Identification of new 2-amino-3-methylimidazo[4,5-f]quinoline urinary metabolites from beta-naphthoflavone-treated mice.

Metabolism of the heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was evaluated in mice with and without 40 mg/kg beta-naphthoflavone (BNF). Following an oral dose of 40 mg/kg (14)C-IQ, a 24-h urine sample was collected. Metabolism was assessed by high-performance liquid chromatography, and metabolites were identified by electrospray ionization mass spectrometry. Three new metabolites were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline (m/z 217, [M + H](+)), 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline (m/z 393, [M + H](+)), and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline (m/z 233, [M + H](+)). These metabolites represented 21% of the total urinary radioactivity recovered. For BNF-treated mice, the abundance of metabolites observed was 5-O-glucuronide > m/z 217 > m/z 393 > 5-sulfate > m/z 233 > N-glucuronide > demethyl-IQ > sulfamate. In control mice, metabolite urinary abundance was 5-O-glucuronide > demethyl-IQ > sulfamate > N-glucuronide > m/z 217 > 5-sulfate. In liver slices from BNF-treated mice, synthesis of m/z 217 and 5-O-glucuronide was significantly reduced by ellipticine, a cytochrome P450 (P450) inhibitor, whereas sulfamate synthesis was significantly increased and demethyl-IQ was unchanged. Liver microsomes from BNF-treated mice produced m/z 217 and demethyl-IQ, with the former inhibited by ellipticine and furafylline, a selective 1A2 inhibitor, and the latter by ellipticine only. Injection (intraperitoneal) of demethyl-IQ into BNF-treated mice resulted in only a 30% conversion to three metabolites that were not observed in urine from animals receiving IQ. Results from BNF-treated mice showed significant IQ metabolism by hepatic P450s. Therefore, differences in metabolism between mice treated with and without BNF may affect IQ tumorigenicity.

Hepatic metabolism of contaminants in the terapontid fish, yellowtail trumpeter (Amniataba caudavittata Richardson).

The yellowtail trumpeter (Amniataba caudavittata) is an estuarine-dependent omnivorous fish found in the Swan-Canning Estuary, Western Australia. Thirty five fish were injected with either the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), the synthetic flavenoid beta-naphthoflavone (BNF), or used as controls. The fish were then sampled at 3 and 7 days postinjection. Induction of the enzyme ethoxyresorufin O-deethylase (EROD) activity was nonsignificant while ethoxycoumarin O-deethylase (ECOD) activity induction differed amongst treatments. A high interindividual variability in the EROD activity was observed. The measurement of sorbitol dehydrogenase in the serum (s-SDH) was elevated (BNF 2.2 times and B[a]P 3.2 times the control fish) demonstrating that liver cell damage had occurred. Increases in biliary metabolites of both B[a]P-type and pyrene-type (19 times and 3.4 times the controls respectively) indicated that detoxification of pyrene-type compounds had taken place. Fish of the Terapontidae family, such as the yellowtail trumpeter, were found to be suitable for biomonitoring the health of the Swan-Canning Estuary. A combination of ECOD activity, s-SDH, and the measurement of biliary metabolites represents a suitable suite of biomarkers for environmental monitoring of the sublethal effects of PAH pollution in these fish.

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

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

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

Upregulation of cytochromes P450 2B in rat liver by orphenadrine.

1 The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. 2 Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)-inducible CYP2B in rat liver. CYP2B protein was increased to approximately 14-fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40-fold. Corresponding increases in the activity of CYP2B-dependent androstenedione 16beta-hydroxylation were measured in microsomes from ORPH and PB-induced rats. 3 Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH-induced rat liver. Consistent with this finding, ORPH was found to activate a PB-responsive enhancer module in constitutive androstane receptor (CAR)-transfected Hep G2 cells. 4 Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post-translational component. In PB-pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. 5 These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post-translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH.

P-glycoprotein in the catfish intestine: inducibility by xenobiotics and functional properties.

The p-glycoprotein (pgp)-mediated multixenobiotic resistance (MXR) mechanism of aquatic animals has been associated with protection against pollution. Recent studies in mammals suggest that intestinal pgp may modulate intestinal bioavailability of dietary xenobiotics. In order to further delineate this mechanism in the catfish, these studies: (1) examined the pgp-related distribution in the intestine and liver of catfish, (2) evaluated the MXR response following exposure to various dietary xenobiotics and a prototypic pgp inducer and (3) evaluated pgp functional activity in membrane vesicles, using prototypic substrates and inhibitors. For this purpose, catfish were exposed in vivo to the pgp inducer vincristine (VIN), and the xenobiotics beta-naphthoflavone (BNF), benzo[a]pyrene (BaP), and 3,4,3',4'-tetrachlorobiphenyl (TCB). Membrane vesicles, prepared from liver and intestine (proximal and distal sections) of control and exposed catfish, were subjected to SDS PAGE, Western Blot, and detection with the pgp C219 monoclonal antibody. Transport activity was evaluated in vitro using the pgp substrate [3H]vinblastine (VBL), and the pgp inhibitor verapamil (VP). Immunoblot studies demonstrated a pgp-related protein of approximately 170 kDa in the intestine and liver of catfish. This protein appears to be very susceptible to degradation, and was present in higher levels in the liver, in comparison to the intestine, where regional differences were not observed. Dietary exposure to the pgp substrate VIN, or the xenobiotics BNF, BaP, and TCB, did not appear to affect pgp-related reactivity. Transport studies with VBL indicate that the pgp-related protein of the catfish intestine displays classic pgp-mediated multidrug resistance (MDR) characteristics, such as energy-dependency, and sensitivity to VP. These studies suggest that the pgp-related protein in the catfish intestine and liver is not only immunochemically, but also functionally related to the mammalian MDR. Moreover, the results presented indicate that pgp-related reactivity and transport in intestinal vesicles of catfish may be influenced by factors including method sensitivity, sample collection, sample preparation, and immunoblot conditions.

Tissue distribution and temperature-dependence of Anguilla anguilla L. EROD activity following exposure to model inducers and relationship with plasma cortisol, lactate and glucose levels.

Anguilla anguilla L. ethoxyresorufin-O-deethylase (EROD) elevation by 2.7 microM beta-naphthoflavone (BNF) 3 days water exposure, or 4 mg/kg ip exposure, was studied in four different organs--liver, kidney, gills, and intestine. The results demonstrated a significant increase in liver EROD activity for the two previous conditions, whereas kidney EROD activity only increased during the intraperitoneal exposure. A. anguilla was also exposed during 8, 16, and 24 h to water contaminated with 2.7 microM BNF or benzo[a]pyrene (BaP) at 20 degrees C and 25 degrees C. Both compounds significantly increased liver EROD activity from 8 up to 24 h. There was no significant difference in liver EROD activity elevation by both compounds, either at 20 degrees C or 25 degrees C. Liver EROD activity was demonstrated to be one of the first warning systems concerning the presence of polycyclic aromatic hydrocarbons (PAHs) in water. A. anguilla 3 h exposure to diesel oil water-soluble fraction (DWSF) significantly increased plasma cortisol and significantly decreased plasma lactate. A prolonged exposure beyond 3 h, i.e. 4 h, 2, 3, 4, and 6 days to the previous conditions demonstrated a significant liver EROD activity elevation from Day 2 up to 6, and a significant increase in erythrocytic nuclear abnormalities (ENA) at Day 6.

Cytochrome P4502E in vivo and in vitro in the dwarf goat: effects of enzyme induction and the applicability of chlorzoxazone as marker substrate.

Cytochrome P4502E activities, inducibility and the applicability of chlorzoxazone as a marker substrate for this enzyme were investigated in female dwarf goats. Goats were treated with either isoniazid or beta-naphthoflavone. Treatment with isoniazid resulted in a 1.4 fold increase of the chlorzoxazone hydroxylation rate in hepatic microsomes. Aniline- and p-nitrophenol hydroxylation rates were increased by roughly the same extent (1.6 and 1.25 fold resp.) and increased levels of cytochrome P4502E apoproteins were found by Western blotting. Treatment with the cytochrome P4501A inducer beta-naphthoflavone resulted in a 2.5 fold induction of the in vitro chlorzoxazone hydroxylation rate, whereas the hydroxylation rates of aniline and p-nitrophenol were not induced. After treatment with isoniazid, chlorzoxazone plasma clearance was increased from 5.0 mL/min/kg to 11.0 mL/min/kg. Chlorzoxazone was almost completely excreted in the urine as conjugated hydroxy metabolites. These results do not support the hypothesis that cytochrome P4502E is of particular importance in goats, as has been suggested earlier. Furthermore, chlorzoxazone has limited value as a marker substrate for this enzyme, since cytochrome P4501A enzymes appear to play an important role in its biotransformation.