Inducibility of cytochrome P450-mediated 7-methoxycoumarin-O-demethylase activity in zebrafish (Danio rerio) embryos.

The zebrafish (Danio rerio) embryo has increasingly been used as an alternative model in human and environmental toxicology. Since the cytochrome P450 (CYP) system is of fundamental importance for the understanding and correct interpretation of the outcome of toxicological studies, constitutive and xenobiotic-induced 7-methoxycoumarin-O-demethylase (MCOD), i.e. 'mammalian CYP2-like', activities were monitored in vivo in zebrafish embryos via confocal laser scanning microscopy. In order to elucidate molecular mechanisms underlying the MCOD induction, dose-dependent effects of the prototypical CYP inducers ß-naphthoflavone (aryl hydrocarbon receptor (AhR) agonist), rifampicin (pregnane X receptor (PXR) agonist), carbamazepine and phenobarbital (constitutive androstane receptor (CAR) agonists) were analyzed in zebrafish embryos of varying age. Starting from 36 h of age, all embryonic stages of zebrafish could be shown to have constitutive MCOD activity, albeit with spatial variation and at distinct levels. Whereas carbamazepine, phenobarbital and rifampicin had no effect on in vivo MCOD activity in 96 h old zebrafish embryos, the model aryl hydrocarbon receptor agonist ß-naphthoflavone significantly induced MCOD activity in 96 h old zebrafish embryos at 46-734 nM, however, without a clear concentration-effect relationship. Induction of MCOD activity by ß-naphthoflavone gradually decreased with progression of embryonic development. By in vivo characterization of constitutive and xenobiotic-induced MCOD activity patterns in 36, 60, 84 and 108 h old zebrafish embryos, this decrease could primarily be attributed to an age-related decline in the induction of MCOD activity in the cardiovascular system. Results of this study provide novel insights into the mechanism and extent, by which specific CYP activities in early life-stages of zebrafish can be influenced by exposure to xenobiotics. The study thus lends further support to the view that zebrafish embryos- at least from an age of 36 h - have an elaborate and inducible biotransformation system.

Comparative effects of dimethylsulfoxide on metabolism and toxicity of carbon tetrachloride and dichloromethane.

The effects of dimethylsulfoxide (DMSO) on the metabolism and toxicity of chlorinated methanes were examined. Male mice were treated with DMSO (1, 2.5 or 5 ml kg(-1), i.p.) prior to challenge with dichloromethane (CH(2)Cl(2)) or carbon tetrachloride (CCl(4)). Blood carboxyhemoglobin elevation resulting from metabolic conversion of CH(2)Cl(2) to carbon monoxide was inhibited dose-dependently by DMSO pretreatment. The elevation of serum aspartate aminotransferase, alanine aminotransferase and sorbitol dehydrogenase activities induced by CCl(4) (0.1 mmol kg(-1)) was not changed in mice pretreated with DMSO at 1 ml kg(-1), but depressed significantly at a greater dose of DMSO. However, DMSO failed to alter the hepatotoxicity of CCl(4) injected at a dose of 0.2 mmol kg(-1). DMSO induced the microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities as early as 2 h following the treatment. Microsomal disposition of CH(2)Cl(2) and CCl(4) was measured using a vial equilibration technique. The disappearance of CH(2)Cl(2) was inhibited competitively by addition of DMSO. But DMSO did not affect the metabolic degradation of CCl(4). The results indicate that DMSO has multiple effects on metabolism and toxicity of xenobiotics. DMSO induces the hepatic metabolizing activity mediated by CYP2E1, but the presence of this solvent in the enzyme site may inhibit directly the enzymatic interaction with a substrate. The toxicological significance of DMSO-induced effects on such an interaction may be variable depending on the properties of each substrate. The invulnerability of CCl(4) metabolism to the effects of DMSO appears to be related to its high affinity for the lipophilic CYP enzyme site.

DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes.

The objectives of this study were to examine DNA demethylase (dMTase) expression in ovarian cancers and evaluate methylation of CpG sites in the promoter of the c-erbB-2 gene and survivin gene exon 1. Forty-three epithelial ovarian cancers and 43 non-cancerous ovarian tissues were studied for dMTase expression by RT-PCR. Genomic DNA was extracted and digested with HindIII and then HpaII. CpG site-sensitive primers were constructed to amplify the promoter of the c-erbB-2 gene and survivin gene exon 1. Immunohistochemical evaluation of ErbB-2 protein and RT-PCR for survivin were also performed. dMTase was positive in 88.4% of ovarian cancers but only in 9.3% of non-cancerous ovaries (P<0.001, Fisher's exact test). The expression was similarly observed in both early stage (stage I+II: 17/19) and advanced stage (stage III+IV: 21/24) groups of ovarian malignancy. It was found that 78.9% of dMTase-positive cancers had both c-erbB-2 promoter and survivin gene exon 1 unmethylated, whereas 40% of dMTase-negative cancers had both sites methylated. In non-cancerous ovaries, these sites were mostly methylated (90.6%) and the difference from cancer cases was highly significant (P<0.001). Immunohistochemical evaluation of ErbB-2 showed significant correlation of unmethylated c-erbB-2 promoter and ErbB-2 expression. The RT-PCR for survivin expression showed that 86% of cancers were positive and six cases were negative. Exon 1 was methylated in 83% of the survivin-negative cases. This is the first report of dMTase expression in ovarian cancers. The correlation of dMTase expression with unmethylation of c-erbB-2 promoter and survivin gene exon 1 suggests that these sites may be targets for demethylation by the enzyme. The up-regulation of oncogenes may be the consequence of epigenetic control of gene expression by the dMTase.

Repression of Acinetobacter vanillate demethylase synthesis by VanR, a member of the GntR family of transcriptional regulators.

Vanillate is converted to protocatechuate by the action of vanillate demethylase encoded by vanAB. Convergent upon and overlapping Acinetobacter vanB is an open reading frame encoding a member of the gntR repressor family and designated vanR. This gene organization differs from that found in a Pseudomonas isolate. An Acinetobacter strain with a knockout mutation in vanR constitutively converted vanillate to protocatechuate. Reverse transcriptase-polymerase chain reaction was used to demonstrate that control of vanAB was exerted at the level of transcription.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Characterization of the effects of tebufelone on hepatic cytochromes P450 in the beagle dog.

Tebufelone (1-[3,5-bis(1,1-dimethylethyl)-4-hydroxy-phenyl]-hex-5-yne-1-one) is an investigational ditertiary butylphenol nonsteroidal anti-inflammatory drug. The purpose of the present study was to assess the effects of tebufelone on hepatocyte ultrastructure and hepatic cytochromes p450 (P450s) in the beagle dog after 2 weeks of oral administration at dose levels of 0, 5, 15, 50, and 100 mg/kg/day (N = 1/sex/dose level). Hepatic tissue was obtained at necropsy for histologic, ultrastructural, and biochemical evaluation. Hepatocellular hypertrophy was observed in only a single tebufelone-treated dog (50 mg/kg). Electron microscopic evaluation, however, revealed marked dose-dependent increases in smooth endoplasmic reticulum in all of the tebufelone treatment groups. Biochemical indicators suggested that tebufelone produced mixed effects on hepatic P450s. p-Nitroanisole O-demethylase and, to a greater extent, ethoxyresorufin O-deethylase activities were decreased with increasing tebufelone dose. The precise mechanism by which tebufelone decreased ethoxyresorufin O-deethylase activity in dogs in unknown, but it was not by competitive inhibition, P450 inactivation, or reduced CYP1A expression. Tebufelone treatment increased NADPH-dependent cytochrome c reductase, total P450, and indicators of CYP2B11 (chloramphenicol covalent binding and immunochemically determined 2B11) and CYP3A12 (erythromycin N-demethylase, triacetyloleandomycin spectral complex formation, testosterone 6 beta-hydroxylase, and immunochemically determined 3A12). The largest increase in the 2B11 and 3A12 markers occurred in the 50 or 100 mg/kg treatment groups. The greatest increase in CYP2B11 markers produced by tebufelone treatment ranged from 2- to 3-fold, whereas the increase in CYP3A12 markers ranged from 5- to 10-fold. The changes in hepatic ultrastructure and increases in CYP2B11 and CYP3A12 markers produced by tebufelone in dogs are similar to that reported for phenobarbital.

Induction of cytochrome P-450 isoenzymes in cultured monkey hepatocytes.

The effect of phenobarbital (PB), beta-naphthoflavone (beta-NF) and rifampicin (Rif) on the drug-metabolizing activity of cultured squirrel monkey hepatocytes was examined. The drug metabolizing activity (e.g. alkoxycoumarin dealkylase or steroid hydroxylase) gradually decreased during the culture period with 40-70% activity remaining at 72 hr. When 0.5 mM PB was added to the culture, the activities of 7-methoxycoumarin O-demethylase (MCOD) and 7-ethoxycoumarin O-deethylase (ECOD) increased to 6-7 fold higher level than those of control at 72 hr. Testosterone 6 beta-hydroxylase (6 beta-OH-T) and testosterone 16 beta-hydroxylase (16 beta-OH-T) activities were approx. 3-fold higher than those of the control. Addition of beta-NF significantly increased the activities of 7-ethoxyresorufin O-deethylase (EROD) and ECOD. Though statistically insignificant, Rif slightly increased 6 beta-OH-T activity. Western blot analysis indicated PB induced production of the CYP 2B and 3A subfamilies, while beta-NF and Rif induced that of the CYP 1A and the CYP 3A subfamily, respectively.

In vitro transcription from the Nectria haematococca PDA1 promoter in an homologous extract reflects in vivo pisatin-responsive regulation.

The PDA1 gene of Nectria haematococca MP VI (anamorph: Fusarium solani) encodes pisatin demethylase. This enzyme detoxifies the isoflavanoid phytoalexin pisatin produced by the plant on which this fungus is pathogenic. Expression of pisatin demethylase activity is induced in a mycelium by pretreatment with pisatin. We have developed homologous in vitro system which accurately initiates transcription from the PDA1 promoter. Transcription levels in vitro reflect the same pisatin-responsive stimulation as measured for PDA1 mRNA in vivo, and are dependent upon sequences in the 5' upstream region of PDA1. Pisatin-responsive transcription from the PDA1 promoter indicates that initiation of transcription is a major regulatory step in the pisatin induction of pisatin demethylase expression.

Scoparone O-demethylase assay is not useful to differentiate the effects of model inducers of cytochrome P-450 in rabbit and guinea pig liver.

Coumarin derivative, scoparone (6,7-dimethoxycoumarin), is regioselectively O-demethylated into isoscopoletin (I) and scopoletin (S). This oxidation is inversely influenced by cytochrome P-450 inducers in the rat such as 3-methylcholantrene (3-MC) and phenobarbital (PB). The I/S ratio is higher than 1.5 with 3-MC treatment whereas it is lower than 0.5 with PB treatment. With regards to this contrasting effect, it has been suggested that the I/S ratio should be useful to differentiate between the effects of these types of inducers. We studied the consequences of in vivo PB and 3-MC treatment on scoparone biotransformation in guinea pig and rabbit. In these two species, at the basal state, scoparone biotransformation was enhanced in comparison to the rat. Moreover, in these untreated animals, two other metabolites were formed. After 3-MC or PB treatment, scoparone metabolism is, in contrast to the rat, inappropriate to differentiate between the P-450 profile of other animals.

Further differentiation of 2'- and 4'-chloroflavones as inducers of hepatic microsomal monooxygenases in rats.

2'-Chloroflavone (CF) and 4'-CF were studied as inducers of cytochrome P-450-mediated monooxygenases in rat hepatic microsomes by comparison with phenobarbital (PB) and 3-methylcholanthrene (MC). As a result of interaction with the substrate 7-ethoxycoumarin (7-EC), 2'-CF-induced microsomes showed a difference spectrum different from usual type I, whereas 4'-CF-induced microsomes showed a typical type I spectrum. 2'-CF-induced microsomes increased the Vmax of aminopyrine (AM) N-demethylase activity about 1.6-fold while not affecting the apparent Km value. In contrast, 4'-CF-induced microsomes decreased the Km of 7-EC O-deethylase activity to about one fourth while significantly increasing the Vmax value. All the activities of AM N-demethylase, 7-EC O-deethylase and 6,7-dimethoxycoumarin O-demethylase in 2'-CF-induced microsomes were inhibited strongly by metyrapone and SKF 525-A, and significantly by diphenhydramine. The three enzyme activities in 4'-CF-induced microsomes were inhibited markedly by alpha-naphthoflavone and significantly by 2-bromo-4'-nitroacetophenone. These results further support the previous proposal that 2'-CF is a PB-type inducer of cytochrome P-450 while 4'-CF is a MC-type inducer.

Selective increase in cytochrome P450 content in hepatic microsomes from rats with ventromedial hypothalamic lesions.

Changes in the components of hepatic microsomal electron transport systems and in drug hydroxylase activities were investigated in ventromedial hypothalamus (VMH) lesioned obese rats. Eight weeks after electrolysis of the bilateral VMH, the content of cytochrome P450 per mg microsomal protein (0.79 +/- 0.07 nmol/mg protein) was significantly higher (P less than 0.02) than that in the sham-operated rats (0.59 +/- 0.02). Cytochrome P450 per whole liver in the VMH-lesioned obese rats had also significantly increased (87 +/- 9 nmol vs 56 +/- 3, P less than 0.02). No significant differences were found in the cytochrome b5 contents, and the activities of NADPH- and NADH-cytochrome c reductases between the VMH-lesioned obese and sham-operated rats. The demethylation activities of aminopyrine (1.04 +/- 0.02 nmol/mg protein/min vs 0.94 +/- 0.02, P less than 0.05) and p-nitroanisole (0.96 +/- 0.02 vs 0.89 +/- 0.02 , p less than 0.02) and the aniline hydroxylase activity (0.22 +/- 0.01 vs 0.16 +/- 0.01, P less than 0.01) were enhanced, but 7-ethoxycoumarin O-deethylase activity was unchanged in the VMH-lesioned obese rats. These results indicate a selective increase in the content of cytochrome P450 among the components of the P450-dependent mixed function oxidase system in the liver of VMH-lesioned obese rats. Our observations suggest that drug metabolism may be enhanced in the hypothalamic obesity.

Clotrimazole induction of cytochrome P-450: dose-differentiated isozyme induction.

Treatment of male rats for 3 days with the N-substituted imidazole, clotrimazole, produced up to a 4-fold induction of hepatic microsomal cytochrome P-450. The monooxygenase activities induced varied with the dose administered. At low doses (less than 25 mg/kg), p-nitroanisole demethylase and aniline hydroxylase activities were induced. Only at higher doses were other monooxygenase activities (erythromycin and ethylmorphine demethylases and cytochrome P-450 metabolic-intermediate complex formation from troleandomycin) induced. Microsomal UDP-glucuronosyltransferase activity toward morphine was induced at low doses in a manner similar to that of p-nitroanisole demethylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomes indicated that low doses of clotrimazole caused the intensification of a 48,000 molecular weight protein band, whereas at high doses, there was a marked intensification of an additional 50,500 molecular weight protein, the same molecular weight band as was intensified in phenobarbital- and dexamethasone-induced microsomes. The observations suggest a phenomenon of "dose-differentiated" isozyme induction for cytochrome P-450.

Promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in mouse hepatocarcinogenesis.

The effects of phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on liver hyperplasia, induction of microsomal enzyme activities, and two-stage hepatocarcinogenesis were evaluated in B6C3F1 female mice. For 4 weeks four groups of mice received PB (500 p.p.m. in the drinking water), TCPOBOP (3 mg/kg i.p. once every week), PB together with TCPOBOP or corn oil vehicle i.p. TCPOBOP induced liver hyperplasia and hypertrophy and increased p-nitroanisole-O-demethylase and aminopyrine-N-demethylase more than PB. Neither chemical changed UDPG-transferase activity. The association of PB and TCPOBOP gave the same effects as TCPOBOP alone. Other four groups of mice were treated with N-nitroso-N-diethylamine at 7 days of age and then, starting from 8 weeks of age, received the above specified weekly treatments for 20 weeks and were then sacrificed. Hepatocellular nodules greater than 150 microns were found in all animals of all groups. Due to increased size of the liver compared to controls, the number of nodules/cm3 decreased after PB and TCPOBOP treatments given alone or together; however the mean volume of nodules and the percentage of liver volume occupied by nodules increased after TCPOBOP but not after BP treatment, and the association of PB and TCPOBOP was even more effective than TCPOBOP alone. Hepatocellular adenomas greater than 2.4 mm in diameter were observed in 5 of 10 TCPOBOP-treated mice (total of 11 nodules) and in 5 of 11 mice that received PB plus TCPOBOP (total of 15 nodules). Hepatocellular carcinomas were seen in one mouse treated with PB and in three mice given PB and TCPOBOP.

Effect of methyltestosterone administration on microsomal drug metabolism in aging rats.

The effects of methyltestosterone administration (100 mg/kg for four days) on the hepatic microsomal drug-metabolizing system from old male rats was investigated. Age-related decreases in cytochrome P-450 content, cytochrome c reductase activity and benzphetamine N-demethylase activity were unaffected by methyltestosterone treatment. Administration of the androgen induced nitroanisole O-demethylase and aniline hydroxylase activities, resulting in a restoration of the latter to levels found in young-adult animals.

Substrate-induction of veratric acid O-demethylase in Nocardia sp.

Nocardia sp. demethylates veratric acid to vanillic and isovanillic acids, and subsequently to protocatechuic acid. The accumulation of specific mRNA observed during incubation of Nocardia sp. cells with substrate, as well as the effect of inhibitors of transcription and translation point to the inducible character of demethylation processes.