Distribution of 14C-ochratoxin A in the rainbow trout (Salmo gairdneri)

The nephrotoxic mycotoxin ochratoxin A was studied in rainbow trout by whole-body autoradiography and scintillation counting using 14C-labelled toxin. After one single intravenous injection of 10 muCi/fish, corresponding to 160 ng toxin/g body weight, the tissue affinity was studied during an eight day period. As soon as 5 min. after injection the concentration of the radioactivity in the blood had dropped to one tenth of that in the kidney and the urinary bladder. The autoradiograms showed two patterns of blackening in the kidney, one diffuse in the pronephros and one very strong spotty blackening in the opistonephros. In addition to the kidney very high concentrations of radioactivity were also noticed in the bile and the pseudobranch. The muscular tissue of treated trouts contained almost no radioactivity during the whole experiment. Chemical analysis revealed that the radioactivity that could be extracted from the organs was mainly ochratoxin A.     

Uptake and biotransformation of 6,7-dimethylquinoline and 6,8-dimethylquinoline by rainbow trout (Salmo gairdneri)

1. 6,7-Dimethylquinoline (6,7-DMQ) is readily taken up by rainbow trout and bioconcentrated in tissue after exposure to ca 1 mg/l for 7.5 h. Mean bioconcentration factors (from water) were 21, 18, 6 and 14 for bile, liver, muscle and carcass respectively. Mean tissue concentrations after 69-96 h depuration were ND, ND, 0.54 and 0.48 micrograms/g for bile, liver, muscle and carcass respectively. 2. Major metabolites, following exposure to 6,7-DMQ, were conjugates (glucuronide or sulphate) of 7-hydroxymethyl-6-methylquinoline and 6-hydroxymethyl-7-methylquinoline. Mean concentration of metabolites in the bile were 500 micrograms/g after 7.5 h exposure to ca 1 mg/l and 1367 micrograms/g after 9.5 h exposure to ca 1 mg/l and 69 h depuration. 3. 6,8-Dimethylquinoline (6,8-DMQ) is also readily bioconcentrated in fish tissue after exposure to ca 1 mg/l. Mean bioconcentration factors (from water) were 23, 20, 13 and 25 for bile, liver, muscle and carcass respectively. Mean tissue concentrations after 7 h exposure to ca 1 mg/l and 63 h depuration were 4.0, 0.67, 0.49, and 3.2 micrograms/g respectively for bile, liver, muscle and carcass. 4. Major metabolites, following exposure to 6,8-DMQ were conjugates (glucuronide or sulphate) of 6,8-dimethyl-5-hydroxyquinoline, 6,8-dimethyl-7-hydroxyquinoline, 6,8-dimethyl-3-hydroxyquinoline and 6-hydroxymethyl-8-methylquinoline. Mean concentration of metabolites in the bile were 1278 micrograms/g after exposure to ca 1 mg/l for 8 h and 1031 micrograms/g after exposure to ca 1 mg/l for 7 h and 63 h depuration.     

O-dealkylation of phenacetin in the olfactory rosette in rainbow trout (Salmo gairdneri)

Olfactory rosettes from trout (Salmo gairdneri) were found to metabolise (14C-ethyl)-phenacetin to 14CO2 in vitro. Based on wet weight, the rate of metabolism was lower than that in liver and gills. Based on protein content, the rate of metabolism was about equal to that in liver and gills. Addition of the cytochrome P-450 inhibitors metyrapone, 9-hydroxyellipticine and piperonyl butoxide significantly decreased the formation of 14CO2 from (14C-ethyl)-phenacetin in olfactory rosettes. Microautoradiography of rosettes incubated with (14C-ethyl)-phenacetin showed the presence of non-extractable metabolites in the sensory and indifferent epithelium. Addition of metyrapone decreased the binding of radioactivity in the epithelia considerably. When olfactory rosettes were incubated with (14C-dimethylamine)-aminopyrine, no formation of 14CO2 was observed. The results are concluded to indicate that cytochrome P-450 dependent enzyme activity is present in the epithelia of the trout olfactory rosette.     

The effect of cadmium on steroidogenesis by testes of the rainbow trout, Salmo gairdneri

Testes of the rainbow trout were incubated in the presence of 0, 0.05, 0.5 and 5 mM cadmium (Cd). Production of those steroids measured was stimulated by 0.05 and 0.5 mM cadmium. Yields of testosterone (T) and 11 beta-hydroxytestosterone (beta) were significantly increased at these concentrations, but there was little effect on glucuronyl transferase and 11 beta-hydroxysteroid dehydrogenase enzyme activities. Cd-induced alteration of the hormonal balance may result in perturbation of reproductive development.     

Toxicity and pharmacokinetics of the antibiotic fumagillin in yearling rainbow trout (Salmo gairdneri)

Yearling trout were administered fumagillin dicyclohexylamine (FDCH), an antibiotic that has shown promise for controlling myxozoan parasites in fish. FDCH was fed at 0.25 or 1 g/kg food at 1.5% body weight per day for 60 days, and gill, liver, kidney, spleen, thymus, intestine, and heart were examined histologically. In both treatment groups the hematopoietic tissue of the kidney and spleen was reduced and hematocrit was significantly lower relative to controls. No alteration was found in liver, intestine, heart, thymus, or gill. FDCH was also administered to trout through an indwelling catheter placed in the dorsal aorta. Plasma FDCH was measured using reverse-phase HPLC, and clearance microconstants were estimated. At the highest doses (60 and 30 mg/kg body wt) fumagillin was lethal within about 6 hr, and histological examination revealed extensive toxic alteration in liver and posterior kidney. Plasma clearance at 6 and 3 mg/kg fit a two-compartment model with a rapid alpha phase (i.e., 20 min) but a prolonged beta phase (5.4 days). Although these fish survived for at least 96 hr, renal tubular alteration remained.     

Effect of permethrin (NRDC-143) on the bioenergetics of rainbow trout,Salmo gairdneri

Growth rate (% live body weight · day⁻¹) of 10-g rainbow trout fed to satiation daily and encouraged to swim continuously at a sustained speed was significantly reduced when exposed to sublethal concentrations of permethrin (0.65 and 1.25 μg·l⁻¹) for 1–6 wk at 7°C. In contrast, growth rates of permethrin-exposed 10-g fish at 12°C and 100-g trout at 7 and 12°C were not significantly different from those for the respective control fish, regardless of length of exposure. Food intake was higher at 12 than at 7°C and did not change appreciably among the 100-g trout on exposure to permethrin. Among the small trout food intake declined on exposure to permethrin but returned to or surpassed that by control fish within 3 wk. Total energy gain by the larger trout over the 6-wk experimental period was not influenced appreciably by permethrin. Energy gain by small trout was inversely related to permethrin concentration and length of exposure and directly related to temperature. The reduction in energy gain among permethrin exposed small trout is attributable largely to an elevation in basal metabolism.     

Bioavailability, disposition and pharmacokinetics of 14C-ormetoprim in rainbow trout (Salmo gairdneri)

1. Uptake, bioavailability, tissue disposition, and elimination of 14C were examined in rainbow trout following intravascular and per os dosing of 14C-ormetoprim (8 mg/kg). 2. Plasma clearance was rapid following a single i.v. dose (t1/2 alpha = 0.54 h, t1/2 beta = 17.5 h) with an apparent volume of distribution (Vss) of 4.85 l/kg. An increase in both t1/2 alpha (0.67 h) and t1/2 beta (36.7 h) was seen following multiple i.v. doses (steady state). 3. Oral dispositional studies revealed the highest concentration of OMP in bile, kidney and liver, indicating significant renal and hepatic extraction. Significant OMP residues were seen in skin (0.90 p.p.m.) and muscle (0.15 p.p.m.) at 38 days. 4. Peak absorption time of orally administered OMP was 12 h with an apparent bioavailability of 87%. 5. Intravascularly administered OMP was 33% and 31% protein-bound at 1 and 6 h respectively, and binding was non-specific and non-saturable by Scatchard analysis.     

Uptake, distribution and retention of zineb and ziram in rainbow trout (Salmo gairdneri)

In short-term static bioaccumulation experiments with 14C-labelled zinc ethylenebisdithiocarbamate (zineb) and zinc dimethyldithiocarbamate (ziram) both compounds were rapidly disseminated through the tissues. Whole-body accumulation was low, with bioconcentration factors less than 100. Whole-body elimination was rapid with 45% and 25% of the initial radioactivity from ziram and zineb, respectively, being retained by the end of the 16-day depuration period. Pigmented tissues appeared to be major distribution sites as well. This may be related to the affinity of the compounds and/or their degradation products to melanin or to complexation with phenoloxidase, a copper-containing enzyme involved in melanin synthesis. Autoradiography also revealed a high labelling of thyroid follicles. The results show that dithiocarbamates are selectively localized in various tissues, reported to be the target organs for their toxic action. The observed differences in toxicokinetics between zineb and ziram may, in part, explain the differences in toxicity to fish between ethylenebisdithiocarbamates and dialkyldithiocarbamates.     

The effects of cadmium on prolactin cell activity and plasma cortisol levels in the rainbow trout (Salmo gairdneri)

Treatment of rainbow trout with cadmium by intraperitoneal injection (4.4 and 7.7 mg·kg⁻¹), exposure in tank water (0.5, 1 and 10 mg·l⁻¹) or incubation of trout pituitary glands in medium containing cadmium (50 mg·l⁻¹) had no consistent effect on prolactin cell activity. Exposure of trout to 0.05 and 0.1 mg·l⁻¹ of cadmium in the tank water produced time-dependent changes in plasma cortisol levels which may reflect the alarm, resistance and exhaustion stages in the response of the fish to the cadmium.     

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout (Salmo gairdneri)

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.     

Uptake,distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri)

Uptake, distribution, and effects of CCl₄ were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl₄ in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl₄ residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of ¹⁴C residue(s) from the tissue samples were most rapid in muscle ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 1.7}\text{hr}$) and relatively prolonged for liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 38.9}\text{hr}$). Maximum liver concentrations of ¹⁴C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl₄ concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl₄.     

Morphologic lesions and acute toxicity in rainbow trout (Salmo gairdneri) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

To determine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on growth, mortality, and morphologic lesions in rainbow trout, juvenile Shasta or Wytheville strain fish, obtained from 4 hatcheries, were administered graded single doses of TCDD, 0.1-125 micrograms/kg, ip. TCDD doses of 25 and 125 micrograms/kg caused 85% lethality 2-4 wk after treatment. At these high doses, death occurred before body weight loss could be detected. A lower dose of 5 micrograms/kg caused decreased growth and cumulative mortality of 20% after 11 wk. Stress associated with netting and weighing the fish at weekly intervals significantly shortened the delay period prior to TCDD-induced lethality. Gross and microscopic lesions were evident in rainbow trout treated with 10 micrograms TCDD/kg, but not in fish treated with 1 or 0.1 microgram/kg. Morphologic lesions occurred consistently in epithelial and lymphomyeloid tissues of TCDD-treated fish. Lymphomyeloid lesions included thymic involution, splenic lymphoid depletion, and hypocellularity of hematopoietic tissues in the head kidney and trunk kidney. In association with decreased hematopoiesis, peripheral leukopenia and thrombocytopenia occurred in Shasta strain yearling trout treated with 1 microgram/kg or more TCDD. Regarding epithelial lesions, all 4 hatchery strains treated with 10 micrograms/kg or more TCDD showed multifocal necrosis of gastric cardiac glandular mucosa, 3 of 4 hatchery strains showed vacuolar inclusions in exocrine pancreatic cells, and 2 of 4 hatchery strains showed fin necrosis. The severity and character of lesions in the liver and gastric mucosa varied markedly between hatchery strains of trout. One hatchery strain showed no hepatic lesions, two showed mild hepatocyte lesions, and one exhibited severe diffuse hepatopathy. In this severely affected hatchery strain, hyaline intracytoplasmic inclusions occurred in hepatocytes at 14 and 34 d after TCDD exposure, and bile-duct hyperplasia occurred at 34 d following TCDD exposure. One of 4 hatchery strains showed atrophy of serous gastric glands and 1 of 4 hatchery strains showed hyperplasia of these same glands at 25 and 34 d, respectively, following TCDD treatment. Thus, lymphomyeloid and epithelial tissues are the primary targets for TCDD-induced pathologic lesions in rainbow trout, and the incidence and severity of these lesions is influenced by the strain of trout used and the hatchery from which the trout were obtained.     

The toxicity and metabolism of the pyrethroids cis-and trans-cypermethrin in rainbow trout,Salmo gairdneri

1. The toxicity of cis-and trans-cypermethrin to rainbow trout was investigated and the concentrations of the two isomers in brain associated with toxic signs (excitability and loss of equilibrium) were determined. cis-Cypermethrin and trans-cypermethrin were equally toxic and showed similar brain levels associated with toxic signs (cis:0.25 μg/g, mean (range 0.07-0-53); trans:0.17 μg/g (0-07-0-31)).

2. Orally administered cypermethrin was less toxic than predicted, probably due to poor intestinal uptake. Toxicity was due to absorption via the gills of unchanged pyrethroid excreted from the intestine into the water.

3. The metabolism of the radiolabelled insecticides, [¹⁴C-cyclopropyl]- and [¹⁴C-benzyl]-cis- and trans-cypermethrin has been investigated in vivo and in vitro.

4. The principal route of elimination in vivo was the bile, with 20-28% dose excreted as biliary metabolites in 24h. No difference in the rates of elimination of the cis and trans isomers was observed.

5. cis-Cypermethrin was metabolized primarily to the glucuronide of 4′-hydroxy-cypermethrin (80% total bile radioactivity), together with dichlorovinyldimethyl-cyclopropanecarboxylic acid and its glucuronide, 3-(4-hydroxyphenoxy)benzoic acid (4′-hydroxy-3BPA) and its ester and ether glucuronides, 3-phenoxybenzoyl glucuronide and 4′-hydroxy-3BPA sulphate were detected. trans-Cypermethrin was metabolized to the same products, but with only 36% as 4′-hydroxy-cypermethrin glucuronide.     

Localization of cytochrome P-450 in the head and trunk kidney of rainbow trout (Salmo gairdneri)

Mature, untreated rainbow trout display a marked sex difference in the level and activity of the P-450-dependent mixed-function oxidase system with males demonstrating higher levels than females. This difference is accentuated in the trunk kidney, is specific for endogenous substrates such as lauric acid and sex steroids, and is correlated with greater levels of the constitutive cytochrome P-450 isozyme, LM2. Using an immunohistochemical technique, the cellular localization of P-450 LM2 and P-450 LM4b, the major beta-naphthoflavone inducible isozyme, in the head and trunk kidney of male, female, and juvenile trout has been demonstrated. Immunostaining for P-450 LM2 was observed in the cytoplasm of cells in the second portion (P2) of the proximal tubules of both male and female trout. In male fish, staining appeared to be more intense and involved most of the P2 segments in a tissue section. In female fish, P2 staining was moderate and involved fewer segments. Low staining intensity for P-450 LM4b was observed in the cytoplasm of cells both in the first portion of the proximal tubules and in P2 of male and female fish. Sexual dimorphism was not apparent. The finding of greater amounts of P-450 LM2 in males and low levels of P-450 LM4b in untreated male and female trout confirms previous biochemical studies. Localization of P-450 isozymes in the proximal tubules is consistent with findings in mammalian species. Moderate immunostaining by anti-P-450 LM4b IgG also was observed in the interrenal cells of the head kidney of male, female, and juvenile trout.     

The erythrocyte transport and transfer of methylmercury to the tissues of the rainbow trout (Salmo gairdneri).

Methylmercury (MeHg) was found to be taken up rapidly and almost completely by trout red blood cells (RBC) both in vitro and in vivo. The binding of MeHg within the RBC was freely reversible both in vitro, as shown by the efflux of MeHg from RBCs suspended in protein solutions, and in vivo following intracardial (i.c.) injection of RBC-bound MeHg. Hemoglobin (Hb) appeared to be the main MeHg transport protein in trout blood since it bound 90% of whole blood Hg following an intragastric dose of Me203HgCl. MeHg, injected i.c. as MeHgS-cysteine, was found to be present in blood bound almost completely to hemoglobin 10 days post-injection. This suggests an ability of hemoglobin to compete for and bind MeHg bound to other sulfhydryl (-SH) compounds. The number of reactive -SH groups per molecule of trout Hb was determined to be 4 by amperometric titration with MeHgCl. The concentration of Hb reactive -SH groups in the trout RBC was calculated to be at least 20 mM. This accounts for the high affinity of the RBC for MeHg.     

Effects of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD) on early life stages of rainbow trout (Salmo gairdneri, Richardson)

Rainbow trout eggs, yolk sac fry and juveniles were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in different concentrations for 96 h. Exposure of eggs to the lowest concentration, used in this study, 0.1 ppt (10-13 glg), resulted in a significant growth retardation for 72 days. At higher concentrations significant numbers of the forthcoming fry developed generalized edemas and died. Histologically, degeneration and necrosis of liver parenchymal cells were observed. Remaining fry showed teratologic changes as foreshortened maxillas and opercular defects. Administration of TCDD to yolk sac fry had similar effects. Juvenile rainbow trout, exposed to 10 and 100 ppt TCCD for 96 h, showed growth retardation and developed slight edematous changes. At 100 ppt all fry had died within 27 days. Histologically, vacuolization of the liver parenchymal cells and intracellular inclusion bodies in liver, pancreas and stomach were observed.     

Regulation of blood haemoglobin and electrolytes in rainbow trout Salmo gairdneri (Richardson) exposed to nitrite

Rainbow trout (30–70 g) were exposed to environmental nitrite for periods varying from 2 to 24 h, and after 12 h blood plasma nitrite concentration was eight times the environmental levels (0.5 mmol 1⁻¹). The rise was followed by an increase in methaemoglobin levels from around 3% to over 60%. After 2 h nitrite exposure the concentrations of plasma potassium, sodium and chloride fell, followed 2 h later by an increase in intra-erythrocyte potassium and sodium concentration with increased red cell volume. Twelve h nitrite exposure led to an increase in the red cell population, the new cells being smaller and containing less haemoglobin. After 24 h exposure, fish fell into two groups: nitrite-intolerant fish with high levels of plasma nitrite and methaemoglobin and nitrite-tolerant fish with low plasma levels. All fish surviving 24 h nitrite exposure had lower plasma potassium levels than unexposed fish.