Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions

It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenic/antiblastic activity. Antitumoral compound effects strongly depend on the biochemical/physiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration. We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant p degrees at different contents of glutathione and cytochrome P-450, molecules able to activate/detoxicate xenobiotics. The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions. The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level. In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450. These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug.     

Effects of cold preservation and reperfusion on microsomal cytochrome P-450-linked monooxygenase system of the rat liver

The routine application of capillary electrochromatography (CEC) is demonstrated by incorporating 75 microns I.D. capillaries packed with 3 microns octadecylsilica (ODS) particles into a commercial CZE instrument. A mixture of several neutral compounds is separated into its components with an average efficiency up to 181 000 plates/m in less than 8 min. Hundreds of consecutive runs are performed over a period of weeks from which it is concluded that the reproducibility of the capacity factors is better than 2% and that CEC separations can be achieved in a reliable and routine manner.     

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes

When male rats were given either a single dose of cadmium (3.58 mg CdCl2.6H2O/kg, i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl2.6H2O/kg, s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg/kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg/kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.     

Cytochrome P450 enzyme activities in the Australian brushtail possum trichosurus vulpesula: a comparison with the rat, rabbit, sheep and chicken

630 neonates with risk factors of perinatal hearing impairment were screened of hearing loss by means of registration of transient otoacoustic emissions before discharge from the newborn nursery. Neonates were screened additionally by means of brainstem evoked response audiometry, if they had bilateral negative emissions. 810 healthy neonates were screened as control group. The investigations were carried out in incubator after the feeding of neonates. The prevalence of a bilateral negative cochlear response was 5.2% in the risk babies and 1.7% in healthy neonates. Neonates are high risk patients for hearing loss if they show craniofacial anomalies including alcohol embryofetopathy, connatal infections, or very low birth weight babies with additional risk factors. The pedaudiological control investigations of the babies with a negative bilateral cochlear response delivered in the risk group 15 cases (2.4%) with an important hearing impairment and in the healthy neonates 2 cases (0.25%) respectively. Prevalence and importance of perinatal hearing impairment explains the necessity of detection in the neonatal period.     

Pharmacokinetics of a synthetic, chemically modified hammerhead ribozyme against the rat cytochrome P-450 3A2 mRNA after single intravenous injections

Modulation of gene expression via nucleic acid sequence-specific intervention represents a new paradigm for drug discovery and development. Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. A 2'-O-allyl-modified hammerhead ribozyme designed to cleave the messenger RNA of cytochrome P-450 3A2 was administered to rats via 0.25 mg intravenous injections to investigate the disposition of this compound. The chemically modified ribozyme binds to serum albumin and can be displaced by phosphorothioate oligonucleotides. A biphasic plasma clearance with a distribution half-life of 12 min and an elimination half-life of 6.5 h was observed. A volume of distribution of 2.1 l/kg indicates perfusion into tissues well beyond the vascular system. The chemically modified ribozyme can be detected intact in the plasma up to 48 h after injection. Metabolic degradation of the chemically modified ribozyme occurs at unmodified ribonucleotides, leaving the 2'-O-allyl-modified sites intact. Recovery of intact chemically modified ribozyme was 1.9% of the administered dose at 12 h along with significant metabolites. The renal clearance of the intact ribozyme is an average 34.3 ml/h. The tissue distribution of the chemically modified ribozyme at 48 h is primarily to kidney and liver but the only detected material is a single 27-mer metabolite that has been cut in the unmodified GAAA region. The brain concentration of the prominent 27-mer metabolite is greater than that observed in the lung or spleen. Examination of tissues reveals no morphological evidence of toxicity. These data strongly support the potential utility of synthetic, 2'-O-allyl-modified hammerhead ribozymes as therapeutic agents in vivo.     

Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18* catalyzing imipramine N-demethylation

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.     

Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue

Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium. In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium. The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture. Very low levels of EROD and MROD activity were found in each culture without induction [EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein]. After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold. When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same. Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities. We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2.     

Influence of tryptophan on the level of hepatic microsomal cytochrome P-450 in well-fed normal, adrenalectomized, and phenobarbital-treated rats

In well-fed normal male rats, either force-feeding of tryptophan or a single injection of phenobarbital produced significant increments in hepatic microsomal cytochrome P-450 and the associated aniline hydroxylase activity. Administration of both tryptophan and phenobarbital together resulted in even greater stimulation than when the compounds were given alone. Adrenalectomy lowered instead the cytochrome P-450 concentration in comparison with that of normal rats, and administration of tryptophan and phenobarbital in this condition produced no significant increment in cytochrome P-450 concentration. In addition, phenobarbital administered either singly or in combination with tryptophan resulted in 80% mortality, which was reduced to zero by pretreatment with cortisol. While in cortisol-treated adrenalectomized rats administration of phenobarbital caused a 56% increment in cytochrome P-450 as compared to controls, tryptophan produced only a minor (9%) increase. In normal, as well as in adrenalectomized rats, tryptophan and phenobarbital administered either idividually or together increased microsomal protein concentration. In normal rats actinomycin-D treatment reduced both cytochrome P-450 and microsomal protein concentrations below that of the non-treated control levels. Further administration of either tryptophan or phenobarbital slightly increased the level of cytochrome P-450, and the two compounds together caused 40 and 21% increments of the same compared to actinomycin-treated and non-treated controls, respectively.     

P-450-dependent metabolism of lauric acid in alcoholic liver disease: comparison between rat liver and kidney microsomes

Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > 25% of ALL-patients. In this study, we have examined whether the chimeric ETV6-AML1 protein could serve as a tumor specific antigen for CTL in HLA-A2.1 individuals. We have identified a nonapeptide (RIAECILGM), encoded by the fusion region of the ETV6-AML1 protein, that binds to HLA-A2.1 molecules and induces specific primary CTL in peripheral blood lymphocytes from healthy donors. These CTL specifically lysed HLA-A2.1 tumor cells endogeneously expressing the ETV6-AML fusion protein. CTL with similar functional capacities were found with high frequencies and cloned from one patient's bone marrow indicating that ETV6-AML1-specific anti-ALL CTL are, at least in some patients, spontaneously stimulated and might participate to host antileukemia defense.     

Biphasic regulation of cytochrome P450 2B1/2 mRNA expression by dexamethasone in primary cultures of adult rat hepatocytes maintained on matrigel

We have demonstrated recently that although rat hepatocytes rapidly lose their cytochrome P450 mRNA content following their introduction into primary culture, hepatocytes cultured on Matrigel, a reconstituted basement membrane, subsequently spontaneously "reexpress" the mRNAs of some constitutive P450 forms (Kocarek et al., Mol Pharmacol 43: 328-334, 1993). In the present study, we used the Matrigel cell culture system to examine the dose-dependent effects of dexamethasone (DEX) treatments on the mRNAs for two of the P450 forms that are reexpressed spontaneously between days 3 and 5 in culture, 2B1/2 and 2C6. Treatment of cultured hepatocytes with low doses of DEX (10(-9) to 10(-8) M) that induced the mRNA for tyrosine aminotransferase, a model glucocorticoid-inducible gene, suppressed the spontaneous appearance of 2B1/2 mRNA while having little or no effect on the level of 2C6 mRNA or on beta-actin mRNA. However, treatment of the hepatocyte cultures with high doses of DEX (10(-6) to 10(-5) M) that induced P450 3A1 mRNA increased the amounts of the 2B1/2 and 2C6 mRNAs (4.1- and 2.4-fold, respectively, at 10(-5) M DEX). In contrast to the suppressive effects on the spontaneous increases in 2B1/2 mRNA, low doses of DEX (10(-8) to 10(-7) M) enhanced the induction of 2B1/2 mRNA by phenobarbital (2.5-fold at 10(-7) M DEX). Treatment of the hepatocyte cultures with triamcinolone acetonide, another potent glucocorticoid, suppressed spontaneous 2B1/2 mRNA expression at low doses, but did not induce 2B1/2 mRNA at high doses. Treatments with steroids of other classes, including dihydrotestosterone, 17 alpha-ethinylestradiol, fludrocortisone or R-5020, failed to suppress 2B1/2 mRNA levels at low doses. Additionally, treatment with RU-486, a glucocorticoid/progestin receptor antagonist, induced 2B1/2 mRNA at high doses (10(-6) to 10(-5) M). The suppressive effects of DEX on spontaneous 2B1/2 mRNA expression observed at low doses are consistent with a classical glucocorticoid-mediated mechanism, while the high-dose inductive effects of DEX appear to be exerted through a nonclassical mechanism, perhaps akin to that for induction of 3A1.     

Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.     

Separation, purification, and properties of multiple forms of cytochrome P-450 from the liver microsomes of phenobarbital-treated mice

Four distinct cytochrome P-450 fractions (A1, A2, C1, and C2) have been separated and purified from the liver microsomes of phenobarbital-treated hybrid mice (B6D2F1/J). Fractions A2 and C2 were highly purified with specific contents of 16.5 and 17.5 nmol of cytochrome P-450/mg of protein, respectively, based on their amino acid compositions. The major hemeprotein bands of A2 and C2 have different minimum molecular weights (50,000 and 56,000, respectively) on polyacrylamide gels in the presence of sodium dodecyl sulfate. All four fractions with respect to their spectral and catalytic properties, thereby demonstrating that mouse liver microsomes from phenobarbital-treated hybrid mice contain at least four forms of cytochrome P-450.