Freshwater Rainbow Trout Research Articles

Cultured branchial epithelia from freshwater rainbow trout: Tools for understanding gill function

Cultured branchial epithelia from freshwater rainbow trout: Tools for understanding gill function

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

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.

Mechanisms of dietary Cu uptake in freshwater rainbow trout: evidence for Na-assisted Cu transport and a specific metal carrier in the intestine

Copper (Cu) is both a vital nutrient and a potent toxicant. The objective of this study was to analyze the mechanistic nature of intestinal Cu transport in rainbow trout using radiolabeled Cu (64Cu) and an in vitro gut sac technique. Reduction of mucosal NaCl levels inhibited Cu transport while increase caused stimulation; Na(2)SO(4) had an identical effect, implicating Na(+) rather than the anion. These responses were unrelated to solvent drag, osmotic pressure or changes in transepithelial potential. The presence of elevated luminal Ag stimulated Cu and Na(+) uptake. Phenamil caused a partial inhibition of both Cu and Na(+) uptake while hypercapnia stimulated Na(+) and Cu transport. Cu uptake was sensitive to luminal pH and inhibited by a tenfold excess of Fe and Zn. These factors had no effect on Na(+ )uptake. On the basis of these results we propose a novel Na(+)-assisted mechanism of Cu uptake wherein the Na(+) gradient stimulates an increase in the H(+) concentration of the brushborder creating a suitable microenvironment for the effective transport of Cu via either DMT1 or Ctr1.

Renal function in the freshwater rainbow trout after dietary cadmium acclimation and waterborne cadmium challenge

Renal function was examined in adult rainbow trout ( Oncorhynchus mykiss) after chronic exposure to a sublethal level of dietary Cd (500 mg/kg diet) for 52 d and during a subsequent challenge to waterborne Cd (10 μg/L) for 72 h. Dietary Cd had no major effects on UFR (urine flow rate) and GFR (glomerular filtration rate) but caused increased renal excretion of glucose, protein, and major ions (Mg 2+, Zn 2+, K +, Na +, Cl − but Ca 2+). However, dietary Cd did not affect any plasma ions except Na + which was significantly elevated in the Cd-acclimated trout. Plasma glucose and ammonia levels fell by 25% and 36% respectively, but neither plasma nor urine urea was affected in Cd-acclimated fish. Dietary Cd exposure resulted in a remarkable increase of Cd load in the plasma (48-fold, ∼ 22 ng/mL) and urine (60-fold, 8.9 ng/mL), but Cd excretion via the kidney was negligible on a mass-balance basis. Clearance ratio analysis indicates that all ions, Cd, and metabolites were reabsorbed strongly (58–100%) in both naïve and dietary Cd exposed fish, except ammonia which was secreted in both groups. Mg 2+, Na +, Cl − and K + reabsorption decreased significantly (3–15%) in the Cd-exposed fish relative to the control. Following waterborne Cd challenge, GFR and UFR were affected transiently, and only Mg 2+ and protein excretion remained elevated with no recovery with time in Cd-acclimated trout. Urinary Ca 2+ and Zn 2+ excretion rates dropped with an indication of renal compensation towards plasma declines of both ions. Cadmium challenge did not cause any notable effects on urinary excretion rates of metabolites. However, a significant decrease in Mg 2+ reabsorption but an increase in total ammonia secretion was observed in the Cd-acclimated fish. The study suggests that dietary Cd acclimation involves physiological costs in terms of renal dysfunction and elevated urinary losses.

Gastrointestinal transport of Ca2+ and Mg2+ during the digestion of a single meal in the freshwater rainbow trout

A diet containing an inert marker (ballotini beads, quantified by X-radiography) was used to quantify the transport of two essential minerals, Ca(2+) and Mg(2+) from the diet during the digestion and absorption of a single meal of commercial trout food (3% ration). Initially, net uptake of Ca(2+) was observed in the stomach followed by subsequent Ca(2+) fluxes along the intestine which were variable, but for the most part secretory. This indicated a net secretion of Ca(2+) along the intestinal tract resulting in a net assimilation of dietary Ca(2+) of 28%. Similar handling of Ca(2+) and Mg(2+) was observed along the gastrointestinal tract (GI), although net assimilation differed substantially between the cations, with Mg(2+) assimilation being close to 60%, mostly a result of greater uptake by the stomach. The stomach displayed the highest net uptake rates for both cations (1.5 and 1.3 mmol kg(-1) fish body mass for Ca(2+) and Mg(2+), respectively), occurring within 2 h following ingestion of the meal. Substantial secretions of both Ca(2+) and Mg(2+) were observed in the anterior intestine, which were attributed to bile and other intestinal secretions, while fluxes in the mid and posterior intestine were small and variable. The overall patterns of Ca(2+) and Mg(2+) handling in the GI tract were similar to those observed for Na(+) and K(+) (but not Cl(-)) in a previous study. Overall, these results emphasize the importance of dietary electrolytes in ionoregulatory homeostasis.

Fooling a freshwater fish: how dietary salt transforms the rainbow trout gill into a seawater gill phenotype

Numerous fish species, including rainbow trout (Oncorhynchus mykiss), are able to inhabit both freshwater and seawater and routinely migrate between the two environments. One of the most critical adjustments allowing such successful migrations is a remodelling of the gill in which a suite of morphological and molecular changes ensure optimal function in the face of reversing requirements for salt and water balance. The remodelling leads to specific freshwater and seawater gill phenotypes that are readily identified by the orientation and/or quantities of specific ion transporters and the presence or absence of specific cell types. The proximate cues promoting gill phenotypic plasticity are unknown. Here, by assessing the consequences of a salt-enriched diet (in the absence of any changes in external salinity) in the freshwater rainbow trout, we demonstrate that internal salt loading alone, is able to induce various elements of the seawater gill phenotype. Specifically, we show upregulation of three ion transport genes, cystic fibrosis transmembrane conductance regulator (CFTR), Na(+)/K(+)/2Cl(-) co-transporter (NKCC1) and Na(+)/K(+)-ATPase, which are essential for ionic regulation in seawater, and the appearance of chloride cell-accessory cell complexes, which are normally restricted to fish inhabiting seawater. These data provide compelling evidence that gill remodelling during migration from freshwater to seawater may involve sensing of elevated levels of internal salt.

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.

Renal responses to acute lead waterborne exposure in the freshwater rainbow trout ( Oncorhynchus mykiss)

The possible nephrotoxic effects of waterborne lead exposure (as Pb(NO 3) 2) were investigated in the freshwater rainbow trout ( Oncorhynchus mykiss). Kidney lead accumulation was time-dependent, increasing upon exposure to 0.57 ± 0.01 mg dissolved Pb L −1 for up to 96 h with a significantly higher burden occurring in the posterior kidney compared to the anterior segment. Urine analyses in trout exposed to 1.20 ± 0.09 mg dissolved Pb L −1 revealed a significant increase in urinary lead excretion rate throughout 96 h of exposure. Urine flow rate and glomerular filtration rate (GFR) were not impacted with the exception of a significant decrease in GFR from 84 to 96 h in lead-exposed trout. Urine pH decreased significantly over time in lead-exposed fish. Correspondingly, urine ammonia excretion rate showed a marked increase from 48 h onwards. In experimental fish, urine glucose excretion was significantly greater by 96 h while urine lactate, urea and protein excretion were not significantly altered by lead exposure. The urine excretion rate of Ca 2+ increased significantly by approximately 43% after only 24 h of lead exposure, and was maintained at a higher rate than controls for up to 96 h. Magnesium excretion increased in a time-dependent fashion, reaching a two- to three-fold rise by 96 h. In contrast, rates of Na + and Cl − excretion were decreased in experimental fish by approximately 30% by 48 h, this trend continuing for the duration of lead-exposure. There were no changes in any of these parameters in similarly treated control fish. Clearance ratio analyses indicated progressive decreases in the net reabsorption efficiencies of the renal system for Ca 2+, Mg 2+, Pb, and glucose, suggesting that the active tubular transport mechanisms for these substances were inhibited by lead exposure, while Na +, K +, Cl −, lactate, and protein reabsorptions were unaffected. Net ammonia secretion increased. We conclude that changes in renal function both reflect and help to minimize some of the associated disturbances in systemic physiology. Lead-induced ionoregulatory toxicity in rainbow trout, particularly the disturbance of Ca 2+ homeostasis, is not exclusively a branchial phenomenon, but is in part a result of disruption of ionoregulatory mechanisms at the kidney. This action of lead outside the gills is critical to consider when developing guidelines for water quality.

Dietary Pb Accumulation in Juvenile Freshwater Rainbow Trout (Oncorhynchus mykiss)

Three different diets amended with lead (Pb) nitrate Pb(NO3)2 (7, 77, and 520 microg Pb/g dry weight) and a Pb-free control diet (0.06 microg Pb/g dry weight) were fed to juvenile freshwater rainbow trout for 21 days. Accounting for measured food consumption, the calculated doses per fish were 0.02, 3.7, 39.6, and 221.5 microg/day, for the control, low, intermediate, and high Pb treatments, respectively. The patterns of Pb accumulation over time were determined in various tissues (gills, liver, kidney, intestine, carcass), red blood cells (RBC), and plasma, as well as feeding, growth, hematological, and ionoregulatory parameters. Pb accumulation occurred in a dose-dependent manner in all tissues except the plasma, where accumulation was minimal. Overall, when fed the highest Pb diet, the intestine exhibited the greatest Pb burden (17.8 microg Pb/g tissue wet weight), with high concentrations also found in the kidney (2.4 microg Pb/g tissue wet weight) and liver (1.9 microg Pb/g) at the highest dietary Pb treatment by day 21. The RBCs accumulated a substantial amount of Pb (1.5 microg Pb/g) when compared to the plasma (0.012 microg Pb/g) in the high treatment group. The percentage of Pb retained in the fish decreased with increasing dietary Pb concentrations. Growth, survival, plasma protein, and hematocrit were not significantly affected by dietary Pb. Plasma Ca2+ levels decreased at the beginning of the experiment, whereas Mg2+ levels decreased during the middle of the experiment in both the intermediate and high dietary treatments. Both the Ca2+ and Mg2+ levels stabilized by day 21. Branchial Ca2+ and Na+ influx rates were not affected by dietary Pb, except on day 8 where Na+ influx rates were significantly elevated. The results of this study show that Pb does accumulate internally from the diet when present at levels within the range reported in contaminated benthic invertebrates in nature. We further identify the intestine as a potential target site of chronic toxicity of Pb via the diet, and RBCs as a reservoir of dietary Pb.

Gastrointestinal processing of Na+, Cl−, and K+during digestion: implications for homeostatic balance in freshwater rainbow trout

The role of the gastrointestinal tract in maintaining ionic homeostasis during digestion, as well as the relative contribution of the diet for providing electrolytes, has been generally overlooked in many aquatic species. An experimental diet that contained an inert reference marker (lead-glass beads) was used to quantify the net transport of Na(+), K(+), and Cl(-) during the digestion and absorption of a single meal (3% ration) by freshwater rainbow trout (Oncorhynchus mykiss). Secretion of Cl(-) into the stomach peaked at 8 and 12 h following feeding at a rate of 1.1 mmol.kg(-1).h(-1), corresponding to a theoretical pH of 0.6 in the secreted fluid (i.e., 240 mmol/l HCl). The majority ( approximately 90%) of dietary Na(+) and K(+) was absorbed in the stomach, whereas subsequent large fluxes of Na(+) and Cl(-) into the anterior intestine corresponded to a large flux of water previously observed. The estimated concentration of Na(+) in fluids secreted into the anterior intestine was approximately 155 mmol/l, equivalent to reported hepatic bile values, whereas the estimated concentration of Cl(-) ( approximately 285 mmol/l) suggested seepage of HCl acid from the stomach in advance of the chyme front. Net absorption of K(+) in the stomach occurred following the cessation of Cl(-) secretion, providing indirect evidence of K(+) involvement with HCl acid production. Overall, 80-90% of the K(+) and Cl(-) contents of the meal were absorbed on a net basis, whereas net Na(+) absorption was negligible. Chyme-to-plasma ion concentration gradients were often opposed to the direction of ion transport, especially for Na(+) and Cl(-).

Physical characterization of high-affinity gastrointestinal Cu transport in vitro in freshwater rainbow trout Oncorhynchus mykiss

This study investigated the transport of copper (Cu) in the gut of trout. Examination of the spatial distribution of Cu along the digestive tract and a physical characterization of the uptake process was carried out using an in vitro gut sac technique and (64)Cu as a tracer. Unidirectional Cu uptake was highest in the anterior intestine followed in decreasing order by the posterior intestine, mid intestine and the stomach. Cu uptake was resistant to hypoxia and appeared to be fueled equally well by Cu(II) or Cu (I) at Cu concentrations typically found in the fluid phase of the chyme in vivo in the trout intestine. Transport demonstrated saturation kinetics (e.g. K (m) = 31.6 microM, J (max) = 17 pmol cm(-2) h(-1), in mid intestine) at low Cu levels representative of those measured in the chyme in vivo, with a diffusive component at higher Cu concentrations. Q (10) analysis indicated Cu uptake is via diffusion across the apical membrane and biologically mediated across the basolateral membranes of enterocytes. The presence of L-histidine but not D-histidine stimulated both Cu and Na uptake suggesting a common pathway for the transport of Cu/Na with L-histidine.

Gastrointestinal assimilation of Cu during digestion of a single meal in the freshwater rainbow trout ( Oncorhynchus mykiss)

Gastrointestinal processing and assimilation of Cu in vivo was investigated by sequential chyme analysis over a 72 h period following ingestion of a single satiation meal (3% body weight) of commercial trout food (Cu content = 0.42 μmol g − 1 ) by adult rainbow trout. Leaded glass ballotini beads incorporated into the food and detected by X-ray radiography were employed as an inert marker in order to quantify net Cu absorption or secretion in various parts of the tract. Cu concentrations in the supernatant (fluid phase) fell from about 0.06 μmol mL − 1 (63 μM) in the stomach at 2 h to about 0.003 μmol mL − 1 (3 μM) in the posterior intestine at 72 h. Cu concentrations in the solid phase were 10 to 30-fold higher than in the fluid phase, and increased about 4-fold from the stomach at 2 h to the posterior intestine at 72 h. By reference to the inert marker, overall net Cu absorption from the ingested food by 72 h was about 50%. The mid-intestine, and posterior intestine emerged as important sites of net Cu and water absorption and a potential role for the stomach in this process was also indicated. The anterior intestine was a site of large net Cu addition to the chyme, probably due to large net additions of Cu-containing fluids in the form of bile and other secretions in this segment. The results provide valuable information about sites of Cu absorption and realistic concentrations of Cu in chyme fluid for future in vitro mechanistic studies on Cu transport in the trout gastrointestinal tract.

Water dynamics in the digestive tract of the freshwater rainbow trout during the processing of a single meal

The temporal effects of feeding and digestion on chyme composition, specifically water and solid content, and net fluxes across the gastrointestinal tract, as well as plasma parameters, were examined in freshwater rainbow trout. A single meal of commercial dry pellets, incorporating ballotini beads as inert reference markers, was employed. Plasma Na+ levels increased by 15-20% at 2 h post-feeding, where Cl- levels did not change. Plasma osmolality was well regulated despite an initial chyme osmolality (775 mOsm) 2.8-fold higher than that in the blood plasma. Chyme osmolality throughout the gastrointestinal tract remained significantly higher than plasma osmolality for the duration of the 72 h period. Solid material was absorbed along the entire intestinal tract, although not in the stomach, necessitating the incorporation of an inert marker. A similar temporal pattern of transit between the ballotini beads (solid phase marker) and 3[H]-PEG 4000 (fluid phase marker), provided support for the use of ballotini beads. Large additions of water to the chyme were seen in the stomach, the largest occurring within 2 h following feeding (7.1+/-1.4 ml kg(-1)), and amounted to approximately 16 ml kg(-1) over the first 12 h. As the chyme entered the anterior intestine, a further large water secretion (3.5+/-0.5 ml kg(-1)) was seen. Thereafter the water fluxes into the chyme of the anterior intestine decreased steadily over time, but remained positive, whereas the mid-intestine exhibited net absorption of water at all time points, and the posterior intestine demonstrated little water handling at any time. The endogenous water that was secreted into the anterior intestine was absorbed along the tract, which showed a net water flux close to zero. However, assuming that the water secreted into the stomach was endogenous in nature, the processing of a single meal resulted in net loss of endogenous water (0.24 ml kg(-1) h(-1)) to the environment, a beneficial consequence of the osmotic challenge offered by the food for a freshwater hyperosmotic regulator.

Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss)

The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

The chronic effects of dietary lead in freshwater juvenile rainbow trout ( Oncorhynchus mykiss) fed elevated calcium diets

This study examined the impact of elevated dietary Ca 2+ on the responses to chronic dietary Pb exposure in juvenile rainbow trout. Trout were fed reference (0.3 μg Pb/g, ∼20 mg Ca 2+/g) and Pb-enriched diets (∼50 or 500 μg Pb/g) in the presence of background Ca 2+ (∼20 mg Ca 2+/g) or (∼60 mg Ca 2+/g) of added Ca 2+ (as CaCO 3) for 42 days. The quantitative order of Pb accumulation in tissues reflected the exposure pathway of Pb via the diet (per tissue wet weight): gut > bone > kidney > liver > spleen > gill > carcass > brain > white muscle. The anterior intestine accumulated the most Pb per tissue wet weight, while the bone accumulated the most Pb per fish weight. Pb concentrations were much higher in the posterior kidney than the anterior kidney. Simultaneous addition of Ca 2+ to the diet had an overall protective effect in all the tissues analysed in reducing Pb accumulation. The RBCs accumulated 100 times more Pb when compared to the plasma, while the whole blood δ-aminolevulinic acid dehydratase was inhibited in the high treatment group without added Ca 2+, by the end of the exposure. Neither plasma Cl −, K +, Mg 2+ nor Na +, K +-ATPase activities in the gills, mid- and posterior intestine were affected. However, there were mild disruptions in plasma Na + and Ca 2+ levels in the elevated Pb and Ca 2+ treatment groups, and a significant up-regulation in Na +, K +-ATPase activity at the anterior intestine in fish fed the high Pb diets with background or added Ca 2+. By day 42, Pb levels in most tissues had either stabilized or started to decrease, indicating some capacity for regulation of accumulated loads. We conclude that elevated dietary Ca 2+ levels will be protective in reducing Pb burdens in freshwater juvenile rainbow trout exposed to environments contaminated with waterborne Pb.

Influence of salinity and fish species on PAH uptake from dispersed crude oil

The use of chemical oil dispersants to minimize spill impacts causes a transient increase in hydrocarbon concentrations in water, which increases the risk to aquatic species if toxic components become more bioavailable. The risk of effects depends on the extent to which dispersants enhance the exposure to toxic components, such as polycyclic aromatic hydrocarbons (PAH). Increased salinities can reduce the solubility of PAH and the efficiency of oil dispersants. This study measured changes in the induction of CYP1A enzymes of fish to demonstrate the effect of salinity on PAH availability. Freshwater rainbow trout and euryhaline mummichog were exposed to water accommodated fractions (WAF), and chemically-enhanced water accommodated fractions (CEWAF) at 0‰, 15‰, and 30‰ salinity. For both species, PAH exposure decreased as salinity increased whereas dispersant effectiveness decreased only at the highest salinity. Hence, risks to fish of PAH from dispersed oil will be greatest in coastal waters where salinities are low.

Photoperiod can be used to enhance growth and improve feeding efficiency in farmed rainbow trout, Oncorhynchus mykiss

The current studies examined whether photoperiod techniques could be successfully transferred to commercial freshwater rainbow trout farming practices, with the key aim being to enhance winter grow-out and production in “open/uncovered systems”. Four experiments were undertaken to assess the effects of photoperiod in relation to: growth and feeding regime in tank reared fry; light intensity and growth in tank reared fry; growth and feeding efficiency in cages; and the use of different lighting technologies (bulb colour temperature) in cages. Commercial field trials conducted over a 3-year period showed that exposure of different developmental stages of rainbow trout to periods of constant light from autumn to spring appeared to enhance growth rates and could improve feed conversion. Furthermore, exposure to higher light intensities appeared to promote greater growth and feeding efficiency in all stages of production. The importance of even light distribution in the culture system rather than a critical light intensity was also evident. The outcome of artificial light regimes has been the ability to increase growth rates by up to 25%, alter stock out times, and reduce production time by as much as 2 months.

Chronic, sublethal nickel acclimation alters the diffusive properties of renal brush border membrane vesicles (BBMVs) prepared from the freshwater rainbow trout

Brush border membrane vesicles (BBMVs) were prepared from the kidneys of rainbow trout exposed acutely (72 h; 13,380 μg Ni L − 1 ), chronically (11 months; 289 μg Ni L − 1 ), or chronically and acutely, to waterborne nickel (Ni). Uptake of 63Ni into renal BBMVs was temperature-dependent and fitted a two component kinetic model composed of a saturable, Michaelis–Menten component prominent at lower Ni concentrations, and a moderate linear diffusive component apparent at higher Ni concentrations. Chronic Ni exposure reduced the permeability of the BBM to Ni, evidenced by a significantly reduced slope of the linear diffusive component of BBMV uptake. Efflux of Ni from 63Ni-loaded renal BBMVs was not significantly altered by acute Ni challenge. Both Ca 2+ and Mg 2+ inhibited uptake of Ni into renal BBMVs when present at a molar ratio to Ni of 1000 : 1. Mg 2+-induced inhibition, however, was concentration-dependent and significant in BBMVs prepared from chronically Ni-acclimated fish at far lower molar ratios of 100 and 10 to 1. The data suggest that subtle, long-term modulation of membrane structure and function in the rainbow trout may be a compensatory response to chronic waterborne Ni exposure. Additionally, the data challenge the assumptions of constancy of the physiological parameters underlying physiologically based approaches to modeling metal toxicity. Such approaches are currently employed to derive water quality criteria for some metals.

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl- -free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA(-) MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout ( Oncorhynchus mykiss)

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant ( K m) for Ni transport within the specifically described vesicular media was calculated as 17.9 ± 1.9 μM, the maximal rate of transport ( J max) was calculated as 108.3 ± 3.7 nmol mg protein −1 min −1, and the slope of the linear diffusive component was calculated as 0.049 ± 0.005 nmol mg protein −1 min −1 per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time ( T 1/2) of 125.2 ± 7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni–histidine complexation was rapid, with a T 1/2 of 12.2 s describing the Ni–histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.