Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites.

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.

Ontogenesis of CYP2C-dependent arachidonic acid metabolism in the human liver: relationship with sudden infant death syndrome.

A modification of the human monooxygenase system have been previously associated with the sudden infant death syndrome (SIDS): the hepatic CYP2C content was markedly enhanced and resulted from an activation of CYP2C gene transcription. To determine the possible consequence of the up-regulation of CYP2C in SIDS, we examined the metabolism of arachidonic acid (AA) an endogenous substrate of CYP2C involved in the physiologic regulation of vascular tone. The overall AA metabolism was extremely low during the fetal period and rose after birth to generate 14,15 epoxyeicosatrienoic acid (EET), 11,12 EET and the sum of 5,6 dihydroxyeicosatrienoic acid (diHETE)+omega/omega-1 hydroxy AA. In SIDS, the accumulation of CYP2C proteins was associated with a significant increase in the formation of 14,15 and 11,12 diHETE, which were shown to be supported by individually expressed CYP2C8 and 2C9 and HETE1 (presumably 15 HETE). This increase was markedly inhibited by addition of sulfaphenazole, a selective inhibitor of CYP2C9. So, we propose that the higher CYP2C content in SIDS stimulates the production of EETs and diHETEs and might have severe pathologic consequences in children.

Metabolism of irinotecan (CPT-11) by CYP3A4 and CYP3A5 in humans.

7-Ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (CPT-11), a DNA topoisomerase I inhibitor, undergoes several metabolic pathways to generate conjugated and unconjugated derivatives that could be excreted from the body. The objective of this study was to determine the oxidative metabolites of CPT-11 recovered in human urine samples and to identify cytochrome P450 (CYP) involved in their formation. In addition to the already known metabolites of CPT-11 [SN-38, SN-38-G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC), and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC)], we isolated three oxidized metabolites from the urine of two children and two adults given CPT-11. M1 and M2 (molecular weight, 602) were hydroxylated, respectively, on the CPT moiety and on the terminal piperidine ring of CPT-11. M3 had a molecular mass of 602, but its urine concentration in patients was too low to establish its chemical structure by liquid chromatography/mass spectrometry. In vitro incubations with cells expressing CYP2C8, CYP2C9, CYP1A1, CYP1A2, or CYP3A7 did not produce any detectable metabolites. Only CYP3A4 produced both APC and NPC, resulting from the oxidation of the piperidinylpiperidine side chain of CPT-11 along with metabolite M2. The metabolism of CPT-11 by CYP3A5 was markedly different because neither APC or NPC nor M2 was produced, whereas only one new metabolite, M4 (molecular weight, 558), was generated by de-ethylation of the CPT moiety. No previous study has reported the presence of the M4 metabolite. Production of APC, NPC, M2, and M4 was prevented by ketoconazole, a specific CYP3A inhibitor. The parameters of CPT-11 biotransformation into M2 and M4 were examined using cell lines expressing, respectively, with CYP3A4 and CYP3A5, indicating that CPT-11 is preferentially metabolized by CYP3A4. In conclusion, CYP3A plays a major role in the metabolism of CPT-11, with some differences of the metabolic profile exhibited by 3A4 and 3A5.

Transcriptional activation of CYP2C, MxA and Fas in sudden infant death syndrome.

The expression of cytochrome P4502C has been shown to be upregulated in sudden infant death syndrome (SIDS) and could be linked to viral infection through the release of interferon alpha and interleukins. MxA is a reliable marker of IFNalpha release and its level was significantly enhanced in SIDS reflecting the release of IFNalpha in response to viral infection. Similarly, the concentration of Fas protein was increased in SIDS (2.6x control) and indicated a stimulation of the Fas gene expression. Accumulation of MxA and Fas proteins were visible in liver and to a lesser extent in lung and kidney. The amount of RNA encoding CYP2C9 (4.4x control), 2C8 (2.5x) and 2C18 (2.3x) was markedly higher in SIDS than in age-matched children and would suggest a transcriptional activation of CYP2C gene expression. Finally, CYP2C genes were shown to be adjacent to two IFN-inducible genes (IFI54 and IFI56) on chromosome 10. We conclude that in SIDS a viral infection leads to the release of IFNalpha which could activate a battery of IFN-inducible genes. This might modify the chromatin structure and facilitate the accessibility to promoter/regulatory sequences of CYP2C and Fas genes close to IFN-inducible gene on chromosome 10.

Expression of CYP2E1 in human lung and kidney during development and in full-term placenta: a differential methylation of the gene is involved in the regulation process.

Methylation of dinucleotide CG residues located in the 5' end of the CYP2E1 gene has been demonstrated to play a role in the control of gene expression in the human developing liver. This study was undertaken to examine the CYP2E1 RNA content of human lung, kidney and full-term placenta and to determine whether the expression of CYP2E1 was controlled by its methylation status in these tissues. CYP2E1 was expressed at a very low level in the lung and kidney at whatever age, and at a variable level in full-term placentas. The restriction profile of genomic DNA was identical in lung and kidney and corresponded to a heavy methylation of HpaII/MspI sites located within the promoter, the first exon and first intron of the CYP2E1 gene. A different pattern of methylation was obtained in full-term placentas, indicating that CpG residues located in the 5' end of the gene were predominantly but not fully demethylated. However, the variable level of CYP2E1 RNA in full-term placentas suggests the involvement of other elements in the regulation process of CYP2E1 in this tissue.

Expression of CYP3A in the human liver--evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth.

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P-450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16alpha hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6beta derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30-40% of the adult activity after one month. CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age. These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.

Biotransformation of taxoids by human cytochromes P450: structure-activity relationship.

The metabolism of paclitaxel and docetaxel by human liver microsomes was investigated in vitro. The main metabolite of paclitaxel formed in vitro was the 6 alpha-hydroxypaclitaxel: its formation largely exceeded the formation of other metabolites hydroxylated on the lateral chain by rat liver microsomes and initially characterized in rat bile. In contrast, in vitro studied showed that the initial metabolite of docetaxel resulted from the hydroxylation of the tert-butyl of the lateral chain at C13 and that the same metabolites were formed in human and animal models. Comparison of individual CYP protein content of human microsomes and catalytic activities with taxoid biotransformation, showed that 2 distinct isoforms were assigned to the 6 alpha-hydroxylation (CYP2C) and to the hydroxylation of the lateral chain (CYP3A4). Chemical and immunological inhibitions confirmed these assumptions. The effect of antineoplastic drugs potentially associated with taxoids during chemotherapy has been tested in vitro on paclitaxel and docetaxel biotransformations. In the therapeutic range, vincristine, vinblastine, doxorubicine and cisplatin elicited a moderate or no inhibition of paclitaxel and docetaxel metabolism, as well as cimetidine, ranitidine and diphenylhydramine used to prevent major side effects associated with taxoid therapy. In patients given barbiturates, the hydroxylation on the lateral chain of paclitaxel and docetaxel was markedly stimulated and resulted from the induction of CYP3A isoforms. These results clearly demonstrated that the biotransformation of paclitaxel and docetaxel by human liver microsomes was supported by 2 distinct CYP proteins and that drug interactions could modify the therapeutic efficiency of taxoids during chemotherapy.

Detection of cytochrome P450 gene expression in human placenta in first trimester of pregnancy.

Human first-trimester placentas were screened for the expression of xenobiotic-metabolizing cytochrome P450 (CYP) genes. mRNAs of CYP1A1, CYP1A2, CYP2C, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were identified by reverse transcriptase-polymearse chain reaction (RT-PCR) in at least some of the six placental samples studied. CYP2A and CYP2B message were absent in all samples. The level of all of these CYP mRNAs was lower compared to the corresponding levels in liver or lung. the catalytic activity marker (7-ethoxyresorufin O-deethylase) was inducible in the placentas by maternal cigarette smoking. Thus, the regulatory system of placental CYP1A1, mediated by the Ah-receptor, appears to be developed as early as the first trimester of pregnancy. Three immunoreactive bands from placental microsomes were detected by an antihuman CYP3A4 antibody, but no functional activity of CYP3A enzymes could be detected. These results show that placental tissue during the first trimester of pregnancy has the potential of expressing several CYP genes, and forms a basis for subsequent analysis of these forms at the protein and functional level.

Metabolism of docetaxel by human cytochromes P450: interactions with paclitaxel and other antineoplastic drugs.

The metabolism of docetaxel by human liver microsomes was investigated in vitro and compared with that of paclitaxel. A main docetaxel metabolite was generated by human liver microsomes in the presence of NADPH: retention time in high pressure liquid chromatography and its ion fragmentation in mass spectrometry were identical to those of the authentic derivative hydroxylated at the butyl group of the C13 side chain. Kinetic measurements and chemical and immunological inhibitions demonstrated that CYP3A was implicated in the hydroxylation of docetaxel: Km (2 microM) and Vm values of docetaxel for human liver microsomes were comparable to those calculated for the formation of metabolite p-hydroxy-phenyl C3' paclitaxel (M4). Docetaxel hydroxylation correlated only with the CYP3A content of microsomes and with CYP3A-dependent 6 beta-hydroxylation of testosterone and 16-hydroxylation of dehydroepiandrosterone. The formation of hydroxydocetaxel was strongly reduced by CYP3A inhibitors such as ketoconazole, midazolam, erythromycin, testosterone, orphenadrine, and troleandomycin, whereas quinidine (CYP2D6), hexobarbital, tolbutamide, and mephenytoin (CYP2C) had no or little effect. The hydroxylation of docetaxel exhibited a highly positive correlation with the formation of metabolite M4 of paclitaxel (r = 0.929, P < 0.0001, n = 12), but not with its 6-hydroxylation (r = 0.48, P > 0.15). Docetaxel abolished the hydroxylation of paclitaxel metabolite M4, but was totally inactive on its 6 alpha-hydroxylation. Conversely, paclitaxel reduced significantly the hydroxylation of docetaxel. We examined in vitro the possible interaction among docetaxel, paclitaxel, and drugs which could be associated during chemotherapy. Cisplatin, verapamil, doxorubicin, vinblastine, and vincristine at concentrations usually recommended did not markedly modify taxoid metabolism. Ranitidine and diphenylhydramine had no effect, but 100 microM cimetidine partially inhibited the formation of 6 alpha-hydroxypaclitaxel. Pretreatment of patients with barbiturates strikingly stimulated docetaxel hydroxylation, whereas no acceleration of docetaxel hydroxylation was noticed in a patient receiving steroids.

Taxol metabolism by human liver microsomes: identification of cytochrome P450 isozymes involved in its biotransformation.

The biotransformation of taxol by human liver was investigated in vitro with microsomes isolated from adult and developing human tissues. In vitro, no metabolism was detected with kidney microsomes, whereas two metabolites were generated by liver microsomes. The most prominent metabolite, termed M5, corresponded to an hydroxylation at the C6 position on the taxane ring, while the other metabolite, termed M4, corresponded to an hydroxylation at the para-position on the phenyl ring at the C3'-position of the C13 side chain. These two taxol derivatives have been shown to be the major metabolites recovered in bile from a patient infused with taxol. Several approaches have been used to identify the cytochrome P450 (CYP) isozymes involved in these reactions. No positive correlation was observed between the in vitro synthesis of these two metabolites, suggesting that two cytochrome P450 isozymes could be involved, although they could not be distinguished by their apparent affinities (Km approximately 15 microM). The formation of metabolite M4 was substantially reduced both by antibody directed against CYP3A and by the addition of CYP3A substrates such as orphenadrine, erythromycin, troleandomycin, and testosterone. Conversely, the formation of metabolite M5 remained unaffected by antibodies against CYP3A and by CYP3A substrates but was sensitive to diazepam inhibition, a preferential substrate of CYP2C. Correlation between CYP2C content or diazepam demethylation and the synthesis of metabolite M5 was highly positive. The formation of metabolite M4 developed during the early postnatal period. In contrast, the synthesis of metabolite M5 rose only after 3 months of age. These data clearly implicate CYP3A in the formation of metabolite M4 and CYP2C in the synthesis of metabolite M5. Microsomes from patients treated with barbiturates and benzodiazepines increased the formation of metabolite M4 to the level of metabolite M5, demonstrating that drug interactions could modify the human metabolism of taxol.

The role of cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver.

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

High levels of expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin.

NAD(P)H:quinone oxidoreductase (NQO1) is a flavoprotein which catalyzes the two-electron reduction of quinones and azo-dyes and thus prevents the formation of free radicals and toxic oxygen metabolites that may be generated by the one-electron reductions catalyzed by cytochrome P450 reductase. Analysis of RNA indicated 20- to 50-fold higher levels of NQO1 gene expression in the liver tumors and in the tissue surrounding the tumors of patients with hepatocarcinoma than in normal individuals. An approximately 50-fold higher level of NQO1 mRNA was also observed in human hepatoblastoma (Hep-G2) cells than in normal liver. By deletion mutagenesis in the human NQO1 gene promoter and subsequent transfection into hepatic and nonhepatic cell lines, a 1.42 kb DNA segment has been identified to contain cis-acting elements responsible for high levels of expression of the NQO1 gene in tumor cells.

Transplacental induction of cytochromes P-450IA1 and P-450IA2 by polycyclic aromatic carcinogens: TCDD-binding protein level as the rate-limiting step.

Induction of cytochromes P-450c and P-450d have been compared in fetal and adult rat liver. A single dose of 3-methylcholanthrene (MC) 16 h before death, resulted in a pronounced accumulation of both mRNAs in adult liver. In contrast, treatment of fetuses with a single dose of MC did not increase the hepatic content in P-450c and P-450d mRNAs, whereas a 3-day treatment gave rise to a considerable accumulation of mRNAs. The fetal rat liver content in 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)-binding protein was extremely low compared to the adult content. Pretreatment of fetuses with phenobarbital potentiated the inducing effect of MC upon P-450c and P-450d mRNA accumulation in relation to the duration of pretreatment, as well as increasing the hepatic content in TCDD-binding protein. Data strongly suggest that the hepatic TCDD-binding protein level is the rate-limiting step in MC induction of P-450c and P-450d in fetal but not in adult rat liver. In addition, phenobarbital and MC are potent inducers of the TCDD-binding protein in the fetal rat liver.

Enhancing effect of a phorbol ester and of retinoic acid on glucocorticoid induction of chenodeoxycholate hydroxylation in hepatoma cultures.

In cultures of the differentiated clones Faza 967, Fao and HF, derived from Reuber hepatoma, physiological doses of glucocorticoid induce chenodeoxycholate 6 beta-hydroxylation, a microsomal cytochrome-P-450-mediated activity (enhanced in liver by phenobarbital and not by benzo[a]anthracene). Whereas 12-O-tetradecanoylphorbol 13-acetate (TPA) alone has no effect the tumor promoter, when added to dexamethasone, enhances this induction. This enhancement, half-maximum with 10 ng/ml TPA, is a function of the dose between 1 ng/ml and 50 ng/ml; 50 ng/ml (80 nM) increase 4-7-fold the induction rate (as measured in cultures by the amount of bile acid hydroxylated per 10(6) cells in 24 h, and in homogenates from treated cells) and 2.5-fold the maximum activity attained by the third day of induction. When added to cultures of the dedifferentiated clone H5, treated with benzo[a]anthracene, TPA does not influence benzo[a]pyrene hydroxylase induction, as shown by the total and relative amounts of the various hydrosoluble benzo[a]pyrene metabolites. TPA does not affect tyrosine aminotransferase induction in dexamethasone-treated Fao cultures. The enhancement is not suppressed by indomethacin, an inhibitor of prostaglandin synthesis. After dexamethasone removal from induced Faza 967 cultures, addition of TPA to the medium does not affect the decay rate of the chenodeoxycholate-hydroxylating activity. Retinoic acid similarly enhances the induction by dexamethasone of chenodeoxycholate hydroxylation, both in treated Faza 967 cultures and in homogenates from treated cultures. The effects of TPA and retinoic acid are additive. These results suggest a possible cooperation at the transcriptional level between transactive factors, involving TPA-mediated alterations, retinoic acid and glucocorticoid receptors. The system described might provide a convenient experimental approach in the study of its mechanism.

Transcriptional control of human cytochrome P1-450 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human tissue culture cell lines.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces aryl hydrocarbon hydroxylase (AHH) activity and human cytochrome P1-450 mRNA in the human breast carcinoma MCF-7 and hepatoblastoma HepG2 tissue culture cell lines. Although AHH activities induced by 100 nM TCDD are comparable in these cell lines, the EC50 values for TCDD differ: EC50 approximately equal to 1 nM for HepG2; EC50 greater than 20 nM for MCF-7. In order to determine the mechanism responsible for this difference in EC50, we have examined putative regulatory factors such as the intracellular TCDD receptor as well as the kinetics of mRNA transcription and accumulation in these cells. TCDD increases transcription of hP(1)450 mRNA in both MCF-7 and HepG2 cells; however, MCF-7 cells require higher concentrations of TCDD to produce transcriptional activation comparable to that observed for HepG2 cells. These data indicate that the difference in EC50 is determined at an early step in the induction of hP(1)450 mRNA. With the use of a sensitive assay based on high-performance anion-exchange liquid chromatography, an intracellular protein which binds TCDD with high affinity was detected in HepG2 cytosolic fractions but not in MCF-7 cells. Thus, the difference in EC50 for TCDD can be correlated to the differences in TCDD binding. We postulate that MCF-7 cells contain a "defective" receptor with decreased affinity for TCDD.

Enhancement of DNA binding, mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice.

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[alpha]pyrene (BP) metabolism and activation and, a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[alpha]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).

Drug-metabolizing enzymes in human foetal liver: partial resolution of multiple cytochromes P 450.

Cytochrome P 450 concentration, related monooxygenase activities (towards aniline, p-nitroanisole, benzphetamine, ethoxycoumarin, benzo(a)pyrene, testosterone, and dehydroepiandrosterone), epoxide hydrolase, and glutathione S-transferase activities were measured in the liver of human foetuses aged from 15 to 38 weeks and compared to adult activities. Different ontogenic patterns seem to exist between monooxygenase activities: if the overall cytochrome P 450 concentration, aniline hydroxylase, benzphetamine demethylase, epoxide hydrolase, and glutathione S-transferase activities reach about the half of adult values as early as 15-25 weeks of gestational age, the metabolism of benzo(a)pyrene, ethoxycoumarin, and testosterone in position 6 beta is very low in these foetuses, whereas the 16 alpha hydroxylation of dehydroandrosterone is higher than in adult human liver. Foetal and adult cytochromes P 450 were resolved by DEAE cellulose chromatography into three different fractions: in reconstitution experiments, the major fraction (A) was active toward aniline, whereas benzphetamine and ethoxycoumarin were mainly metabolized by the two other fractions (Ba and Bb). Results show that multiple cytochromes 450 are present in foetal liver and are able to catalyze, with a lower molecular activity, the same reactions as in adult human liver.

Biotransformation of chloroform by rat and human liver microsomes; in vitro effect on some enzyme activities and mechanism of irreversible binding to macromolecules.

The effects of chloroform on some rat microsomal enzyme activities were studied in vitro. Maximum inhibition of oxygen consumption, NADPH oxidase and NADPH-cytochrome c reductase was observed at 0.5 mM chloroform; prior metabolization of CHCl3 by microsomal monooxygenases increased inhibition by about 50% at 0.2-0.5 mM chloroform. Higher concentrations produced a paradoxical reversal of inhibition, whereas p-nitroanisole demethylase was steadily inhibited by about 50% up to 10 mM chloroform. Irreversible binding of 14CHCl3 was confirmed to depend on chloroform metabolization by monooxygenases. The increased irreversible binding due to phenobarbital induction is accompanied by a diminished affinity towards chloroform as shown by increased KM of irreversible binding, and a higher spectral dissociation constant KS. Aminoacids with nucleophilic functions (histidine, cysteine) partially prevented the irreversible binding of chloroform metabolites to microsomes; non-volatile radioactive derivatives were recovered in trichloracetic acid supernatants when microsomes were incubated with cysteine, but not with histidine. Phosgene has been demonstrated as a biological metabolite of chloroform: its possible reactions with nucleophilic groups of macromolecules, water and added aminoacids partly explain these experimental data. Similar results were obtained with human microsomes, showing that chloroform hepatotoxicity in man could involve the same mechanisms.