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.

In vitro investigations of the metabolism of Victoria pure blue BO dye to identify main metabolites for food control in fish

Although banned, dyes, such as Victoria pure blue BO (VPBO), are illicitly used in aquaculture to treat or prevent infections due to their therapeutic activities. The present study examined the formation of phase I and phase II metabolites derived from VPBO using trout liver microsomes and S9 proteins. The well-known malachite green (MG) dye was also studied as a positive control and to compare its metabolism with that of VPBO. First, we optimised the incubation conditions for the detection of VPBO and MG metabolites by studying the formation of cytochrome P450 (CYP) substrates. Using the determined conditions (2 h at 20 °C), we incubated VPBO with trout microsomal and S9 fractions induced with β-naphtoflavone, and analysed the supernatant in a LC-LTQ-Orbitrap-HRMS system. The in vitro assays led to the detection of 16 VPBO metabolites from Phase I reactions, arising in particular from reactions with CYP1A. No metabolites were detected from Phase II reactions. The main metabolite detected, deethyl-VPBO, was CID-fragmented to determine its chemical structure, and thus recommend a potential biomarker for the control of VPBO in farmed fish foodstuffs.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency.2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow.3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout.4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

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.

The integration of in vivo and in vitro metabolism assays to investigate the stereoselective behaviors of benalaxyl in rainbow trout (Oncorhynchus mykiss)

The bioconcentration and elimination of racemic benalaxyl (BX) in trout liver microsomes and in juvenile rainbow trout (Oncorhynchus mykiss) were investigated to determine whether the fish can bioconcentrate and degrade this fungicide enantioselectively. Both enantiomers of BX were extracted with organic solvents and evaluated using high-performance liquid chromatography. In the microsomes, BX degradation followed first-order kinetics, and the S−(+) enantiomer of BX was eliminated twice as rapidly as the R−(−) enantiomer, resulting in residues enriched for R−(−)−BX. In vivo experiment, chiral analysis showed an obvious selective bioconcentration of BX based on statistically altered enantiomer fractions (EFs) in the fish compared with the values in the water. The R−(−)−BX was initially preferentially bioconcentrated by rainbow trout and then dissipated more slowly than its antipode. The mean half-lives for individual enantiomers were calculated as 31.6 h for R−(−)−BX and 20.3 h for the S−(+)−form. The results of the study showed that the degradation of BX enantiomers was stereoselective in rainbow trout.

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.

Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): Comparison of basal‐level enzyme activities with rat and determination of xenobiotic intrinsic clearance in support of bioaccumulation assessment

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.

Cross-Species Comparison of Conazole Fungicide Metabolites Using Rat and Rainbow Trout (Onchorhynchus mykiss) Hepatic Microsomes and Purified Human CYP 3A4

Ecological risk assessment frequently relies on cross-species extrapolation to predict acute toxicity from chemical exposures. A major concern for environmental risk characterization is the degree of uncertainty in assessing xenobiotic biotransformation processes. Although inherently complex, metabolite identification is critical to risk assessment since the product(s) formed may pose a greater toxicological threat than the parent molecule. This issue is further complicated by differences observed in metabolic transformation pathways among species. Conazoles represent an important class of azole fungicides that are widely used in both pharmaceutical and agricultural applications. The antifungal property of conazoles occurs via complexation with the cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell wall synthesis. This mode of action has cause for concern regarding the potential adverse impact of conazoles on the broad spectrum of CYP-based processes within mammalian and aquatic species. In this study, in vitro metabolic profiles were determined for thirteen conazole fungicides using rat and rainbow trout (Oncorhynchus mykiss) liver microsomes and purified human CYP 3A4. Results showed that 10 out of the 13 conazoles tested demonstrated identical metabolite profiles among rat and trout microsomes, and these transformations were well conserved via both aromatic and aliphatic hydroxylation and carbonyl reduction processes. Furthermore, nearly all metabolites detected in the rat and trout microsomal assays were detected within the human CYP 3A4 assays. These results indicate a high degree of metabolic conservation among species with an equivalent isozyme activity of human CYP 3A4 being present in both the rat and trout, and provides insight into xenobiotic biotransformations needed for accurate risk assessment.

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.

Comparative in vitro metabolism of benzo[ a]pyrene by recombinant zebrafish CYP1A and liver microsomes from β-naphthoflavone-treated rainbow trout

The zebrafish ( Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from β-naphthoflavone (BNF)-treated rainbow trout ( Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with V max values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [ 33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Occurrence of pharmaceutical products in a municipal effluent and toxicity to rainbow trout ( Oncorhynchus mykiss) hepatocytes

Pharmaceutical and personal care products (PPCPs) are found in municipal effluents and represent the major sources of contamination for the aquatic environment. A preliminary chemical analysis of wastewater identified several compounds associated with PPCPs, including caffeine, ibuprofen, naproxen, oxytetracycline, novobiocin, carbamazepine, gemfibrozil, bezafibrate, trimethoprim, sulfamethoxazole, and sulfapyridine. The purpose of this study was to examine the cytotoxic and oxidative effects of these products and other wastewater-related products (i.e., coprostanol, cotinine, estradiol-17 β, nonylphenol, and cholesterol) in primary cultures of rainbow trout hepatocytes. The redox activity of various PPCPs in trout ( Oncorhynchus mykiss) liver microsomes was investigated in vitro by tracking the rate of oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the formation of lipid peroxidation (LPO) after a 60-min incubation period. In addition, primary cultures of rainbow trout hepatocytes were exposed to various drugs identified in the municipal effluent for 48 h at 15 °C. Our results show that most PPCPs (83%) accelerated the rate of NADPH oxidation in the presence of microsomes and 72% of them increased LPO in microsomal membranes. LPO levels were significantly correlated ( R = 0.5 ; P < 0.05 ) with the number of functional groups on the molecule's backbone (i.e., number of O, S, N, P/number of C and H) and negatively so ( R = - 0.44 ; P < 0.05 ) with the octanol/water partition coefficient, suggesting that nucleophilicity and hydrophobicity are related to oxidative activity for these compounds. Exposure of trout hepatocytes to these products leads in many cases to decreased cell viability, increased CYP3A-related monooxygenase activity (benzylether resorufin dealkylase), and LPO. No induction of CYP1A1-related activity (7-ethoxyresorufin O-deethylase) was observed. Moreover, municipal effluent extracts (ethanol) were able to increase all the above responses in a dose-dependent manner. These results suggest that the basic redox properties of PPCPs could influence oxidative metabolism in liver cells and lead to oxidative damage. These products have the potential to produce a toxic response in aquatic organisms and the above biomarkers were shown to respond readily to PPCPs in aquatic organisms.

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE?FPLC with ICP?(ORS)MS detection

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.

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.

Biotransformation of N-ethyl perfluorooctanesulfonamide by rainbow trout (Onchorhynchus mykiss) liver microsomes.

Rainbow trout (Onchorhynchus mykiss) liver microsomes were incubated with N-ethyl perfluorooctanesulfonamide [N-EtPFOSA, C8F17SO2NH(C2H5)], to examine the possibility of in vitro biotransformation to perfluorooctane sulfonate (PFOS, C8F17SO3-) and perfluorooctanoate (PFOA, C7F15COO-). Incubations were performed by exposing trout liver microsomes to N-EtPFOSA at 8 degrees C in the dark. Reaction mixtures were analyzed after incubation periods of 0, 2, 4, 8, 16, and 30 h for N-EtPFOSA, PFOS, PFOA, and perfluorooctanesulfonamide (PFOSA, C8F17SO2NH2), a suspected intermediate. Amounts of PFOS and PFOSA were found to increase with incubation time, but only background levels of PFOA were detected. Three possible reaction pathways are proposed for the conversion of N-EtPFOSA to PFOS: (i) direct conversion of N-EtPFOSA to PFOS by deethylamination accompanied by conversion of the sulfone group to sulfonate, (ii) deethylation of N-EtPFOSA to PFOSA, followed by deamination to form PFOS, and (iii) direct hydrolysis of N-EtPFOSA. These findings represent the first report indicating a possible biotransformation of a perfluorosulfonamide to PFOS in fish and may help to explain the detection of PFOS, which is relatively involatile, and thus not likely to undergo atmospheric transport, in biota from remote regions.

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.

Rate and capacity of hepatic microsomal ring-hydroxylation of phenol to hydroquinone and catechol in rainbow trout ( Oncorhynchus mykiss)

Rainbow trout ( Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol at 11 and 25 °C by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and catechol (CAT). An HPLC method with integrated ultraviolet and electrochemical detection was used for metabolite identification and quantification at low (pmol) formation rates found in fish. The Michaelis–Menten saturation kinetics for the production of HQ and CAT over a range of phenol concentrations were determined at trout physiological pH. The apparent Km's for the production of HQ and CAT at 11 °C were 14±1 and 10±1 mM, respectively, with Vmax's of 552±71 and 161±15 pmol/min per mg protein. The kinetic parameters for HQ and CAT at 25 °C were 22±1 and 32±3 mM (Km) and 1752±175 and 940±73 pmol/min per mg protein (Vmax), respectively. The calculated increase in metabolic rate per 10 °C temperature rise ( Q 10) was 2.28 for HQ and 3.53 for CAT production. These experiments assess the potential for metabolic bioactivation in fish through direct quantification of putative reactive metabolites at the low, but toxicologically significant, chemical concentrations found in aquatic organisms. This work initiates a series of studies to compare activation pathway, rate, and capacity across fish species, providing a basis for development of biologically-based dose response models in diverse species.

Characterisation of head kidney and liver microsomal 17-hydroxyprogesterone 21-hydroxylase activity in rainbow trout (Oncorhynchus mykiss) and the lack of regulatory effects of ACTH

The formation of 11-deoxycortisol from 17-hydroxyprogesterone was studied in rainbow trout (Oncorhynchus mykiss) head kidney and liver microsomes. The Km for head kidney microsomal 17-hydroxyprogesterone 21-hydroxylase measured in pooled microsomes from juvenile rainbow trout was 3.75 μM, and the Vmax was 41.4 pmol/(mg protein × min). The Km for liver microsomal 17-hydroxyprogesterone 21-hydroxylase was 31.0 μM, and the Vmax was 51.0 pmol/(mg protein × min). The intrinsic clearance values suggest that liver, in addition to head kidney, could significantly contribute to the formation of 11-deoxycortisol in rainbow trout. Progesterone inhibited both the head kidney and the liver microsomal 17-hydroxyprogesterone 21-hydroxylase activity competitively. The Ki for progesterone in head kidney was 1.8 μM and in liver 144 μM. The different Km and Ki values for microsomal 21-hydroxylation suggest that 21-hydroxylation of 17-hydroxyprogesterone in head kidney and liver is catalysed by different enzyme systems. Treatment of rainbow trout by adrenocorticotrophic hormone (ACTH) for 1.5 or 3 h (single injections) or three weeks (injections every other day) did not affect head kidney or liver17-hydroxyprogesterone 21-hydroxylase activity. ACTH may thus not regulate this enzyme activity. The increase in plasma cortisol levels 1.5 and 3 h after ACTH injection but not following repeated injections suggests that the hormone regulates the initial secretion of cortisol, but does not have a long-term regulatory effect upon cortisol production in rainbow trout.

Biochemical Responses of Fish Sac Fry and a Primary Cell Culture of Fish Hepatocytes Exposed to Polychlorinated Naphthalenes

Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment. Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited. The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes. A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac). The exposure was terminated 2 weeks later. The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups. Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells. Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ. Staining in control sac fry was weak or absent. Exposure of the primary cell culture of trout hepatocytes to a low doses (</=10 ng/ml) of the chlorinated naphthalenes increased significantly CYP1A-associated EROD activity and CYP1A mRNA content, HxCN-mix being the most potent and thereafter HpCN and Hallovax 1014. The higher doses (50-100 ng/ml) of each naphthalene also inhibited EROD activity. However, the content of CYP1A mRNA or the intensity of the CYP1A protein band (58 kDa) recognized by anti-trout CYP1A peptide antibodies were not decreased with increasing polychlorinated naphthalene (PCN) concentration, indicating that the inhibition was not due to reduced protein synthesis. Furthermore, in vitro analyses of the inhibitory potential of PCNs on CYP1A activity with trout liver microsomes suggested that these naphthalene preparations may be CYP1A substrates and act as competitive inhibitors of CYP1A catalyst. Our results demonstrate that highly chlorinated naphthalenes are potent modulators of fish CYP1A enzyme and suggest that hepatocytes and tubular epithelial cells are the cell types that may be vulnerable to their metabolic products for cell injury in fish sac fry.

Comparative metabolism of dibenzo[a,l]pyrene by liver microsomes from rainbow trout and rats

In order to assess the differences in the ability of fish and rat liver to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), we have investigated the metabolism of dibenzo[a,l]pyrene (DB[a,l]P), a highly potent carcinogenic PAH, by liver microsomes from 3-methylcholanthrene-treated Shasta rainbow trout ( Oncorhynchus mykiss) and rats. Rat liver microsomes metabolized DB[a,l]P at a slightly higher rate (1.3-fold) than trout liver microsomes. Compared to benzo[a]pyrene (B[a]P), DB[a,l]P was metabolized at a significantly lower rate by both rat and trout liver microsomes. Although the microsomes from the two species metabolized DB[a,l]P to qualitatively similar metabolites, they showed significant differences in the profile of the metabolites formed. The proportion of DB[a,l]P-11,12-diol, the proximate carcinogen of DB[a,l]P, formed by trout microsomes was over two-fold greater (32.6%) than the corresponding value for rat microsomes (15.6%). Unlike rat microsomes, trout microsomes metabolized DB[a,l]P to its K-region diol (8,9-diol) to a small extent (26.1 vs 3.6%). As previously noted with B[a]P, trout liver, compared to rat liver, appears to be more efficient in forming the proximate carcinogenic metabolite of DB[a,l]P but less efficient in producing its K-region diol, a non-carcinogenic metabolite.