In vitro analysis of the bioavailability of six metals via the gastro-intestinal tract of the rainbow trout (Oncorhynchus mykiss)

Abstract

An in vitro gut sac technique was used to compare the uptake rates of essential (copper, zinc and nickel) and non-essential metals (silver, cadmium and lead) at 50 μmol L⁻¹ each (a typical nutritive level in solution in chyme) in the luminal saline in four sections of the gastro-intestinal tract (stomach, anterior, mid and posterior intestines) of the freshwater rainbow trout. Cu, Zn, Cd and Ag exhibited similar regional patterns: on an area-specific basis, uptake rates for these metals were highest in the anterior intestine, lowest in the stomach, and approximately equal in the mid and posterior intestinal segments. When these rates were converted to a whole animal basis, the predominance of the anterior intestine increased because of its greater area, while the contribution of the stomach rose slightly to approach those of the mid and posterior intestines. However, for Pb and Ni, area-specific and whole organism transport rates were greatest in the mid (Pb) and posterior (Ni) intestines. Surprisingly, total transport rates did not differ appreciably among the essential and non-essential metals, varying only from 0.025 (Ag) to 0.050 nmol g⁻¹ h⁻¹ (Ni), suggesting that a single rate constant can be applied for risk assessment purposes. These rates were generally comparable to previously reported uptake rates from waterborne exposures conducted at concentrations 1–4 orders of magnitude lower, indicating that both routes are likely important, and that gut transporters operate with much lower affinity than gill transporters. Except for Ni, more metal was bound to mucus and/or trapped in the mucosal epithelium than was transported into the blood space in every compartment except the anterior intestine, where net transport predominated. Overall, mucus binding was a significant predictor of net transport rate for every metal except Cd, and the strongest relationship was seen for Pb.

Introduction

There is increasing interest in the uptake and potential toxicity of metals through the gastro-intestinal tract of fish (see Clearwater et al., 2002, Bury et al., 2003, Meyer et al., 2005, for recent reviews). Essential metals such as copper and zinc are required for the normal growth and physiological function of fish, and the diet can be their main supply. Nickel is similarly thought to be essential, based on evidence in terrestrial vertebrates, though definitive evidence is not yet available for fish and other aquatic animals (see Muyssen et al., 2004, for a recent critical review). Non-essential metals such as silver, cadmium and lead have no known physiological function in fish, but can also be acquired from the diet. Both types of metals can originate from a variety of sources—for example, from domestic and agricultural/aquacultural sources for copper and zinc; industrial processes for copper, zinc, nickel, cadmium and lead; mining, forestry and waste disposal for cadmium; natural leaching and photographic processing for silver; geological weathering, smelting, coal burning, batteries and paint for lead (e.g. Pratap et al., 1989, Farag et al., 1994; World Health Organisation, 1995; Purcell and Peters, 1998, Wood, 2001).

In fish, some studies have indicated that the anterior intestine is a site of high absorptive capacity for copper, zinc and cadmium uptake compared to other parts of the tract, while the stomach in particular has been considered to exhibit low copper, zinc and cadmium absorption (Pentreath, 1976, Shears and Fletcher, 1983, Hardy et al., 1987, Clearwater et al., 2000, Clearwater et al., 2002, Chowdhury et al., 2004). However, at least with respect to copper, other studies have reached different conclusions, variously emphasizing the importance of the stomach, the mid and posterior intestines (Handy et al., 2000, Kamunde et al., 2002, Nadella et al., 2006a, Nadella et al., 2006b). An early study (Hodson et al., 1978) indicated that dietary lead was not absorbed in trout, though more recent studies have demonstrated that lead can be absorbed through the gastro-intestinal tract (Crespo et al., 1986, Mount et al., 1994, Alves et al., 2006). Based on accumulation patterns in dietary studies, the anterior intestine appears to be the most important site for lead uptake in trout (Alves and Wood, 2006) and for nickel uptake in whitefish (Ptashynski and Klaverkamp, 2002). Silver is also absorbed from the diet in trout (Galvez and Wood, 1999, Galvez et al., 2001), though the regional sites of uptake have not been investigated. Overall, while it is clear that the gastro-intestinal tract is an important site of metal uptake in fish, no clear picture has emerged as to the regional distribution of uptake capacity for the different metals in a single species.

At a mechanistic level, metal transport via the gastro-intestinal tract likely involves several steps (Campbell et al., 2005). The first step is binding of the metal to the mucus in the lumen, which may either facilitate or retard its uptake (Part and Lock, 1983, Whitehead et al., 1996). The second step involves the transfer of the metal from the lumen into the mucosal epithelium across the apical membranes of the enterocytes, while the third step involves the export of the metal out through the basolateral membranes of the enterocytes into the blood or extracellular fluid. Alternately or additionally, it is possible that metals may move through paracellular channels, thereby bypassing cellular transport mechanisms (Bronner, 1998, Foulkes, 2000).

In recent years, isolated gut sac techniques have proven to be a very powerful approach for studying transport processes in the fish gastro-intestinal tract (e.g. Grosell and Jensen, 1999, Grosell et al., 1999, Grosell et al., 2001, Grosell et al., 2005, Bury et al., 2001). The preparations generally absorb Na⁺ and Cl⁻ on a net basis, and this process constrains an accompanying osmotic absorption of water. Treatments which inhibit ion transport result in reduced fluid transport (e.g. Grosell et al., 1999). Recently, Nadella et al. (2006b) have demonstrated that in vitro preparations from the gastro-intestinal tract of trout remain viable for at least 4 h, transporting both a metal (copper) and fluid at a steady rate over this duration. The technique allows manipulation of both the luminal (mucosal) and serosal media, and the sampling of mucus, the mucosal epithelium (i.e. the enterocytes), and the blood side (muscle tissue and serosal saline), thereby enabling the study of three steps in the transport process (mucus binding; accumulation in the mucosal epithelium; and transport to the blood side). In the present study, we have used this approach to compare the transport of three essential (copper, zinc, nickel) and three non-essential metals (cadmium, silver, lead) in various segments (stomach, anterior, mid and posterior intestines) of the trout gastro-intestinal tract. All metals were presented individually at the same concentration (50 μmol L⁻¹), and fluid transport rate was monitored as an indicator of possible toxicity to ion transport processes.

Our study was designed as a first step to understanding metal uptake in the fish gastro-intestinal tract under standardized conditions, with the long term goal of future mechanistic analyses and ionic competition studies (e.g. Nadella et al., 2006b, Nadella et al., 2007), an approach that has been very successful in modeling waterborne metal toxicity at fish gills (e.g. Playle et al., 1993, Di Toro et al., 2001, Playle, 2004, Niyogi and Wood, 2004). Based on the general belief that nutritive (essential) metal uptake in the fish gut may be facilitated by specific carriers (Bury et al., 2003), we hypothesized that essential metals would be taken up at a higher rate than non-essential metals. We further hypothesized that there would be regional differences in the uptake of various metals, due to differential distribution of these specific carriers or differences in passive permeability. Finally, we hypothesized that the rate of metal accumulation in the various compartments (mucus, mucosal epithelium, blood side) would differ among the various metals. For example, mucus might facilitate the uptake of essential metals and retard the uptake of non-essential metals, while the latter might reduce their own uptake by inhibiting specific transporters. In this regard, cadmium has been shown to have a high affinity for epithelial mucus (Part and Lock, 1983), to increase the rate of mucus secretion (Gardner and Yevich, 1970, Glover and Hogstrand, 2003), and to inhibit basolateral Ca²⁺-ATPase in fish enterocytes (Schoenmakers et al., 1992).