Effect of cortisol and urea on flavin monooxygenase activity and expression in rainbow trout,Oncorhynchus mykiss

Expression of flavin-containing monooxygenase(s) (FMO) correlates with salinity exposure in certain species of euryhaline fish, such as the rainbow trout, Oncorhynchus mykiss. The mechanism(s) by which salinity regulates FMO is unclear. Adult rainbow trout were infused through the dorsal aorta with either cortisol or urea. At 500 ng/ml, cortisol caused a significant increase in FMO-catalyzed thiourea oxidase activity in gill and liver microsomes. FMOI expression, however, was significantly increased by the high cortisol dose only in gill microsomes. The levels of TMAO and urea were not altered by cortisol. In the liver, urea infusion caused an increase in hepatic FMO activity. FMO expression and activity correlated with elevated tissue urea levels, but TMAO concentrations were not related. These results indicate that FMO expression and activity may be partially controlled by the osmoregulatory/stress hormone. cortisol, and concentrations of the organic osmolyte, urea, in the rainbow trout.     

Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in rainbow trout

In order to elucidate the metabolic fate of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in fish and to thereby facilitate the assessment of the risks posed by this environmental toxin, we determined the whole body half-life, tissue distribution and metabolism of [³H TCDF in rainbow trout (Onchorynchus mykiss), treated orally. A whole body biphasic elimination pattern resulted in the excretion of 60% of the administered chemical during the first 3 days, after which a much slower elimination-rate (half-life = 14 days) was observed. Significant amounts of water-soluble metabolites were found in both bile and liver. Of the TCDF-derived radioactivity in bile, approximately 50% represented glucuronide conjugates, predominantly 4-hydroxy-2,3,7,8-TCDF: substantial amounts of the sulfate conjugate of this same metabolite were also present. Except at early time points, muscle contained the predominant fraction of TCDF-derived radioactivity, amounting to 25–65% of the total radioactivity present in the fish. More than 95% of the radioactivity present in muscle represented unmetabolized TCDF.     

Characterization and induction of cytochrome P-450 in the rainbow trout kidney

Cytochrome P-450 monooxygenases catalyze the biotransformation of a great variety of foreign, as well as endogenous, lipid-soluble compounds to more water-soluble products. As in mammals, highest concentration of cytochrome P-450 in fish is found in the liver. However, previous studies have indicated that fish kidney contains relatively high cytochrome P-450-mediated activities.^1,2 We have therefore prepared and characterized subcellular fractions from the kidney of rainbow trout suitable for studies on cytochrome P-450-dependent reactions. Furthermore, as in the liver, several cytochrome P-450-mediated reactions in the kidney were induced following treatment of the fish with β-naphthoflavone.     

Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes

Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17beta-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 10(-7) M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish.     

脂肪营养对封闭循环水养殖道氏虹鳟生长、相关代谢酶及基因表达的影响

为探讨饲料脂肪营养对封闭循环水养殖道氏虹鳟(Oncorhynchus mykiss)生长、脂肪代谢及相关酶基因表达的影响,作者采用单因素设计,即4个脂肪水平——12%、15%、18%、21%(以Ⅰ、Ⅱ、Ⅲ、Ⅳ组表示),共4个处理组,每处理3重复,每重复25尾鱼。试验鱼为初始体质量(333.25±20.71)g的道氏虹鳟幼鱼,试验期77 d。结果表明,与后3组相比,Ⅰ组试验鱼增质量率显著提高17.20%~33.17%,Ⅰ和Ⅱ组饲料系数显著低于Ⅲ和Ⅳ组6.20%~20.56%(P0.05);脂肪合成酶类FAS活力随饲料脂肪水平升高而降低,脂肪分解酶类LPL、HL及L-CPTⅠ活力变化特征与之相反(P0.05)。其中,Ⅰ组FAS活力显著高于Ⅲ、Ⅳ组7.5%、8.7%;L-CPTⅠ活力显著低于后3组13.08%~16.35%;Ⅳ组LPL活力显著或极显著高于前3组18.8%~44.7%(P0.05或P0.01);I组肝脏FAS的mRNA表达量比后3组显著高48.29%~55.15%(P0.05);LPL呈相反特征,Ⅳ组显著高于前3组36.72%~113.59%(P0.05或P0.01);Ⅰ组肌肉FAS mRNA表达量显著高于Ⅳ组66.82%(P0.05);LPL表达丰度随脂肪水平升高先下降后显著上升(P0.05)。该结果特征与肝脏中该两种酶活力变化具有同步性。研究发现,随饲料脂肪水平变化,肌肉FAS mRNA表达量变化比肝脏更明显且规律性更强,而肝脏LPL mRNA表达量变化较肌肉更明显。本试验初步确定,在封闭循环水养殖模式下,330 g左右道氏虹鳟饲料蛋白水平在45%时,脂肪水平以不超过12%为宜。     

The effect of estrogen on cadmium distribution in rainbow trout (Oncorhynchus mykiss)

The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.     

Interaction of CYP1A1 inducers in liver of rainbow trout and eelpout

The hepatic CYP1A1 (ethoxyresorufin-O-deethylase (EROD) and protein level) in rainbow trout and eelpout was induced by isosafrole, β-naphthoflavone, 3,3′,4,4′-tetrachlorobiphenyl and mixtures of two of the compounds. A potentiation effect of the CYP1A1 response was observed when isosafrole was given together with β-naphthoflavone, but not when isosafrole was given with 3,3′,4,4′-tetrachlorobiphenyl.     

Comparative oxygen radical formation and toxicity of BDE 47 in rainbow trout cell lines

The polybrominated diphenyl ethers (PBDEs) constitute a class of flame retardants whose residues have markedly increased in fish and human tissues during the last decade. In particular, the levels of certain PBDE congeners in salmon have raised concern regarding potential risks associated with dietary PBDE exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro cell models. We conducted a comparative study of oxyradical production and cell injury in rainbow trout gill (RTgill-W1) and trout liver cells (RTL-W1) exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47), a predominant BDE residue found in fish tissues such as salmonids. Exposure to low micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 and RTL-W1 cell viability as measured by alamarBlue assay. The dose-response of BDE toxicity differed among the two cell lines, with the RTL-W1 liver cells showing greater resistance to toxicity at lower BDE 47 doses, but a more dramatic loss of viability relative to gill cells when challenged with higher (50 microM) doses. The sensitivity of the trout liver cells at higher BDE 47 exposures was reflected by a higher basal production of oxygen radical production by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence that was markedly enhanced in the presence of BDE 47, suggesting an overwhelming of trout liver cell antioxidant defense pathways. Collectively, our data indicate that RTgill-W1 and RTL-W1 liver cells are sensitive to BDE 47-mediated cell injury through a mechanism that may involve oxidative stress. Our data also provide an in vitro basis for potential tissue differences in BDE 47-mediated cell injury.     

Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitellogenic rainbow trout

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre⁻¹ for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.     

Tag loss and movement of stocked yearling rainbow trout (Oncorhynchus mykiss) in southwestern Australia

Rainbow trout, Oncorhynchus mykiss, have been stocked in southwestern Australia since the 1930s. Trials at a research station maintained 868 tagged yearling O. mykiss (age: 14 months; standard length: 208 mm) for up to 375 days. The mean tag loss rate was 0.12% ± 0.040% per day for both single- and double-tagged fish. The tag loss rate was used to interpret tag return data from 7030 single-tagged yearlings stocked into three river systems in southwestern Australia. Recaptures indicated that O. mykiss survived into their second season in all rivers, with the maximum time at liberty of 21 months (longevity of at least 35 months). Upstream and downstream movements of O. mykiss increased with time, although 29% of recaptures were reported from the stocking site. Significantly more O. mykiss were recaptured from the Warren River, and differences remained even when corrected for stocking levels; angler effort had a marginal effect on return rates. Higher return rates from the Warren River may be due to availability of suitable habitat (stream cover, cooler water) for O. mykiss. Results allow the stocking regime to be reviewed to improve fishery performance.     

Transient induction of 7-ethoxyresorufin-O-deethylase (EROD) activity by medium change in the rainbow trout liver cell line, RTL-W1.

Cytochrome P4501A (CYP1A) metabolizes a wide array of lipophilic xenobiotics. In fish liver, CYP1A is constitutively expressed at low levels, but xenobiotics can strongly induce CYP1A expression via a receptor-mediated pathway. While induction of hepatic CYP1A in teleosts by xenobiotics is well investigated, very little is known on the regulation of constitutive CYP1A expression and its induction by factors other than xenobiotics. In the present study we show that in the rainbow trout liver cell line, RTL-W1, CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity can be induced by a change of the culture medium, in the absence of xenobiotics. The increase in cellular EROD levels is of transient nature. Experiments with cell incubation solutions supplemented with various medium components indicate that photooxidized tryptophan is the agent causing the increase of EROD activity after medium change.     

Food and energy in the diet of brown and rainbow trout from Lake Benmore,New Zealand

The diet of brown trout (Salmo trutta) and rainbow trout (S. gairdnerii) was studied in specimens from Lake Benmore, a deep, oligo‐trophic lake in South Island, New Zealand. Between November and July, both species fed mainly on small molluscs (Potamopyrgus antipodarum, Physa sp., and Gyraulus corinna) gleaned from the littoral weed beds. Energy values for the three species of mollusc were determined: P. antipodarum, 6000 J g^‐1; G. corinna, 5500 J g^‐1; Physa sp., 9800 J g^‐1. Potamopyrgus antipodarum yielded little energy to the fish, unless its shell broke during passage through the gut. Physa sp. was the most profitable mollusc, irrespective of shell breakage and Potamopyrgus antipodanim the least profitable. The mean energy value per snail for G. corinna and Physa sp. eaten by rainbow trout was 25–30% less than for snails eaten by brown trout, possibly because rainbow trout ingested empty shells from the sediment surface. Rainbow trout extracted about 20% more energy than brown trout from unbroken shells. In July, 84% of the brown trout switched to predation of common bullies, Gobio‐morphus cotidianus, probably as a result of bully reproductive behaviour. Rainbow trout did not show the same change, apparently because they were feeding in deeper water where few bullies were available. The low‐energy diet and its possible connection with growth rate are discussed.     

The role of iron chelates in the NAD(P)H-dependent oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) by rainbow trout and pacific salmon microsomal fractions

Uninduced microsomes from three different species of the genus Oncorhynchus catalyzed a NAD(P)H-dependent oxidation of the radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA). This reaction was stimulated by the addition of iron-EDTA, hemoglobin and myoblobin but inhibited by catalase and benzoate. Superoxide dismutase (SOD) did not substantially inhibit the oxidation in the presence of iron-EDTA. This hydroxyl radical (·OH)-generating system was mainly NADH-dependent which is similar to observations in aquatic invertebrates but in contrast to mammalian systems. This study demonstrates that this radical-generating activity is wide ranging and requires consideration in evaluating xenobiotic metabolism.     

Bioconcentration and distribution of 4-tert-octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss)

Branched chain alkylphenols are weak oestrogen mimics which are present in the aquatic environment and have been implicated in the feminisation of fish. This study reports the biotransformation, bioconcentration and tissue distribution of the xenoestrogen 4-tert-octylphenol (t-OP) in juvenile rainbow trout. Fish were exposed for 10 days to a concentration of 4 micrograms/l of [14C] t-OP in a flow-through system and were sampled after 1, 4, 7 and 10 days of exposure. t-OP residues were extracted from all tissues and analysed by radio-high-performance liquid chromatography. After 1 day of exposure radioactive residues were detected in all tissues and reached steady state conditions in the whole fish after 4 days of exposure. The concentration of t-OP residues were highest in bile, followed by faeces, pyloric caeca, liver and intestine. In these tissues the majority of alkylphenol was in the form of two metabolites which were identified by gas chromatography-mass spectroscopy as the glucuronide conjugates of t-OP and t-octylcatechol. t-OP accumulated as the parent compound in fat with a bioconcentration factor (BCF) of 1190, and in brain, muscle, skin, bone, gills, and eye with BCFs of between 100 and 260. This study suggests that exposure to water-borne alkylphenols results in rapid conjugation and elimination of the chemical via the liver/bile route, but that high amounts of the parent xenoestrogen can accumulate in a variety of other fish tissues.     

Environmental estrogens: dose–response relationships for vitellogenin formation and reproductive toxicity in male rainbow trout

The appearance of the egg-yolk protein vitellogenin (Vg) in plasma of male fish is a sensitive indicator of exposure to estrogenic compounds. We have been studying the kinetics of Vg formation and excretion in rainbow trout with a goal towards developing an integrated pharmacokinetic–pharmcodynamic (PK–PD) model to quantitatively relate cumulative estrogenic exposure of fish to the expression and appearance of Vg in plasma. We administered graded doses of ethynylestradiol (EE2), o,p-DDT, DDD and DDE and octylphenol to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of the xenobiotics and Vg were simultaneously quantified using ELISA and GC–MS or GC–ECD. In separate experiments, sexually mature trout were exposed to graded water concentrations of EE2 for 3 months and various parameters indicative of the functional status of the male reproductive system determined. These parameters included tissue-somatic indices, histopathological evaluation, spermatocrit, sperm motility (quantified using computer-assisted-motion analysis) and viability of semen based on fertilization assays using eggs harvested from untreated trout. Results from fertilization assays indicated that 12 week exposure to EE2 concentrations of 10 and 100 ng/l caused a 50% reduction in the fertilization rate of semen harvested from exposed trout. PK–PD modeling strategies proved valuable tools for linking chemical exposures to Vg formation.