Mercury levels in blood, urine and hair in a nation-wide sample of Spanish adults.

Mercury (Hg) is among the top 10 environmental chemicals of major public health concern (WHO). The Minamata Convention on Mercury (United Nations Environment Program, 2017), commits signing countries to control anthropogenic mercury emissions and reduce human exposure. Human biomonitoring (HBM) programs, are the most straight-forward approaches to get information on the actual exposure levels in the population and assess over time. We report here the results of a HBM study in a nationwide cross-section of Spanish adults (18-65y) as baseline values obtained before the Minamata Convention entered into force. Subsequent follow-ups will show if the Convention has been successful. The study includes 1880 blood samples, 1704 urine samples and 577 hair samples from all Spanish regions collected and analysed under a strictly quality controlled and quality assured protocol. The EU-DEMOCOPHES project demonstrated that fish and seafood are the major sources of mercury exposure and that the Spanish as well as the Portuguese populations have higher levels than other European countries. The data from the present study confirms this pattern at national level and that inhabitants in coastal regions have higher values than from inland regions. The geometric mean (GM) for blood is 6.35 µg Hg/l, in urine is 1.11 µg Hg/l and for hair is 1.91 µg Hg/g. In an international comparison these values are not exceptional. Spanish concentrations fall into the group of Easter Mediterranean populations. Although information on gender, age, occupational sector, geographical area, sampling period and frequency of fish consumption is reported in the tables, the purpose of this paper has not been to analyse the determinants of exposure in detail but to provide baseline data for future assessments and for regional authorities.

Determination of cadmium and zinc in Alzheimer's brain tissue using inductively coupled plasma mass spectrometry.

In this work, brain tissue was taken from Alzheimer's Disease (AD) subjects (n=11), 'normal' subjects (n=10) and from subjects with senile involutive cortical changes (SICC) (n=6). Concentrations of Cd and Zn were determined in all samples, using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The brain tissue was selected and obtained from the Netherlands Brain Bank. Samples were taken in each case, from both hemispheres of the superior frontal gyrus, the superior parietal gyrus, the medial temporal gyrus, the hippocampus and the thalamus of the same brain.Cd which is known to have no essential role in the brain was found to follow, as expected, a lognormal distribution of concentrations in 'normal' subjects (Shapiro-Wilk's test (0.98) (p<0.18)). For the Alzheimer's Disease subjects and SICC subjects, the data tends to follow a lognormal distribution, rather than a normal distribution, but is still significantly different from it (Shapiro-Wilk's test (0.97) (p<0.03); (0.93) (p<0.0067), respectively)). In the case of Zn concentrations, the data tends to follow a normal distribution for the 'normal' subject group, even though the data is significantly different from it (Shapiro-Wilk's test (0.95) (p<0.001)). Whereas in the Alzheimer's Disease and SICC subject groups, the data follows a normal distribution (Shapiro-Wilk's test (0.98) (p<0.21); (0.97) (p<0.2002), respectively)). When comparing age-matched groups, for all regions and both hemispheres, no significant differences (p>0.1) for Cd were found between 'normals' and Alzheimer's Disease subjects and Alzheimer's Disease subjects and SICC but at a low level of significance, lower concentrations of Cd were found in the SICC group compared to the 'normals'. For all regions and both hemispheres, Zn was found to be significantly decreased in the Alzheimer's Disease group, compared to the 'normal' and SICC groups. Zn concentrations were also found to be significantly decreased in the 'normals' compared to the SICC group. It is also of interest that Cd negatively correlates with the scale of tangles in both 'normals' (p<0.001) and Alzheimer's Disease subjects (p<0.01). In the SICC subjects Cd correlates negatively with the tangles but not significantly so (p>0.1).

7-Ethoxyresorufin O-deethylase induction in rainbow trout gill epithelium cultured on permeable supports: asymmetrical distribution of substrate metabolites.

The induction of 7-ethoxyresorufin O-deethylase (EROD) has been measured in cultured epithelia from rainbow trout gills. Epithelia incubated with water on the apical side and culture media at the basolateral side were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (betaNF), benzo[k]fluoranthene (B(k)F), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126) from the water. EROD activity was measured as the formation of resorufin from 7-ethoxyresorufin over time in intact epithelia. The EC(50) values obtained after 24 h of exposure (mean+/-S.D.) were for TCDD (n=9) 4.1+/-3.2x10(-11) M, for betaNF (n=6) 1.6+/-3.8x10(-9) M, for B(k)F (n=4) 5.4+/-3.0x10(-9) M and for PCB#126 (n=4) 6.15+/-10.1x10(-9) M. When assaying for EROD activity, it was found that the resorufin concentrations differed between the apical and the basolateral compartments, indicating an asymmetrical distribution of the enzymatically formed resorufin molecules. Generally, the resorufin concentration was highest in the basolateral compartment, but there were differences between epithelia obtained from different fish individuals. Of a total of 13 preparations 10 had the highest resorufin concentration in the basolateral compartment, while in three preparations, the resorufin was uniformly distributed or slightly higher in the apical compartment. The reasons for this asymmetrical distribution of substrate metabolites are not known, and the addition of multidrug resistance inhibitors (verapamil and cyclosporin A) did not alter the asymmetrical pattern. The transepithelial electrical resistance (TER) was also measured to diagnose the tightness of the epithelia. The change from culture media to experimental water (containing TCDD, betaNF, or DMSO as control) in the apical compartment resulted in a large increase in TER, followed by a decline, measured after 24 h. The cytochrome P450 1A (CYP1A) inducers had no effect on the TER and were judged, therefore, not to affect the tightness of the epithelia.

Intracellular vesicular trafficking in the gill epithelium of urea-excreting fish.

Most teleost fish are ammoniotelic, and relatively few are ureotelic, in which the majority of nitrogenous waste is excreted as urea. This study aimed to determine whether the gill ultrastructure of ureotelic fish might have specific, unique characteristics compared with ammoniotelic fish. The gill morphology was studied in three closely related species of the family Batrachoididae: Opsanus beta, the gulf toadfish; Opsanus tau, the oyster toadfish; and Porichthys notatus, the plainfin midshipman, because prior studies have demonstrated that the two former species are ureotelic and excrete urea in unique, short daily pulses, whereas the latter is ammoniotelic. Ultrastructural studies demonstrated significant trafficking of dense-cored vesicles (50-200 nm) between the Golgi apparatus and the apical membrane of epithelial cells surrounding gill filaments and lamellae in these two Opsanus spp. The material constituting the core of these vesicles was intensely stained by lead salt and was unloaded externally when vesicles contacted the apical membrane. Another characteristic of these urea-secreting fish was the presence of numerous large, black-stained lysosomes, which contained cored vesicles, suggesting a second destination for the dense-cored vesicles. As a working hypothesis, the present data suggest that the urea-transporter protein, recently found in toadfish gills, is inserted in the vesicle. Subsequently, it could serve to either sequester cytosolic urea that ultimately is secreted into the water after contact of these vesicles with the pavement cell apical membrane, or it could allow facilitated diffusion of urea across the plasma membrane following insertion into the membrane. As further comparative evidence, the ammoniotelic P. notatus exhibited neither the vesicular trafficking nor the population of lysosomes both found in Opsanus spp.

Intracellular pH regulation and buffer capacity in CO2/HCO3-buffered media in cultured epithelial cells from rainbow trout gills.

The influence of a CO2/HCO3(-)-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pHi) was monitored by continuous spectrofluorometric recording from cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH = 7.70 were transferred to normal CO2/HCO3(-)-buffered medium ¿PCO2 = 3.71 mmHg, [HCO3-] = 6.1 mmol l(-1), extracellular pH (pHe) = 7.70¿, they exhibited a brief acidosis but subsequently regulated the same pHi (approximately 7.41) as in HEPES. Buffer capacity (beta) increased by the expected amount (5.5-8.0 slykes) based on intracellular [HCO3-], and was unaffected by most drugs and treatments. However, after transfer to high PCO2 = 11.15 mmHg, [HCO3-] = 18.2 mmol l(-1) at the same pHe = 7.70, the final regulated pHi was elevated (approximately 7.53). The rate of correction of alkalosis caused by washout of this high PCO2, high-HCO3- medium was unaffected by removal of extracellular Cl-. Removal of extracellular Na + lowered resting pHi and greatly inhibited the rate of pHi recovery from acidosis. Bafilomycin A1 (3 micromol l(-1)) had no effect on these responses. However amiloride (0.2 mmol l(-1)) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pHi, the latter differing from the response in HEPES where amiloride also lowered resting pHi. Similarly 4-acetamido-4'- isothiocyanatostilbene-2,2'-disulfonic acid, sodium salt (0. 1 mmol l(-1)) did not affect resting pHi but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl(-1) also slowed the rate of recovery but greatly increased beta by an unknown mechanism; when this was taken into account, H+ extrusion rate was unaffected. These results are consistent with the presence of Na+ -(HCO3)N co-transport and/or Na+-dependent HCO3(-)/Cl(-) exchange, in addition to Na+/H+ exchange, as mechanisms contributing to "housekeeping" pHi regulation in gill cells in CO2/HCO3(-) media, whereas only Na+/H+ exchange is seen in HEPES. Both Na(+)-independent Cl-/HCO3(-) exchange and V-type H(+)-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.

Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells.

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7+/-2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the 'external environment' and exhibit ultrastructural characteristics similar to those found in the 'chloride cells' of freshwater fish gills. After 6-9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 k capomega cm(2), indicative of electrically 'tight' epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9+/-0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na(+)/K(+)-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (-6 to -15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na(+) and Cl(-) fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl(-) uptake and Na(+) extrusion. Unidirectional Ca(2+) fluxes were only 0.5-1.0 % of Na(+) and Cl(-) fluxes; active net Ca(2+) uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater 'chloride cells'.

Procedures for the preparation and culture of 'reconstructed' rainbow trout branchial epithelia.

Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.

Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture.

The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.

Regulation of Cl- secretion in seawater fish (Dicentrarchus labrax) gill respiratory cells in primary culture.

1. Primary cultures of sea bass (Dicentrarchus labrax) gill cells grown on permeable membranes form a highly differentiated tight epithelium composed of respiratory-like cells. This preparation was also found to provide a functional model for investigating the hormonal regulation of Cl- secretion. 2. In control conditions, i.e. in the absence of hormones or other stimuli, the cultured epithelium showed a short-circuit current (Isc) of 8.8 +/- 0.4 microA cm-2, a transepithelial potential (Vt) of 28.6 +/- 0.6 mV (serosal side positive), and a transepithelial resistance (Rt) of 5026 +/- 127 Omega cm2. Addition of 50 nM PGE2 caused a stimulation of Isc, Vt and transepithelial conductance, Gt. The increase in Isc was probably due to the elevation in Cl- secretion, since it could be correlated with the stimulation of serosal to mucosal 36Cl- flux. Application of the neurohypophyseal peptide arginine vasotocin (AVT; 50 nM) or the beta-adrenergic agonist isoproterenol (isoprenaline; 0. 5 microM) evoked a stimulation in Cl- secretion, as was shown by the increases in Isc and Gt. The excitatory effect of isoproterenol followed by the inhibitory action of propranolol, a beta-adrenergic antagonist, suggested the presence of beta-adrenergic receptors. Noradrenaline (0.1 microM) elicited a reduction in Isc, Vt and Gt, which was counterbalanced by the addition of phentolamine, an alpha-adrenergic antagonist. This suggested an activation of alpha-adrenergic receptors. 3. This study provides evidence for hormonal control of the Cl- secretion in sea bass gill respiratory cells in culture, involving AVT, prostaglandin (PGE2), and beta- and alpha-adrenergic receptors.

Urea and water permeability in the ureotelic gulf toadfish (Opsanus beta).

The permeability of toadfish gills and skin to urea and water has been measured in order to investigate the mechanisms behind the pulsatile excretion of urea previously described in this species. A perfused gill preparation was used in the gill studies and isolated pieces of skin mounted in an Ussing chamber in the skin studies. Simultaneously, urea and water permeability was measured in vivo in free swimming fish. In vivo the nonpulsing urea permeability was exceptionally low compared to other teleosts, while the tritiated water permeability was similar to that of other teleosts. The urea permeability increased 30-fold during a pulse while water permeability stayed unaffected. Compared to in vivo, tritiated water permeability was approximately 50% lower in the gills and the skin when measured directly in the isolated preparations. The urea permeability was almost identical between the three preparations. Four out of 20 perfused gill preparation showed a spontaneous urea pulse during perfusion. Several treatments were tested to elicit the pulse artificially but without success. Hormones and drugs tested were: arginine-vasotocin (AVT), 10(-10) M; adrenaline, 10(-7) M; isoprenaline, 10(-5) M; acetylcholine, 10(-7) and 10(-6) M; serotonin, 10(-7) and 10(-6) M; adenosine, 10(-6) M; cortisol, 10(-7) M; and combinations of AVT, adrenaline, and cortisol. Adrenaline and isoprenaline increased tritiated water permeability without affecting urea permeability. Gradually increasing the ammonia levels in the perfusate from 0.1 mM to 1.6 mM caused a slight increase in water permeability but a marked and progressive increase in urea permeability. No indications of an ammonia trapping mechanism in the gills were found. There was no effect of AVT (10(-10) mol l-1) in the urea permeability of the skin preparation while cortisol (10(-7) M) led to a modest increase in urea permeability. Based on a comparison between the in vivo and in vitro preparations used here, we conclude that the urea pulse in a urea-pulsing toadfish occurs through the gills and not the skin. We still do not know which internal mechanism or signal triggers the urea pulse in the toadfish.

Determination of trace elements in porcine brain by inductively coupled plasma-mass spectrometry, electrothermal atomic absorption spectrometry, and instrumental neutron activation analysis.

Methods have been developed for the analyses of trace metals in various areas of porcine brains, (temporal, parietal, frontal cortex, both right and left hemispheres). Determinations were carried out using inductively coupled plasma-mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ETAAS). The elements investigated were Li, Mn, Cu, Zn, Cd, Hg, and Pb by ICP-MS and Cu, Cd, and Mn by ETAAS. For determination by ICP-MS, a method of standard additions calibration coupled with internal standards was used, and for ETAAS, standard additions calibrations were prepared. The accuracy of all methods was determined using NIST and IAEA certified reference material. A small number of pig brains were analyzed by instrumental neutron activation analysis for Cr, Co, Cs, Fe, Rb, Se, Sc, Sb, and Zn using the comparator method of analysis. Four separate NIST standard reference materials have been used to examine the validity of the comparator method.

Nitrogen excretion and the cardiorespiratory physiology of the gulf toadfish, Opsanus beta.

Gulf toadfish, Opsanus beta, are facultatively ureotelic and can excrete the majority of their nitrogenous waste as urea. Urea excretion occurs in "pulses." The hypothesis that pulsatile urea excretion reflects sudden, transient, generalized increases in the branchial conductance was investigated by the simultaneous monitoring of cardiorespiratory variables, oxygen uptake, and whole-body urea, ammonia, and/or 3H2O effluxes. The direct monitoring of both expired branchial water and water exiting a respirometer demonstrated that urea pulses were derived from the gills. No significant changes in ventilation or cardiac frequency, oxygen uptake, or ammonia efflux were observed during natural urea pulses, refuting the hypothesis that pulsatile urea excretion reflects pulsatile increases in the generalized diffusive properties of the gill for solute transfer. An alternative model for pulsatile urea excretion postulates that the gill urea permeability is increased periodically by the insertion and/or activation of specific urea transporters into gill cell membranes. Pulsatile urea excretion was abolished by pretreatment with the cytoskeletal-disrupting agent colchicine; colchicine may block trafficking of urea transporter-containing vesicles. Exocytosis of water following the fusion of vesicles with gill cell membranes could explain the significantly elevated 3H2O efflux observed during urea pulses.

Pulsatile urea excretion in gulf toadfish (Opsanus beta): evidence for activation of a specific facilitated diffusion transport system.

When toadfish are made ureotelic by a crowding/confinement protocol, they excrete approximately 90 % of their urea nitrogen (urea-N) production in large, irregular pulses (1-2 pulses per day) from the gill region. We investigated three hypotheses as to the mechanism of pulsatile excretion: (i) the presence of an active reabsorptive 'back-transport' mechanism that is periodically inhibited to allow urea-N excretion to occur; (ii) the periodic occurrence of a generalized, non-specific increase in gill permeability; and (iii) the presence of a specific facilitated diffusion transport system that is periodically activated. Exposure of toadfish during non-pulse periods to treatments designed to block a 'back-transport' mechanism (Na+-free sea water or the urea analogues 30 mmol l-1 thiourea or 30 mmol l-1 acetamide in the external water) did not stimulate a leakage of urea-N, thereby opposing the first hypothesis. The second hypothesis was opposed by several results. Neither injection of the potent branchial vasodilator L-isoprenaline (10(-5) mol l-1) nor infusion of NH4Cl, the latter at levels known to stimulate urea-N efflux in perfused gills, had any effect on urea-N excretion. Furthermore, during natural pulse events, when the normally very low gill permeability to urea (3x10(-7) cm s-1) increased at least 35-fold, there was no accompanying increase in permeability to either 3H2O (1.5x10(-5) cm s-1) or the paracellular marker [14C]PEG-4000 (10(-8) cm s-1). However [14C]thiourea permeability (1.5x10(-7) cm s-1) increased approximately fivefold, in support of the third hypothesis. Furthermore, when 30 mmol l-1 urea was placed in the external water, a concentration (60 000 micromol-N l-1) approximately three times that of blood (20 000 micromol-N l-1), each efflux pulse event (measured with [14C]urea) was accompanied by a net uptake, such that blood urea-N levels rose rather than fell. A proportional 1:1 relationship between influx per unit external concentration and efflux per unit internal (i.e. plasma) concentration indicated a fully bidirectional transport system. The simultaneous presence of 60 mmol l-1 thiourea in the external water inhibited the influx component by 73 %, further supporting this conclusion. These data, together with recent molecular, morphological and endocrinological evidence, strongly suggest that pulsatile urea-N excretion is caused by the periodic activation of a facilitated urea transporter in the gills, similar to the vasopressin-regulated urea transporter in the mammalian kidney.

Urea and water permeability in dogfish (Squalus acanthias) gills.

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss).

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.

Influence of water quality on the accumulation of methyl 203mercury in gill tissue of minnow (Phoxinus phoxinus).

Effects of pH and concentrations of Cl and Ca on the uptake of methyl mercury (MeHg) in the gills of the minnow (Phoxinus phoxinus) were studied. Chloride concentration and pH in the water affect the speciation of MeHg. Ca was included because it affects the permeability of the gills and could therefore indirectly affect the amount of MeHg accumulated in the tissue. The species formed differ in hydrophobicity, as reflected in their octanol/water partition coefficients (Pow). Both a reduction in pH and an increase in Cl- concentration increased the Pow of MeHg. Ca had no effect on speciation. The accumulation of MeHg in the gill tissue increased with decreasing pH (from pH 7.0 to pH 3.9). Accumulation also increased as Cl- concentrations were increased from 10(-7) to 10(-1) M at pH 7.0. An increase in Ca concentration did not alter the accumulation of MeHg beyond a decrease in MeHg accumulation as Ca increased from 20 to 50 microM. We conclude that, of the water-quality factors studied, those affecting chemical speciation were most important in determining the MeHg uptake. The Ca concentration appears to be of minor importance.

Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills.

Uptake rate constants of different classes of hydrophobic organic chemicals have been determined in isolated perfused gills of rainbow trout (Oncorhynchus mykiss) as an alternative for studies in vivo. The uptake rate constants have been compared to those determined in guppy, Poecilia reticulata, in vivo. The organic chemicals which have been used are anthracene, hexabromobenzene, octachloronaphthalene, octachlorodibenzo-p-dioxin, phenol, polychlorinated anisoles, polychlorinated benzenes, polychlorinated biphenyls, and tetrachloroveratrole. Uptake rate constants in guppy are higher than those in rainbow trout gills, and show relatively high variation in both gills and guppy. When uptake rate constants in each study are normalized for that of pentachlorobenzene (pCBz), variation is significantly reduced both in perfused gills and in guppy. All allometric relationship is derived between weight and uptake rate constant. Uptake rate constants determined in one fish can thus be used for prediction of those in other fishes. When a reference chemical, such as pCBz, is included, the gill perfusion experiments can be highly suitable to determine uptake rate constants of organic chemicals, which can be extrapolated to fish of different sizes.

Kinetics of copper and nitrite in rainbow trout (Oncorhynchus mykiss): The isolated perfused head preparation as alternative to in vivo assays.

Isolated perfused preparations permit the use of physiological routes of exposure for the study of mechanisms of toxicity under conditions where the architecture remains intact and the potential influences of other organs are eliminated. The perfusion of gills in the isolated head preparation has been used extensively in aquatic toxicology. Quantitative comparisons between in vivo systems and this preparation are required for the extrapolation of results. This paper compares data on uptake and elimination rates obtained in vivo on rainbow trout with those provided by the perfusion of gills in the isolated head preparation. The use of compartmental models allowed calculations of equivalent rates, based on relative changes in the total amount of chemical. Similar absorption rates of nitrites, 3.0 +/- 0.7 v. 4.4 +/- 1.1 for in vivo and in vitro systems, respectively, were observed. The in vivo rate of nitrite elimination was 0.057 +/- 0.024; the concentration changes observed in the in vitro experiments on nitrite elimination were too low to estimate accurate rates, but data suggest, at least, a similar order of magnitude. For copper, the in vitro elimination rate, 0.33 +/- 0.04, is two orders of magnitude higher than in vivo, 0.0035 +/- 0.0005. This difference could be explained by the binding of copper to proteins and cells, suggesting this binding as the limiting factor for the elimination of copper. The mass balance method proposed in this paper was useful to compare gill perfusion data and in vivo compartmental assessments under quantitative estimations.