Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 62
Filter
1.
Toxicology ; 300(1-2): 31-45, 2012 Oct 09.
Article in English | MEDLINE | ID: mdl-22659317

ABSTRACT

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0-300mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman(®) qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300mg/kg/day). Hepatic PROD activity increased 2-3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia.


Subject(s)
Thyroxine/antagonists & inhibitors , Triclosan/adverse effects , Animals , Animals, Newborn/blood , Animals, Newborn/metabolism , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Female , Fetus/chemistry , Fetus/drug effects , Glucuronosyltransferase/drug effects , Glucuronosyltransferase/metabolism , Liver/drug effects , Liver/enzymology , Pregnancy , Radioimmunoassay , Rats , Rats, Long-Evans , Thyrotropin/blood , Thyroxine/blood , Triclosan/analysis , Triclosan/blood , Triiodothyronine/blood
2.
Toxicol Appl Pharmacol ; 254(1): 48-55, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21569787

ABSTRACT

Serum total thyroxine (T4) level was markedly decreased, without significant increases in the levels of hepatic T4-UDP-glucuronosyltransferase (T4-UGT) and serum thyroid-stimulating hormone, 3 days after treatment with 2,2',4,4',5,5'-hexachlorobiphenyl (CB153) (100mg/kg, ip) in both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-sensitive C57BL/6 and TCDD-resistant DBA/2 mice. Likewise, in either strain of mice, no CB153-mediated changes in the binding levels of [(125)I]T4 to serum proteins, such as transthyretin, albumin, and thyroxine binding globulin, were observed, while in CB153-pretreated C57BL/6 mice, but not in CB153-pretreated DBA/2 mice, the levels of biliary [(125)I]4T and [(125)I]T4-glucuronide at 90-120 min after injection of [(125)I]T4 slightly increased, as compared with those in the corresponding control mice. Concerning tissue distribution of [(125)I]T4, liver-selective increases in the [(125)I]T4 accumulation by CB153-pretreatment were observed in both C57BL/6 and DBA/2 mice, and the hepatic levels of [(125)I]T4 in the C57BL/6 and DBA/2 mice became more than 44% and 34% of the [(125)I]T4 dosed, respectively. The present findings indicated that the CB153-mediated decreases in the level of serum total T4in C57BL/6 and DBA/2 mice occur mainly through an increase in the accumulation of T4 in the liver.


Subject(s)
Polychlorinated Biphenyls/pharmacology , Thyroxine/blood , Animals , Blotting, Western , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Thyrotropin/blood , Thyroxine/antagonists & inhibitors , Triiodothyronine/blood
3.
Pharm Biol ; 49(2): 152-60, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21110733

ABSTRACT

CONTEXT: Byrsocarpus coccineus Schum. and Thonn. (Connaraceae) is a scandent shrub widely employed as a medicinal remedy for various disease conditions in West Africa. OBJECTIVE: This study evaluated fractions of B. coccineus for modulation of cytochrome P450 (CYP) enzyme activity, cytokine production, and proliferation. MATERIALS AND METHODS: The BROD (benzyloxyresorufin O-debenzylase) and BFCOD (benzyloxy-4-[trifluoromethyl]-coumarin O-debenzyloxylase) assays were used to evaluate effect on CYP2B1/2 and CYP3A4 enzyme activity. Effects on cytokine production and proliferation of HT29 cells were investigated using interferon expression assay and MTT (3-3[4,5-dimethyl-2-thiazolyl]-2-5-diphenyl-2H-tetrazolium bromide) assay, respectively. RESULTS: Fractions derived from the organic solvent extraction of B. coccineus produced significant (p<0.05) stimulation of human hepatic CYP2B1/2 activity in the BROD assay. The greatest effects were elicited at 1 ng/mL corresponding to ∼ 3-fold stimulation of enzyme activity. Enhancement of CYP3A4 enzyme activity was also observed in the BFCOD assay. Other fractions from the organic extract showed significant antiproliferative effects on HT29 cells at 100 µg/mL. Fractions obtained from the aqueous extract of B. coccineus (1 µg/µL) significantly stimulated the expression of IFNα2a and IFNß in peripheral blood mononuclear cells (PBMC), causing a maximum 26-fold increase of IFNα2a-transcript. DISCUSSION AND CONCLUSION: The effect on CYP suggests that B. coccineus may reduce the therapeutic efficacy of co-administered drugs. This justifies the need for proper education of patients by healthcare practitioners on the outcomes of drug-herb interactions. This study identifies several in vitro activities that could underlie the attributed uses of this plant in traditional African medicine (TAM).


Subject(s)
Connaraceae/chemistry , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP3A/drug effects , Plant Extracts/pharmacology , Cell Proliferation/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP3A/metabolism , Cytokines/biosynthesis , Cytokines/drug effects , Gene Expression Regulation/drug effects , HT29 Cells , Humans , Interferon alpha-2 , Interferon-alpha/drug effects , Interferon-alpha/genetics , Interferon-beta/drug effects , Interferon-beta/genetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Medicine, African Traditional , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Recombinant Proteins
4.
Hypertension ; 55(6): 1461-7, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20439821

ABSTRACT

Cytochrome P450 1B1, expressed in vascular smooth muscle cells, can metabolize arachidonic acid in vitro into several products including 12- and 20-hydroxyeicosatetraenoic acids that stimulate vascular smooth muscle cell growth. This study was conducted to determine whether cytochrome P450 1B1 contributes to angiotensin II-induced rat aortic smooth muscle cell migration, proliferation, and protein synthesis. Angiotensin II stimulated migration of these cells, measured by the wound healing approach, by 1.78-fold; and DNA synthesis, measured by [(3)H]thymidine incorporation, by 1.44-fold after 24 hours; and protein synthesis, measured by [(3)H]leucine incorporation, by 1.40-fold after 48 hours. Treatment of vascular smooth muscle cells with the cytochrome P450 1B1 inhibitor 2,4,3',5'-tetramethoxystilbene or transduction of these cells with adenovirus cytochrome P450 1B1 small hairpin RNA but not its scrambled control reduced the activity of this enzyme and abolished angiotensin II- and arachidonic acid-induced cell migration, as well as [(3)H]thymidine and [(3)H]leucine incorporation. Metabolism of arachidonic acid to 5-, 12-, 15-, and 20-hydoxyeicosatetraenoic acids in these cells was not altered, but angiotensin II- and arachidonic acid-induced reactive oxygen species production and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activity were inhibited by 2,4,3',5'-tetramethoxystilbene and cytochrome P450 1B1 small hairpin RNA (shRNA) and by Tempol, which inactivates reactive oxygen species. Tempol did not alter cytochrome P450 1B1 activity. These data suggest that angiotensin II-induced vascular smooth muscle cell migration and growth are mediated by reactive oxygen species generated from arachidonic acid by cytochrome P450 1B1 and activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase.


Subject(s)
Angiotensin II/pharmacology , Cell Movement/drug effects , Cytochrome P-450 CYP2B1/metabolism , Muscle, Smooth, Vascular/drug effects , Reactive Oxygen Species/metabolism , Superoxides/metabolism , Animals , Aorta/cytology , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Cytochrome P-450 CYP2B1/drug effects , DNA/metabolism , Muscle, Smooth, Vascular/growth & development , Muscle, Smooth, Vascular/physiology , Proteins/metabolism , Rats , Renin-Angiotensin System/physiology , Sensitivity and Specificity
5.
J Pharmacol Exp Ther ; 333(3): 663-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20200115

ABSTRACT

The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence (296)FFAGTSSTTLR(308) in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 A, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.


Subject(s)
Acetylene/analogs & derivatives , Cytochrome P-450 CYP2B1/drug effects , Threonine/drug effects , Acetylene/pharmacology , Amino Acid Sequence , Capillary Electrochromatography , Catalytic Domain/drug effects , Cytochrome P-450 CYP2B1/chemistry , Databases, Protein , Escherichia coli , Heme/chemistry , Heme/metabolism , Hydrolysis , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Recombinant Proteins/metabolism , Spectrum Analysis , Threonine/chemistry , Trypsin
6.
J Nutr ; 139(12): 2252-6, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19812219

ABSTRACT

Alk(en)yl sulfides have been found to be responsible for the anticancer, antithrombotic, and antioxidant effects of garlic. We sought to identify the most potent structure of sulfides that exhibits a hepatoprotective effect against carbon tetrachloride (CCl(4))-induced acute liver injury in rats. Rats were pretreated with diallyl trisulfide (DATS) i.g. at a dose of 500 micromol/kg body weight for 5 d. On d 6, CCl(4) was administered i.g. at a dose of 2.5 mL/kg body weight. Twenty-four hours after CCl(4) administration, rats were killed and plasma and liver samples collected. DATS pretreatment significantly suppressed the CCl(4)-induced elevation of plasma aspartate aminotransferase and alanine aminotransferase activities (P < 0.05). Histological observations supported the hepatoprotective effects. Western blot and spectrophotometric analyses indicated that DATS suppressed cytochrome P450 2E1 activity and its protein level and elevated those of glutathione S-transferase. Dipropyl trisulfide (DPTS), which is a saturated alkyl chain analogue of DATS, did not affect CCl(4)-induced liver toxicity or drug-metabolizing enzymes. These results suggest that hepatoprotective activity of trisulfides is due to their regulation of drug-metabolizing enzymes. Furthermore, the effects of 6 kinds of alk(en)yl trisulfides, including DATS and DPTS, on phase II enzyme activity were examined in rats. Alk(en)yl trisulfides were administered i.g. (500 micromol/kg body weight) to rats for 5 d. Only the allyl group-containing DATS and allyl methyl trisulfide enhanced these activities.


Subject(s)
Allyl Compounds/pharmacology , Carbon Tetrachloride Poisoning/prevention & control , Liver/pathology , Sulfides/pharmacology , Alanine Transaminase/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/drug effects , Aspartate Aminotransferases/metabolism , Carbon Tetrachloride Poisoning/pathology , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP2E1/drug effects , Cytochrome P-450 CYP2E1/metabolism , Liver/drug effects , Liver/enzymology , Male , Rats , Rats, Wistar , Soybean Oil/pharmacology , Thiobarbituric Acid Reactive Substances/metabolism , Weight Gain
7.
Drug Metab Lett ; 3(2): 108-14, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19601872

ABSTRACT

Drug candidates with the propensity to induce rat CYP1A1 or 2B1 isoforms are believed to possess a greater tendency to induce hepatic tumors in oncogenicity studies. We have previously published on a manual rat liver slice assay that showed a satisfactory relationship between in vitro CYP2B1 m-RNA induction using real time PCR and the ex vivo pentoxyresorufin O-dealkylase (PROD) activity in liver microsomes prepared from rats treated daily via the oral route for 14 consecutive days with inducers or non-inducers. We now describe this automated in vitro high throughput liver slice technique to screen out drug candidates that are potent rodent CYP1A1 and/or CYP2B1 inducers. A good concordance between in vitro and in vivo data was observed for both CYP1A1 (100 %) and CYP2B1 (90%) isoforms. Automation of key steps has enabled us to increase the annual screening throughput from 200 (manual) to 1500 compounds. The increase in throughput allowed the quick development of structure-induction relationships (SIR's) for multiple drug discovery programs in a facile manner.


Subject(s)
Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP2B1/drug effects , Enzyme Induction/drug effects , Administration, Oral , Animals , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/metabolism , Drug Discovery/methods , Liver/enzymology , Male , Microsomes, Liver/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
8.
J Toxicol Environ Health A ; 71(8): 521-32, 2008.
Article in English | MEDLINE | ID: mdl-18338287

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion that are commonly inhaled by workers in the dusty trades. Many PAHs are metabolized by cytochrome P-4501A1 (CYP1A1), which may facilitate excretion but may activate pulmonary carcinogens. PAHs also stimulate their own metabolism by inducing CYP1A1. Recent studies suggest that respirable coal dust exposure inhibits induction of pulmonary CYP1A1 using the model PAH beta-naphthoflavone. The effect of the occupational particulate respirable crystalline silica was investigated on PAH-dependent pulmonary CYP1A1 induction. Male Sprague-Dawley rats were exposed to intratracheal silica or vehicle and then intraperitoneal beta-naphthoflavone, a CYP1A1 inducer, and/or phenobarbital, an inducer of hepatic CYP2B1, or vehicle. Beta-naphthoflavone induced pulmonary CYP1A1, but silica attenuated this beta-naphthoflavone-induced CYP1A1 activity and also suppressed the activity of CYP2B1, the major constitutive CYP in rat lung. The magnitude of CYP activity suppression was similar regardless of silica exposure dose within a range of 5 to 20 mg/rat. Phenobarbital and beta-naphthoflavone had no effect on pulmonary CYP2B1 activity. Both enzymatic immunohistochemistry and immunofluorescent staining for CYP1A1 indicated that sites of CYP1A1 induction were nonciliated airway epithelial cells, endothelial cells, and the alveolar septum. Using immunofluorescent colocalization of CYP1A1 with cytokeratin 8, a marker of alveolar type II cells, the proximal alveolar region was the site of both increased alveolar type II cells and decreased proportional CYP1A1 expression in alveolar type II cells. Our findings suggest that in PAH-exposed rat lung, silica is a negative modifier of CYP1A1 induction and CYP2B1 activity.


Subject(s)
Air Pollutants/adverse effects , Cytochrome P-450 CYP1A1/metabolism , Dust , Particulate Matter/adverse effects , Pulmonary Alveoli/metabolism , Silicon Dioxide/adverse effects , Silicosis/physiopathology , Animals , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Disease Models, Animal , Enzyme Induction/drug effects , Enzyme Inhibitors/administration & dosage , Inhalation Exposure/adverse effects , Male , Occupational Exposure/adverse effects , Pulmonary Alveoli/pathology , Rats , Rats, Sprague-Dawley , beta-Naphthoflavone/administration & dosage
9.
Environ Toxicol Chem ; 27(1): 220-5, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18092871

ABSTRACT

As the active metabolites of polychlorinated biphenyl (PCBs), hydroxylated polychlorinated biphenyls (OH-PCBs) are found in wildlife and human tissues. They have been proposed as main contributors for endocrine disruption of PCBs in living organisms. In this study, mono-ortho PCB 156 and its hydroxylated metabolites 4'-OH-PCB 159, 4'-OH-PCB 121, and 4'-OH-PCB 72 were selected to investigate the toxic effects on rat hepatoma H4IIE cell line and rat thyroid follicle FRTL-5 cell line at concentrations of 1, 10(2), 10(4) nM. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) activities were determined with micro-EROD/PROD to indicate cytochrome P4501A1 (CYP1A1) and cytochrome P4502B (CYP2B) induction in the H4IIE cell after exposure for 72 h. To assess thyroid disruption of these compounds, thyroglobulin concentrations also were detected inside FRTL-5 cell with immunocellularchemistry and in its medium with radioimmunoassay after exposure for 24 h. Significant inductions of EROD activity by PCB156 at 10(2) and 10(4) nM (p < 0.05) were observed, but no effects by the three OH-PCBs in H4IIE cell line. 7-Pentoxyresorufin-O-dealkylase activities were induced only by 10(4) nM of PCB156 and the three OH-PCBs (p < 0.05). Meanwhile, significant increases of thyroglobulin concentrations were observed in the medium of FRTL-5 cell exposed to 4'-OH-PCB 121 and 4'-OH-PCB 72 at all of the test concentrations (p < 0.05), but not to the other compounds. The results demonstrated that mono-ortho PCBs mainly could be metabolized to hydroxylated metabolites through CYP1A1 instead of CYP2B. Moreover, after being metabolized, OH-PCBs still sustained the ability to induce PROD activity and did exhibit the disruption on thyroglobulin synthesis/excretion in rat cells.


Subject(s)
Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP2B1/drug effects , Polychlorinated Biphenyls/pharmacology , Thyroglobulin/biosynthesis , Animals , Cell Line , Cell Line, Tumor , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/metabolism , Dose-Response Relationship, Drug , Endocrine Disruptors/pharmacology , Hydroxylation , Liver/enzymology , Metabolism , Polychlorinated Biphenyls/metabolism , Rats , Thyroglobulin/metabolism
10.
Toxicol Appl Pharmacol ; 225(3): 329-36, 2007 Dec 15.
Article in English | MEDLINE | ID: mdl-17904175

ABSTRACT

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 microM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.


Subject(s)
Cytochrome P-450 CYP2B1/drug effects , Docosahexaenoic Acids/pharmacology , Down-Regulation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Phenobarbital/pharmacology , Receptors, Cytoplasmic and Nuclear/drug effects , Transcription Factors/drug effects , Animals , Binding Sites , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B1/metabolism , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Fatty Acids, Omega-3/pharmacology , Fatty Acids, Omega-6/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Immunoprecipitation , Male , Phenobarbital/administration & dosage , Protein Transport , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/metabolism , Time Factors , Transcription Factors/metabolism
11.
Toxicol Appl Pharmacol ; 225(3): 278-92, 2007 Dec 15.
Article in English | MEDLINE | ID: mdl-17919674

ABSTRACT

Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD(50)) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.


Subject(s)
Behavior, Animal/drug effects , Cytochrome P-450 Enzyme System/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Hexachlorocyclohexane/toxicity , Insecticides/toxicity , Administration, Oral , Animals , Brain/drug effects , Brain/metabolism , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP2E1/drug effects , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Female , Hexachlorocyclohexane/administration & dosage , Insecticides/administration & dosage , Liver/drug effects , Liver/metabolism , Male , Maternal Exposure/adverse effects , Motor Activity/drug effects , Pregnancy , Prenatal Exposure Delayed Effects/chemically induced , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Wistar
12.
Toxicol In Vitro ; 21(7): 1276-86, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17513084

ABSTRACT

We investigated the effects of two 5-HT(6) receptor antagonists on rat primary hepatocytes using a combined biochemical and toxicogenomics approach. Both compounds share the same pharmacological target, but displayed strikingly different toxicity profiles in pre-clinical animal studies: While R7199 caused hepatic steatosis in rats, no hepatotoxicity was observed with R0074. Here, we partially reproduced the steatosis findings seen in vivo using primary rat hepatocytes. Biochemical analyses and gene expression results generally supported the findings observed in the animal model and also allowed the differentiation of both compounds with regards to hepatotoxic potential. In particular, the induction of Cyp 2B and Cyp 3A1 directly correlates to the findings in the livers of treated animals. The effects on genes of the steroideogenic pathway relate to the deregulation of cholesterol homeostasis. We also observed the inhibition of beta-oxidation, indicating impaired lipid metabolism. Hence, gene expression analysis in combination with biochemical parameters can provide additional insight into the possible mechanisms underlying adverse events.


Subject(s)
Receptors, Serotonin/drug effects , Serotonin Antagonists/toxicity , Toxicogenetics/methods , Animals , Aryl Hydrocarbon Hydroxylases/drug effects , Aryl Hydrocarbon Hydroxylases/metabolism , Cholesterol/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP3A , Enzyme Induction/drug effects , Gene Expression Profiling , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Homeostasis/drug effects , Lipid Metabolism/drug effects , Liver/pathology , Male , Oxidation-Reduction/drug effects , Rats , Rats, Wistar
13.
Biopharm Drug Dispos ; 28(3): 125-33, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17295362

ABSTRACT

The possible reason for the significantly greater AUC of oral warfarin with oral oxolamine in male Sprague-Dawley rats was evaluated. After oral administration of warfarin at a dose of 2 mg/kg to male rats with oxolamine at doses of 10 and 50 mg/kg, the AUC values of warfarin were significantly greater than the controls (254 and 330 versus 180 microg h/ml). However, the AUC values of warfarin were not affected by oxolamine in female rats. This could be due to inhibition of CYP2B1, 2C11 and 3A2 by oxolamine in male rats, since warfarin was metabolized via CYP1A1, 2B1, 2C6, 2C11 and 3A2 in rats and CYP2B1 is male dominant, and CYP2C11 and 3A2 are male specific. Therefore, phenytoin, torasemide and clarithromycin (mainly metabolized via CYP2B1/2, 2C11 and 3A2 in rats, respectively) were administered intravenously to male rats with or without oral oxolamine. After oral oxolamine at doses of 10 and 50 mg/kg, the AUC of phenytoin was significantly greater (1280 and 1640 versus 938 microg min/ml), however, the AUC values of torasemide and clarithromycin were independent of oxolamine. The above data suggest that the significantly greater AUC of oral warfarin with oral oxolamine could be due to inhibition of CYP2B1/2 by oxolamine in male rats.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anticoagulants/pharmacokinetics , Cytochrome P-450 CYP2B1/metabolism , Oxadiazoles/pharmacology , Warfarin/pharmacokinetics , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Area Under Curve , Clarithromycin/pharmacokinetics , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Female , Male , Oxadiazoles/administration & dosage , Phenytoin/pharmacokinetics , Protein Binding , Rats , Rats, Sprague-Dawley , Sex Factors , Sulfonamides/pharmacokinetics , Torsemide
14.
Eur J Clin Invest ; 36(9): 647-53, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16919048

ABSTRACT

Artemisinin (a sesquiterpene lactone endoperoxide) has become important in multi-drug treatment of malaria. There is evidence that artemisinin induces drug metabolism which could result in drug-drug interactions. The objective of this study was to characterize the inductive properties of artemisinin on drug-metabolizing cytochrome P450 (CYP450) enzymes. The possibility of artemisinin to induce CYP450 was studied in artemisinin-treated (orally for four days) and vehicle-treated rats using reverse transcriptase polymerase chain reaction (RT-PCR). The effect on enzymatic activities in mouse microsomes from multiple artemisinin administration (intraperitonally) to mice were also studied as well as the effect on the expression in mouse primary hepatocytes and HEK293 cells. Increased CYP2B1 mRNA levels in rats could be seen after artemisinin treatment as well as a weak but reproducible increase in the intensity of CYP1A2. Administration of artemisinin to mice up-regulated hepatic CYP2B10-dependent, and to a lesser extent, CYP2A5-dependent enzyme activities. In primary hepatocyte culture, artemisinin significantly increased the CYP2B10 mRNA levels whereas the CYP2A5 mRNA levels were increased to a lesser extent. No significant changes were seen in the levels of other CYP enzymes. Artemisinin was an activator of constitutive androstane receptor (CAR) but not pregnane X receptor (PXR) in HEK293 cells. The results demonstrate that the drug exerts its effects on drug metabolism via the CAR receptor that results in up-regulation of genes such as the Cyp2b. The weaker up-regulation of CYP2A5 might also be CAR-dependent or alternatively, a consequence of artemisinin toxicity. The results of this study are of importance when predicting potential drug-drug interactions in multi-drug therapies with artemisinin.


Subject(s)
Anti-Infective Agents/administration & dosage , Artemisinins/administration & dosage , Cytochrome P-450 Enzyme System/drug effects , Receptors, Cytoplasmic and Nuclear/drug effects , Sesquiterpenes/administration & dosage , Transcription Factors/drug effects , Administration, Oral , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Cells, Cultured , Constitutive Androstane Receptor , Cytochrome P-450 CYP1A2/drug effects , Cytochrome P-450 CYP2A6 , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P450 Family 2 , Drug Interactions , Enzyme Induction/drug effects , Hepatocytes , Male , Mice , Mixed Function Oxygenases/metabolism , Pregnane X Receptor , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Steroid/drug effects , Reverse Transcriptase Polymerase Chain Reaction/methods
15.
Cancer Sci ; 97(9): 896-904, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16805852

ABSTRACT

To elucidate the mechanism underlying suppression by curcumin of esophageal carcinogenesis induced by NMBA, we evaluated the CYP level and mutagenic activation of environmental carcinogens, by immunoblot analyses and Ames preincubation test, respectively, and bilirubin, 4-nitrophenol and testosterone UDPGT activities in F344 rats treated with curcumin and/or NMBA. No significant alterations in the hepatic levels of constitutive CYP proteins, mutagenic activation by liver S9 or hepatic UDPGT activities were produced by subcutaneous treatment with 0.5 mg/kg NMBA for 5 weeks and/or feeding of 0.05% and 0.2% curcumin for 6 weeks. In contrast, gavage of 0.2% curcumin decreased esophageal CYP2B1 and 2E1 by up to 60%, compared with vehicle control. Similarly, intragastric treatment with 270 mg/kg curcumin decreased esophageal and gastric CYP2B1 and CYP2E1, but not in lung, kidney or intestine. Conversely, large intestinal CYP2B1 was 2.8-fold higher in the treated rats than in control rats. Mutagenic activities of NOC, including NMBA, in the presence of esophagus and stomach S9 were markedly decreased in the treated rats, whereas those in the presence of large intestine S9 were 2.2-3.0-fold above control. These results show that modifying effects of curcumin on esophageal carcinogenesis can be attributed to a decrease in metabolic activation of NMBA by esophageal CYP2B1 during the initiation phase, without the contribution of metabolic activation and inactivation by liver. Further, the present findings suggest the potential of curcumin for modification of gastric and intestinal carcinogenesis initiated with NOC.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinogens/metabolism , Curcumin/pharmacology , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2E1/drug effects , Nitrosamines/metabolism , Animals , Bilirubin/metabolism , Blotting, Western , Cytochrome P-450 CYP2B1/analysis , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP2E1/analysis , Cytochrome P-450 CYP2E1/metabolism , Dimethylnitrosamine/analogs & derivatives , Dimethylnitrosamine/metabolism , Esophagus/drug effects , Esophagus/enzymology , Glucuronosyltransferase/drug effects , Glucuronosyltransferase/metabolism , Intestines/drug effects , Intestines/enzymology , Liver/drug effects , Liver/metabolism , Male , Mutagenicity Tests , Nitrophenols/metabolism , Rats , Rats, Inbred F344 , Stomach/drug effects , Stomach/enzymology , Testosterone/metabolism , UDP-Glucuronosyltransferase 1A9
16.
Mar Environ Res ; 62 Suppl: S200-4, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16716393

ABSTRACT

The possibility that certain Mediterranean cetaceans are subject to toxicological hazard due to organochlorines and emerging contaminants, such as polybrominated diphenyl ethers (PBDEs) with endocrine disrupting capacity, was investigated using non-lethal methods. The need for new biomarkers for EDCs and for a "cell model" to explore the different susceptibilities to several classes of ECDs, including emerging contaminants, led us to culture fibroblasts of different cetacean species ("dolphins in test tubes"). We then explored interspecies and gender susceptibility to OC-EDCs and PBDEs using qualitative and semi-quantitative evaluation of target proteins, such as CYP1A and CYP2B in cultured cetacean fibroblasts (Stenella coeruleoalba, Tursiops truncatus and Balaenoptera physalus), by western blot and immunofluorescence techniques.


Subject(s)
Cetacea/physiology , Cytochrome P-450 CYP2B1/drug effects , Hydrocarbons, Brominated/toxicity , Hydrocarbons, Chlorinated/toxicity , Phenyl Ethers/toxicity , Water Pollutants, Chemical/toxicity , Animals , Antibodies , Blotting, Western/methods , Cells, Cultured , Female , Fibroblasts/cytology , Fluorescent Antibody Technique, Indirect/methods , Male , Mediterranean Sea , Sex Factors , Species Specificity
17.
Mol Carcinog ; 45(2): 118-25, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16329150

ABSTRACT

Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabolism, resulting in metabolites that lead to formation of TAM-DNA adducts. We have investigated the effect of CYP3A2 antisense (AVI-4472) exposure on CYP3A2 transcription, enzyme activity, translation, and TAM-DNA adducts, in livers of rats administered TAM (50 mg/kg body weight [bw]/day) for 7 days. The study design included administration of 0, 0.5, 2.5, or 12.5 mg AVI-4472/kg bw/day for 8 days, beginning 1 day before TAM exposure. The specific activity of CYP3A2 was increased after TAM administration, and decreased significantly (approximately 70%) in the presence of 12.5 mg AVI-4472. CYP3A2 protein levels, determined by immunoblot analysis, showed a similar pattern. Hepatic TAM-DNA adduct levels were measurable in all TAM-exposed groups. However, when rats were co-treated with 2.5 and 12.5 mg AVI-4472/kg bw/day, statistically significant (approximately 50%) reductions in TAM-DNA adduct levels (2.0-2.8 adducts/10(8) nucleotides) were observed compared to rats treated with TAM alone (5.1 adducts/10(8) nucleotides). Rat toxicology U34 arrays (Affymetrix) were used to investigate the modulation of gene expression patterns on co-administration of TAM with AVI-4472. Results indicated that several CYP genes were down regulated although no significant induction of CYP3A2 was observed in the TAM-exposed rats co-treated with AVI-4472. Overall the data suggest the utility of antisense technology in the redirection of TAM metabolism thereby lowering TAM genotoxicity in rat liver.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Antisense Elements (Genetics)/genetics , Aryl Hydrocarbon Hydroxylases/drug effects , Aryl Hydrocarbon Hydroxylases/genetics , DNA Adducts/genetics , Membrane Proteins/drug effects , Membrane Proteins/genetics , Tamoxifen/pharmacology , Animals , Antisense Elements (Genetics)/pharmacology , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP3A , Dose-Response Relationship, Drug , Liver/drug effects , Liver/physiology , Male , Membrane Proteins/metabolism , Rats , Rats, Sprague-Dawley , Tamoxifen/metabolism , Toxicity Tests/methods
18.
Comb Chem High Throughput Screen ; 8(4): 341-6, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16101010

ABSTRACT

Recent studies have suggested that both constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B and 3A through a mechanism called cross-talk. In this study we intend to determine if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen structurally diverse compounds was evaluated by using rat precision-cut liver slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs in rat liver slices was quantified by real-time polymerase chain reaction. Rat PXR activation was measured by induction of luciferase activity in rat PXR reporter-gene system. Linear regression analysis of the fold of induction of mRNA in liver slices and the fold of luciferase activity in rat PXR reporter-gene system shows that a reasonable correlation (r2 = 0.6) exists between the CYP3A induction and the rat PXR activation. A much lower correlation was observed between CYP2B induction and the rat PXR activation (r2 = 0.1). The results from this study suggest that the PXR may play a major role in the induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay may be useful in a high-throughput screening to predict CYP3A induction in rats.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Biological Assay , Liver/metabolism , Oxidoreductases, N-Demethylating/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/genetics , Animals , Aryl Hydrocarbon Hydroxylases/drug effects , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP3A , Drug Evaluation, Preclinical/methods , Enzyme Activation/drug effects , Genes, Reporter , In Vitro Techniques , Liver/drug effects , Luciferases/drug effects , Luciferases/genetics , Luciferases/metabolism , Male , Oxidoreductases, N-Demethylating/drug effects , Oxidoreductases, N-Demethylating/genetics , Pregnane X Receptor , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/drug effects , Receptors, Steroid/metabolism
19.
J Toxicol Environ Health A ; 67(11): 875-88, 2004 Jun 11.
Article in English | MEDLINE | ID: mdl-15205042

ABSTRACT

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.


Subject(s)
Aryl Hydrocarbon Hydroxylases/drug effects , Inhalation Exposure , Lipid Peroxidation/drug effects , Vehicle Emissions/toxicity , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/drug effects , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP2E1/drug effects , Cytochrome P-450 CYP2E1/metabolism , Immunoblotting , Kidney/drug effects , Kidney/enzymology , Liver/drug effects , Liver/enzymology , Lung/drug effects , Lung/enzymology , Male , Microsomes/drug effects , Microsomes/enzymology , Motorcycles , Rats , Rats, Wistar
20.
Int J Occup Med Environ Health ; 17(3): 347-53, 2004.
Article in English | MEDLINE | ID: mdl-15683155

ABSTRACT

OBJECTIVES: Hexabromobenzene (HBB) is a flame retardant, which added to polymers, plastics, textiles, wood or paper, decreases the amount of carbon monoxide and heat release during fire. HBB is also formed as a result of decabromodiphenyl oxide pyrolysis or natural decabromobiphenyl ether debromination as the effect of photolysis. 1,2,4,5-Tetrabromobenzene (1,2,4,5-tetraBB) is a compound formed in the body as a metabolite of HBB. Both these compounds may appear in the environment and human tissue. The purpose of the study was to estimate the effect of repeated administration of HBB and 1,2,4,5-tetraBB on the levels of selected cytochromes in rat liver. MATERIALS AND METHODS: The investigated compounds were administered intragastrically in three different doses for 7, 14, 21 or 28 days. Relative liver mass was estimated as well as total concentration of cytochromes P-450 and EROD (CYP 1A) and PROD (CYP 2B) activity in rat liver. Concentration of cytochromes P-450 was determined in microsomal fraction (using the spectrometric method). EROD and PROD were detected by fluorimetric method. RESULTS: Repeated administration of 1,2,4,5-tetraBB and HBB (in the highest dose) was found to increase relative liver mass. After 1,2,4,5-tetraBB administration, total liver concentration of cytochromes P-450 increased even by several times, depending on the volume and number of doses. Less pronounced alterations were found after repeated administration of HBB. Exposure to HBB resulted in a tenfold increase in EROD activity (after 14-28 days) and a significantly lower increase in PROD activity. 1,2,4,5-TetraBB increased EROD activity by 2-3 times and PROD activity by maximum 2 times. CONCLUSIONS: Following the experiments, it may be stated that HBB and 1,2,4,5-tetraBB are inductors of microsomal enzymes system. 1,2,4,5-TetraBB more than HBB increases the level of total concentration of cytochromes and induces isoform CYP 2B (PROD). Administration of HBB resulted in the increase in CYP 1A (EROD) activity comparable to that after 3-methylcholanthrene.


Subject(s)
Bromobenzenes/pharmacology , Cytochrome P-450 Enzyme System/drug effects , Flame Retardants/pharmacology , Liver/enzymology , Animals , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP2B1/drug effects , Dose-Response Relationship, Drug , Female , Liver/drug effects , Rats , Rats, Wistar , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...