Trout Liver Microsomes Research Articles

Biotransformation of 4-methoxyphenol in rainbow trout ( Oncorhynchus mykiss) hepatic microsomes

Rainbow trout liver microsomes were used to study the O-demethylation and ring hydroxylation of 4-methoxyphenol (4-MP) (4-hydroxyanisole) at 11 and 25°C by directly measuring the production of the primary metabolite hydroquinone (HQ), 4-methoxycatechol (4-MCAT), and additional metabolites. An HPLC method with integrated ultraviolet (UV) and electrochemical detection (ECD) was developed for metabolite identification and quantification at low concentrations. Sample handling with appropriate buffers, solvents, low temperature and light prevented loss of extremely labile metabolites. Saturation kinetics for the production of HQ via O-demethylation of 4-MP (0.66–40 mM) was never achieved, with substrate solubility being the limiting factor. The linear rate of HQ formation at 11°C was 22.0±2.2 (coefficient±S.E., r 2=0.91) pmol min −1 per mg protein per mM substrate, and at 25°C was 34.0±1.3 ( r 2=0.99) pmol min −1 per mg protein per mM substrate. The second major microsomal metabolite 4-MCAT was also identified, with linear rates of ring hydroxylation determined to be 19.0±1.6 ( r 2=0.94) and 17.2±0.5 ( r 2=0.99) pmol min −1 per mg protein per mM substrate at 11 and 25°C, respectively. Unlike HQ production, the rate of 4-MCAT production was found to be similar at the two temperatures when linear formation rates were corrected for the effect of temperature on substrate and product solubility at 11°C. Measurement of `freely dissolved' fraction was essential to the accurate determination of ring hydroxylation and O-demethylation reaction rates in rainbow trout microsomes incubated at physiological temperature. Experimental conditions were shown to affect dissolved 4-MP and HQ at 11°C (verified using microdialysis) while not altering substrate and product levels at 25°C. Small but detectable levels of 1,4-benzoquinone were observed in 4-MP microsomal incubations. 1,2,4-Trihydroxybenzene was also detected, with possible routes of production through hydroxylation of HQ or O-demethylation of 4-MCAT. A metabolic scheme for bioactivation of 4-MP is proposed and the significance of observed metabolic conversions in rainbow trout microsomes discussed in relation to aquatic toxicity of 4-MP.

Carbonyl reduction of an anti-insect agent imidazole analogue of metyrapone in soil bacteria, invertebrate and vertebrate species

Carbonyl reduction to the respective alcohol metabolites of the anti-insect agent imidazole analogue of metyrapone, NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) and its parent compound metyrapone was characterized in subcellular fractions from soil bacteria, as well as insect, invertebrate and teleost species. The enzymes involved in this metabolic step were characterized with respect to their cosubstrate specificities, inhibitor susceptibilities, and immunological crossreactivities with antibodies directed against previously described bacterial and mammalian hydroxysteroid dehydrogenases/carbonyl reductases (HSD/CR). All fractions investigated rapidly reduced metyrapone, with highest specific activities found in insect, invertebrate and vertebrate fractions. Except for the insect fractions, all species examined reduced the NKI compound. Cosubstrate dependence and inhibitor specificities suggest that the enzymes described belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR). Immunological crossreactions to the previously established subgroup of HSD/CRs were found in trout liver microsomes and insect homogenates, but not in all bacterial extracts or earthworm microsomes. These findings suggest that the high CR activities found in these fractions belong to different subgroups of SDR or AKR.

Effects of docosahexaenoic (22:6n-3), tetracosapentaenoic (24:5n-3) and tetracosahexaenoic (24:6n-3) acids on the desaturation and elongation of n-3 polyunsaturated fatty acids in trout liver microsomes

The effects of long chain n-3 polyunsaturated fatty acids (PUFA) on the desaturation and elongation systems involved in the conversion of 18:3n-3 to 24:6n-3 were investigated. Microsomes were prepared from the livers of rainbow trout and incubated with 14C-labelled 18:3n-3 and cofactors required for elongation and/or desaturation in the presence of 22:6n-3, 24:5n-3 or 24:6n-3. The formation of 24:6n-3 was significantly inhibited in the presence of 50 μM 22:6n-3, 24:5n-3 or 24:6n-3, whereas the amount of radiolabelled 20:5n-3 formed was inhibited by only 24:5n-3 or 24:6n-3 at the same concentration. When malonyl-CoA was omitted from the incubation system to allow the measurement of desaturation in the absence of elongation, the Δ6 desaturation of 14C-18:3n-3 to 14C-18:4n-3 was inhibited by approximately 25% in the presence of 24:5n-3 or 24:6n-3 but was not affected by 22:6n-3. The Δ5 desaturation of 14C-20:4n-3 was not affected by the presence of any of the long chain PUFA and no significant effect of 18:3n-3, 22:6n-3 or 24:6n-3 on the Δ6 desaturation of 24:5n-3 to 24:6n-3 was observed. To permit the measurement of individual elongation reactions, KCN was included in the incubation medium to inhibit desaturation and 14C-labelled 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3 and 22:5n-3 were examined as substrates. 18:4n-3 and 22:5n-3 were more extensively used for elongation than 18:3n-3, 20:4n-3 and 20:5n-3. The presence of 22:6n-3, 24:5n-3 or 24:6n-3 in the incubation system had no effect on any of the specific elongations of any of the substrates examined. It is concluded that, in the conversion of 18:3n-3 to 24:6n-3 by trout liver microsomes, the Δ6 desaturation of 18:3n-3 may be subjected to direct feedback inhibition and that 24:5n-3 may be preferred over 18:3n-3 as a substrate for Δ6 desaturation.

Catalytic hydrogenation of polyunsaturated biological membranes: effects on membrane fatty acid composition and physical properties

The relationship between phospholipid saturation and membrane physical structure in a complex, highly polyunsaturated biological membrane (trout liver microsomes) has been studied by the graded and specific hydrogenation of polyunsaturated fatty acids. The homogeneous catalyst Pd(QS) 2 caused rapid and effective hydrogenation, increasing the proportion of saturated fatty acids from 20–30% up to 60%, without loss or fragmentation. Long chain, polyunsaturated fatty acids (20:5 ω3, 22:6 ω3) were rapidly converted to a large number of partially hydrogenated isomers, and ultimately to the fully saturated C20 or C22 fatty acids. C18 mono- and di-unsaturates showed slower rates of hydrogenation. Increased saturation was closely associated with an increased membrane physical order as determined by the fluorescence anisotropy probe, 1,6-diphenyl-1,3,5-hexatriene. However, extensive hydrogenation led to highly ordered membranes exhibiting a gel-liquid crystalline phase transition between 30 and 60°C. Polyunsaturated membranes can thus be converted into partially or substantially saturated membranes with measurable phase structure without direct alteration of other membrane components. This offers a less equivocal means of assessing the influence of polyunsaturation upon membrane structure and function.

In Vitro Metabolism of the N-Alkyl-N-(5-isothiazolyl)- and N-(Alkylisothiazolin-5-ylidene)phenylacetamides. Evidence of Proinsecticidal Activity

In vitro studies have demonstrated that N-(4-chloro-3-methyl-5-isothiazolyl)-N-methyl-2-[p-((α,α,α-trifluoro-p-tolyl)oxy]phenyl]acetamide (2a) undergoes NADPH-dependent metabolism, which is catalyzed by monooxygenase enzymes, in rat liver microsomes. The primary metabolite in rat was found to arise from ring-methyl hydroxylation, while N-demethylation to give N-(4-chloro-3-methyl-5-isothiazolyl)-2-[p-[(a,a,a-trifluoro-p-tolyl)oxy]phenyl]acetamide (1) was also observed to occur, but at a slower rate. In microsomal proteins prepared from tobacco budworm midgut tissues, the reverse was observed, as 1 is the predominant metabolite, while ring-methyl hydroxylation occurs at a slower rate. The overall rate of metabolism in trout liver microsomes was found to be 50-fold slower than in rat and afforded 1 as the predominant metabolite. Metabolism studies conducted on the N-alkyl-N-(5-isothiazolyl)- and N-(alkyl-isothiazolin-5-ylidene)phenylacetamides (2 and 3) have shown that the ring-alkylated isomers 3 were converted to 1 more rapidly than isomers 2 in all three species. In general, the rate of conversion to 1, or bioactivation, increased with increasing radical or carbocation stability of the alkyl group in rat and trout liver. In tobacco budworm, however, bioactivation was highest in the ethyl and n-propyl analogues. The ratio of bioactivation in tobacco budworm to that in trout, used as a predictor of selectivity, was observed to be highest with the methyl group.

Inhibitory effects of long chain n-3 polyunsaturated fatty acids on the desaturases and elongases of trout liver microsomes: Henderson, R. J., Buzzi, M., Burkow, I. C., Bayer, A. and Park, M. T. N.E.R.C. Unit of Aquatic Biochemistry, University of Stirling, Stirling, FK9 4LA. Scotland, N.I.L.U. & University of Tromso, Tromso, Norway

Inhibitory effects of long chain n-3 polyunsaturated fatty acids on the desaturases and elongases of trout liver microsomes: Henderson, R. J., Buzzi, M., Burkow, I. C., Bayer, A. and Park, M. T. N.E.R.C. Unit of Aquatic Biochemistry, University of Stirling, Stirling, FK9 4LA. Scotland, N.I.L.U. & University of Tromso, Tromso, Norway

Glucuronidation of Thyroxine and 3,5,3′-Triiodothyronine by Hepatic Microsomes in Rainbow Trout, Oncorhynchus mykiss

We studied the glucuronidation of thyroxine (T 4) and 3,5,3′-triiodothyronine (T 3) in the hepatic microsomal fraction of rainbow trout, Oncorhynchus mykiss. Uridine diphosphoglucuronosyl transferase (UDPGT) activity toward T 4 depended on both protein concentration (linear up to about 0.75 mg/ml) and time (linear up to 60 min). The optimal pH for glucuronidation was 6.8–7.8 units for T 4 and ≥ 8.5 units for T 3. At a common pH of 7.8, the apparent K m and V max values were, respectively, 6.0 μM and 42 nmol/mg protein/hr for T 4, and 1.1 μM and 4.3 nmol/mg protein/hr for T 3. At concentrations of 100 μM, T 4 inhibited T 3-glucuronidation, but T 3 did not inhibit T 4-glucuronidation. T 4-glucuronidation was inhibited by 3,3′, 5′-triiodothyronine (rT 3) and tetraiodothyroacetic acid (Tetrac); T 3-glucuronidation was inhibited by rT 3, T 4, Tetrac, and triiodothyroacetic acid. Therefore, analogues with two outer-ring iodines were the most effective inhibitors, showing that outer-ring iodine substitution influences UDPGT substrate specificity. Following a 15-min pre-incubation at 24°C, UDPGT thermal stability was higher for T 4 than T 3. Polyoxyethylene 10 cetyl ether (Brij 56) maximally stimulated UDPGT activity for both T 4 and T 3 about two-fold at 0.0125% (w/v) detergent, suggesting that the UDPGTs are transmembrane proteins with the active site facing the lumen of the microsomal vesicles. Clofibrate did not affect either T 4- or T 3-UDPGT activity. On a per mg microsomal protein basis, T 4-glucuronidation in the rat liver exceeded that in the trout 3-fold. We conclude that (a) trout liver microsomes have more than one form of UDPGT for the glucuronidation of T 4 and T 3 and (b) these trout UDPGTs share several properties of with those of the rat.

In VitroMetabolism of 7,12-Dimethylbenz[a]anthracene by Rainbow Trout Liver Microsomes and Trout P450 Isoforms

Liver microsomes from juvenile trout metabolized DMBA to unknown highly polar metabolites (X) and to DMBA-t-5,6-diol, DMBA-t-8,9-diol, 7-OHM-12-MBA, 7M-12-OHMBA, 2-OH- DMBA, 4-OH-DMBA, and trace amounts of DMBA-t-3,4-diol. Treatment of trout with β-naphthoflavone (BNF) and isosafrole (ISF) increased the formation of these products except for the hydroxymethyl derivatives of DMBA. The production of DMBA-t-3,4-diol, 2-OH-DMBA, and 4-OH-DMBA was much greater in BNF-induced liver microsomes than that in ISF-induced liver microsomes. In contrast, the yield of DMBA-t-8,9-diol and 7-OHM-12-MBA was greater in ISF-induced microsomes than that in BNF-induced microsomes. Trout CYP1A1 (P450 LM4b) purified from BNF-treated trout catalyzed the formation of the same metabolites generated by BNF-induced microsomes in the presence of added human microsomal EH. The constitutive forms of P450 isolated from untreated trout such as P450s LMC3, LMC4, and LMC5, CYP2M1 (P450 LMC1), and CYP2K1 (P450 LMC2) did not produce any of the DMBA metabolites (except for DMBA-t-8,9-diol by CYP2K1) generated by the trout microsomes. Generation of DMBA–DNA and DMBA–protein adductsin vitrowas enhanced by treatment of trout with BNF and by ISF to a lesser extent. Formation of adducts and DMBA diols by BNF-induced liver microsomes and by trout CYP1A1 was completely blocked by the CYP1A inhibitor ellipticine (100 μm). These results suggest that the BNF-inducible trout P450 (CYP1A), not the constitutive P450s, is the major catalyst for the biotransformation of DMBA to metabolites that bind to macromolecules.

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.

Role of molecular species catabolism in the temperature-induced restructuring of phosphatidylcholines in liver microsomes of thermally-acclimated rainbow trout (Oncorhynchus mykiss)

Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-(14)C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.

Effect of divalent metal ions on the microsomal aniline hydroxylase system of rainbow trout.

The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/ min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25 degrees C. The activity of the aniline hydroxylase system was competitively inhibited by Hg+2, Ni+2, Cd+2, and Zn+2, while Cu+2 and Fe+3 seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC50 values at fixed aniline concentration were estimated to be 0.45 mM for Hg+2, Ni+2, and Cd+2, 1.8 mM for Zn+2 and Fe+3, and 1.3 mM for Cu+2. Eadie-Hofstee plots gave identical Vmax values of approximately 0.046 nmol/min per mg of protein while K(m) values were increased in the presence of Hg+2, Ni+2, Cd+2, and Zn+2, indicating competitive inhibition. Both K(m) and Vmax values were affected by Fe+3 and Cu+2, suggesting noncompetitive inhibition. Ki values extracted from the Dixon plots were determined to be 0.23, 0.43, and 0.65 mM for Hg+2, Ni+2, and Cd+2, respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The Ki values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions.

Effect of divalent metal ions on the microsomal aniline hydroxylase system of rainbow trout

The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25°C. The activity of the aniline hydroxylase system was competitively inhibited by Hg+2, Ni+2, Cd+2, and Zn+2, while Cu+2 and Fe+3 seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC50 values at fixed aniline concentration were estimated to be 0.45 mM for Hg+2, Ni+2, and Cd+2, 1.8 mM for Zn+2 and Fe+3, and 1.3 mM for Cu+2. Eadie-Hofstee plots gave identical Vmax values of approximately 0.046 nmol/min per mg of protein while Km values were increased in the presence of Hg+2, Ni+2, CD+2, and Zn+2, indicating competitive inhibition. Both Km and Vmax values were affected by Fe+3 and Cu+2, suggesting noncompetitive inhibition. Ki values extracted from the Dixon plots were determined t be 0.23, 0.43, and 0.65 mM for Hg+2, Ni+2, and Cd+2, respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The Ki values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions. © 1997 John Wiley & Sons, Inc.

Effects of acclimation and assay temperature on outer‐ and inner‐ring thyroxine and 3,5,3′‐triiodo‐L‐thyronine deiodination by liver microsomes of rainbow trout, Oncorhynchus mykiss

AbstractWe studied effects of temperature on the deiodinating enzymatic pathways governing hepatic production of 3,5,3′‐triiodo‐L‐thyronine (T3) from L‐thyroxine (T4) in rainbow trout. Kinetics of outer‐ring deiodination (ORD) and inner‐ring deiodination (IRD) of T4 and T3 were measured in vitro at 4°, 12°, and 18°C using liver microsomes of trout that were acclimated for 21 days at 4°, 13°, and 18°C and fed on temperature‐compensated food rations. For all temperatures the Vmax (level of functional enzyme) and Vmax/Km (index of enzyme efficiency) values for the T3‐generating T4ORD pathway greatly exceeded values for the T3‐degrading T3IRD pathway; T4IRD and T3ORD pathways were negligible. The main effects of temperature occurred between 4°C and 12°/13°C; T4ORD Vmax/Km rose from 0.48 at 4°C to 1.62 at 12°/13°C and to 1.83 at 18°C. This response correlated with in vivo deiodination rates measured previously. T4ORD apparent Km did not change over the temperature acclimation range, suggesting an absence of temperature‐induced isozymes. However, Km was depressed at the assay temperature of 4°C, indicating a direct effect of temperature to enhance enzyme‐T4 substrate affinity. T4ORD Vmax was insensitive to assay temperature but was depressed in trout acclimated at 4°C. We conclude that from 4° to 18°C T4ORD is the main thyroid hormone deiodinating pathway in trout liver. T4ORD activity increases more than threefold from 4° to 13°C. This is due to a direct effect of temperature to decrease Km and to an acclimatory increase in Vmax. © 1995 Wiley‐Liss, Inc.

Comparative metabolism of 2-acetylaminofluorene by rainbow trout and rat liver subcellular fractions

In order to assess the cause of the resistance of rainbow trout ( Oncorhynchus mykiss), Shasta strain, to the hepatocarcinogenic effects of 2-acetylaminofluorene (AAF), we have investigated the metabolism of this model chemical by subcellular fractions from trout liver. Treatment of trout with 3-methylcholanthrene (3-MC) enhanced the rate of metabolism of AAF by liver microsomes. The major AAF metabolites produced by trout liver microsomes at saturating substrate concentration (34.3 and 19.5 μM AAF for control and 3-MC-induced microsomes, respectively) were 7-hydroxy-AAF and 5-hydroxy-AAF which represented 75 and 21% of total AAF metabolites, respectively for control microsomes, and 79 and 18% of total metabolites, respectively for induced microsomes. With both control and 3-MC-induced microsomes, N-hydroxy-AAF (the proximate carcinogen of AAF) was a minor metabolite accounting for less than 1% of total AAF metabolites. At a low substrate concentration (0.18 μM AAF), 7-hydroxy-AAF formation accounted for 95 and 91% of total metabolites for control and 3-MC induced microsomes, respectively, and a slight relative increase in N-hydroxy-AAF production (1.5–2.5%) was observed. Liver microsomes from untreated male Sprague-Dawley rats (a species susceptible to hepatocarcinogenesis by AAF) formed N-hydroxy-AAF in greater proportions (14-fold) and ring-hydroxylated metabolites in smaller proportions compared to trout liver microsomes. The activities of cytosolic N-hydroxy-AAF sulfotransferase and acyltransferase in trout liver (24 and 17 pmol/mg h −1, respectively) were found to be substantially lower than those reported for Sprague-Dawley rats. The low activity of hepatic enzymes implicated in the metabolic activation of AAF (AAF-N-hydroxylase, N-hydroxy-AAF-sulfotransferase, and N-hydroxy-AAF acyltransferase) coupled with a relatively high capacity of the trout liver for ring-hydroxylation (detoxification) of AAF may explain, in part, the resistance of rainbow trout to the hepatocarcinogenic action of AAF.

Retinoic acid hydroxylation in rainbow trout ( Oncorhynchus mykiss) and the effect of a coplanar PCB, 3,3′,4,4′-tetrachlorobiphenyl

When liver microsomes of rainbow trout ( Oncorhynchus mykiss) were incubated with [ 14C]-labeled retinoic acid plus NADPH, high performance liquid chromatography revealed the presence of radioactive peaks having elution times identical to those of 4-hydroxy- and 4-oxoretinoic acid. In the absence of NADPH, the radioactive peaks were not detected which is consistent with cytochrome P450-dependent 4-hydroxylation. In trout injected intraperitoneally with 5 μg/g of 3,3′,4,4′-tetrachlorobiphenyl (TCB) and sacrificed 56 days later, the hydroxylation rate was higher ( P < 0.0005) compared with control trout. No difference was observed between TCB-treated and control groups 7 days after injection. In contrast, cytochrome P4501-dependent ethoxyresorufin- O-deethylase activity was significantly higher in the TCB-treated group, both at 7 and 56 days. Liver retinoid stores were not affected by TCB treatment. These results clearly demonstrate that fish metabolize retinoic acid through a hydroxylation step which can be accelerated by a cytochrome P4501-inducing coplanar PCB. Given the pronounced biological activity of retinoic acid and closely related retinoids, increased retinoic acid metabolism could explain some of the effects of PCBs in fish.

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.

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on the Epidermal Growth Factor Receptor in Hepatic Plasma Membranes of Rainbow Trout (Oncorhynchus mykiss)

Time- and dose-dependent alterations in epidermal growth factor receptor (EGF-R) ligand binding and protein kinase activity were observed in hepatic plasma membranes of TCDD-treated rainbow trout. Trout were dosed by a single ip injection of TCDD in a corn oil vehicle. A single ip injection of TCDD (10 μg TCDD/kg body wt) caused a maximal reduction of EGF binding to hepatic plasma membranes by 10 days post-treatment and remained reduced until Day 40. EROD activity in the liver microsomes of TCDD-treated trout increased relative to that in untreated fish over the course of the study. Protein kinase C and tyrosine kinase activity as well as EGF-receptor phosphorylation was greater in livers of treated fish than in those of control fish within 5 days but returned to control levels by 40 days postinjection. In a dose-response study. EGF binding was reduced in a dose-dependent manner with an ED50 of 0.17 μg TCDD/kg wet wt while EROD activity was induced with an ED50 of 0.79 μg TCDD/kg. The reduction in EGF binding was correlated to an increase in EROD activity, protein kinase C activity, and tyrosine kinase activity but was negatively correlated to EGF-receptor phosphorylation. Of the parameters examined in both the time-course and dose studies, protein kinase C was the best predictor of the reduction of EGF binding to hepatic plasma membranes of rainbow trout. The results from this study are consistent with the hypothesis that the mode of action of TCDD on the EGF receptor is in part mediated through the protein kinase C activity. It also suggests that the toxic mode of action of TCDD is similar in rainbow trout and mammals.

Immunological characterization of flavin-containing monooxygenases from the liver of rainbow trout ( Oncorhynchus mykiss): sexual- and age-dependent differences and the effect of trimethylamine on enzyme regulation

Polyclonal antibodies raised against flavin-containing monooxygenase (FMO) enzymes purified from pig liver and rabbit lung were used in conjunction with N, N-dimethylaniline (DMA) N-oxidase to better characterize FMO from the liver of rainbow trout ( Oncorhynchus mykiss). Two proteins reacted with polyclonal antibodies raised against pig liver FMO (PL-1 and PL-2) and anti-rabbit lung FMO (RL-1 and RL-2). Although there was no difference in DMA N-oxidase observed between sexually mature male and female trout liver microsomes, RL-2 and PL-2 were significantly less than RL-1 and PL-1, respectively, in sexually mature females. FMO activity and protein content increased as fish aged. DMA oxidase and FMO isozymes were unaltered after pretreatment with the endogenous substrate trimethylamine. Since antibodies to the purified mammalian enzymes react with proteins of similar MW in trout, some forms of FMO appear to be structurally conserved through evolution.

Effect of 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) on the Epidermal Growth Factor Receptor in Hepatic Plasma Membranes of Rainbow Trout

Time- and dose-dependent alterations in epidermal growth factor receptor (EGF-R) ligand binding and protein kinase activity were observed in hepatic plasma membranes of TCDD-treated rainbow trout. Trout were dosed by a single ip injection of TCDD in a corn oil vehicle. A single ip injection of TCDD (10 μg TCDD/kg body weight) caused a maximal reduction of EGF binding to hepatic plasma membranes by 10 days post-treatment which remained reduced until Day 40. EROD activity in the liver microsomes of TCDD-treated trout increased relative to untreated fish over the course of the study. Protein kinase C and tyrosine kinase activity as well as EGF receptor phosphorylation was greater in livers of treated than in those of control fish within 5 days but returned to control levels by 40 days postinjection. In a dose-response study, EGF binding was reduced in a dose-dependent manner with an ED5O of 0.17 μg TCDD/kg wet weight while EROD activity was induced with an ED5O of 0.79 μg TCDD/kg. The reduction in EGF binding was correlated to an increase in EROD activity, protein kinase C activity, and tyrosine kinase activity but was negatively correlated to EGF receptor phosphorylation. Of the parameters examined in both the time course and dose studies, protein kinase C was the best predictor of the reduction of EGF binding to hepatic plasma membranes of rainbow trout. The results from this study are consistent with the hypothesis that the mode of action of TCDD on the EGF receptor is in part mediated through the protein kinase C activity. It also suggests that the toxic mode of action of TCDD is similar in rainbow trout and mammals.

Characterization of cytochrome P450 dependent mixed-function oxidase system of gilthead seabream ( sparus aurata; sparidae) liver

1. 1. The average cytochrome P450 content of gilthead seabeam liver microsomes was found to be 0.078 ± 0.012 ( n = 4) nmol/mg protein, which was about 50%, 24% and 13% of that measured in trout, sturgeon and scup liver microsomes, respectively. 2. 2. Gilthead seabream liver microsomes contained a relatively higher amount of cytochrome b5, as 0.32 ± 0.05 ( n = 4) nmol/mg protein. 3. 3. Effects of microsomal protein amount and temperature ranging from 15 to 30°C on aniline 4-hydroxylation and ethylmorphine N-demethylation activities were examined. No significant differences were observed between the enzyme activities at these temperatures. 4. 4. The liver microsomal aniline 4-hydroxylase activity of gilthead seabream in the 1990 fall (0.016–0.021 nmol p-aminophenol/min/mg protein) was 4.5–6 times higher than that in the 1991 spring (0.003–0.004 nmol p-aminophenol/min/mg protein). 5. 5. Liver microsomal ethylmorphine N-demethylase activity measured in 1991 spring (0/371) nm ol formaldehyde/min/mig protein) was 41% higher than that in 1990 fall (0.263 nmol formaldehyde/min/mg protein).