100 g L -1 in some marine annelids (Mayer et al. 1997; Mayer et al. 2001), a value that is 3 to 5 orders of magnitude higher than is typical for fresh or marine waters. Gut fluids vary in ph, ranging from the acidic milieu found in vertebrates such as most fish to the near-neutral ph of detritivorous invertebrates. Gut fluid is thus an environmentally intense aqueous medium, one quite different from the conditions that particulate metals are likely to encounter outside the consumer's gut. In some invertebrates, especially crustaceans and mollusks, digestion can occur in both extracellular guts and intracellular lysosomes. While similar to guts in their reliance on enzymes, lysosomes can differ in terms of other variables important for metal dissolution. For example, lysosomal processing involves a ph-lowering step that exposes food substrates to potential metal dissolution not likely to be found in the more neutral gut fluid. However, some studies have also shown that assimilation efficiencies in benthic animals correlate with the extent to which metals desorb from sediment particles into water that has a ph comparable to that of an adult bivalve gut, i.e. about ph 5.5 (Gagnon and Fisher 1997; Griscom et al. 2000). In many organisms, particle sorting occurs after ingestion but prior to intracellular digestion; this process is especially well-developed in mollusks. Suspension- and deposit-feeding bivalves characteristically combine extracellular digestive processing in the stomach with intracellular digestion in the digestive diverticula. Particles are subjected to complex sorting processes in their

13 Chapter 3 p.10 stomachs, either before or after initial extracellular digestion, resulting in a fraction of the particles being transported to the diverticula for prolonged intracellular digestion. Sorting is based on particle size and nutritional quality. Much of the digestion in lamellibranch bivalves can occur in the diverticula, but the composition and fraction of material subjected to intracellular digestion is controlled by several factors. The role of intracellular digestion decreases as food density and ingestion rate increase (Kofoed et al. 1989; Decho and Luoma 1991). In general, the most nutritious particles are more likely to be digested intracellularly. For example, Mercenaria mercenaria (a clam) shunts digestible diatoms to the diverticula, whereas indigestible cyanobacteria are diverted from the stomach directly to the intestine for rapid egestion (Bricelj et al. 1984). Nutritious algae are subjected to initial digestion in the stomach, effectively separating membranes from cytosolic fractions; membranes are then rapidly defecated, whereas the cytosolic material is phagocytized by the cells of the diverticula. Most guts harbor intestinal microflora, whose community structure and function is poorly known. Hindgut fermentation occurs in some fish and some invertebrates, converting refractory vascular plant material to short-chain fatty acids (Horn 1998). Bacteria are also hypothesized to be involved in other digestive functions, such as enzyme secretion (e.g., cellulase and chitinase Horn 1992, 1998). Bacteria might contribute to O 2 consumption in guts, decreasing the redox potential and thus facilitating reductive metal dissolution The reaction Thus, the stage is set for the reaction between the gut milieu and particulate metals ingested by the organism. If metals are contained within the target organic matrices that constitute the

14 Chapter 3 p.11 digestible portion of the animal's food, enzyme hydrolysis can be expected to solubilize the metals, or at least expose them to the solution phase in a manner that will allow for ligandexchange reactions 1. This type of solubilization probably dominates during digestion of live cells or detrital biochemicals that are used for nutrition by detritivores. Metals associated with the surfaces of organic or inorganic detritus are more likely to partition to the solution phase via ligand-exchange reactions. The high concentration of dissolved organic matter in many animal guts, particularly the proteinaceous material with its plethora of carboxyl and N- and S- containing functional groups, will favor the complexation of the exposed or dissolved metals by the dissolved organic pool. For example, gut fluids from 19 different invertebrate species were able to dissolve 65 Zn pre-adsorbed on marine sediments to an extent strongly correlated with their amino acid content (Mayer et al. 2001). The extent of the transfer of these metals to dissolved organic ligands depends on a complex competition between the metal-binding sites in the ingested matrix and those in the gut fluid. The outcome of this competition is determined by a combination of the binding-site strengths and concentrations of the ligands in the ingested matrix versus those of the dissolved organic matter in the gut fluid. Unfortunately, the outcome of this competition is difficult to predict because the distributions of these binding sites are poorly understood. More binding-site distributions (e.g., Chen and Mayer 1998b; Haitzer et al. 2002) need to be determined for gut fluid and environmental matrices. The distribution of metals among gut-fluid ligands also probably affects subsequent metal absorption at the gut epithelium. In fact, the redistribution of metals onto ligands in the gut fluid might result in an intermediary 1 Once free in solution, metal-ligand complexes of the type M-L can exchange their ligand "L" for another competing ligand "X" present in solution, as follows: M-L + X M-X + L. Such reactions can occur by dissociative or associative mechanisms (Morel and Hering 1993).

15 Chapter 3 p.12 speciation that decreases the influence of the original particulate metal speciation on net gut assimilation and subsequent pathways. Lowering the ph and E H to levels below those of the external environment might also redistribute adsorbed metals more extensively into the dissolved state. These partitioning transfers will be constrained by kinetic considerations. Most animals have finite gut residence times ranging from minutes to tens of hours (Fange and Grove 1979; Penry and Jumars 1990), which may prevent some reactions from achieving an equilibrium of the sort described above. For example, complete digestion of food usually is slow. If digestion is slow compared to gut residence time, the incomplete digestion might prevent metals held within a cellular or tissue matrix from being exposed to the solution phase and thus limit their opportunity to react with ligands in gut fluid. Another kinetic constraint might be the chemical lability or rate of dissociation of various metal-ligand complexes. Empirically, because slow reaction kinetics of digestive processes and metal dissolution (e.g., from sediments) can require many hours to attain equilibrium (Mayer et al. 1995; Chen and Mayer 1999a), factors affecting gut residence time (e.g., taxon, food quality) are likely to influence metal bioavailability. For example, some animals do not maintain sufficiently intense digestive capability to extensively digest cell membranes in their gut lumen during extracellular digestion; hence, assimilation efficiencies for cell membranes are low (Reinfelder and Fisher 1991). Intracellular digestion glands in some animals (e.g., adult bivalves) can provide extended residence times for particles amenable to this pathway, allowing for membrane-associated particles to become more fully assimilated (Decho and Luoma 1991).

16 Chapter 3 p Possible modeling approaches Although model predictions of metal concentrations in the tissues of a number of aquatic animals have shown remarkable closeness to independent measurements of these concentrations, ultimately the power to model the digestive uptake of metals in animal guts will depend on a better understanding of the chemical processes occurring during digestion, from both kinetic and thermodynamic perspectives. The successful toxicokinetic models involving digestive uptake are derived from analogous bioenergetic and pharmacokinetic models (e.g., Landrum et al. 1992;Wang and Fisher 1997) and, while useful in many situations, only include simple aggregate uptake and elimination terms. They do not yet incorporate mechanistic modeling of the metal transfer processes occurring at the molecular scale, transferring metals from ingested substrates to dissolved phases in gut fluids, or perhaps most importantly, determining the chemical form of the metal that eventually gets transported across the gut lining at absorptive sites. These molecular extensions, which will require intensive chemical characterization work to be successful, will be necessary to "tune" models for changing binding site energy distributions in ingested substrate and dissolved ligands, each of which may vary with animal species or physiological state. Coupling ligand-based models (e.g., Chen and Mayer 1999a) to chemical reactor models of gut physiology (Jumars 2000a,b) may provide useful predictive capability for metal transfer among phyletic or functional groupings. It may also be possible to develop general models of dietborne metal bioavailability for teleosts. Different models would probably be required for omnivores (e.g., channel catfish (Ictalurus punctatus)), herbivores (e.g., Tilapia sp.) and carnivores (e.g., seabass (Dicentrarchus labrax)); in most carnivores or omnivores the diet will be exposed to acid digestion, possibly followed by

17 Chapter 3 p.14 fermentative digestion (e.g., in pyloric caecae) and then an alkaline digestion phase, whereas in many herbivores the diet will only be exposed to fermentative and alkaline digestion. When attempting to apply these models to a particular species, the investigator would have to take into account digestive specializations. For example, the Labridae, a large group of marine fishes, are almost all carnivorous and yet they are also stomach-less (Horn 1989). An unusual example is the Lake Magadi tilapia (Alcolapia grahami), which feeds in extremely alkaline waters and has the ability to allow ingested water to bypass the acidic stomach (Bergman 2001). These specializations probably have a large effect on the bioavailability and toxicity of dietborne metals, but we are unaware of any research on this subject. 3.2 Metal uptake pathways in the intestine Intestinal metal uptake is much more complex than transepithelial uptake at the gills. Because of this complexity, and the general consensus that branchial metal uptake is more important for acute toxicity, uptake of metals across the alimentary tract is relatively poorly characterized for most species. Metal uptake across the fish intestine is better understood than for the guts or digestive glands of invertebrates. In fish, uptake follows several phases starting with the diffusion of metals into an unstirred boundary layer, created by the rheology of gut fluid as it passes along the intestine (Fig. 3.2). Subsequently, the diffusing metal encounters a layer of mucus that coats the intestinal epithelium. Thus, the initial binding of metal to the intestinal wall might not be to the epithelial cells themselves, but to the mucopolysaccharides that constitute this mucus layer. Many factors may stimulate mucus production, including exposure to metals such as Cd, Cu and Zn. It follows

18 Chapter 3 p.15 that mucus is a powerful regulatory checkpoint for metal uptake (Handy et al. 2000; Glover and Hogstrand 2002a). After short-term metal exposures, the amount of the metal present in intestinal mucus relative to total retained metal might differ among metals and exposure concentrations. For example, during exposures of African catfish (Clarias gariepinus), European flounder (Platichthys flesus) or rainbow trout (Oncorhynchus mykiss) intestines to dissolved metal, 40 to 70% of the retained metal was bound to mucus (Handy et al. 2000; Glover and Hogstrand 2002a; Hogstrand et al. 2002). These processes described for metal transit into and through the diffusive boundary layer and the mucus layer are largely analogous to those occurring outside the epithelial layer of gills. Even the initial interaction of metals at the surface of the gut epithelium is analogous to the interaction at the branchial epithelium. Intestinal epithelial cells are joined together by tight junctions that are relatively impermeable to ions and water (Nonnotte et al. 1995), although the intestinal epithelium is less tight than the gill epithelium. Thus, to be absorbed, metals normally must pass through the cells that form the intestinal epithelium. Some of these cells have transporters and channels that mediate uptake of nutrients. A critical difference between the gill and gut epithelia of fish is that the former currently is known only to have transporters for inorganic ions, whereas the latter contains transport proteins for both inorganic and organic nutrients. Some of these ion transporters are involved in the uptake of Cu, Fe and Zn (which can be nutritionally essential elements or toxicants, depending on their dose) and some other toxicologically relevant metals, all of which gain entry to the epithelial cells via these ion transporters (Bury et al. 2002). Alternatively, some metals probably enter the epithelium bound to amino acids, where uptake is aided by the respective amino acid transporter (Bingham et al.

19 Chapter 3 p ; Glover and Hogstrand 2002b; Glover et al. 2003). Thus, the variety of processes through which metals can be absorbed through gut epithelia is much more complex than in fish branchial epithelia, in which normally only free metal ions (i.e., aquo ions) are transported. Inside the cell, the metal can be bound to metallochaperones, such as metallothionein, cox-17 and glutathione. The purpose of these proteins is to transport the metal to the appropriate intracellular compartment or to the basolateral membrane (i.e., the side of the cell facing away from the gut lumen), where export proteins mediate transport of nutritional metals and other ions (Handy et al. 2000; Bury et al. 2002). These basolateral proteins might coincidentally extrude non-essential elements (Bury et al. 1999, 2002). 4. Potential effects of dietborne metals on the digestive tract before they are assimilated across the gut wall The major gut functions include: (a) secretion of digestive enzymes and hormones; (b) dissolution of food; (c) absorption of dissolved nutrients (e.g., amino acids, sugars, fatty acids, etc); and (d) peristalsis to move food along the gastrointestinal tract. Exposure to dietborne metals has the potential to alter all of these processes. Because these functions are controlled by processes that occur in the gut lumen or in the epithelial walls on the mucosal side of the gut lumen (i.e., on the side in contact with the ingested material), adverse effects might arise without measurable metal accumulation in the gut tissue or other internal organs. Therefore, lack of metal bioaccumulation is not sufficient evidence for lack of dietborne metal toxicity.

20 Chapter 3 p Digestive enzymes and intestinal microorganisms Because metals bind readily to proteins, the potential exists for ingested metals to alter the function of digestive enzymes. For example, Chen and Mayer (1998a) and Chen et al. (2002) reported enzyme inhibition in gut fluids of marine invertebrates when Cu was added at concentrations that might be expected to be solubilized from highly contaminated sediments. At low concentrations of added Cu, the metal attaches to gut-fluid ligands that are not essential to enzyme function. However, at a critical concentration Cu binds to ligands that affect enzyme activity, and enzyme activity decreases. This inhibition might be due to direct binding onto active sites of the enzyme, or other processes such as cross-linking that disrupt protein conformation. The threshold concentration appears to be similar within a gut fluid for a number of different hydrolytic enzymes within a given invertebrate species, suggesting that the inhibition is nonspecific. However, the threshold concentration varies considerably among different invertebrate species and correlates positively with the concentration of total amino acids in the gut fluid (Chen et al. 2002). Thus, animals with gut fluids that contain high concentrations of amino acids are able to dissolve more metal from ingested substrates, reaching higher metal concentrations in the gut fluid; paradoxically, their digestive enzymes are less affected by these high metal concentrations (presumably because the free-metal ion concentration is low). Acidity also protects enzymes from Cu inhibition, perhaps via competition by non-inhibitory protons (H + ions) for the Cu-binding sites. Thus, animals with a lower gut ph are less likely to be affected. This inhibition of digestive reactions has only been demonstrated in vitro, and its existence and impacts in vivo and in field populations remain to be demonstrated. If it were important, inhibition of digestion might contribute considerably to dietborne metal toxicity. Such effects

21 Chapter 3 p.18 might be manifested as overall starvation or, in less contaminated circumstances, as reduced growth. The wide range of threshold concentrations sets the stage for a strong selective pressure in animal communities, with animals having less aggressive digestive systems (e.g., echinoderms in benthic communities) being more susceptible to dietborne metal toxicity. We are only aware of four studies on the effects of metals on digestive enzymes in fish. Contrary to expectation, all studies found that metals stimulated at least some enzyme activity (i.e., waterborne Hg stimulated trypsin: Gupta and Sastry 1981; dietborne Zn stimulated trypsin: Brafield and Koodie 1994) or did not alter enzyme activity (i.e., a combination of several elevated dietborne metals (Cd, Cu, Pb and Zn) did not alter aminopeptidase, maltase and trehalase: Elderkin 1997); only one study also showed inhibition of digestive enzymes (waterborne Cd inhibited trypsin, aminotripeptidase and glycylglycine dipeptidase: Sastry and Gupta 1979). Similar results have been observed for invertebrates, in that low concentrations of added Cu increased invertebrate enzyme activities relative to unspiked gut fluids, and only inhibited them at a critically high threshold value (Chen et al. 2002). Intestinal microorganisms and their role in fermentative digestion have only recently been characterized in a few species of fish (Horn 1998). High concentrations of dietborne metals might be toxic to these microorganisms, thereby decreasing their beneficial effect on digestion and the nutritional status of the fish. This effect would probably be most important for herbivorous fish, which benefit most from the contribution of intestinal microorganisms. However, it is difficult to generalize about the role of intestinal microorganisms in digestive physiology because their influence will differ among fish species and will also change according to the host's diet and physiological status (e.g., life-stage). These issues have been explored in

22 Chapter 3 p.19 deposit-feeding invertebrates (Plante et al. 1990; Plante and Jumars 1992), but not in fish. Although metal uptake itself might be influenced by the presence of intestinal microorganisms, this will depend on the metal, its concentration and mineral form, and other ligands present in the intestinal contents. 4.2 Mucus secretion The mucus that coats the intestinal epithelium of fish appears to play an important role in metal absorption. Several metals including Cd, Cu and Zn stimulate massive production of mucus in the intestine (Handy et al. 2000; Glover and Hogstrand 2002a). The secreted mucus moderates Zn uptake by stimulating absorption at low Zn concentrations and decreasing the uptake at higher concentrations (Glover and Hogstrand 2002a). Although such a regulation of metal uptake could be of obvious benefit in a pulsed-exposure scenario, it might also have the undesirable effect of impairing uptake of other nutrients. Additional research is needed to evaluate if metalstimulated mucus secretion affects nutrient assimilation. 4.3 Gut motility, hormone secretion and nutrient absorption Fish that are fed some metal-contaminated diets have exhibited decreased gut motility, and sometimes their guts have become impacted (Mount et al. 1994; Woodward et al. 1995). These symptoms might be related to damage of the enteric nervous system by dietborne metals. The enteric nervous system is a division of the autonomic nervous system that coordinates gastrointestinal function in all vertebrates. Laboratory studies indicate that exposure to dietborne

23 Chapter 3 p.20 metals can affect serotonin receptors in the enteric nervous system of brown trout (Hotchkiss 2003). There is more general evidence from mammals that metals could interfere with hormones and neurotransmitters important in gut motility (Table 1). An important aspect of these mechanisms of toxicity is that they could occur without significant metal bioaccumulation in internal organs. In general, and in addition to their potential effects on digestive enzymes (Section 4.1), dietborne metals might decrease the efficiency of digestion by, for example, preventing the secretion of hormones essential to gut function and inhibiting the absorption of nutrients (Table 1). The consequences of this inhibition would be less energy intake for physiological maintenance, locomotion, growth and/or reproduction. Such effects might not be detectable in a fish being fed a generous ration in a laboratory, but could be significant in the natural environment where sufficient rations might be difficult to obtain. 4.4 Inhibition of metal uptake Interactions among different cations in fish intestines can substantially influence metal uptake, and are probably explained by competition for specific transporters. In mammals, Cd can be transported into cells by CRT1, which is a Cu transporter (Lee et al. 2002), and by DMT1 (aka Nramp2; DCT1; Slc1 1a2), which is a ferrous (Fe 2+ ) ion importer (Elisma and Jumarie 2001). These transporters are present in fish and are believed to be expressed in the fish intestine (Bury et al. 2002). Although information about functional properties of homologues to these proteins in fish and other aquatic species is missing, similar functions of the piscine homologues would explain the observations that Cd inhibits absorption of both Cu and Fe in fish (Handy 1992).

24 Chapter 3 p.21 Moreover, Cu and Fe in the intestinal lumen also inhibit Cd uptake (Handy 1992). Zinc absorption in the rainbow trout intestine is inhibited by Cu, probably because of competition for one of the Zn transporters at the brushborder membrane (Glover and Hogstrand 2003). Uptake of Zn is also influenced by Cd, but Cd only depresses the Zn accumulation rate in the intestinal tissue and has no effect on Zn uptake into post-intestinal compartments (Glover and Hogstrand 2003). In contrast, over the ranges encountered in typical fish intestines, changes in Mg or Na concentrations have little effect on intestinal metal uptake (Glover and Hogstrand 2003; Handy et al. 2002; Hogstrand et al. 2002), but dietborne Cu can influence waterborne Cu uptake (Kamunde et al. 2001). In winter flounder 8 different metals (Cd, Co, Cr, Cu, Fe, Hg, Mg, Ni) inhibited intestinal Zn uptake (Shears and Fletcher 1983). Note however that interactions are not always straightforward: for example, low or high concentrations of Ca stimulated Zn uptake across the brushborder membrane (a layer of microvilli facing the intestinal lumen) in rainbow trout, but intermediate Ca concentrations strongly inhibited Zn transport (Glover and Hogstrand 2003). Given that metals interact in such complex ways in the intestinal tract, it is important to consider that, with a few exceptions (Farag et al. 1994; Mount et al. 1994; Woodward et al. 1995; Farag et al. 1999), most laboratory studies on dietborne metal bioavailability and toxicity have used diets containing high concentrations of a single metal.

25 Chapter 3 p Influence of exposure pathway on the internal distribution of metals and their toxicity If the internal distribution of metals were determined by equilibrium processes, this distribution would be independent of the exposure pathway and route of metal uptake. Such a situation might be approached in small, morphologically simple animals. However, for more complex organisms, where the internal metal distribution is determined by non-equilibrium (though perhaps steady-state) processes (Mason and Jenkins 1995), one might reasonably expect the internal fate of metals absorbed via the gut to differ from the fate of metals absorbed via other epithelial membranes (e.g., gills or integument surfaces), with these differences perhaps being more marked for higher organisms with well-developed circulatory systems. In addition, for a given organism and a particular route of uptake, the internal fate of the metal might vary as a function of the physical speciation of the ingested metal and the chemical form of the assimilated metal. In this section we consider how the exposure pathway affects the internal distribution of metals in invertebrates and vertebrates, and we review the experimental evidence indicating that these differences in pharmacodynamics lead to differences in metal-induced effects. 5.1 Invertebrates A number of studies have examined the tissue distribution of metals in marine invertebrates following exposure to waterborne or dietborne metals. Overall, these two metal uptake pathways result in different metal distributions, with metal accumulated from waterborne exposure often located to a much greater extent on animal surfaces such as the chitinous exoskeletons of crustacean zooplankton or the shells of bivalve mollusks (Fisher et al. 1983; Fisher and Teyssié 1986; Fisher et al. 1996). One could speculate that this different tissue distribution might be

26 Chapter 3 p.23 used as a diagnostic tool for evaluating the principal metal sources for invertebrates collected from natural waters. Thus, if an invertebrate had a tissue distribution of metals similar to that following uptake from the dissolved phase, it could be inferred that the metal was accumulated principally from the dissolved phase. Conversely, a metal distribution substantially similar to that following dietborne exposure would suggest that ingested metal was the main source for that invertebrate. Fisher and Teyssié (1986) and Fisher et al. (1996) found examples of different metal distributions in mussel (Mytilus galloprovincialis) tissue following uptake of Ag, Am, Cd, Co, Pb and Zn from water and from food for exposure periods ranging from 5 h to 5 d. Less than 7% of these metals was associated with the shell of the mussels when food was the sole source of the metal, whereas up to 48% of the whole-body metal load was bound to the shell in mussels that had accumulated metal from the dissolved phase. Similarly, gills and especially byssal threads had higher metal contents in mussels that accumulated waterborne metal than in mussels that ingested metal, whereas the digestive tract typically had 10% of the whole-body burden in animals following exposure to waterborne metals. In contrast, mussels that ingested their metal through diet (in this case, marine diatoms) typically had 50% of their whole-body metal load in the digestive tract. In addition, the biological half-life of these metals (i.e., the time required to depurate 50% of the accumulated metal) was determined in individual tissue compartments, as well as in whole mussels, in animals that were allowed to depurate in the field for up to 4 months (Fisher et al. 1996). Again, differences were detected for each metal following different uptake pathways. Biological half-lives differed greatly among the metals and tissues, but the overall pattern was that, for each metal tested, metal accumulated from water tended to be lost more

27 Chapter 3 p.24 slowly from many tissues than metal accumulated from the diet. Although the detailed mechanisms underlying these differences in biological half-lives are not known, this pattern probably reflects the extent to which metals accumulated from the two different pathways partitioned into the most slowly exchanging biochemical compartments within the various mussel tissues. Waterborne metal accumulated by crustacean zooplankton, such as marine copepods and freshwater cladocerans, tends to bind to chitinous exoskeletons and be concentrated there to a much greater extent than metal accumulated from diet, which is more enriched in the "soft parts" of these animals (Fisher et al. 1983; Reinfelder and Fisher 1994b; Hook and Fisher 2001a, 2001b). When exoskeletons are shed by animals during molting, metals obtained from the dissolved phase are rapidly lost from the animals (Fisher et al. 1983). In marine gelatinous zooplankton like appendicularians (a genus of pelagic tunicates in the Class Larvacea), metals accumulated from water are located principally in the mucopolysaccharide houses of these animals, which are cast off and replaced numerous times per day; metals accumulated through a diet composed of phytoplankton can be delivered into the main trunk of the animal (Gorsky et al. 1984; Fisher et al. 1991). 5.2 Fish As in the case of invertebrates, metal partitioning in fish differs according to the route of uptake, and the partitioning changes over time. In the short term, metals tend to accumulate in high concentrations in the gills of fish after waterborne exposure and in the intestine after dietborne exposure (McGeer et al. 2000; Handy 1996; Clearwater et al. 2002). Waterborne metals tend to

28 Chapter 3 p.25 transfer rapidly from the gill after an acute metal exposure, and they then circulate via the blood to all other internal organs including muscle. In contrast, dietborne metals first accumulate in the intestinal tissues and from there circulate via the portal system directly to the liver before they reach any other organs. Because the liver has a strong tendency to accumulate many metals, and because during dietborne exposure the metals are delivered to the liver in the first pass, dietborne metals are preferentially accumulated in this tissue (see Hogstrand and Haux 1991). After leaving the intestine or gills, Cd, Cu, Pb and Zn tend to partition to different organs, depending on the metal. For example, Cu tends to accumulate in the liver and bile, whereas Cd accumulates in the kidney and liver (Kumada et al. 1980; Haesloop and Schirmer 1985; Kraal et al. 1985; Hatakeyama and Yasuno 1987; Dallinger and Kautzky 1985; McGeer et al. 2000; Clearwater et al. 2002). The target organ for metal toxicity in fish is probably different for acutely toxic exposures (e.g., gill for waterborne metal exposures, intestine for dietborne metal exposures) than for chronically toxic conditions. Little is known about the target organ(s) for chronically toxic waterborne metal exposures; for chronically toxic dietborne metal exposures the target organs may depend on the metal (e.g., liver and bile for Cu, kidney for Cd). When fish are exposed to dietborne metals, the partitioning of the metals after uptake can also be affected by the ligands present in the intestinal lumen. For example, Glover and Hogstrand (2002b) used an in vivo intestinal perfusion preparation of rainbow trout to investigate the influence of various components in the intestinal fluid on Zn uptake. Presentation of Zn as a Zn-cysteine complex (Zn[cys] 2 ) stimulated absorption of Zn, compared with the inorganic form

29 Chapter 3 p.26 of the metal. In contrast, Zn-histidine was absorbed at the same rate as Zn 2+. The Zn-histidine complex tended to accumulate in the subepithelial layers of the intestine whereas the Zn-cysteine complex tended to accumulate in blood plasma and red blood cells. These results demonstrate that the form of a metal present in the intestine can influence the accumulation rate and affect its subsequent distribution in the body. Differences in metal partitioning after waterborne or dietborne exposure have functional consequences because, for example, the route of exposure influences acclimation to waterborne metal (Cu, Miller et al. 1993; Cd, Szebedinszky et al. 2001). In rainbow trout, pre-exposure to dietborne Cd resulted in enhanced tolerance of subsequent waterborne exposure; in contrast, preexposure to waterborne Cd did not result in enhanced tolerance of subsequent waterborne exposure, even though the metal load in the gill was similar (Szebedinszky et al. 2001). In addition, dietborne Cd exposure caused much higher Cd burdens in the liver, kidneys and intestinal tissues than did waterborne exposure. However, the daily dietborne Cd dose was around 30 mg kg -1 d -1 (trout were fed 2% body weight d -1, food contained 1500 mg Cd kg -1 food), i.e. much higher than the daily waterborne dose (if a ventilation rate of 200 ml min -1 kg -1 body weight is assumed, trout exposed to 2 g Cd L -1 received a daily waterborne dose of only 0.6 mg Cd kg -1 body weight d -1 see Clearwater et al. (2002) for discussion of the waterborne metal dose). As in the case of invertebrates, metal depuration occurs at different rates depending on the route of exposure. For example, rainbow trout depurated Cd more rapidly after dietborne exposure than after waterborne exposure (Kumada et al. 1980), whereas guppies (Poecilia reticulata) depurated Pb more rapidly after waterborne exposure than after dietborne exposure (Vighi 1981).