P450IA1 mRNA Research Articles

Cytochrome P450IA1 is rapidly induced in normal human keratinocytes in the absence of xenobiotics.

Cytochrome P450IA1 is a polycyclic aromatic hydrocarbon (PAH)-responsive monooxygenase enzyme with no known endogenous inducer or substrate. We investigated the effect of suspension on P450IA1 gene (CYP1A1) expression in cultured human keratinocytes without the addition of xenobiotics. To prohibit adhesion and trigger differentiation, human keratinocytes or dermal fibroblasts were suspended in medium made semisolid with methylcellulose. Following suspension, we observed dramatic increases (> 100-fold) in steady state P450IA1 mRNA in keratinocytes within 1 h; however, dermal fibroblasts were nonresponsive. This effect was not dependent on methylcellulose itself and could be achieved by suspension in medium alone or in Percoll solution. The induction of P450IA1 mRNA was independent of exogenous calcium or serum concentrations, agents commonly used to signal differentiation. Adherent keratinocytes overlaid with methylcellulose exhibited slight morphological changes accompanied by increased P450IA1 mRNA. The activity of the P450IA1 enzyme was found to parallel Northern analysis data. Changes in keratinocyte adhesion or shape also affect other PAH-responsive genes suggesting Ah receptor involvement. We report a novel mechanism for cell type-specific induction of CYP1A1 expression without the addition of xenobiotic inducers. These findings suggest a possible endogenous role for P450IA1 in stratified squamous epithelia.

Suppression of cytochrome P450IA1 by interleukin-6 in human HepG2 hepatoma cells

The effects of interleukin-6 (IL-6), the major inducer of the acute-phase reaction, on the expression of cytochrome P450IA1 (CYPIA1) were examined using human HepG2 hepatoma cells. Treatment of cells with IL-6 decreased the level of 3-methylhcholanthrene-induced CYPIA1 protein and its mRNA. Nuclear runoff analysis revealed that the effect of IL-6 was largely transcriptional. IL-6 treatment of HepG2 cells increased mRNA for microsomal heme oxygenase, the rate-limiting enzyme in heme.catabolism, suggesting that the suppressive effect of IL-6 on CYPIA1 mRNA may be due to a loss of heme. Consistent with this hypothesis, simultaneous treatment of cells with Sn-mesoporphyrin, an inhibitor of heme oxygenase, prevented the IL-6-mediated suppression of CYPIA1. These findings suggest that the suppression of P450IA1 mRNA by IL-6 appears to occur, at least in part, from the decline in free heme content as a result of the induction of heme oxygenase. Our results raise the possibility that other physiological as well as environmental stimuli which affect cellular heme concentrations may also modulate the expression of P450s.

Induction of cytochrome P450IA1 and its recombinant construct in H4IIE rat hepatoma cells

1. 1. Our previous studies have shown that benzo(a)pyrene (BP), 3-methylcholanthrene (3MC) and tetrachlorodibenzofuran (TCDBF) can induce the expression of the cytochrome P450IA1 mRNA in the rat hepatoma cell line, H4IIE, although the kinetics of induction differed. 2. 2. In the present study, by using biochemical, immunochemical and recombinant DNA approaches, the effects of these inducers have been examined on the steady state level of endogenous cytochrome P450IA1 protein and on induction of chloramphenicol acetyltransferase activity (CAT) in the H4IIE cells transfected with pMC 1CAT (a recombinant construct consisting of CAT linked to 5' upstream DNA sequence of the rat cytochrome P450IA1 gene). 3. 3. From 7-ethoxyresorufin O-deethylase activity (EROD) and immunochemical analysis of cytochrome P450IA1, the optimal concentrations of BP, 3MC and TCDBF for induction in the H4IIE cells were determined as 1, 0.1-1 and 0.1 μ M, respectively. 4. 4. The elevated expression of the protein was more sustained in the TCDBF-exposed cells than in the BP or 3MC-treated cells. 5. 5. After 1.5 hr of treatment, little if any detectable P450IA1 protein was observed in the H4IIE cells although a considerable amount of mRNA was present. 6. 6. In addition, no cytochrome P450IA2 protein was detected in the control or induced H4IIE cells. 7. 7. H4IIE cells were transfected by pMC 1CAT, and the induction ratio of CAT expression in the transfected H4IIE cells after BP, 3MC or TCDBF treatment was 10-, 17- and 40-fold, respectively. 8. 8. These results indicate that the rat H4IIE cell line offers a valid homologous system for studies of the regulation of the rat cytochrome P450IA1 gene.

L-gulono-gamma-lactone oxidase is not induced in rats by xenobiotics stimulating L-ascorbic acid biosynthesis.

L-Ascorbic acid (AsA) is synthesized from D-glucose in rats; the terminal step of this synthetic pathway is catalyzed by L-gulono-gamma-lactone oxidase (GLO). In this study, we examined the effects of phenobarbital (PB) and 3-methylcholanthrene (MC), both of which are known to stimulate AsA biosynthesis in rats, on the hepatic levels of GLO activity, GLO mRNA, and AsA. Firstly, the existence of GLO mRNA was examined in the liver, kidney, lung, small intestine, spleen, testis, and prostate from a male rat; and GLO mRNA was found to be present only in the liver, in which GLO activity was also detected. The intraperitoneal injection with PB (100 mg/day/kg body weight, once a day for 2 days) or MC (20 mg/day/kg body weight, once) significantly elevated the hepatic level of AsA and the urinary excretion of AsA in rats (5-week-old males). The hepatic levels of cytochrome P-450IIB1 mRNA and cytochrome P-450IIB2 mRNA and those of cytochrome P-450IA1 mRNA and cytochrome P-450IA2 mRNA were also elevated in the rats treated with PB and MC, respectively, indicating a normal response of these animals to these compounds. However, the level of GLO mRNA and the activity of GLO in the liver tended to be slightly decreased by the administration of PB or MC, though the differences were not significant. Thus it is clear that the treatment with PB or MC stimulates the biosynthesis of AsA by increasing the activity of some enzyme(s) participating in the synthesis prior to GLO.

Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis.

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.

The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes.

Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulsed treatment with the receptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF) and beta-naphthoflavone (BNF) gave the same maximal induction response, the effect of BNF was transient compared to effect of TCDF. This was most likely due to metabolism of BNF as exemplified by the fact that a P450IA1 enzyme suicide-inhibitor, 1-ethynylpyrene, could prolong the induction response following a short BNF treatment. The TCDF induction of a reporter gene construct under the control of the -1140 to +2435 part of the CYPIA1 gene transiently transfected into HK was effectively inhibited by the dioxin-receptor antagonist alpha-naphthoflavone (ANF). In addition, ANF inhibited the accumulation of TCDF-activated nuclear receptors with capacity to bind to a xenobiotic response element. Interestingly, ANF could also suppress already maximally induced P450IA1 mRNA levels. The data demonstrate that the stability of the ligand influences the long-term effects on gene expression and that the effect of stable ligands may be masked due to receptor antagonist presence. In addition, the results support the hypothesis that a constant low level of activated nuclear receptors is required to maintain induced P450IA1 expression.

Hamster Cytochrome P-450 IA Gene Family, P-450IA1 and P-450IA2 in Lung and Liver: cDNA Cloning and Sequence Analysis1

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

The Ah receptor, cytochrome P450IA1 mRNA induction, and aryl hydrocarbon hydroxylase in a human lymphoblastoid cell line

The immunosuppressive and carcinogenic effects of aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and 3-methylcholanthrene (MC) on B lymphocytes of adult rodents and the induction of cytochrome P450IA1 and aryl hydrocarbon hydroxylase (AHH) in human mitogenactivated lymphocytes and B-lymphoblastoid cell lines are believed to be mediated by the Ah receptor. However, there has not been a direct demonstration or characterization of the Ah receptor in denned populations of any of these cells. We report here the detection and characterization of an abundant, high-affinity B lymphocyte Ah receptor in the AHH-inducible human B lymphoblastoid cell line BCR-5. Our results represent the first characterization of a human lymphocyte receptor in a well-defined lymphocyte population. Sucrose density gradient analysis of BCR-5 cytosols incubated with [ 3H]TCDD revealed a characteristic 9 S specific binding peak. The maximum concentration of Ah receptor was about 200 fmol/mg protein. Specific binding to the Ah receptor was also detected with [ 3H]MC and, to a lesser extent, with [ 3H]benzolalpyrene. The apparent binding affinity ( K d ) for [ 3H]TCDD (determined by saturation analyses) was about 5 nM. A specific [ 3 H] TCDD-Ah receptor complex which sedimented at 5 S was extracted from nuclei of BCR-5 cells incubated at 37° with [ 3H]TCDD. The Ah receptor of BCR-5 cells is thus similar in characteristics to that identified in other cell lines. When BCR-5 cells were exposed in culture for 24 hr to increasing concentrations of benz[ a]anthracene there was a concentrationdependent increase in induction and a good correlation ( r = 0.98) between the level of induced AHH activity and the relative abundance of cytochrome P450IA1 mRNA. The human B lymphoblastoid cell line BCR-5, therefore, has a complete regulatory mechanism for Ah receptor-mediated induction of cytochrome P450IA1 that is essentially the same as that which has been well established in many rodent species. The accessibility of human blood lymphocytes and the ease of establishment of B lymphoblastoid cell lines from any donor provide a source of pure cultures of human B lymphocytes which can be grown continuously in vitro for the study of mechanisms related to Ah receptor-mediated cytochrome P450IA1 induction, immunosuppression and carcinogenesis.

Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon

Ingestion of broccoli or other cruciferous vegetables inhibits the induction of cancer by chemicals and modifies some cytochrome P-450 enzyme activities. The effect of dietary broccoli on the levels of P450IA and IIB mRNA and proteins in rat liver and colon has been studied. Rats were fed a ten percent broccoli diet for 7 days. The expression of the cytochrome P-450 forms was altered to a different extent in the liver and colon. The level of total P450IA mRNA in the liver was increased by the broccoli together with the P450IA1 and IA2 proteins. Colonic P450IA1 mRNA and protein were induced by the broccoli diet, whereas only P450IA2 protein and not mRNA was detectable in colon, but the protein level was unaffected by the broccoli diet. Liver P450IIB and IIE1 proteins were increased by the broccoli diet, whereas the level of P450IIB mRNAs was not affected. In contrast, the P450IIB mRNA levels were reduced but the protein levels were increased in colon and we suggest that a feedback mechanism caused the decrease of the P450IIB mRNAs levels. Because the ratio between activation and deactivation may be an important risk determinant, we conclude that the protective effect of the broccoli diet on chemically induced tumors in rodents may be caused by the broccoli-induced changes in P450IA and IIB associated enzyme activities.

Omeprazole and aryl hydrocarbon hydroxylases: Should we be worried?

Diaz and colleagues have carefully studied the effects of omeprazole on the expression of cytochrome P-450 in primary cultures of human hepatocytes. When omeprazole was added to the culture medium in varying concentrations, there was an increase in P-450IA2 protein and mRNA concentrations, an increase in de novo synthesis of P-450IA2 protein, and an increase in microsomal catalytic activities characteristic of P-450IA2 (phenacetin deethylase and acetanilide hydroxylase). Omeprazole treatment also resulted in an increase in both enzymatic activity characteristic of P-450IA1 (ethoxyresorufin deethylase and benzpyrene hydroxylase) and concentration of P-450IA1 mRNA. In contrast, omeprazole appeared to have no significant effect on expression of other P-450s within the P-450II or P-450III families in the hepatocytes. To validate these in vitro observations, liver biopsy specimens were obtained from five patients before and after a 4-day course of pharmacological doses of omeprazole. In each patient, omeprazole treatment appeared to result in a two- to eightfold increase in P-450IA2 immunoreactive protein and P-450IA1 and P-450IA2 enzymatic activities. The authors conclude that omeprazole is an inducer of P-450IA2 and probably P-450IA1 in human liver. Induction of these enzymes could potentiate the bioactivation of carcinogens or the hepatotoxicity of some drugs such as acetaminophen.

Serum and extracellular calcium modulate induction of cytochrome P-450IA1 in human keratinocytes.

Culture conditions allowing for cytochrome P-450IA1 induction by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in normal human keratinocytes (HK) were investigated. HK grown in serum-free low extracellular Ca2+ (0.1 mM) medium did not accumulate P-450IA1 mRNA in response to TCDF. If, however, the cultures were pretreated for more than 24 h with either serum or elevated extracellular Ca2+ (2.0 mM), induction of P-450IA1 was obtained by TCDF. Serum and elevated Ca2+ concentrations were found to be additive in this respect. When analyzing HK derived from five individuals, no apparent difference was found in the relative induction of P-450IA1 by increasing concentrations of TCDF, giving an EC50 of approximately 2 nM. The permissive effect of serum and elevated Ca2+ could be conferred to a reporter gene by the -1140 to +2435 part of the human CYPIA1 gene. Culture conditions allowing for P-450IA1 induction correlated with conditions that induced mRNA corresponding to the differentiation specific enzyme epidermal transglutaminase. This finding, together with the known differentiation promoting effects of serum and elevated Ca2+, suggest that terminal differentiation is necessary for P-450IA1 induction in HK by Ah receptor ligands.

Evidence for increased translational efficiency in the induction of P450IIE1 by solvents: Analysis of P450IIE1 mRNA polyribosomal distribution

The potential for enhanced translational processing of P450IIE1 mRNA during the early phase of P450IIE1 induction by pyridine or acetone was assessed by hybridization analysis of polyribosomal P450IIE1 mRNA distribution in rat hepatic tissue. Optical absorbance profiles of polyribosomal fractions exhibited an apparent shift at 5 h following pyridine administration relative to control. Slot and Northern blot analyses for P450IIE1 mRNA in the cytoplasmic extracts isolated from 5 h pyridine-treated rats demonstrated a shift in distribution of P450IIE1 message toward heavier polyribosomal fractions and Northern blot analysis suggested the presence of different populations of P450IIE1 mRNA. Slot blot analyses also demonstrated a shift in the polyribosomal distribution of P450IIE1 mRNA at 12 h following pyridine treatment; in contrast, hybridization analysis for P450IA1 revealed no shift in polyribosomal distribution of P450IA1 mRNA. Acute acetone administration to animals also resulted in a similar shift in polyribosomal distribution of P450IIE1 mRNA as compared to control. These data suggest that P450IIE1 mRNA shifts toward larger polyribosomes following acute exposure of animals to pyridine or acetone and provide evidence that induction of P450IIE1 at early times following acute pyridine or acetone administration involves enhanced translational efficiency through increased loading of ribosomes on P450IIE1 mRNA.

Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities

The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B 2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase activities (ERDE) in placental tissues from smoking mothers, whereas the anti-PB-B 2 (to phenobarbital-induced rat liver P-450) was without significant inhibitory effect. Inhibition of 7-ethoxycoumarin O-deethylase (ECDE) by the anti-BNF-B 2 was dependent on maternal smoking: the enzyme from non-smokers was not significantly inhibited, whereas the enzyme from smokers was variably inhibited by 15–60 per cent. The monoclonal antibodies towards the major 3-methylcholanthrene-inducible and phenobarbital-inducible rat liver P-450s (Mab 1-7-1 and 2-66-3, respectively) behaved similarly, except the inhibition was somewhat stronger if present. Antibody raised against rat liver NADPH-cytochrome P-450 oxido-reductase did not inhibit any activity studied. In immunoblotting experiments, the anti-reductase recognized the protein in human placental microsomes. However, neither anti-BNF-B 2, anti-PB-B 2 or Mab 1-7-1 or Mab 2-66-3 detected any proteins in human placental microsomes, regardless of smoking status. Northern blot hybridization analysis of placental RNA samples showed that only P-450IA1 mRNA existed in the placentas of smoking mothers with detectable ERDE activity. Despite the discrepancy between protein blotting and immunoinhibition data all other findings support the conclusion that maternal cigarette smoking induces the expression of CYPIA1 gene (and not CYPIA2), resulting in an increased synthesis of P-450IA1 protein and increased AHH, ERDE and ECDE activities in human placenta.

Selective expression of cytochrome P450 isozymes by 4- n-alkyl-methylenedioxybenzenes in rat lung cells

Rat lung preparations were analyzed for cytochrome P450 expression as a function of pretreatment with 1 mmol/kg/day of various substituted n-alkyl-methylenedioxybenzenes (MDBs). Lung P450s were quantitated by Western blotting and visualized by immunocytochemical techniques using polyclonal antibodies directed against P450IA1/IA2 and P450IIB1/IIB2. Results demonstrated that n-hexyland n-octyl-MDB (but not n-butyl-MDB) increased P450IA1 in lung microsomes (50 pmol/mg microsomal protein) compared to untreated controls and n-butyl-MDB-treated animals (6 pmol/mg). However, treatment with all of these MDBs concomitantly decreased P450IIB1 in rat lung microsomes, from 40 pmol/mg in untreated control lungs to below the detection limit of this method (<2 pmol/mg). In vitro metabolism assays with rat lung microsomes, utilizing 7,12-dimethylbenz[ a]anthracene as substrate, confirmed the increase in P450IA1 by selected MDBs and the concomitant decrease of catalytically active P450IIB1. Immunocytochemistry of rat lungs with these same IgGs indicated selective cellular responses. n-Hexyl-MDB (but not n-butyl-MDB) increased P450IA1-like immunoreactivity, but the increase was specifically localized to Clara cells. Immunocytochemical results further showed that P450IIB1-like immunoreactivity disappeared entirely from alveolar type II cells, yet appeared unchanged in Clara cells relative to untreated controls. Increases in P450IA1 due to treatment with n-hexyl-MDB were associated with increases in P450IA1 mRNA as indicated by Northern blot experiments. In contrast, the decreased levels of P450IIB1, consequent to treatment with n-alkyl-MDBs, were not associated with altered levels of pulmonary P450IIB1 mRNA as determined by Northern blotting and solution hybridization assays. These results imply that n-alkyl-MDBs regulate pulmonary cytochromes P450 by both transcriptional and post-transcriptional mechanisms.

Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.

Differences in the modulation of P4501A1 and epoxide hydratase expression by benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse embryo versus mouse hepatoma-derived cell lines

Polycyclic aromatic hydrocarbon (PAH)-induced C3H/10-T1/2/CL8 mouse embryo fibroblasts (10T1/2) and mouse hepatoma-derived Hepa 1c1c7 cells (Hepa-1), exhibit comparable total cytochrome P450 levels and total PAH-metabolizing activities but very different distributions of PAH metabolites. Based on anti-P450IA1-IgG inhibition data, P450IA1 contributes essentially all PAH metabolism in Hepa-1 microsomes but is not involved in PAH metabolism by 10T1/2 cells. In addition, the microsomal epoxide hydratase (EHm) in Hepa-1 cells is far less effective in dihydrodiol (diol) formation compared to that in 10T1/2 microsomes [Pottenger, L.H. and Jefcoate, C.R. Carcinogenesis, 11, 321-327 (1990)]. In the present study, the levels of expression of P450IA1 and EHm proteins and the corresponding mRNAs, both prior to and following exposure to benz[a]anthracene (BA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been correlated with microsomal PAH metabolism by each cell type. In 10T1/2 cells, P450IA1 protein (56 kd) and mRNA (2.6 kb) were detectable at extremely low levels in only two of five cell preparations and then only after maximum induction by TCDD and BA. Thus although 10T1/2 cells contain functional Ah receptors, their capacity to induce P450IA1 is highly suppressed, representing at most 2% of the total P450. TCDD (10 nM) was 4-fold more effective than BA (10 microM) in inducing P450IA1 mRNA, while the levels of immunodetectable protein were comparable. An even greater discrepancy between P450IA1 mRNA and protein levels was seen in BA-induced Hepa-1 cells, where a 4-fold increase in mRNA was paralleled by a 20-fold increase in protein. This difference is probably due to the greater effect of BA depletion on mRNA compared to protein levels. In 10T1/2 cells, BA and TCDD were equally effective at increasing expression of an unidentified 1.9 kb mRNA sequence that blotted very weakly with the P450IA1 cDNA probe. The expression of this mRNA was independent from that of P450IA1. A similar band was visible in Hepa-1 cells less than 1% of the P450IA1 mRNA. EHm mRNA was almost 3-fold higher in 10T1/2 compared to Hepa-1 cells and was unaffected by cell treatments. In Hepa-1 cells, BA and TCDD elevated EHm protein and hydrating activity to levels comparable to those expressed in 10T1/2 cells. It is, therefore, suggested that the relative ineffectiveness of Hepa-1, compared to 10T1/2 EHm, to hydrate low levels of PAH-epoxides is due to differences between the two proteins or their disposition in the microsomal membrane.

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals: Phenobarbital and TCDD induce the same form of cytochrome P450

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different “classes”. Both 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P 1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducerreceptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD ( Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.

2‐(4′‐Chlorophenyl)benzothiazole is a potent inducer of cytochrome P450IA1 in a human and a mouse cell line

Two benzothiazole derivatives, 2-(4'-chlorophenyl)benzothiazole (CPBT) and 2-(4'-formylphenyl)benzothiazole (FPBT) were studied for their ability to induce aryl hydrocarbon hydroxylase activity in a mouse and a human cell line. In both the mouse hepatoma cell line, Hepa-1, and the human choriocarcinoma cell line, JEG-3, a high aryl hydrocarbon hydroxylase activity was observed after treatment with CPBT. In contrast, FPBT had a very weak inducing capacity in both cell lines. The maximal induction by CPBT was several times (e.g. in Hepa-1 about fourfold on average) greater than that observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific cDNA probe for mouse cytochrome P4501A1 gene was used to quantify mRNA levels in Hepa-1 cells. CPBT increased cytochrome P450IA1 mRNA to a level of 88% of that induced by TCDD. Immunoblot analysis with monoclonal antibody 1-7-1, directed against rat liver cytochrome P450IA1 and P450IA2, showed that the amount of P450IA1 is substantially increased in Hepa-1 cells after treatment with CPBT. The observation that CPBT competed TCDD off its specific cytosolic binding site suggests a receptor-mediated induction of cytochrome P450IA1 mRNA. An in vitro activation effect did not explain the exceptionally high hydroxylase activity. The results show that CPBT is a more efficient inducer of aryl hydrocarbon hydroxylase than TCDD in Hepa-1 and JEG-3 cells and that the induction is supported by P450IA1. The discordant effect of CPBT on mRNA and aryl hydrocarbon hydroxylase activity suggests that post-translational modifications of P450IA1 account for a major part of the increased enzyme activity.

Inducibility of rat liver cytochrome P-450IA1 (P-450c) mRNA during the partial inhibition of protein synthesis

Inducibility of rat liver cytochrome P-450IA1 (P-450c) mRNA during the partial inhibition of protein synthesis