-
[show abstract]
[hide abstract]
ABSTRACT: Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40 μM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24 h of treatment. By contrast, the aliphatic ITCs (40 μM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40μM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 06/2012; 50(8):2822-30. · 2.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome P450 (CYP) 1B1 and CYP2J have been studied in various mammals, but not in pig. The sequences encoding pig CYP1B1 and CYP2J34 were isolated from liver cDNA by RACE and sequenced. The open reading frames of pig CYP1B1 showed a higher sequence homology to bovine 1B1 (89%) than to dog 1B1 (88%) or to human 1B1 (85%). On the other hand, the coding sequence of pig CYP2J34 showed a similar homology (83-85%) to CYP2J of these species. From the substrate recognition sites (SRS 1-6) analysis of the deduced proteins, it was found that the porcine CYP1B1, unlike CYP2J34, completely shared the six SRS with the bovine counterpart. RT-PCR analysis of CYP1B1 and CYP2J34 expression in ten porcine tissues revealed that CYP1B1 was principally expressed in adrenal gland, whereas CYP2J34 was predominantly expressed in small intestine. These results further support the pig as an useful model for human.
Research in Veterinary Science 05/2011; 92(3):438-43. · 1.65 Impact Factor
-
Biotechnology Letters 03/2011; · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.
Biotechnology Letters 02/2011; 33(6):1201-6. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The protective effect of a powder of grain (Lisosan G) against cisplatin-induced toxicity in rats was studied. Male rats were fed with Lisosan G before injection of cisplatin and four days later they were killed and blood was collected along with hepatic, renal and testicular tissues. The results showed that cisplatin treatment increased plasma blood urea nitrogen, creatinine and hydrogen peroxide and decreased cytochrome P450 content in renal and hepatic tissues. It also reduced the plasmatic testosterone level and caused a depletion of testicular 17α-progesterone hydroxylase activity. In the group fed with Lisosan G and treated with cisplatin blood urea nitrogen and creatinine returned to the control level indicating a protective effect of Lisosan G. It was also observed that the ones fed with Lisosan G were able to attenuate the decrease in the P450-dependent activities and the activities of antioxidant enzymes as well. Lisosan G protected the testicular 17α-progesterone hydroxylase activity and increased the plasma testosterone level compared to animals treated only with cisplatin. Our results showed a protective effect of Lisosan G against the cisplatin induced toxicity. The protective effect of Lisosan G could be associated mainly with the attenuation of the oxidative stress and the preservation in antioxidant enzymes.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 10/2010; 49(1):233-7. · 2.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome P450 (CYP) 2Js have been studied in various mammals, but not in sheep, as an animal model used to test veterinary drug metabolism. Sheep CYP2J was cloned from liver messenger RNA (mRNA) by RACE. The cDNA, after modification at its N- and C-terminals, was expressed in Escherichia coli and the sheep CYP2J protein, purified by chromatography, was 80% homologous to human and monkey CYP2J2. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that CYP2J mRNA was expressed in liver, cortex, respiratory and olfactory mucosa, heart, bronchi, lung, spleen, small intestine and kidney. The purified enzyme was catalytically active towards aminopyrine, all-trans-retinoic acid, and particularly arachidonic acid forming 20-HETE, 19-HETE, and 18-HETE (about 86% of the total) and 14,15-, 11,12-, 8,9-, and 5,6-EETs (cis-epoxyeicosatrienoic acids; about 14% of total), with a regioselectivity similar to that shown by the mammalian CYP2J2s.
Xenobiotica 12/2009; 40(2):109-18. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The presence and inducibility of specific CYPs (1A1, 1A2, 1B1, 2B22, 3A22, 3A29 and 3A46) and the related transcriptional factors (AhR, CAR, PXR, and HNF4alpha) were investigated, at activity and/or transcriptional level, in liver, respiratory and olfactory mucosa of control and beta-naphthoflavone (betaNF)-treated pigs an agonist of AhR, or rifampicin (RIF), an agonist of PXR. Experiments with real-time PCR showed that CYP1A1 mRNA was enhanced by betaNF, although at different extent, in liver, respiratory and olfactory tissues, whereas mRNAs of CYP1A2 and 1B1 were increased only in liver. Accordingly, in microsomes of both nasal tissues, the transcriptional activation of CYP1A1 was accompanied by an induction of ethoxyresorufin deethylase activity (a marker of this isoform) but not of methoxyresorufin demethylase activity (a marker of CYP1A2). The rifampicin treatment resulted in a transcriptional activation of CYP2B22 and CYP3As genes in liver but not in respiratory and olfactory mucosa. In parallel, the marker activity of CYP2B (ethoxy 4-(trifluoromethyl)coumarin deethylase) and CYP3As (6beta-testosterone hydroxylase and benzyloxyquinoline debenzylase) were induced in liver microsomes but not in the nasal ones. Considering the transcriptional factors, the basal expression of AhR mRNA was found to be as high in liver as in both nasal tissues but not susceptible to induction by betaNF. Also PXR mRNA was found, aside liver, well expressed in the nasal tissues, whereas CAR and HNF4alpha mRNAs were barely detected. In any case, these transcripts appeared to be enhanced by RIF treatment. Our results demonstrated that in the respiratory and olfactory mucosa of pig, although the presence of AhR, only CYP1A1, but not 1A2 and 1B1 resulted to be inducible by betaNF. Similarly, it was observed that in these nasal tissues, although the presence of PXR, neither CYP2B22 nor any CYP3A resulted to be inducible by RIF. Thus, the regulation mechanism of CYP1A2, 1B1, 2B22, 3A22, 3A29, and 3A46, in the nasal mucosa involves tissue-enriched transcriptional factors others than AhR, CAR, PXR, and HNF4alpha, which are fundamental in liver.
Toxicology 07/2009; 260(1-3):47-52. · 3.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Porcine cytochrome P450 (CYP) 1A2 was purified to electrophoretic homogeneity from the hepatic microsomes of beta-naphthoflavone-treated male pigs. In a reconstituted system, this enzyme showed a good catalytic activity towards caffeine, acetanilide, and methoxyresorufin, all known markers of mammalian CYP1A2. Using 3'- and 5'-rapid amplification of coding DNA (cDNA) ends (RACE), we amplified from the liver RNA of control pigs a full-length 1827 bp cDNA containing an open reading frame of 1548 bp which encoded a putative CYP1A2 protein of 516 amino acids and an estimated Mr of 58 380 Da. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that the messenger RNA (mRNA) of CYP1A2 was expressed in liver, heart and nasal mucosa but not in lung, small intestine, kidney and brain. Using the pCW vector containing a N-terminal modified cDNA, pig CYP1A2 was expressed in Escherichia coli. 3-[(3-Chloroamidopropyl)dimethylmmonio]-1-propane-sulfonate (CHAPS)-solubilized E. coli preparations expressing CYP1A2 produced a functionally isoform which, in a reconstituted system, was catalytically active toward ethoxyresorufin and methoxyresorufin showing K(m)'s similar to those obtained with CYP1A2 purified from pig liver or human recombinant CYP1A2. Taken together, these results demonstrate that domestic pigs have a functionally active CYP1A2 gene well expressed in the liver with biochemical properties quite similar to those corresponding to the human enzyme.
Xenobiotica 11/2008; 38(12):1453-70. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It was previously found that fenarimol, vinclozolin or acephate, three of the most used pesticides worldwide, provoked a marked perturbation of murine cytochrome P450 (CYP)-linked monooxygenases. Here, to more closely mimic human exposure, it was investigated whether different pesticide combinations administered i.p. in male Swiss Albino CD1 mice in single or repeated fashion (daily, for three consecutive days), affect CYP-dependent oxidations. The four simulated mixtures showed a complex pattern of CYP induction and suppression, especially after repeated injection. For example, while fenarimol alone was the most inducing agent--reaching a 79-fold increase over control in testosterone 2alpha-hydroxylase--followed by vinclozolin and acephate, coadministration with the former markedly reduced induction. Coadministration with vinclozolin, determined various positive and negative modulations. An increase of CYP2B1/2 and CYP3A1/2-associated oxidases and a decrease of ethoxycoumarin metabolism was observed in the acephate and vinclozolin mixture. An equivalent or reduced CYP expression, if compared to double combinations, was seen using the complete mixture. Taken as a whole, the unpredictability of the recorded effects with simple mixtures, shrinks the misleading extrapolation performed on a single pesticide. If reproduced in human, such changes, altering either endogenous metabolism or biotransformation of ubiquitous toxins, might have public health implications.
Food and Chemical Toxicology 02/2008; 46(1):34-42. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An adequate folate intake minimizes the risk of various cancers and other disorders such as vascular diseases and neural tube defects. However, meta-analyses revealed difficulties in supporting the relationship between folate intake and the risk of cancer. Interestingly, there have been no reports to date on the potential ability of folate to modulate xenobiotic metabolising enzymes (XMEs), the inhibition of bioactivating Phase-I XMEs and/or induction of detoxifying Phase-II XMEs being one of the most evoked cancer chemopreventive strategies. Here, several CYP-dependent oxidations were studied in liver sub-cellular preparations from Sprague-Dawley rats receiving rodent chow supplemented with folic acid daily, for 1 or 2 consecutive months. Using either specific substrates as probes of different CYP isoforms or the regio- and stereo-selective metabolism of testosterone as a multibiomarker, we found that folic acid markedly inactivated most of the Phase-I XME analysed; up to 54% for the CYP1A1-linked deethylation of ethoxyresorufin in males, and up to 86% for the testosterone 2alpha-hydroxylase (CYP2C11) in females, after 2 months treatment. The Phase-II marker glutathione S-transferase significantly increased (~107%) after 1 month of supplementation in females only. These changes, if reproduced in humans might have public health implications. These data suggest caution in performing folate chemoprevention trials before its overall toxicological characterization has been fully addressed.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2008; 637(1-2):16-22. · 2.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.
Journal of Biochemical and Molecular Toxicology 02/2007; 21(1):32-40. · 1.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effect of acute and chronic dioxane administration on hepatic, renal, pulmonary and nasal mucosa P450 enzymes and liver toxicity were investigated in male rats. The acute treatment consisted of two doses (2 g/kg) of dioxane given for 2 days by gavage, whereas the chronic treatment consisted of 1.5% of dioxane in drinking water for 10 days. Both the acute and chronic dioxane treatments induced cytochrome P450 2B1/2- and P450 2E1-dependent microsomal monooxygenase activities (pentoxyresorufin O-depentylase and p-nitrophenol hydroxylase) in the liver, whereas in the kidney and nasal mucosa, only the 2E1 marker activities were enhanced. In addition in the liver, an induction of 2alpha-testosterone hydroxylase (associated with the constitutive and hormone-dependent P450 2C11) was also revealed, whereas the hepatic P450 4A-dependent omega-lauric acid hydroxylase was not enhanced by any dioxane treatment. These inductions were mostly confirmed by western blot analysis of liver, kidney and nasal mucosa microsomes. In the lung, no alteration of P450 activities was observed. To assess the mechanism of 2E1 induction, the hepatic, renal and nasal mucosa 2E1 mRNA levels were also examined. Following two kinds of dioxane administration, in the liver the 2E1 induction was not accompanied by a significant alteration of 2E1 mRNA levels, while both in the kidney and nasal mucosa the 2E1 mRNA increased about 2- to 3-fold, indicating an organ-specific regulation of this P450 isoform. Furthermore, dioxane was unable to alter the plasma alanine aminotransferase activity and hepatic glutathione (GSH) content, examined as an index of toxicity, when it was administered into rats with P450 2B1/2 and 2E1 preinduced by phenobarbital or fasting pretreatment. These results support the lack of or a poor formation of reactive and toxic intermediates during the biotrasformation of this solvent, even when its metabolism was enhanced by P450 inducers. The chronic administration of dioxane was also unable to induce the palmitoyl CoA oxidase, a marker of peroxisome proliferation, excluding this as a way to explain its toxicity. Thus, although the mechanism of dioxane carcinogenicity remains unclear, the present results suggest that the induction of 2E1 following a prolonged administration of dioxane might provide oxygen radical species, and thereby contribute to its organ-specific toxicity.
Archive für Toxikologie 03/2005; 79(2):74-82. · 4.67 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.
Aquatic Toxicology 09/2004; 69(3):259-70. · 3.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 1. 4-Biphenylaldehyde (4-BA) and 9-anthraldehyde (9-AA) were examined as substrates for cytochrome P450 (CYPs) enzymes in rat and human. Both aldehydes were oxidized by CYPs to fluorescent carboxylic acids, which can be assayed with a high sensitivity by an easy fluorimetric method. 2. With liver microsomes from control and induced rats, the oxidation of both 9-AA and 4-BA followed simple Michaelis-Menten kinetics. Only microsomes from rats pretreated with phenobarbital (a strong inducer of P4502B1/2) could increase (about threefold) the oxidation rates (V(max)) of both aldehydes above the control values, which were 6.7+/-1.1 and 3.3+/-0.6 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively. On the other hand, the (K)(m)'s, which were similar for both aldehydes (about 25 micro M), did not change significantly with any inducer. The use of purified rat CYP1A1, 2E1, 2B1 and 2C11 in a reconstituted system showed that only 2B1 and 2C11 could oxidize both substrates with a high turnover. 3. In human liver microsomes, the oxidation rates of these aldehydes (1.6+/-0.2 and 0.42+/-0.1 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively) were lower than those of rat but with similar K(m)'s(20-26 microm). 4. The oxidation of these aldehydes was also determined with cDNA-expressed CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 and with a characterized bank of 14 human liver microsomes. In a reconstituted system, only CYP2B6, 2A6, 3A4 and with a lower turnover 2C9 oxidized both substrates. 5. Among the CYP marker activities of the 14 human samples, good correlations were only observed between CYP3A-dependent 6 beta-testosterone hydroxylase and the oxidation of 4-BA (r = 0.74) or 9-AA (r = 0.80) and between the oxidation of 4-BA versus 9-AA (r = 0.74). Weak correlations were also found between the 2B6-linked S-mephenytoin N- demethylase and the oxidation of 4-BA (r = 0.58) or 9-AA (r = 0.65). 6. Inhibition experiments revealed that the oxidation of these aldehydes was inhibited by ketoconazole, 8-methoxypsoralene and sulphophenazole, selective inhibitors for P4503A6, 2A6 and 2C9, respectively. 7. In summary, based on the use of cDNA-expressed CYPs, correlation analysis and chemical inhibition, the metabolism in human liver microsomes of these aldehydes appears primarily catalysed by CYP3A, although CYP2A6, 2B6 and 2C9 may play a role. 9-AA and particularly 4-BA, owing to the high rate of its metabolism, may be two novel useful fluorescent probe substrates for assaying CYP activities in various species.
Xenobiotica 02/2003; 33(1):1-11. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.
Aquatic Toxicology 02/2003; 62(1):27-33. · 3.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effect of beta-naphthoflavone (beta-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, beta-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, beta-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg beta-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3-7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. beta-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose-response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg beta-NF and was the most sensitive measurement for CYP 1A-like induction. beta-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 10/2001; 130(1):133-44. · 2.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Praziquantel (PZQ) is a broadly effective trematocide and cestocide, widely employed in veterinary and human medicine. In view of several differences in both its pharmacokinetic profile in different animal species and in the cytochrome P450-dependent system between ruminant and nonruminant species, the present study was undertaken to determine the pharmacokinetics of this drug, its effects on the P450 system and the involvement of cytochrome P450 in its metabolism in 3-month-old lambs infested by Fasciola hepatica. Following both oral and i.m. administration, PZQ disposition was best described by a linear one-compartment open model with a rapid absorption and elimination. Although the PZQ dose used by the i.m. route was only half of that used by the oral route, the mean PZQ plasma concentration was higher after i.m. than after oral treatment. Oral treatment with 30 mg/kg/day of PZQ did not modify the mono-oxygenase activities tested, whilst the administration of PZQ at a dose of 60 mg/kg/day for 2 days caused a significant decrease in the P450 3A-dependent erythromycin N-demethylase and 6beta testosterone hydroxylase activities. From the incubation of microsomes from lambs not treated with PZQ, a single metabolite (PZQ 11b-OH or PZQ 1-OH) was identified by GC/MS analysis. By selective inhibition of the 3A subfamily performed with triacetyloleandromycin, the production of this metabolite declined by about 90% suggesting a prominent role of P450 3A isoforms in this oxidation. These features indicate that agents or drugs which are able to modulate P450 3A-dependent catalysis may interfere with the metabolism, bioavailability and therapeutic effects of PZQ.
Journal of Veterinary Pharmacology and Therapeutics 09/2001; 24(4):251-9. · 1.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, several P450-dependent monoxygenase activities in the liver, kidney, and nasal mucosa of ring-necked pheasants were examined. In addition, the presence and inducibility of P450 isoenzymes in the hepatic and renal tissues of pheasants were examined by using typical substrates and inducers of P450s along with polyclonal antibodies raised against mammalian isoforms. Anti-rat P450 1A1 recognized in microsomes of both pheasant liver and kidney a protein that was markedly induced by beta-naphthoflavone and accompanied by an increase of various monooxygenases, in particular, methoxyresorufin-O-demethylase (MROD) activity. Anti-rat P450 2E1 revealed in microsomes of the pheasant liver but not in kidney an immunoreactive protein that was slightly induced by acetone but not accompanied by an increase of para-nitrophenol hydroxylase activity. On the other hand, acetone treatment caused an induction of other hepatic monoxygenases including MROD, erythromycin N-demethylase, and 6beta-testosterone hydroxylase. These two latter activities, known to be markers for 3A isoenzymes in rodents, were also enhanced in pheasant liver by phenobarbital but not by dexamethasone. The treatment with these two inducers also lacked to point out hepatic and renal proteins immunorelated to P450 3A or 2B subfamily, suggesting that these isoforms may be not expressed in pheasant. On the other hand, anti-rat P450 2C11 recognized two immunorelated proteins in the liver of both control and treated pheasants. The treatment with clofibrate, a mammalian inducer of 4A subfamily, induced both in liver and kidney of pheasant: i) a protein that cross-reacted with anti rat P450 4A1 and ii) the (omega) and (omega-1) lauric acid hydroxylase activities, known to be associated in mammals to this P450 subfamily. In the nasal mucosa of pheasant, a protein immunorelated to P450 2A and some monooxygenase activities (i.e., 7-ethoxycoumarin O-deethylase) linked, in mammals, to this isoform have been found; by contrast a protein immunoreactive with anti P450 2G1 was not found. In conclusion, the immunochemical properties and monooxygenase activities of constitutive and inducible P450s in pheasants were different not only from those of mammals but also from those of chickens. The findings of the present work also suggest that the P450 induction profiles might provide a potential biomarker of pheasant exposure to chemicals or environmental pollutants in the wild-field or in the stock-farm.
Toxicology and Applied Pharmacology 10/2000; 167(3):237-45. · 4.45 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome P450 (CYP) enzymes of nasal tissue are relatively resistant to induction by classical inducers. In the present study, the effects of starvation on the expression of CYP1A, 2A, 2B, 2C, 2E, 2G, and 3A subfamilies in the nasal mucosa of rat were studied. Fasting for 72 hr caused an increase in 2E1-dependent p-nitrophenol hydroxylase and 1A-dependent ethoxy- (or methoxy) resorufin dealkylase activities, but did not affect either 2A-linked coumarin hydroxylase or the testosterone hydroxylase activity, the latter reaction being a marker of several CYPs including 2G1. Whereas increases in 2E1- and 1A- associated catalytic activities were accompanied by a concomitant increase in the corresponding apoproteins as determined by immunoblotting, immunoactive protein bands reactive with antibodies raised against rat 1A1, 2B1, 2C11, 3A1 or rabbit nasal 2A10/11 and 2G1 were not altered. Fasting also increased CYP2E1 and CYP1A2 on the mRNA level, but did not alter CYP1A1 mRNA as determined by hybridization with cDNA probes selective for these cytochromes. A reiterative administration of chlormethiazole, a specific inhibitor of 2E1 in liver, strongly inhibited many CYPs, including 2E1, 1A2, 2G1, and 2A in the nasal mucosa, but did not influence expression of 2B or 3A as determined by immunoblotting or catalytic activities. The chlormethiazole-mediated inhibition of 1A1 and 2E1 was demonstrated to be at the mRNA level. These results suggest that fasting induces the gene expression of 2E1 and 1A2 and that the mechanisms involved in the regulation of CYPs in the nasal mucosa are tissue-specific. The inducibility of the above-mentioned isoforms may have a significant role in the clearance of drugs and bioactivation of inhaled compounds.
Biochemical Pharmacology 07/2000; 59(11):1425-32. · 4.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The induction of a variety of drug-metabolizing enzymes by six anthraquinones (AQs) has been investigated in the liver and small intestine of rat. In the liver, the intragastric administration for 3 days of 100 mg/kg 9,10-anthraquinone (9,10-AQ). 1-hydroxy-AQ, 1,4-dihydroxy-AQ, but not 1,2-dihydroxy-AQ and 2-carboxy-AQ, resulted in a significant induction of the UDP-GT, DT-diaphorase, P450 1A-linked monooxygenase activities and in particular the methoxyresorufin-O-demethylase (MEROD), an activity dependent on P450 1A2. Immunoblot analysis indicated that 1-hydroxy-AQ and 1,4-dihydroxy-AQ induced P450 1A2 but not 1A1 and 9,10-AQ induced both P4501A2 and P4502B. Northern blotanalysis, using a cDNA probe for CYP 1A1 and CYP 1A2, confirmed that the AQs induce CYP 1A2 but not 1A1 mRNA. In the mucosa of small intestine, none of the above-mentioned enzymatic activities were enhanced following AQ administration. The induction mechanism of the hepatic enzymes by AQs is not known and it deserves a further study as it might be independent from the activation of the Ah-receptor as reported for other tricyclic compounds. The results from inhibition experiments showed that the hydroxylated AQs were strong inhibitors of P450 1A2-dependent monooxygenases. This suggests that long-term ingestion of certain AQs, may affect the toxicity of other components present in the diet through the hepatic induction or inhibition of P450 1A2.
Chemico-Biological Interactions 05/2000; 126(1):63-77. · 2.46 Impact Factor
