In vitro metabolism of isoline, a pyrrolizidine alkaloid from Ligularia duciformis, by rodent liver microsomal esterase and enhanced hepatotoxicity by esterase inhibitors. (49/208)

Isoline, a major retronecine-type pyrrolizidine alkaloid (PA) from the Chinese medicinal herb Ligularia duciformis, was suggested to be the most toxic known PA. Its in vitro metabolism was thus examined in rat and mouse liver microsomes, and its toxicity was compared with that of clivorine and monocrotaline after i.p. injection in mice. Isoline was more rapidly metabolized by both microsomes than clivorine and monocrotaline and converted to two polar metabolites M1 and M2, which were spectroscopically determined to be bisline (a deacetylated metabolite of isoline) and bisline lactone, respectively. Both metabolites were formed in the presence or absence of an NADPH-generating system with liver microsomes but not cytosol. Their formation was completely inhibited by the esterase inhibitors, triorthocresyl phosphate (TOCP) and phenylmethylsulfonyl fluoride, but not at all or partially by cytochrome P450 (P450) inhibitors, alpha-naphthoflavone and proadifen (SKF 525A), respectively. These results demonstrated that both metabolites were produced by microsomal esterase(s) but not P450 isozymes. The esterase(s) involved showed not only quite different activities but also responses to different inhibitors in rat and mouse liver microsomes, suggesting that different key isozyme(s) or combinations might be responsible for the deacetylation of isoline. Isoline injected i.p. into mice induced liver-specific toxicity that was much greater than that with either clivorine or monocrotaline, as judged by histopathology as well as serum alanine aminotransferase and aspartate aminotransferase levels. Isoline-induced hepatotoxicity was remarkably enhanced by the esterase inhibitor TOCP but was reduced by the P450 inhibitor SKF 525A, indicating that rodent hepatic esterase(s) played a principal role in the detoxification of isoline via rapid deacetylation in vivo.  (+info)

Effect of cytochrome P450 (CYP) inducers on caffeine metabolism in the rat. (50/208)

Our previous studies, carried out using rat cDNA-expressed cytochrome P450 (CYP) isoforms, liver microsomes and specific CYP inhibitors, showed that the 1-N- and 3-N-demethylation of caffeine at a therapeutic concentration was predominantly catalyzed by CYP1A2 and CYP2C, its 7-N-demethylation was governed by P450s of the CYP2C subfamily, while its 8-hydroxylation was specifically mediated by CYP1A2. The present study was aimed at corroborating the above-described results using another experimental model, i.e. a study of caffeine metabolism in the liver microsomes and specific CYP inducers. Animals received one of the following inducers: beta-naphthoflavone (100 mg/kg i.p. for 4 days), phenobarbital (10 mg/kg for 6 days or 100 mg/kg i.p. for 4 days), pregnenolone 16alpha-carbonitrile (100 mg/kg i.p. for 4 days) or 15% ethanol ( approximately 11 g/kg in drinking water for 6 days). Sixteen hours after the last dose of an inducer liver microsomes were prepared and the caffeine metabolism and CYP isoform activities (testosterone 2alpha-, 2beta-, 6beta-, 7alpha-, 16beta-hydroxylation and warfarin 7-hydroxylation) were investigated. beta-Naphthoflavone (mainly a CYP1A inducer and CYP2C11 inhibitor) potently accelerated the metabolism of caffeine, the effect on 7-N-demethylation being the weakest. Moreover, the influence of beta-naphthoflavone on caffeine metabolism was more potent at the substrate concentration of 100 microM than 800 microM, in particular in the case of 7-N-demethylation and 8-hydroxylation. Pregnenolone-16alpha-carbonitrile (mainly a CYP3A inducer and CYP2C11 inhibitor) moderately induced 8-hydroxylation only. Phenobarbital (an inducer of CYP2B and other CYPs and a CYP2C11 inhibitor) moderately stimulated the metabolism of caffeine, but practically did not affect 7-N-demethylation. Ethanol (mainly a CYP2E1 inducer) modestly increased the rates of the N-demethylation reactions. The presently obtained data confirm the pivotal role of CYP1A2 in the metabolism of caffeine, as well as the involvement of CYP3A in the 8-hydroxylation of caffeine and that of CYP2C11 in its 7-N-demethylation.  (+info)

Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine. (51/208)

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.  (+info)

Signaling pathway for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages. (52/208)

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.  (+info)

Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish. (53/208)

Polycyclic aromatic hydrocarbons (PAHs) are pollutants created by the incomplete combustion of carbon, and are increasing in the environment largely due to the burning of fossil fuels. PAHs occur as complex mixtures, and some combinations have been shown to cause synergistic developmental toxicity in fish embryos, characterized by pericardial edema and craniofacial malformations. Previous studies have indicated that in the zebrafish model, this toxicity is mediated by the aryl hydrocarbon receptor 2 (AHR2), and enhanced by inhibition of CYP1A activity. In this study, we further examined this interaction of the model PAH and AHR agonist beta-naphthoflavone (BNF) with and without the AHR partial agonist/antagonist and CYP1A inhibitor alpha-naphthoflavone (ANF) to determine (1) whether ANF was acting as an AHR antagonist, (2) what alterations BNF and ANF both alone and in combination had on mRNA expression of the AHR regulated genes cytochrome P450 (cyp) 1a, 1 b 1, and 1 c 1, and the AHR repressor (ahrr2) prior to versus during deformity onset, and (3) compare CYP1A enzyme activity with mRNA induction. Zebrafish embryos were exposed from 24-48 or 24-96 hpf to BNF, 1-100 microg/L, ANF, 1-150 microg/L, a BNF+ANF co-exposure (1 microg/L+100 microg/L), or a DMSO solvent control. RNA was extracted and examined by quantitative real-time PCR. Both BNF and ANF each individually resulted in a dose dependent increase CYP1A, CYP1B1, CYP1C1, and AHRR2 mRNA, confirming their activities as AHR agonists. In the BNF+ANF co-exposures prior to deformity onset, expression of these genes was synergistic, and expression levels of the AHR regulated genes resembled the higher doses of BNF alone. Gene induction during deformities was also significantly increased in the co-exposure, but to a lesser magnitude than prior to deformity onset. EROD measurements of CYP1A activity showed ANF inhibited activity induction by BNF in the co-exposure group; this finding is not predicted by mRNA expression, which is synergistically induced in this treatment. This suggests that inhibition of CYP1A activity may alter metabolism and/or increase the half-life of the AHR agonist(s), allowing for increased AHR activation. This study furthers a mechanistic understanding of interactions underlying PAH synergistic toxicity.  (+info)

Aryl hydrocarbon receptor mediates laminar fluid shear stress-induced CYP1A1 activation and cell cycle arrest in vascular endothelial cells. (54/208)

AIMS: We investigated the mechanisms of shear stress (SS)-induced activation of cytochrome P450 (CYP) 1A1 and cell cycle arrest with regard to the role of the aryl hydrocarbon receptor (AhR), since AhR mediates the expression of CYP1A1 induced by polycyclic aromatic hydrocarbons (PAHs) and is thought to be involved in the regulation of cell growth and differentiation. METHODS AND RESULTS: Human umbilical vein endothelial cells (ECs) were exposed to laminar SS and thereafter collected to evaluate the expression, activity, and transcription of CYP1A1 and the expression of AhR and cell cycle-related proteins. A physiological level of laminar SS (15 dynes/cm(2)) markedly increased the expression level and enzymatic activity of CYP1A1. SS stimulated CYP1A1 promoter activity without influencing mRNA stability. Loss of two functional xenobiotic response elements (XREs) in the 5'-flanking region of the CYP1A1 gene suppressed the SS-induced transcription of CYP1A1. Laminar SS stimulated the expression and nuclear translocation of AhR. alpha-Naphthoflavone, an AhR antagonist, and a small interfering RNA (siRNA) for AhR significantly suppressed SS-induced CYP1A1 expression. The siRNA also abolished SS-induced cell cycle arrest, the expression of the cyclin-dependent kinase inhibitor p21(Cip1), and dephosphorylation of retinoblastoma protein. CONCLUSION: Laminar SS stimulated the transcription of CYP1A1 through the activation of AhR in a way that is similar to the effects of PAHs. AhR was also involved in cell cycle arrest induced by SS. Our results suggest that sustained activation of AhR exposed to blood flow plays an important role in the regulation of EC functions.  (+info)

Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein. (55/208)

We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (K(D)=0.48 microM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or alpha-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator.  (+info)

BRCA1 transcriptional activity is enhanced by interactions between its AD1 domain and AhR. (56/208)

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