A possible mechanism for 2,2',4,4',5,5'-hexachlorobiphenyl-mediated decrease in serum thyroxine level in mice.

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.

Biological effects of Byrsocarpus coccineus in vitro.

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).

Covalent modification of Thr302 in cytochrome P450 2B1 by the mechanism-based inactivator 4-tert-butylphenylacetylene.

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.

Diallyl trisulfide protects rats from carbon tetrachloride-induced liver injury.

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.

Crystalline silica is a negative modifier of pulmonary cytochrome P-4501A1 induction.

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.

Induction of hepatic cytochrome P4501A1/2B activity and disruption of thyroglobulin synthesis/secretion by mono-ortho polychlorinated biphenyl and its hydroxylated metabolites in rat cell lines.

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.

An in vitro study on 5-HT6 receptor antagonist induced hepatotoxicity based on biochemical assays and toxicogenomics.

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.

Development of new-tools to investigate toxicological hazard due to endocrine disruptor organochlorines and emerging contaminants in Mediterranean cetaceans.

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.

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey (Callithrix jacchus).

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo-p-dioxin or tetrabromodibenzo-p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYP1A1, CYP1A2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYP1A2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYP1A1 is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYP1A1 and CYP1A2 in liver, but only CYP1A1 in lung. No induction of CYP1A1 or CYP1A2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYP1A2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYP1A2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a Ki of 10 microM. In the lung the induction of CYP1A1 was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYP1A1 does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices.

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.

Effects of a vegetables-fruit mixture on liver and colonic 1,2-dimethylhydrazine-metabolizing enzyme activities in rats fed low- or high-fat diets.

The effects of a vegetables-fruit mixture (19.5% w/w) were studied on hepatic (h) and colonic (c) 1,2-dimethylhydrazine (DMH)-metabolizing enzyme activities (ethoxy- and pentoxyresorufine-O-deethylation, NDMA-demethylase, glutathione-S-transferase, UDP-glucuronyltransferase) in rats fed low- or high-fat diets (20 or 40 energy%). A vegetables-fruit mixture added to the diets resulted in altered enzyme activities in animals either treated or not treated with DMH. Remarkably, the vegetables-fruit mixture given to DMH-treated rats decreased glutathione-S-transferase (h) and increased NDMA-demethylase activities (c), whereas the mixture given to controls increased glutathione-S-transferase (h) and decreased NDMA-demethylase activities (c). The high-fat diets only modulated enzyme activities in animals not treated with DMH.

Molecular non-genetic biomarkers of effect related to methyl thiophanate cocarcinogenesis: organ- and sex-specific cytochrome P450 induction in the rat.

We used selective biochemical markers of effect to evaluate some non-genotoxic cocarcinogenic properties of methyl thiophanate (MTH) associated with cytochrome P450 (CYP) changes. Several CYP-dependent reactions were monitored in the liver, kidney and lung microsomes of male and female Sprague-Dawley rats treated (i.p.) with a single (285 or 570 mg/kg body weight) or repeated (daily 285 or 570 mg/kg body weight for three consecutive days) doses of this pesticide. No significant changes in absolute or relative liver, kidney and lung weights were observed after MTH injection. Highly specific substrates were used as probes of different isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences, was observed, particularly in the liver (CYP3A, 2B1), kidney (CYP1A1, 2E1) and lung (CYP3A, 1A1). In the liver, an increase up to 29-fold in the 2B1-like activity, probed by the O-dealkylation of pentoxyresorufin, was observed at lower dose in both sexes, and the induction of CYP 1A2-mediated methoxyresorufin O-demethylase activity (up to 3.6-fold) was recorded at the higher dose in males. In the kidney, the O-deethylation of ethoxyresorufin (CYP1A1-linked) was increased up to 28.2-fold and the CYP2E1-dependent p-nitrophenol hydroxylases were enhanced up to 6.3-fold in females receiving higher multiple MTH administration. In the lung, the CYP3A-associated activity was the most induced oxidases, as exemplified by the marked increase in the O-demethylation of aminopyrine (up to 3.6-fold) in males. A weak, although significant, reduction of CYP2B1-linked oxidases was also observed in repeated treatment in the kidney (males) and lung (females). These results suggest that the induction of CYP-catalyzed drug metabolism by prolonged exposure to MTH may result in accelerated metabolism of coadministered drugs with important implications for their disposition Together with an alteration of endogenous metabolism, the adverse effects associated with CYP changes such as toxicity/cotoxicity, cocarcinogenicity and promotion may also have clinical consequences.

Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes.

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.

Induction of CYP2B1/2 and nicotine metabolism by ethanol in rat liver but not rat brain.

A higher proportion of alcoholics than non-alcoholics smoke (>80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4-6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.

Differential effects of two lots of aroclor 1254 on enzyme induction, thyroid hormones, and oxidative stress.

Aroclor 1254 is a commercial mixture of polychlorinated biphenyls (PCBs), which is defined as being 54% chlorine by weight. However, the congener composition varies from lot to lot. Two lots which have been used in toxicity studies, 124-191 and 6024 (AccuStandard), were analyzed for their congener composition. Lot 6024 has approximately 10 times the dioxin toxic equivalents (TEQ) of lot 124-191. The purpose of this study was to determine if the difference in the TEQ of the two lots explains the different in vivo responses seen on a weight basis. Male Long-Evans rats (70 days old) were treated orally with a single dose of 0-1,000 mg/kg of each lot. Hepatic ethoxy-, methoxy-, and pentoxyresorufin O-deethylase (EROD, MROD, and PROD, respectively) activities as well as serum thyroxine (T(4)) concentrations and measures of oxidative stress were determined 4 days after treatment. Results, on a weight basis, indicate that lot 6024 led to a greater induction of EROD, MROD, and PROD but not total T(4) reduction. The differences in TEQ between the lots explained the differential induction of EROD and MROD but did not account for the induction of PROD nor decreases in T(4). PROD induction is not due to dioxin-like congeners, whereas the decrease in serum T(4) levels may involve multiple mechanisms. Effects on the antioxidants ascorbic acid and uric acid were seen only at the highest mass dose for both lots and were not explained by the difference in TEQ. These results illustrate that the differences in the TEQ explain the differences in the strict dioxin-like effects (EROD, MROD induction), but the non-dioxin-like congeners cause other effects that are not associated with the aryl hydrocarbon receptor (e.g., PROD). In addition, supra-additive effects also occur in the mixture (T(4), oxidative stress). Thus, current results demonstrate that overall toxicity cannot be predicted on the basis of the TEQ values. It is also critical that the lot number is reported in studies conducted with Aroclor 1254 because the congener composition and therefore the effects observed can be very different.

Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2.

Although the induction of drug-metabolizing systems by phenobarbital has been recognized for about 40 years, the mechanism by which cytochrome P450 gene expression is increased is still not well understood. A 163-bp fragment at about -2.2 Kb in CYP2B2 has been shown to mediate phenobarbital induction in primary rat hepatocytes (Trottier, et al. [1995] Gene 158:263-268) and by an in situ transient transfection assay in rat liver (Park, Y., et al. [1996]. J. Biol. Chem. 271:23725-23728). Deletion mutations of this fragment indicated that the 88-bp stretch from -2258 to -2170 was the minimal sequence that could mediate phenobarbital induction in the in situ system if single copies of the deleted fragments fused to the CYP2C1 proximal promoter were assayed. If three copies of the fragments were present, 5' and 3' deletions defined a minimal 37-bp core fragment, which, although necessary for phenobarbital responsiveness, was not sufficient unless additional sequence was present at either end, suggesting that redundant elements were present in the two flanking regions. Site-specific mutagenesis of an NF-1 site within the 88-bp fragment and linker scanning mutagenesis across the fragment indicated that the NF-1 site and a region to the 5' side of the site contributed to the magnitude of the response, but neither the NF-1 mutations nor any of the linker scanning mutations eliminated the response to phenobarbital. Mutation in a region 3' of the NF-1 site resulted in elevated basal expression without substantial effects on phenobarbital-induced expression. Binding of NF-1 to the 37-bp core fragment was established by gel-shift competition studies and by supershifts of the protein-DNA complexes by antisera to NF-1. Additional protein-DNA complexes were detected in the regions flanking the NF-1 site. These studies indicate that the CYP2B2 phenobarbital-responsive enhancer contains multiple constitutive and phenobarbital-responsive elements. Binding of nuclear proteins from control or phenobarbital-treated animals in vitro to this region was very similar. The only difference detected was a complex that was substantially reduced by phenobarbital treatment and mapped to the 3' side of the NF-1 site.

The contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazine neuroleptics.

The aim of the present study was to determine optimum conditions for studying promazine and perazine metabolism in rat liver microsomes, and to investigate the influence of specific cytochrome P-450 inhibitors on 5-sulfoxidation and N-demethylation of these neuroleptics. Based on the developed method, the metabolism of neuroleptics in liver microsomes was studied at linear dependence of product formation on time, and protein and substrate concentrations (incubation time: 10 min; concentration of microsomal proteins: promazine-0.7 mg ml(-1), perazine-0.5 mg ml(-1); substrate concentrations: promazine-25, 40 and 75 nmol ml(-1), perazine-20, 35, 50 nmol ml(-1)). A Dixon analysis of the metabolism of neuroleptics showed that quinine (a CYP2D1 inhibitor), metyrapone (a CYP2B1/B2 inhibitor) and alpha-naphthoflavone (a CYP1A1/2 inhibitor) affected, whereas erythromycin (a CYP3A inhibitor) and sulfaphenazole (a CYP2C inhibitor) did not change the neuroleptic biotransformation. N-Demethylation of promazine was competitively inhibited by quinine (K(i)=20 microM) and metyrapone (K(i)=83 microM), while that of perazine-by quinine (K(i)=46.5 microM), metyrapone (K(i)=46 microM) and alpha-naphthoflavone (K(i)=78.8 microM). 5-Sulfoxidation of promazine was inhibited only by quinine (K(i)=28.6 microM), whereas that of perazine-by quinine (K(i)=10 microM) and metyrapone (K(i)=96 microM). The results obtained are compared with our previous findings of analogous experiments concerning thioridazine, and with the data on other phenothiazines and species. In summary, it is proposed that N-demethylation of the mentioned phenothiazine neuroleptics in the rat is catalyzed by the isoenzymes CYP2D1, CYP2B2 and CYP1A2 (CYP1A2 does not refer to promazine). 5-Sulfoxidation of these drugs may be mediated by different isoenzymes, e.g. CYP2D1 (promazine and perazine), CYP2B2 (perazine) and CYP1A2 (thioridazine). Isoenzymes belonging to subfamilies CYP2C and CYP3A do not seem to be involved in the metabolism of the investigated neuroleptics in the rat. The results obtained point to the drug structure and species differences in the contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazines.

Induction of liver monooxygenases by beta-myrcene.

Beta-Myrcene (MYR) is an acyclic monoterpene found in the essential oils of a variety of useful plants such as lemongrass (Cymbopogon citratus), hop, verbena, bay and others. MYR and essential oils containing this olefinic monoterpene are widely used as flavoring food additives, as fragrances in cosmetics and as scents in household products. The present study was undertaken to investigate the induction of liver monooxygenases by MYR. Female Wistar rats were treated by gavage with MYR (1000 mg/kg body weight) or corn oil (vehicle) for 1 or 3 consecutive days. Activities of ethoxycoumarin-O-deethylase (ECOD) and alkoxy-resorufin O-dealkylases (methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD) and benzyloxy-resorufin-O-dealkylation (BROD)) were determined fluorimetrically in the hepatic microsomal fraction. Exposure to MYR, either for 1 or 3 days, produced marked (13-34-fold) increases in the activities of PROD and BROD and only minor changes in ECOD, EROD and MROD. Since PROD and BROD are metabolized mainly by CYP2B isoenzymes, these results suggest that MYR induces this phenobarbital-inducible P450 subfamily. The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting. Levels of apoproteins CYP2B1/2B2 were increased 8.2-fold after treatment with MYR (1000 mg/kg body wt, 3 days). Results from this study therefore indicate that MYR is an inducer of isoenzymes belonging to CYP2B subfamily.

Maternal and developmental toxicity of polychlorinated diphenyl ethers (PCDEs) in Swiss-Webster mice and Sprague-Dawley rats.

Polychlorinated diphenyl ethers (PCDEs) are industrial byproducts found in many ecosystems at low levels. PCDEs are not markedly toxic to adult rodents, but their developmental toxicity has not previously been examined. We evaluated the maternal and perinatal toxicity of nine PCDE congeners to outbred mice when compounds were administered from gestation day (GD) 6 through GD 15. 2,2',4,4',5,6'-hexaCDE and 2,3',4',6-tetraCDE decreased the number of pups born per female mated and the number of pups surviving per litter born. 2,2',4,4',5,5'-hexaCDE and 2,2',4,5,6'-pentaCDE decreased the number of litters born per female mated, without decreasing postnatal survival. The other PCDEs did not decrease survival either pre- or postnatally. None of the PCDEs caused absence of Harderian glands in surviving offspring at the doses administered. Neither induction of cytochromes P450 nor tissue residues of individual congeners correlated well with developmental toxicity. Three PCDEs were also evaluated in outbred (Sprague-Dawley) rats: 2,2',4,5,6'-pentaCDE and 2,3',4',6-tetraCDE, because of their toxicity to mice; 2,2',4,4',5,5'-hexaCDE, because it should exhibit PCB-like toxicity. Each congener was administered at three dose levels from GD 6 through GD 15. 2,2',4,5,6'-pentaCDE decreased the number of litters born at 100 mg/kg/day, and the survival of pups in litters carried to term, at both 50 and 100 mg/kg per day. Postnatal weight gain was also reduced. In contrast to its action in mice, 2,3',4',6-tetraCDE decreased neither the numbers of litters born nor postnatal survival of rat offspring, but did suppress postnatal weight gain at least through PD 5. As in mice, induction of cytochromes P450 was not well correlated with the developmental toxicity of individual congeners.

Induction of cytochromes P450 2B and 2E1 in rat liver by isomeric picoline N-oxides.

Pyridine derivatives are widely used solvents and precursors for the synthesis of chemicals of industrial importance. Oxidized metabolites have been implicated in the observed toxicity of pyridines and are known to induce drug-metabolizing enzymes in rat liver. In this study the three isomeric picoline (methylpyridine) N-oxides, as major oxidized metabolites of 2-, 3- and 4-picoline, were evaluated as inducers of cytochrome P450 (CYP) enzymes in rat liver. After a single dose of 100 mg/kg 24 h before sacrifice the 3- and 4-isomers were effective inducers of microsomal substrate oxidations associated with the phenobarbital-inducible CYPs 2B; upregulation of CYP2B protein was confirmed by immunoblotting. In contrast, the 2-isomer did not increase CYP2B protein or activity in rat liver but CYP2E1 protein expression was upregulated by the isomers to 160-200% of control. The three chemicals increased aniline 4-hydroxylation activity in rat liver, which is consistent with induction of CYPs 2B or 2E1 and 4-nitrophenol 2-hydroxylation activity was increased in microsomal fractions from 3- and 4-picoline N-oxide-treated rats. The activities of several other CYPs were also determined and CYP1A-dependent 7-ethylresorufin O-deethylation was increased (to approximately 6- and 2-fold of control) by the 3- and 4-isomer, respectively, whereas the activity of CYP3A-mediated androstenedione 6beta-hydroxylation was decreased by the agents--most notably by the 2-isomer. During NADPH-supported oxidation of CCl4, lipid peroxidation was increased in microsomes from 3- and 4-picoline N-oxide-pretreated rats and was modulated in vitro by the CYP2B inhibitor orphenadrine, but not by the CYP2E1 inhibitor 4-methylpyrazole. These findings establish that particular isomers of picoline N-oxide rapidly upregulate CYP2B or, to a lesser extent, CYP2E1 and implicate CYP2B in the enhanced lipid peroxidation observed in microsomes from rats treated with 3- and 4-picoline N-oxides. Such induction process may contribute to the hepatotoxicity of pyridines by enhancing the capacity for microsomal lipid peroxidation.