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1.
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.  相似文献   

2.
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.  相似文献   

3.
本文研究了不同盐度驯化方式下虹鳟(Oncorhynchus mykiss,(99.44±0.26)g(简写为99 g))和两种规格硬头鳟(Oncorhynchus mykiss,(99.01±0.61)g(简写为99 g)和(394.50±1.16)g(简写为395 g))的血清抗氧化酶活性变化。实验设置4种盐度驯化方式:淡水对照组(T0组);从淡水直接过渡到盐度30(T30组);从淡水直接过渡至盐度14,然后以升盐速度2/d(T2组)和6/d(T6组)至盐度30。盐度驯化结果显示:99 g硬头鳟和虹鳟血清中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)活性均受盐度驯化方式、时间及其交互作用影响显著。T30组99 g虹鳟和99 g硬头鳟的SOD、GSH-Px和MDA活性在0.5、1、4、8、15和40 d这6个取样点均显著高于对照组(P<0.05)。盐度驯化40 d后,T2组99 g虹鳟SOD活性、GSH-Px活性和MDA含量与对照组无显著差异,99 g硬头鳟GSH-Px活性与对照组无显著差异,T6组99 g虹鳟和99 g硬头鳟的SOD和GSH-Px活性显著高于对照组。T2组395 g硬头鳟SOD活性、过氧化氢酶(CAT)活性和MDA含量与对照组相比无显著差异(P>0.05),而T6和T30组395 g硬头鳟血清中SOD活性、CAT活性、GSH-Px活性和MDA含量显著高于对照组(P<0.05)。研究结果表明,T2组渐变盐度方式更适合虹鳟和硬头鳟盐度驯化,395 g硬头鳟对盐度驯化的适应能力优于99 g硬头鳟。  相似文献   

4.
Alterations to hepatic xenobiotic metabolizing enzymes (XMEs) is an important biomarker of contaminant exposure in aquatic toxicology. Measurement of XMEs in fish liver slices in vitro is an emerging tool for examining enzyme activity and response within the intact 3-D architecture of the liver tissue. We examined integrated phase I/phase II, and phase II metabolism of XMEs from liver slices in control and B[a]P-treated rainbow trout and channel catfish. Fluorescent assay substrates to measure rates of metabolism included 7-methoxycoumarin (7-MC), 7-ethoxycoumarin (7-EC) and 7-hydroxycoumarin (7-HC). Time-dependent increases in metabolism, and a lower rate of 7-MC metabolism compared with 7-EC metabolism, were observed at all time points for both fish species. In rainbow trout, B[a]P pretreatment caused a 10-fold increase in phase I metabolism of both 7-MC and 7-EC, and a 1.6-fold increase in phase II metabolism of 7-HC. Phase I activity in channel catfish was not notably altered by B[a]P pretreatment. However, B[a]P pretreatment in channel catfish caused a 48% decrease in phase II metabolism of 7-HC. These results indicate differences in baseline and B[a]P-altered XME profiles between rainbow trout and channel catfish.  相似文献   

5.
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.  相似文献   

6.
Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioactivation of the carbamate insecticide, aldicarb, to a more potent cholinesterase inhibitor, it would be predicted that euryhaline fish exposed to aldicarb at higher salinity should have greater levels of FMO and, hence, be more susceptible to aldicarb toxicity. It was not surprising, therefore, that mature female Japanese medaka (Oryzias latipes) were significantly more susceptible to aldicarb toxicity at higher salinity. A direct correlation was observed between salinity and FMO expression, but salinity had no significant effect on the uptake of aldicarb. These data suggest that FMOs are upregulated by salinity and may be responsible for the enhanced toxicity of aldicarb at higher salinities.  相似文献   

7.
The effects of isosafrol (ISF) or β-naphthoflavone (βNF) treatments on cytochrome P450 (P450) levels in rainbow trout liver were investigated using immunochemical and catalytic techniques. The discrepancies in catalytic activities and ELISA quantification of rainbow trout P4501A1 protein levels between ISF- and βNF-treated fish indicate that important differences exist between the responses induced by βNF and ISF treatments in the rainbow trout liver.  相似文献   

8.
Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.  相似文献   

9.
In fish, as well as in mammals, it is well known that the cytochrome P450-dependent oxidative metabolism of xenobiotics can generate DNA-reactive species. Moreover, this metabolism is known to be inducible by several compounds of environmental significance, such as polychlorobiphenyls, polycyclic aromatic hydrocarbons (PAHs) and dioxins. Consequently, we studied the relationship between the degree of induction of the cytochrome P4501A, expressed as that of 7-ethylresorufin O-deethylase (EROD) activity, and the level of DNA-adducts, using the post-labelling assay, in the liver of rainbow trout exposed to benzo(a)pyrene (a representative PAH). The results showed a significant 2- to 4-fold increase in EROD activity 2, 4 and 8 days after treatment, paralleled by an increase in DNA-adduct levels. This work further emphasizes the involvement of cytochrome P4501A in the metabolism of benzo(a)pyrene into genotoxic metabolites in rainbow trout.  相似文献   

10.
Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM4b)* appears to be a P-448 type of cytochrome. A minor form (LM4a), having properties very similar to LM4b, was also obtained. In addition, a P-450 form (LM2) was isolated, with properties quite different from LM4a or LM4b, including a high rate of aflatoxin B1 (AFB1) metabolism (Williams & Buhler, 1983c). Antibodies to all three forms were obtained from rabbits. The IgGs prepared against LM4a and LM4b both cross-reacted (forming lines of identity) equally well with both antigens on Ouchterlony plates. Rat P-448 cross-reacted (without lines of identity) with both LM4a- and LM4b-IgG. LM4b-IgG was much more effective than LM4a-IgG for inhibition of LM4a or LM4b reconstituted benzo[a]pyrene (BP) hydroxylase, suggesting that these two antibodies recognize different antigenic sites. The LM2-IgG did not cross-react with any of the other rat or trout cytochromes P-450 examined. Levels of LM2 and LM4b in microsomes from untreated, polychlorinated biphenyl (PCB), phenobarbital (PB) or BNF-treated trout were estimated with an immunological technique involving electrophoresis on SDS-PAGE, followed by transfer to nitrocellulose and staining with either LM 2 - or LM4b -IgG. The ratio of in microsomes from PCB- or BNF-treated rainbow trout was much higher than 1, whereas the reverse was true with microsomes from untreated rainbow trout. These results are consistent with previous observations (Vodicnik et al., 1982) that pretreatment with BNF induced the synthesis of a P-448 type cyytochrome, presumably responsible for the great increase in the metabolism and activation of BP seen in these fish. Conversely, pretreatment with PB did not affect the levels of either LM2 or LM4b. This specific immunological technique should make it possible to assay the levels of these P-450 and P-448 isozymes in various strains of rainbow trout and other species of fish. In addition, the effect of age, sex, diet and exposure to P-450 and P-448 inducers could be examined and, perhaps, utilized to predict the relative risk of certain populations to pollutants activated by these different isozymes.  相似文献   

11.
Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.  相似文献   

12.
Effects on the hepatic cytochrome P450 1A1 system were investigated in juvenile rainbow trout i.p. injected with three different aromatic containing fractions: kerosene, light gas oil or heavy gas oil, originated from distilled North Sea crude oil. Kerosene treatment resulted in no effect on the P450 1A1 system, light gas oil injection caused a weak induction of EROD activities and heavy gas oil treatment resulted in a prominent induction of EROD activities as well as accumulation of CYP1A1 mRNA and P450 1A1 protein levels. The effects of heavy gas oil were compared with effects of β-napthoflavone (β-NF) on the P450 1A1 system. It was obvious that important discrepancies seemed to exist between EROD activities and corresponding CYP1A1 mRNA and P450 1A1 levels in rainbow trout treated with either heavy gas oil or β-NF i.e. heavy gas oil treatment resulted in higher specific EROD activities (EROD/P450 1A1) compared to β-NF. GC-MS analyses revealed that liver and bile from heavy gas oil treated rainbow trout in addition to naphthalene also contained polycyclic aromatic hydrocarbons such as phenanthrenes, anthracene, pyrenes, fluoranthene benz(a)anthracene and chrysene, while none of these compounds were detected in control trout.  相似文献   

13.
Our goal was to study the involvement of cytochrome P450 genes in estrogen metabolism and the extent to which the potentially carcinogenic 4-hydroxyestradiol metabolite is formed by channel catfish (Ictalurus punctatus; CC). Estradiol metabolism and ethoxyresorufin-O-deethylase (EROD) activity were assessed in several tissues from fish collected from three variably contaminated sites in the Mississippi River Delta, from laboratory control fish, and from fish exposed to 20 mg/kg benzo(a)pyrene (BaP) i.p. for 4 days. Liver EROD activity was induced by BaP, but Delta fish EROD activities were not statistically higher than activities in control fish. Gill microsomal EROD activity was also induced by BaP, but activities were 8- to 77-fold lower than those from liver. The predominant estrogen metabolites formed by CC liver, gill and gonad microsomes were 2-hydroxyestradiol and estrone as detected by GC/MS. Liver and gill 2-hydroxyestradiol formation was induced in BaP-dosed fish. The trends in hydroxyestradiol formation in field collected fish were more variable. In all fish liver microsomes there was more 2- compared to 4-hydroxyestradiol formed. However, BaP-treatment increased the 4:2 hydroxyestradiol ratio from 0.04 in control fish to 0.2 in BaP-exposed fish, suggesting that BaP induces the formation of the potentially genotoxic estrogen metabolite. No detectable 4-hydroxyestradiol was produced by gill and gonad microsomes. These results will ultimately help in determining which fish P450 genes are susceptible to environmental contamination and may be involved in estrogen genotoxicity.  相似文献   

14.
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.  相似文献   

15.
The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in fish, and is readily biotransformed by most organisms studied. In fish, both the cytochrome P450 (CYP) and the flavin monooxygenase systems (FMO) are involved in bioactivating aldicarb to aldicarb sulfoxide, which is a more potent inhibitor of acetylcholinesterase (AChE). Channel catfish (Ictalurus punctatus), along with many other fresh water species, do not express FMO and are relatively resistant to the effects of aldicarb. This project examined the toxicity, AChE inhibition, metabolism, and toxicokinetic of aldicarb in channel catfish, and compared these values with an aldicarb-sensitive species, rainbow trout, which expresses FMO. Studies of in vitro and in vivo aldicarb biotransformation in catfish suggest that a low rate of bioactivation (10 times slower Vmax), resulting in less initial conversion to the activated metabolite, aldicarb sulfoxide, may be a contributing factor to resistance of channel catfish to aldicarb toxicity. These data are supported by toxicokinetic and enzyme inhibition studies. This work demonstrates that differences in FMO expression among fish species may have significant influence on toxicity resulting from exposure to some xenobiotics.  相似文献   

16.
The incidence of hepatoma, epidermal and other forms of cancerous growths in fish is well documented (Halver, 1967; Matsushima & Sugimura, 1976; Dawe et al., 1964). In fish, as in mammals, cancer may be a result of metabolically activated carcinogens. In mammals the primary enzyme system involved in the activation of environmental carcinogens is the cytochrome P-450 linked mixed-function oxidase (MFO). The hepatic microsomes of the species offish studied contain variable levels of cytochrome P-450. Liver microsomes of the trout Salmo trutta lacustris are surprisingly active in metabolising benzo-[a]pyrene (BP) and other compounds preferentially metabolised by polycyclic aromatic hydrocarbon (PAH)-specific cytochrome P-450. This finding may be significant, because it is apparent that the detrimental effects of PAHs require metabolic activation.We have studied the production of reactive intermediates of BP by following their binding to DNA and by measuring the specific nucleotide-BP metabolite complexes by chromatography. Untreated S. trutta liver microsomes catalyse the production of reactive intermediates of BP which bind to nucleotides of DNA at a rate that is 3–4 times higher than that catalysed by control rat liver microsomes. The most prominent DNA-BP metabolite adducts produced by trout liver microsomes are the nucleoside adduct of BP-7, 8-diol-9,10-epoxide and 9-OH-BP-4,5-oxide and other phenol oxides. In contrast to the trout, another fish species, roach (Rutilus rutilus), has extremely low activity. Although the in vitro binding of BP to DNA is not strictly correlated to in vivo binding or biological effects, our results show that reactive intermediates are formed by trout liver and thus the prerequisite for chemical carcinogenesis or mutagenesis is ful filled. This is further supported by the fact that in Ames's test, trout liver preparations produce mutagenic products from promutagens.  相似文献   

17.
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.  相似文献   

18.
The aim of this study was to examine the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, given intraperitoneally) alone and together with bleached kraft pulpmill effluents (BKME) on fish biotransformation reactions and testosterone levels in mature male rainbow trout (Salmo gairdneri Richardson). The results show extremely high induction in liver and kidney monooxygenase (PSMO) activities in 3 weeks caused by TCDD alone or TCDD together with BKME. BKME alone did not cause significant effects. 7-Pentoxyresorufin O-dealkylase activity, previously shown to be an indicator of phenobarbital-type induction, was shown to be highly inducible by TCDD in fish. Plasma testosterone levels, although decreased in another subacute exposure to BKME, showed only slight changes due to TCDD and TCDD + BKME.  相似文献   

19.
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.  相似文献   

20.
Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis × M. crysops), and bluegill (Lepomis macrochuris) (n = 8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.  相似文献   

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