Quantification of cytochrome P4501A1 and catalytic activities in liver microsomes of isosafrol- and β-Naphthoflavone-treated rainbow trout (Oncorhynchus mykiss)

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.     

7-ethylresorufin O-deethylase activity and level of DNA-adducts in trout treated with benzo(a)pyrene

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.     

Effects of petroleum hydrocarbons on the hepatic cytochrome P450 1A1 system in rainbow trout

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.     

Using cytochrome P450 to monitor the aquatic environment: Initial results from regional and national surveys

We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAA's Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.     

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.     

Nomenclature for hydrocarbon-inducible cytochrome P450 in fish

A growing number of studies concerning organic chemical pollutant induction of cytochrome P450 mixed function oxidases in fish stimulate a need for common terminology. Similar names for proteins might be based on similarities in function, in regulation, and in their structure. Under the current guidelines for P450 proteins, classification is based on measured or inferred amino acid sequence. At present, a single teleost hydrocarbon-inducible P450 (rainbow trout) can conclusively be termed 1A1: a second (scup P450E) can be so termed on the basis of partial sequence. The lack of primary sequence information makes it difficult to assign a specific identity to hydrocarbon inducible P450s in other fish. Nevertheless, the sum of information on catalytic activities, introduction and immunological cross-reactivities provides a weight of evidence sufficient to assume (but not prove) gene family (I) and subfamily (A) identities for hydrocarbon-inducible proteins in many species. Reference to these proteins as P4501 or IA (or CYPI or IA) in fish species where sequence data are lacking is suggested as more appropriate than reference to them as P450IAI (or CYPIAI). Additional primary sequence data are required to further define orthologous relationships among P450IA genes in fish, and to determine whether IAI is a correct designation for a hydrocarbon-inducible P450 in many species.     

Comparison of xenobiotic metabolizing enzymes of Tilapia with those of other fish species and interspecies relationships between gene families

Baseline data for hepatic xenobiotic metabolizing biomarker enzyme activities were obtained for artificially reared tilapia Oreochromis niloticus, and were compared with those of the plaice (Pleuronectes platessa) and rainbow trout (Onchorynchus mykiss). Basal activities exhibited species variations with notably higher CYP1A and phenol UGT activities and lower GST activity in plaice than the freshwater species. Interspecies relationships between gene families determined by immunoblotting and substrate-activity profiles demonstrated the presence of homologous CYP1A and CYP3A enzymes in all three species, alpha class GSTs in plaice and trout, mu and pi class GSTs in trout and theta class GSTs in plaice and tilapia. CYP1A of tilapia was induced by 3-MC or PBO treatment, whilst CYP3A was induced by PCN treatment.     

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

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.     

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.     

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.     

盐度变化对虹鳟和硬头鳟抗氧化酶活性的影响

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。     

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.     

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.     

The use of polyclonal antibodies raised against rat and trout cytochrome P450 CYP1A1 orthologues to monitor environmental induction in the channel catfish (Ictalurus punctatus)

Induction of hepatic cytochrome P450-dependent microsomal mono-oxygenase by xenobiotics is a well-established phenomenon in teleost fish. As in laboratory mammals, fish possess multiple forms of cytochrome P450 that display overlapping substrate specificity. One such isoform, CYP1A1, which has been cloned and sequenced from rainbow trout, has been shown to be orthologous to rat CYP1A1 and, as in mammals, is inducible up to several hundred-fold by planar aromatic hydrocarbons, PCBs and dioxins. It has been suggested that induction of CYP1A1 orthologues might provide a sensitive biomonitor for environmental pollution by mixtures of such compounds. In the current study, polyclonal antibodies directed against CYP1A1 purified from rat and trout liver were used to monitor induction of the CYP1A1 orthologue in hepatic microsomes from the fresh water species, the channel catfish (Ictalurus punctatus). Catfish from a local fish farm were induced in the laboratory by three daily injections of 50 mg/kg of the PCB mixture Aroclor 1254 and compared with fish taken from a site in central Arkansas—the Bayou Meto, known to be polluted with dioxin. Hepatic microsomal activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD) were measured and Western blot analysis carried out with the two antibodies. EROD was elevated in both the Aroclor-treated fish and in the Bayou Meto fish compared with untreated fish farm controls; smaller but significant increases were observed in PROD. Spearman's rank correlations of 0·74 and 0·89 were observed between EROD and immunoquantified cross-reactivity towards the rat CYP1A1 and trout CYP1A1 antibodies.     

Role of biotransformation in determining aldicarb toxicity in fish

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 V_max), 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.     

Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

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.