Regulation of hepatic metallothionein in estradiol-treated rainbow trout

Metallothionein has previously been shown to be regulated during the period of exogenous vitellogenesis (Olsson et al. (1987); Fish Physiol, Biochem., 3(1), 39–47). Following estradiol injection of rainbow trout it has been shown that hepatic metallothionein remains at basal levels until the vitellogenin mRNA levels begin to decline and Zn is released from high-molecular-weight proteins (Olsson et al. (1989); Biochem.J., 257, 555–559), In the present study the effects of estradiol treatment on the metal-inducibility of metallothionein have been investigated. Estradiol-treated fish that were injected with low doses of cadmium or zinc did not respond by induction of metallothionein.     

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.     

Estrogenicity of xenobiotics in rainbow trout (Oncorhynchus mykiss) using in vivo synthesis of vitellogenin as a biomarker

The estrogenicity of several xenobiotics was evaluated using in vivo vitellogenin (Vtg) synthesis in immature rainbow trout as a biomarker. 17β-estradiol, DES and ethinyl estradiol were tested as positive controls. The xenobiotic compounds tested were technical nonylphenol, bisphenol A, butylbenzylphthalate (BBP) and dibutylphthalate (DBF). Measurements of the Vtg concentration was performed with a direct sandwich ELISA. 17β-estradiol, DES and ethinyl estradiol caused up to 100 000-fold increases in the Vtg-levels. Nonylphenol and bisphenol A had the highest estrogenic potency of the xenobiotics increasing the vitellogenin level approximately 300-fold while BBP was only weakly estrogenic, increasing the concentration about 3 times. DPB did not raise the vitellogenin contration above the detection limit.     

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.     

Turnover of hepatic hemoproteins in rainbow trout

The hepatic mixed function oxidase system in the fish differs from that in mammals in its responses to the two classic mammalian inducers. The cytochrome P-448-type inducers (polycyclic aromatic hydrocarbons) stimulate monooxygenase activity, but phenobarbital, a P-450-type inducer, does not.¹ We have compared the effects of phenobarbital (PB) and polychlorinated biphenyls (PCB) on the turnover of hepatic microsomal hemoproteins in trout (PCB's are P-448- and P-450-type inducers in mammals, which in fish induce only cytochrome P-448). We show here that neither PCB nor PB treatment changed the turnover rate. However, both the rates of synthesis and degradation were much slower than in the rat.     

Possible DDT mortality in young rainbow trout

An unusually high mortality of eggs and fry of rainbow trout (Salmo gairdneri Richardson) from Lake Taupo in 1966 was possibly caused by excessive exposure to DDT and its metabolites. The egg and fry mortality (44.6%), and DDT level in the fry (4.63 ppm), was compared with other similar cases of possible DDT poisoning in young salmonids, both in New Zealand and North America. Other possible adverse effects on young salmonids exposed to DDT were discussed.     

Metabolism of 2-acetylaminofluorene by rainbow trout

In order to examine factors that may contribute to the reported resistance of rainbow trout, Shasta strain, to the well-known hepatocarcinogenic effects of 2-acetylaminofluorene (AAF), the in-vitro and in-vivo metabolism of [¹⁴C]AAF in trout has been examined. Trout (compared to rat) liver microsomes metabolized AAF more efficiently, producing 3-fold larger amounts of ring-hydroxylated metabolites (7-hydroxy-AAF and 5-hydroxy-AAF), but 5-fold less N-hydroxy-AAF. Freshly isolated trout hepatocytes extensively metabolized AAF to form the same ring-hydroxylated metabolites and their respective glucuronide and sulfate conjugates. N-OH-AAF (plus its conjugates) and covalently-bound AAF derivatives amounted, respectively, to < 1% and 1.4–1.6% of total metabolites. Liver DNA of trout treated with AAF contained a single AAF-DNA adduct identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (the major persistent AAF-DNA adduct found in rat liver). The level of this adduct (12 attomoles/μg DNA) was about 1000-fold lower than the level of AAF-DNA adduct previously reported in rat liver. The data show that trout liver, compared to rat liver, is considerably less efficient in metabolizing AAF to carcinogenic metabolites, and more efficient in forming nontoxic products, thus possibly explaining, in part, the resistance of trout to AAF-induced hepatocarcinogenesis.     

Oxidative damage in rainbow trout caged in a polluted river

Sewage treatment works (STWs) are a common source of chemicals entering into the aquatic environment. In order to assess effects of these effluents on oxidative stress parameters in aquatic organisms, we caged rainbow trout at five sites: upstream, near an STW effluent, and three sites downstream in the river Viskan in western Sweden for 14 days during autumn, 2006. We then measured protein carbonyls in plasma as well as 20S proteosome activity and lipid peroxidation products, i.e. MDA and 4-HNE, in liver samples. Levels of both lipid and protein oxidative damage products were elevated in fish caged near the STW effluent while 20S activity showed no differences. This argues that complex mixtures of chemicals entering into the aquatic environment do have deleterious effects on fish. Additionally, oxidative stress parameters can serve as a biomarker in aquatic organisms.     

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.     

Growth hormone effect on xenobiotic-metabolizing activities of rainbow trout

In recent years several studies reported the regulation by growth hormone (GH) of the expression of a variety of P450 forms in mammals. However the effect of GH on the xenobiotic-metabolizing enzymes of fish are still unknown. The aim of this work was to investigate the effects of ovine GH—a growth hormone known to be efficient in trout—on the cytochrome P450 level and on aryl hydrocarbon hydroxylase, aminopyrine-N-demethylase, glucuronyl transferase and glutathione transferase activities in trout. The GH-implanted trout (n = 50) each received a single cholesterol pellet containing ovine GH and were compared to control animals (n = 50) receiving a single cholesterol pellet without GH. After 15 days fish were killed and the liver and gills were excised for the measurement of xenobiotic-metabolizing enzyme activities. GH treatment significantly decreased the level of hepatic cytochrome P450 and the activities of cytochrome P450 dependent monooxygenases. In contrast, no significant effect of the treatment was observed on the glutathione transferase and UDP-glucuronyl transferase activities. Moreover, GH treatment had no effect on branchial phase I and phase II enzyme activities. This study provides evidence that GH level significantly affects the expression of several members of the hepatic cytochrome P450 family in trout.     

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.     

Organochlorine insecticides in rainbow trout from three North Island lakes

From 12 rainbow trout (Salmo gairdneri Richardson) taken from each of three lakes (Rerewhakaaitu, Opouri and Okataina) in the Rotorua district, N.Z., samples of liver, skeletal muscle and, from mature fish, the gonads were examined for organochlorine insecticides.

Lindane, heptachlor and heptachlor epoxide were not detected in any of the samples. Traces of dieldrin were detected only in trout taken from Rerewhakaaitu Lake. In all fish examined, pp'‐DDT, pp’DDD and pp’‐DDE were detected in all three tissues. The highest concentrations were found in some of the ovaries of fish from Rerewhakaaitu Lake, the catchment area of which had been extensively topdressed with DDT. Although most of the catchment area of Lake Okataina had never been topdressed with DDT, fish from this lake contained small amounts of DDT and its metabolites.     

Xenobiotic metabolizing enzyme activities in aggregate culture of rainbow trout hepatocytes

Rainbow trout hepatocytes were isolated by a two step perfusion technique and maintained either in monolayer culture for 5 days or in aggregate culture for 30 days. Cytochrome P450 content decreased from day 0 to day 5 in both culture systems, and then was preserved at the same level after one month in aggregated cells. 7-Ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyl transferase were not significantly different in freshly isolated cells and in 30-day aggregated hepatocytes, whereas a substantial increase in glutathione S-transferase was observed. Two-day exposure of cells to β-naphthoflavone led to a significant increase in EROD activity in both culture systems, especially after one month of aggregation (10-fold increase). According to these results, aggregate culture of rainbow trout hepatocytes seems to be a promising in vitro model to investigate the biotransformation pathways in fish and their regulation by endogenous and exogenous compounds.     

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.     

Thermal marking of rainbow trout (Oncorhynchus mykiss) otoliths

A small‐scale experiment was done to test the feasibility of thermally marking hatchery‐reared rainbow trout (Oncorhynchus mykiss Walbaum) fry that are released into rivers and impoundments in New South Wales (NSW), Australia. Fry of rainbow trout were exposed to two different 48‐h thermal cycles each of a cold and warm water period. One thermal regime consisted of a cold water period during which the temperature was reduced from 14 to 8°C for 18 h followed by a return to 14°C for 30 h. For the second thermal treatment, water temperature was reduced to 4°C for 10 h followed by a period of 38 h at 14°C. Thermal cycles were repeated 4 and 8 times for each thermal regime, respectively. Following a growth period after treatment, obvious marks were visible on all treated otoliths as distinct from control otoliths. The 10°C differential treatment created the most visible patterns and growth of these fish was not significantly different from control fish. This marking method could be applied to normal hatchery practices to evaluate the effectiveness of large‐scale rainbow trout stockings in NSW.     

Unique monooxygenation pattern indicates novel flavin-containing monooxygenase in liver of rainbow trout

Vertebrate flavin-containing monooxygenases (FMOs) have only been isolated from mammalian organisms. However, many FMO substrates include pesticides which may adversely affect fish and other aquatic organisms residing in adjacent waterways to treated fields. Although FMO activities have been identified in fish, the exact isoform profile is uncertain. Utilizing prochiral methyl tolyl sulfides (MTS) and isoform-selective antibodies, an attempt was made to identify specific FMO isoforms which may be involved in sulfoxidation reactions which have been shown to bioactivate thioether pesticides, such as aldicarb. Rainbow trout hepatic microsomes treated with detergent to eliminate cytochrome P450 contributions catalyzed the formation of the sulfoxide of MTS in 75% S enantiomeric excess. These catalytic results contrast activities of the five other FMO isoforms including FMO1 (> 98% R) and FMO3 (50% R). Benzydamine N-oxidation was also observed as were methimazole, thiourea, and aldicarb sulfoxidation reactions. Antibodies to FMO1 recognized a single protein of 60 kDa in trout liver microsomes, while anti-FMO3 antibodies only slightly reacted with a 55-kDa microsomal protein. These results indicate a novel isoform profile in rainbow trout liver implicating either a mixture of competing FMO isoforms or a FMO1-like isoform displaying unique catalytic activity.     

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.