Rainbow Trout Liver Microsomes Research Articles

Cytochrome P450 Inhibition by Antimicrobials and Their Mixtures in Rainbow Trout Liver Microsomes In Vitro.

Antimicrobials are ubiquitous in the environment and can bioaccumulate in fish. In the present study, we determined the half-maximal inhibitory concentrations (IC50) of 7 environmentally abundant antimicrobials (ciprofloxacin, clarithromycin, clotrimazole, erythromycin, ketoconazole, miconazole, and sulfamethoxazole) on the cytochrome P450 (CYP) system in rainbow trout (Oncorhynchus mykiss) liver microsomes, using 7-ethoxyresorufin O-deethylation (EROD, CYP1A) and 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD, CYP3A) as model reactions. Apart from ciprofloxacin and sulfamethoxazole, all antimicrobials inhibited either EROD or BFCOD activities or both at concentrations <500 µM. Erythromycin was the only selective and time-dependent inhibitor of BFCOD. Compared with environmental concentrations, the IC50s of individual compounds were generally high (greater than milligrams per liter); but as mixtures, the antimicrobials resulted in strong, indicatively synergistic inhibitions of both EROD and BFCOD at submicromolar (~micrograms per liter) mixture concentrations. The cumulative inhibition of the BFCOD activity was detectable even at picomolar (~nanograms per liter) mixture concentrations and potentiated over time, likely because of the strong inhibition of CYP3A by ketoconazole (IC50 = 1.7 ± 0.3 µM) and clotrimazole (IC50 = 1.2 ± 0.2 µM). The results suggest that if taken up by fish, the mixtures of these antimicrobials may result in broad CYP inactivation and increase the bioaccumulation risk of any other xenobiotic normally cleared by the hepatic CYPs even at biologically relevant concentrations. Environ Toxicol Chem 2022;41:663-676. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Development of a microplate method for the determination of hepatic UDP-glucuronosyltransferase activity in rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis niloticus)

Hepatic glucuronidation represents an important phase II biotransformation reaction in both mammals and fish. The kinetic characteristics of uridine 5′-diphosphate (UDP) glucuronosyltransferases (UDPGTs) in rainbow trout liver microsomes were examined using p-nitrophenol (p-NP) as an aglycone and UDP-glucuronic acid (UDPGA) as a glucuronyl donor according to an existing protocol. The kinetic data obtained with varying concentrations of p-NP best fit the Hill equation and UDPGT activity was successfully induced following an i.p. injection of β-naphthoflavone (β-NF). The assay was subsequently adapted to a microplate method for determination of UDPGT activity in microsomal samples obtained from rainbow trout as well as Nile tilapia. In contrast to rainbow trout, UDPGT activity of Nile tilapia was best described by Michaelis-Menten kinetics. Based on the linearity of p-NP glucuronide formation, a p-NP concentration of 0.60 mM and a UDPGA concentration of 6.89 mM were determined to be suitable for assaying UDPGT activity in samples from rainbow trout and Nile tilapia. The microplate method offers several advantages over the historical assay; most notably it enables the observation of successive kinetics which ensures that enzyme activity is calculated in the most linear (initial) rate of the reaction. It also provides practical advantages in terms of ease-of-use and efficiency. This may be relevant to researchers investigating exposure of wild or farmed fish to environmental or feed-borne contaminants which are substrates of UDPGTs.

Effects of salinity acclimation on the endocrine disruption and acute toxicity of bifenthrin in freshwater and euryhaline strains of Oncorhynchus mykiss

The pyrethroid insecticide bifenthrin is frequently detected at ng/L concentrations in tributaries of the San Francisco Bay Delta. The estuary is also experiencing increasing salinity through climate change and water redirection. To evaluate the impacts of hypersaline conditions on bifenthrin toxicity in anadromous salmonids of the San Francisco Bay Delta (CA, USA), a 14-d laboratory exposure was performed using 2 strains of Oncorhynchus mykiss (rainbow trout and steelhead) acclimated to freshwater and to 8 g/L and 17 g/L salinity. The fish were then exposed to nominal concentrations of 0 µg/L, 0.1 µg/L, and 1.5 µg/L bifenthrin. Rainbow trout exhibited significant mortality following exposure to 1.5 µg/L (1.07 ± 0.35 µg/L measured) bifenthrin in freshwater. Elevated levels of Na⁺ /K⁺ adenosine triphosphatase α1A mRNA subunit expression was observed in the gill of rainbow trout acclimated to hypersaline conditions relative to freshwater animals. No significant difference was noted in Na⁺ /K⁺ adenosine triphosphatase subunit levels in brains of either strain in freshwater or hypersaline conditions. Likewise, significant differences were not observed in plasma vitellogenin or steroid hormone concentrations in either strain whether maintained in freshwater or saltwater. Saltwater acclimation significantly reduced nicotinamide adenine dinucleotide phosphate-catalyzed biotransformation of bifenthrin in liver microsomes of rainbow trout but not of steelhead. The present study showed that, relative to steelhead, rainbow trout have different responses to bifenthrin acute toxicity as well as different rates of hepatic bifenthrin biotransformation and regulation of Na⁺ /K⁺ adenosine triphosphatase subunits in gills. These data indicate that significant differences exist between the strains and that animal life history may have important effects on the susceptibility of each strain to environmental contaminants.

Microsomal biotransformation of chlorpyrifos, parathion and fenthion in rainbow trout ( Oncorhynchus mykiss) and coho salmon ( Oncorhynchus kisutch): Mechanistic insights into interspecific differences in toxicity

Rainbow trout often serve as a surrogate species evaluating xenobiotic toxicity in cold-water species including other salmonids of the same genus, which are listed as threatened or endangered. Biotransformation tends to show species-specific patterns that influence susceptibility to xenobiotic toxicity, particularly organophosphate insecticides (OPs). To evaluate the contribution of biotransformation in the mechanism of toxicity of three organophosphate (phosphorothionate) insecticides, (chlorpyrifos, parathion and fenthion), microsomal bioactivation and detoxification pathways were measured in gills, liver and olfactory tissues in juvenile rainbow trout ( Oncorhynchus mykiss) and compared to juvenile coho salmon ( Oncorhynchus kisutch). Consistent with species differences in acute toxicity, significantly higher chlorpyrifos bioactivation was found in liver microsomes of rainbow trout (up to 2-fold) when compared with coho salmon. Although bioactivation to the oxon was observed, the catalytic efficiency towards chlorpyrifos dearylation (detoxification) was significantly higher in liver for both species (1.82 and 0.79 for trout and salmon, respectively) when compared to desulfuration (bioactivation). Bioactivation of parathion to paraoxon was significantly higher (up to 2.2-fold) than detoxification to p-nitrophenol in all tissues of both species with rates of conversion in rainbow trout, again significantly higher than coho salmon. Production of fenoxon and fenthion sulfoxides from fenthion was detected only in liver and gills of both species with activities in rainbow trout significantly higher than coho salmon. NADPH-dependent cleavage of fenthion was observed in all tissues, and was the only activity detected in olfactory tissues. These results indicate rainbow trout are more sensitive than coho salmon to the acute toxicity of OP pesticides because trout have higher catalytic rates of oxon formation. Thus, rainbow trout may serve as a conservative surrogate species for the evaluation of OP pesticides in coho salmon.

Impacts of hypersaline water on the biotransformation and toxicity of fenthion on rainbow trout ( Oncorhynchus mykiss), striped bass ( Morone saxatilis X Morone chrysops) and tilapia ( Oreochromis mossambicus)

Hypersaline water derived from agricultural practices primarily in arid landscapes can impact fisheries in streams receiving run-off from fields. Previous studies have indicated significant elevation in the toxicity of thio-ether pesticides by hypersaline water in certain species of euryhaline fish due to enhanced formation of sulfoxide metabolites which may be more toxic than the parent compounds. The objectives of this study were to evaluate the effects of salinity on the toxicity of the thio-ether organophosphate pesticide, fenthion in three species of euryhaline fish: rainbow trout ( Oncorhynchus mykiss), striped bass ( Morone saxatilis X Morone chrysops), tilapia ( Oreochromis mossambicus) and determine whether a relationship was observed between toxicity and enantioselective sulfoxidation. Stereochemical formation and total sulfoxide formation did not mirror acute toxicity in the three species exposed in fresh or hypersaline conditions. Mortality of striped bass and rainbow trout due to fenthion exposure was enhanced by hypersaline treatments and a trend towards increased toxicity was observed in tilapia. In liver microsomes of rainbow trout, inhibition of cytochrome P450 (CYP) caused a respective 7- and 3-fold increase in sulfoxide formation in liver microsomes fresh and saltwater fish, and a significant increase in the formation of the (+) R-sulfoxide. CYP inhibition also caused a significant elevation of the (+) R-sulfoxide in freshwater striped bass, but not in hypersaline-treated bass. The results indicated contribution by CYP in the sulfoxidation of fenthion as well as the formation of other metabolites in all three species. In summary, hypersaline conditions impacted fenthion toxicity under conditions that appeared to be independent of fenthion sulfoxide formation indicating a much more complex mechanism of action for compounds with phosphorothioate and thio-ether structural features potentially involving multiple oxidative pathways.

Effects Of Dichlorvos And Characterization Of Microsomal Nadph-Cytochrome P450 And NADH-ferricyanide Reductase In Rainbow Trout (Oncorhynchus Mykiss) Liver

The kinetics of oxidation of rainbow trout (Oncorhynchus mykiss) NADPH-cytochrome P450 reductase (NCPR) by cytochrome-c and NADH-ferricyanide reductase (NFR) by K3Fe(CN)6 were studied. NCPR and NFR activities from liver microsomes of rainbow trout (Oncorhynchus mykiss) had a specific activity of 0.0246 and 0.0350 nmoles/min/mg protein, respectively. The maximal rate of NCPR reaction was found at cytochrome-c concentrations above 2.1 mM and in the presence of cyt c. Vmax and Km were 0.0083 nmoles/min/mg and 1.14 mM. The maximal rate of NFR reaction was at K3Fe(CN)6 concentrations higher than 2.0 mM and in the presence of K3Fe(CN)6. Vmax and Km values were 1.04 nmoles/min/mg and 0.352 mM, respectively. The inhi- bition by dichlorvos (DDVP) on NCPR and NFR activity was not exhibited over the range 1.0-5.0 mM DDVP.

Metabolism of chrysene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of chrysene, a four-ring symmetrical carcinogenic polycyclic aromatic hydrocarbon (PAH), by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom-dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized chrysene at the rate of 30.1 and 82.2 pmol/mg protein/min, respectively. Benzo-ring diols (1,2-diol and 3,4-diol) were the major chrysene metabolites formed by liver microsomes from control and 3-MC-treated fish. However, the control microsomes produced a considerably higher proportion of chrysene 1,2-diol (benzo-ring diol with a bay region double bond) plus 1-hydroxychrysene, than 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in attacking the 1,2- position of the benzo-ring. On the other hand, 3-MC-induced microsomes did not show such a regioselectivity in the metabolism of chrysene. Control bullhead liver microsomes, compared to control rat liver microsomes, produced a considerably higher proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Like rat liver microsomes, bullhead liver microsomes produced only trace amounts of the K-region diol. Chrysene 1,2-diol and 3,4-diol formed by the liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R-enantiomers. Chrysene is metabolized by bullhead liver microsomal enzymes to its benzo-ring diols with a relatively lower degree of stereoselectivity compared to benzo[a]pyrene (a five-ring PAH), but with a higher degree of stereoselectivity compared to phenanthrene (a three-ring PAH). The data of this study, together with those from our previous studies with phenanthrene, benzo[a]pyrene and dibenzo[a,l]pyrene (a six-ring PAH), indicate that the regioselectivity in the metabolism of PAHs by brown bullhead and rainbow trout liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring dihydrodiols does.

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.

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.

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.

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.

Channel catfish liver monooxygenases: Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases

Multiple drugs and pesticides are used in the aquaculture of channel catfish in the Southeastern United States. However, little is known regarding the enzymatic metabolism of these chemicals in the fish. Western blots, utilizing polyclonal antibodies raised against five purified rainbow trout liver cytochrome P450 enzymes, revealed at least two protein bands that were approximately 50 kDa (CATL-1) and 53 kDa (CATL-2). Anti-trout LMC3 and LMC4 only hybridized with the 53 kDa protein, whereas anti-trout LMC1, LMC2, and LMC5 recognized both proteins. Cytochrome P450-catalyzed activities (testosterone and progesterone hydroxylases) associated with LMC1 and LMC5 were also found in catfish liver microsomes. These data suggest that at least two constitutive forms of cytochrome P450 are present in the liver of juvenile channel catfish. Western blots utilizing antibodies raised against rabbit-lung flavin-containing monooxygenases (FMO) showed hybridization with two proteins from rainbow trout liver microsomes, but no cross-reaction with nucrosomes from catfish liver. N, N,-Dimethylaniline N-oxidase and methimazole oxidase were observed in microsomes from trout, but were absent in catfish liver microsomes prepared in three different laboratories. Consequently, FMO do not appear to be present in liver microsomes from channel catfish or they are rapidly degraded during tissue homogenization.

Response of xenobiotic metabolizing enzymes in rainbouw trout (Oncorhynchus mykiss) to endosulfan, detected by enzyme activities and immunochemical methods

A single i.v. injection (75 μg/ kg body weight) of the insecticide endosulfan was administered to gonadally immature rainbow trout ( Oncorhynchus mykiss). Experiments were done with both technical and analytical grade endosulfan. In liver microsomes prepared from fish killed 24 h after administration, the cytochrome P-450-dependent monooxygenase activities of 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), and aldrin epoxidase (AE) were measured. In addition, NADPH-cytochrome c reductase (NCCR) was analyzed, and the content of a specific cytochrome P−450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anticod P−4501 A1 IgG. Technical grade endosulfan significantly induced EROD and AE to 160 and 144%, respectively, of the corresponding control values, while analytical grade endosulfan significantly increased only AE activity to 147% and tended to induce EROD to 162%. AHH and NCCR were not affected. The antibodies to cod P−4501 A1 recognized a single protein band ( M 1 = 58 Da) in the rainbow trout liver microsomes. The ELISA absorbances of this protein in the technical and analytical grade endosulfan treated fish were 151 and 138%, respectively, of the corresponding values in the controls. These results were supported by corresponding results from a 14 days study, where rainbow trout were exposed to technical grade endosulfan (8.3 μg/l water). In this study glutathione S-transferase (GST) activity towards 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also included, however, no effect on GST was observed.

Response of xenobiotic metabolizing enzymes of rainbow trout (Oncorhynchus mykiss) to the mono-ortho substituted polychlorinated PCB congener 2,3?,4,4?,5-pentachlorobiphenyl, PCB-118, detected by enzyme activities and immunochemical methods

The mono-ortho-substituted polychlorinated PCB congener 2,3',4,4',5'-pentachlorobiphenyl (PCB-118) was administered i.p. (30 mg/kg body weight) to gonadally immature rainbow trout (Oncorhynchus mykiss), of both sexes. In liver microsomes prepared from fish killed 4 days after administration, the cytochrome P450-dependent monooxygenase activities of 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), and aldrin epoxidase (AE) were measured. In addition, NADPH-cytochrome c reductase (NCCR) was analyzed, and the content of a specific cytochrome P450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anticod P450IA1 IgG. The monooxygenase parameters EROD and AHH were significantly induced to 558 and 268%, respectively, of the corresponding control values, while NCCR and AE activities were not affected. The antibodies to cod P450IA1 recognized a single protein band (Mr = 58,000 D) in the rainbow trout liver microsomes. The ELISA absorbance of this protein in the PCB-118 treated fish was 401% of the corresponding value in the controls. These results demonstrate that PCB-118 is an effective inducer of the subfamily cytochrome P450IA1 in rainbow trout liver microsomes.

Monooxygenase activity of fish liver in biomonitoring aquatic environment.

The hepatic biotransformation activity of four cultivated freshwater fish, Amur carp (Ctenopharyngodon idella), carp (Cyprinus carpio), rainbow trout (Salmo gairdneri) and tench (Tinca tinca) were studied one day after catching the fish from ponds and a river in the south of GDR and in fish kept for two weeks in clean filtrated and deacidified water. Aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities fell to about 20% during the stay in clean water in rainbow trout liver microsomes. The reductions were smaller in the other species studied.

The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate, a zwitterionic detergent, has been shown to exhibit superior membrane protein solubilizing characteristics as compared to nonionic detergents. Replacement of NP-40 with CHAPS in isoelectric focusing of rainbow trout liver microsomes has increased resolution markedly. The two-dimensional electrophoretic system described will allow effective resolution of up to 300 μg of crude microsomal protein. CHAPS exhibits no effect on the stability or type of pH gradient when compared to NP-40 during isoelectric focusing in the presence of urea.

Seasonal variation of hepatic biotransformation in female and male rainbow trout ( Salmo gairdneri)

1. 1. Seasonal and sex variations in the cytochrome P450 content and monooxygenase and glucuronidation activities were observed in the liver microsomes of rainbow trout cultivated in North-European waters. 2. 2. During the summer the specific monooxygenase activities lowered but the total calculated capacity remained unchanged (measured at 18°C) or increased (measured at environmental temperatures) due to liver growth. 3. 3. During water cooling in autumn the specific monooxygenase activity as well as the total capacity showed an ideal temperature compensation and remained unchanged if measured at environmental temperatures but increased considerably if measured at 18°C. 4. 4. Before the spawning time cytochrome P450 and the monooxygenase activities lowered in both sexes but more in females revealing a temporary sex difference which disappeared after spawning. 5. 5. The specific UDP glucuronosyltransferase activity lowered considerably during the cooling of environmental temperature, and thereafter an increase in both sexes was observed towards the spawning time (significantly higher in females) and coming summer.

In vitro metabolism of permethrin isomers by carp and rainbow trout liver microsomes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIn vitro metabolism of permethrin isomers by carp and rainbow trout liver microsomesAndrew H. Glickman, Toshio Shono, John E. Casida, and John J. LechCite this: J. Agric. Food Chem. 1979, 27, 5, 1038–1041Publication Date (Print):May 1, 1979Publication History Published online1 May 2002Published inissue 1 May 1979https://doi.org/10.1021/jf60225a008RIGHTS & PERMISSIONSArticle Views127Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (488 KB) Get e-Alerts Get e-Alerts