Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor.
(1/208)
Some flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and cause cell cycle arrest. The dependency of the cytostatic effects of five flavonoids (flavone, alpha-naphthoflavone, apigenin, 3'-methoxy-4'-nitroflavone and 2'-amino-3'-methoxyflavone) on a functional AHR was examined in AHR-containing rat hepatoma 5L cells and an AHR-deficient cell line (BP8) derived from the 5L line. The potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was cytostatic to the 5L line due to the induction of a G(1) arrest and dramatically elevated steady-state levels of CYP1A1 mRNA. TCDD affected neither the proliferation nor CYP1A1 mRNA contents of BP8 cells. With the exception of apigenin, the flavonoids under study induced G(1) arrest in both 5L and BP8 cells when used at concentrations at which they functioned as AHR agonists, but not antagonists. Apigenin-treated 5L and BP8 cultures primarily arrested in G(2)/M. The AHR-containing murine hepatoma cell line 1c1c7 arrested following exposure to AHR agonist concentrations of flavone and alpha-naphthoflavone, but not TCDD. Unlike the G(1) arrest observed in 5L cultures, the latter two flavonoids caused principally G(2)/M arrest in 1c1c7 cells. These studies demonstrate that the cytostatic activities of flavonoids do not require the AHR and the site of checkpoint arrest with a specific flavonoid can vary with cell type. (+info)
Antioxidant protection against PCB-mediated endothelial cell activation.
(2/208)
Certain environmental contaminants such as polyhalogenated aromatic hydrocarbons may be implicated in diseases of the vasculature by compromising normal functions of vascular endothelial cells. We have shown previously that 3,3',4,4'-tetrachlorobiphenyl (PCB 77), an aryl hydrocarbon (Ah) receptor agonist, can cause disruption of endothelial barrier function. This was supported by an increase in oxidative stress as measured by enhanced 2',7'-dichlorofluorescein (DCF) fluorescence and activation of the oxidative stress-sensitive transcription factor NF-kappaB. We have now tested the protective effects of antioxidants vitamin E (alpha-tocopherol) and pyrrolidine dithiocarbamate (PDTC) on endothelial cell activation induced by PCB 77. Only vitamin E completely blocked PCB 77-mediated endothelial barrier dysfunction. This protective effect by vitamin E was associated with a decrease in both oxidative stress, as measured by DCF fluorescence, as well as in NF-kappaB activation. Furthermore, vitamin E decreased PCB 77-mediated production of the inflammatory cytokine IL-6. Although pretreatment of endothelial cells with PDTC prevented the induction of NF-kappaB by PCB 77, this inhibition was not associated with a decrease in DCF levels or protection against endothelial barrier dysfunction. Pretreatment with alpha-naphthoflavone (alpha-NF), an Ah receptor partial antagonist and specific inhibitor of cytochrome P450 1A, partially protected against PCB 77-induced endothelial barrier dysfunction. This observation was paralleled by the fact that alpha-NF did not fully antagonize the PCB-induced increase in DCF in endothelial cells. Furthermore, PCB-mediated induction of NF-kappaB and production of IL-6 were only partially blocked by alpha-NF. Of all the tested compounds (vitamin E, PDTC and alpha-NF), vitamin E was most potent in blocking PCB 77-mediated endothelial cell activation. These data give an insight into the potential use of vitamin E and related antioxidants to limit PCB-mediated cell injury and into the use of alpha-NF to explore mechanisms underlying the injurious potential of Ah receptor agonists. (+info)
Human cytochrome P-450 metabolism of retinals to retinoic acids.
(3/208)
Retinoic acids have important pleiotropic biological effects and thus the potential for human cytochrome P-450s (CYPs) to mediate retinoic acid synthesis was investigated. We examined the retinoic acid synthetic activity of human cDNA-expressed CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A4+ cytochrome b(5) (b(5)), 3A5, and 4A11, expressed individually in insect cells together with NADPH-P-450 reductase. Only CYP1A1, 1A2, 1B1, and 3A4+b(5) converted all-trans-retinal (20 microM) to all-trans-retinoic acid with turnover numbers of 0.53, 0.18, 0.20, and 0.41 nmol/min/nmol P-450, respectively. With 9-cis-retinal as substrate, CYP1A2 exhibited a turnover number of 1.58 nmol/min/nmol P-450 whereas CYP1A1, 2C19, and 3A4+b(5) had turnover numbers of 0.40, 0.27, and 0.41 nmol/min/nmol P-450, respectively. For CYP3A4 activities with both retinals, b(5) was required. Kinetic analyses revealed that CYP1A1, 1A2, and 3A4+b(5) with all-trans-retinal had apparent K(m) values of 55, 356, and 255 microM, and V(max) values of 2.0, 8.3, and 6.3 nmol/min/nmol P-450, respectively, and with 9-cis-retinal had K(m) values of 77, 91, and 368 microM, and V(max) values of 2.7, 9.7, and 7.6 nmol/min/nmol P-450, respectively. The 9-cis retinoic acid synthetic activity of a group of 12 human liver microsomes correlated only with the CYP1A2 activity (r = 0.96), implicating CYP1A2 in human liver microsomal metabolism of 9-cis- retinal to 9-cis-retinoic acid. These studies have indicated that human CYPs are capable of catalyzing retinal to retinoic acid metabolism, but the physiological relevance of this metabolism is still unclear. (+info)
A novel 4 S [3H]beta-naphthoflavone-binding protein in liver cytosol of female Sprague-Dawley rats treated with aryl hydrocarbon receptor agonists.
(4/208)
beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo. (+info)
Dual role of human cytochrome P450 3A4 residue Phe-304 in substrate specificity and cooperativity.
(5/208)
The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by alpha-naphthoflavone, exhibiting a 2-fold decrease in the K(m) value for progesterone 6beta-hydroxylation in the presence of 25 microM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained the V(max) and decreased the K(m) for progesterone 6beta-hydroxylation, minimized stimulation by alpha-naphthoflavone, and decreased the rate of alpha-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the DeltaA(max) for spectral binding of alpha-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and alpha-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen. (+info)
Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay.
(6/208)
A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites. (+info)
Metabolism of the antidepressant mirtazapine in vitro: contribution of cytochromes P-450 1A2, 2D6, and 3A4.
(7/208)
The metabolism of the antidepressant mirtazapine (MIR) was investigated in vitro using human liver microsomes (HLM) and recombinant enzymes. Mean K(m) values (+/-S.D., n = 4) were 136 (+/-44) microM for MIR-hydroxylation, 242 (+/-34) microM for N-demethylation, and 570 (+/-281) microM for N-oxidation in HLM. Based on the K(m) and V(max) values, MIR-8-hydroxylation, N-demethylation, and N-oxidation contributed 55, 35, and 10%, respectively, to MIR biotransformation in HLM at an anticipated in vivo liver MIR concentration of 2 microM. Recombinant CYP predicted a 65% contribution of CYP2D6 to MIR-hydroxylation at 2 microM MIR, decreasing to 20% at 250 microM. CYP1A2 contribution increased correspondingly from 30 to 50%. In HLM, quinidine and alpha-naphthoflavone reduced MIR-hydroxylation to 75 and 45% of control, respectively, at 250 microM MIR. A >50% contribution of CYP3A4 to MIR-N-demethylation at <1 microM MIR was indicated by recombinant enzymes. In HLM, ketoconazole (1 microM) reduced N-desmethylmirtazapine formation rates to 60% of control at 250 microM. Twenty percent of MIR-N-oxidation was accounted for by CYP3A4 at 2 microM MIR, increasing to 85% at 250 microM, while CYP1A2 contribution decreased from 80 to 15%. Ketoconazole reduced MIR-N-oxidation to 50% of control at 250 microM. MIR did not substantially inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP1E2, and CYP3A4 activity in vitro. Induction/inhibition or genetic polymorphisms of CYP2D6, CYP1A2, and CYP3A4 may affect MIR metabolism, but involvement of several enzymes in different metabolic pathways may prevent large alterations in in vivo drug clearance. (+info)
Influence of beta-naphthoflavone on 7,12-dimethylbenz(a)anthracene metabolism, DNA adduction, and tumorigenicity in rainbow trout.
(8/208)
Metabolism, DNA adduction, and tumor induction by 7, 12-dimethylbenz(a)anthracene (DMBA) were examined in cultured trout liver cells and in vivo in trout. Modulating CYP1A1 activity indicated this enzyme plays a significant role in metabolizing DMBA to water-soluble compounds in isolated trout liver cells. The major DMBA metabolites identified in trout liver cells were 10-, 11-, 8,9-, and 5,6-DMBA dihydrodiols, and DMBA, 2- or 3- or 4-phenol; 7-OH-methyl-12-methyl-benz(a)anthracene and 12-OH-methyl-7-methyl-benz(a)anthracene were minor metabolites. A very small amount of DMBA-3,4-dihydrodiol was detected, and polar metabolites, which did not migrate with any DMBA metabolite standards, were observed. Incubating trout hepatocytes with DMBA-3, 4-dihydrodiol produced three prominent, nonpolar adducts indistinguishable from those in mouse embryo cells. However, DMBA-DNA adducts, formed in trout in vivo or in trout liver cells exposed to DMBA, were predominantly more polar than those formed in mouse embryo fibroblasts, and levels of DMBA-DNA adducts formed in trout liver cells were not significantly altered by modulating CYP1A1 activity. No significant repair of DMBA-DNA adducts was detected in cultured trout liver cells over a 48-h period, supporting previous studies indicating that fish are less efficient than mammals in repairing polyaromatic hydrocarbon DNA adducts. Compared to animals receiving DMBA alone, beta-naphthoflavone pretreatment in vivo did not affect hepatic CYP1A1, DMBA-DNA adducts, nor hepatic tumor response; but did significantly reduce tumor response in two other target organs. These results collectively indicate that DMBA bioactivation to DNA-binding metabolites in trout liver cells and mouse embryo cells predominantly involve different metabolic pathways to form the DNA-binding intermediates. (+info)