Cytochrome P450 1A1 mRNA Levels Research Articles

Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype.

This study investigated the relationship in human placenta between polycyclic aromatic hydrocabon (PAH)-DNA adduct levels and two biomarkers of cytochrome P4501A1 (CYP1A1): gene induction evidenced by CYP1A1 mRNA, and a genetic polymorphism, the CYP1A1 MspI RFLP. CYP1A1 codes for an inducible enzyme system that catalyzes the bioactivation of PAHs. Prior research found a high correlation in human lung tissue between CYP1A1 activity and DNA damage from PAHs. The CYP1A1 Mspi RFLP has been linked in some studies to risk of lung cancer. The relationships in human placenta between DNA damage, CYP1A1 activity and genotype have not been well characterized and may be relevant to risks from transplacental PAH exposure. The study cohort consisted of 70 newborns from Krakow, Poland, a city with elevated air pollution, and 90 newborns from nearby Limanowa, an area with lower air pollution but greater indoor coal use. Contrary to results seen previously in lung tissue, CYP1A1 mRNA was not significantly correlated with PAH-DNA adduct levels in the placenta. Smoking (self-reported maternal and infant plasma cotinine) was significantly associated with CYP1A1 mRNA levels (P < 0.01), but not with PAH-DNA adduct levels. Placental PAH-DNA adduct levels were significantly higher in infants with the CYP1A1 MspI restriction site compared with infants without the restriction site (P < 0.01), implicating a genetic factor in inter-individual variation in DNA damage in human placenta. Further studies are needed to determine the relevance of this finding to risk of transplacental carcinogenesis.

Disruption of endogenous regulator homeostasis underlies the mechanism of rat CYP1A1 mRNA induction by metyrapone.

The transcriptional induction of the cytochrome P-450 1A1 (CYP1A1) gene by xenobiotics such as polyaromatic hydrocarbons is dependent on their interaction with the aryl hydrocarbon receptor. Administration of the structurally unrelated compounds metyrapone (a cytochrome P-450 inhibitor) or dexamethasone (a glucocorticoid) to male rats does not induce hepatic CYP1A1 mRNA. However, administration of both metyrapone and dexamethasone to male rats results in the induction of hepatic CYP1A1 mRNA expression. The induction response is mimicked in vitro in cultured rat hepatocytes by the addition of metyrapone and dexamethasone to a serum-free culture medium, suggesting that these compounds act directly on the liver in vivo to effect hepatic CYP1A1 mRNA induction. An examination of the characteristics of CYP1A1 induction by metyrapone and dexamethasone in combination in vitro indicate that at least 6 h of treatment is required for detectable levels of CYP1A1 mRNA to accumulate in hepatocytes. In contrast, beta-naphthoflavone, which is known to bind to the aryl hydrocarbon receptor to effect CYP1A1 gene expression, induces detectable levels of CYP1A1 mRNA within 2 h of treatment. CYP1A1 mRNA is also induced when hepatocytes are treated with metyrapone in combination with the protein synthesis inhibitor cycloheximide but not with dexamethasone in combination with cycloheximide, indicating that CYP1A1 mRNA induction is strictly dependent on the presence of metyrapone and suggesting that the metyrapone-associated induction of CYP1A1 mRNA is dependent on a loss of a constitutively expressed protein that functions to suppress CYP1A1 gene expression. The role of dexamethasone in metyrapone-associated induction of CYP1A1 is probably mediated through the glucocorticoid receptor since the glucocorticoid receptor antagonist RU486 reduces the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination. Increasing the levels of the photosensitizer riboflavin present in the culture medium 10-fold and exposure to light increases the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination in vitro, suggesting that photoactivation of inducing medium constituent(s) might be required for induction. Failure to induce CYP1A1 mRNA by co-administration of metyrapone and dexamethasone in hepatocytes cultured in a balanced salt solution with or without photoactivation indicates that induction is dependent on a photoactivated component of the culture medium and not on metyrapone or dexamethasone alone. The addition of tryptophan in the presence of riboflavin to the balanced salt solution restores CYP1A1 mRNA induction by metyrapone alone and induction is increased when medium is exposed to light, indicating that induction is dependent on tryptophan photoactivation in vitro. Metyrapone failed to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for specific binding to the aryl hydrocarbon receptor in rat liver cytosolic fractions. These results suggest that CYP1A1 might be induced in rats by metyrapone through an indirect mechanism associated with an elevation in the level of an endogenously generated inducer such as photoactivated product(s) of tryptophan and not because of metyrapone's interacting with the aryl hydrocarbon receptor. The dependence of CYP1A1 induction on dexamethasone or cycloheximide suggests that derepression by a glucocorticoid receptor-modulated negative-acting factor of CYP1A1 gene expression might be critical to induction by metyrapone.

Glycine N-Methyltransferase Is an Example of Functional Diversity: ROLE AS A POLYCYCLIC AROMATIC HYDROCARBON-BINDING RECEPTOR

The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 269, 5750-5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 microM B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway.

A Comparison of Cytochrome P4501A (CYP1A) mRNA Inducibility in Four Species of Atlantic Coast Anadromous Fishes

Quantification of levels of cytochrome P4501A (CYP1A) gene expression in sentinel species of fishes has been proposed as a management tool to evaluate contamination of aquatic systems. Based on preliminary studies, we hypothesized that differences in CYP1A mRNA inducibility among individuals, populations, or species might lead to spurious conclusions when using this approach in environmental monitoring programs. To address this possibility, we quantitated and compared CYP1A mRNA induction levels in four species of common Atlantic Coast estuarine fish: smooth flounder, hogchoker, striped bass, and Atlantic tomcod, which were treated with model chemicals (beta naphthoflavone (β-NF), or benzo[a]pyrene at 10 ppm) known to induce CYP1A mRNA, or were exposed to contaminated environments. Species-specific CYP1A DNA probes were generated from PCR (polymerase chain reaction) amplification of genomic DNA using conserved oligonucleotide primers, and, along with cloned rainbow trout and Atlantic tomcod CYP1A cDNA probes were used to quantify CYP1A mRNA levels in northern blot analyses. Successful PCR amplification of CYP1A hybridizable DNA fragments was observed for all four species. Results from northern blot analyses showed large differences in CYP1A mRNA induction among species; only Atlantic tomcod exhibited significant induction of CYP1A mRNA for both chemically treated (97-fold) and environmentally exposed fish (34-fold). Significant, although lower, levels of induction were observed in β-NF treated (14-fold) smooth flounder, but not in environmentally exposed smooth flounder. Only low levels (not significant) of CYP1A gene induction were detected in hogchokers and striped bass. We conclude that CYP1A mRNA inducibility differed significantly among fish taxa perhaps due to differences in regulation of gene expression, suggesting that careful selection of sentinel species should be exercised prior to the use of CYP1A mRNA induction in environmental monitoring programs. However, the significance of differences in CYP1A mRNA inducibility in relation to higher level biological endpoints has yet to be determined.

Regulation of sucrase-isomaltase and hexose transporters in Caco-2 cells: a role for cytochrome P-4501A1?

Involvement of cytochrome P-4501A1 (CYP1A1) in the regulation of sucrase-isomaltase and hexose transporters was analyzed in low (TC7)- and high (PF11)-glucose-consuming Caco-2 clones. CYP1A1 mRNA is elevated in exponentially growing cells concomitantly with high rates of glucose consumption and high levels of GLUT-1 and GLUT-3 mRNA. After confluency, CYP1A1 is not detectable in TC7 cells; this is associated with a decreased glucose consumption, a downregulation of GLUT-1 and GLUT-3, and an upregulation of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. In PF11 cells CYP1A1 mRNA remains elevated, along with high glucose consumption, high levels of GLUT-1 and GLUT-3, and minimal expression of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. Exposure of TC7 cells to inducers of CYP1A1 results in high levels of CYP1A1 mRNA, a 10-fold increase of glucose consumption after confluency, an upregulation of GLUT-1 and GLUT-3, and a downregulation of sucrase-isomaltase, GLUT-2, and, to a lesser extent, SGLT-1 and GLUT-5. These results suggest that activation of CYP1A1, whether spontaneous or drug induced, is involved in the variations of glucose utilization and in the associated modifications of expression of sucrase-isomaltase and hexose transporters.

Glycine N-Methyltransferase Is a Mediator of Cytochrome P4501A1 Gene Expression

Cytochrome P4501A1, the isozyme most closely approximating aryl hydrocarbon hydroxylase activity under conditions of induction, is thought to be regulated by several trans-acting factors, including the 4S polycyclic aromatic hydrocarbon-binding protein; this protein has recently been identified as glycine N-methyltransferase (Raha et al. (1994) J. Biol. Chem. 269, 5750-5756). Previous studies had shown that partially purified liver preparations containing the 4S binding protein interacted with 5′-flanking regions of the cytochrome P4501A1 gene. Consequently, the ability of the 4S binding protein to serve as a mediator in the regulation of the cytochrome P4501A1 gene was investigated further. Introduction of an antisense 24-mer oligonucleotide to glycine N-methyltransferase cDNA into rat hepatoma H4IIE cells by lipofectin resulted in a 60% reduction in the benzo(a)pyrene-mediated induction of ethoxyresorufin-O-deethylase activity and protein over the sense and scrambled antisense oligonucleotide controls. In addition, the antisense oligonucleotide caused a marked reduction in the steady-state level of cytochrome P4501A1 mRNA; no such effect was observed with the sense oligonucleotide. Introduction of GNMT polyclonal antibodies into H4IIE cells by a streptolysin-O permeabilization technique markedly reduced both benzo(a)pyrene-binding and benzo(a)pyrene-induced ethoxyresorufin-O-deethylase activities, but had no effect on 2,3,7,8-tetrachlorodibenzo-p-dioxin induction. Collectively, these findings suggest that, in addition to the Ah (dioxin) receptor, glycine N-methyltransferase appears to be both a polycyclic aromatic hydrocarbon-binding protein and a mediator of the induction of the cytochrome P4501A1 gene by polycyclic hydrocarbons such as benzo(a)pyrene.

Regulation of Cytochrome P4501A1 Gene Expression in Vascular Smooth Muscle Cells Through Aryl Hydrocarbon Receptor-Mediated Signal Transduction Requires a Protein Synthesis Inhibitor

The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 μM benzo[a]pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 μg/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzofuran (2 μM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 μM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.

A Biomarker Approach to Assessing Xenobiotic Exposure in Atlantic Tomcod from the North American Atlantic Coast

We determined levels of hepatic cytochrome P4501A (CYP1A) mRNA, hepatic DNA adducts, and fluorescent aromatic compounds (FACs) in bile, a measure of exposure to polyaromatic hydrocarbons, in Atlantic tomcod from six river systems ranging from highly polluted to relatively pristine on the northeast North American coast (the Hudson River, New York; the St. Lawrence River, Quebec; the Miramichi River, New Brunswick; the Saco and Royal rivers, Maine; and the Margaree River, Nova Scotia). Hudson River tomcod showed the greatest response for all parameters, and tomcod from the Margaree River exhibited the least response. Tomcod from the Miramichi River exhibited marked induction of CYP1A mRNA but low levels of hepatic DNA adducts and biliary FACs, whereas fish from the St. Lawrence River showed no induction of CYP1A mRNA and moderately elevated levels of DNA adducts and biliary FACs. In tomcod from the Hudson and Miramichi rivers, the levels of CYP1A mRNA were 28 times and 14 times, respectively, as great as the levels in fish from the St. Lawrence, Saco/Royal, and Margaree rivers. Mean levels of DNA adducts varied from 120 nmol adducts/mol bases in Hudson River tomcod to &lt; 3 nmol adducts/mol bases in fish from the Miramichi and Margaree rivers. Concentrations of FACs in the bile of tomcod from the Hudson and St. Lawrence rivers were 8 and 1.8 times, respectively, as great as the concentrations in tomcod from the Miramichi River and Margaree River. In tomcod from the Hudson River, all three biomarkers were markedly elevated; in the St. Lawrence River two biomarkers were elevated, in the Miramichi River one was elevated, but no biomarker was substantially elevated in fish from the Saco/Royal and Margaree rivers. Elevated levels of hepatic DNA adducts and biliary FACs in tomcod from the Hudson River suggest increased exposure to PAHs, consistent with previous studies.

Regulation of Cytochrome P4501A1 in Teleosts: Sustained Induction of CYP1A1 mRNA, Protein, and Catalytic Activity by 2,3,7,8-Tetrachlorodibenzofuran in the Marine Fish Stenotomus chrysops

Cytochrome P4501A1 (CYP1A1) is known to play important roles in the activation and detoxification of carcinogens and other toxicants in vertebrate animals, including fish. Although extensively studied in mammalian systems, the regulation of CYP1A forms in other vertebrates is less well understood. We examined the time course and dose-response relationships for induction of CYP1A1 mRNA, protein, and catalytic activity by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in the marine fish Stenotomus chrysops (scup). The time course of CYP1A1 induction was determined following a single ip dose (10 nmol/kg) of 2,3,7,8-TCDF. Hepatic ethoxyresorufin O-deethylase activity was increased after 1 day, reached a maximum by 8 days, and was still elevated 14 days after treatment. The content of immunodetectable CYP1A1 protein in liver was elevated on Day 1 and continued to increase through 14 days. CYP1A1 protein content was also strongly induced in heart and gill beginning at 2 days after treatment and extending through Day 14. Hepatic CYP1A1 mRNA was strongly induced by 1 day after dosing and remained elevated through 14 days. The sustained induction of CYP1A1 mRNA by 2,3,7,8-TCDF contrasts with the transient induction seen previously in fish treated with nonhalogenated inducers and most likely reflects differences in persistence of the inducers. Dose-response studies indicated that induction of CYP1A1 mRNA, protein, and catalytic activity occurred following doses of 2,3,7,8-TCDF as low as 0.4 nmol/kg (120 ng/kg), within the range of whole-body contents of this congener measured in fish from contaminated environments. The estimated dose producing half-maximal CYP1A1 induction in scup was approximately 2-10 nmol/kg, suggesting that the sensitivity of these fish to induction may be as great as or greater than that of rats. In contrast to previous results obtained with 3,3′,4,4′-tetrachlorobiphenyl (TCB) and β-naphthoflavone, which appear to inhibit or inactivate CYP1A1 in fish and other vertebrates, there was a good correlation among levels of CYP1A1 mRNA, protein, and catalytic activity in individual fish following various doses of 2,3,7,8-TCDF. The difference in response to 2,3,7,8-TCDF versus 3,3′,4,4′-TCB may reflect differences in the inducing potencies of the two compounds relative to their similar potencies as inhibitors of CYP1A1 catalytic activity. In additional studies to evaluate structure-activity relationships for CYP1A1 induction by chlorinated dibenzofurans in fish, scup were treated with 2,3,6,8-tetrachlorodibenzofuran (2,3,6,8-TCDF). At 10 or 50 nmol/kg, 2,3,6,8-TCDF was inactive as an inducer of CYP1A1 mRNA, protein, or catalytic activity. Overall, these results illustrate temporal and dose-dependent aspects of CYP1A1 induction in fish that are highly inducer-specific; the results also indicate that fundamental features of CYP1A1 regulation appear to be conserved in fish and mammals, two widely divergent groups of vertebrates.

Induction and clearance of cytochrome P4501A mRNA in Atlantic tomcod caged in bleached kraft mill effluent in the Miramichi River

Levels of cytochrome P4501A mRNA were measured in Atlantic tomcod caged in the effluents of a bleached kraft pulp and paper mill on the Miramichi River, NB, Canada and two sites downstream, and compared to levels in fish caged upstream of the mill and in unexposed laboratory-maintained tomcod. Additionally, the rate of clearance of CYP1A mRNA in tomcod exposed at the mill and transferred to clean water was compared to that observed in tomcod treated in the laboratory with a single dose of 2,3,7,8-TCDD (0.5 μg/kg). Levels of CYP1A mRNA in tomcod caged upriver of the mill were low and comparable to those observed in untreated laboratory control fish. Levels of CYP1A mRNA were elevated up to 11-fold in tomcod caged at the mill site and a gradient in levels of gene expression was seen at the two downstream sites. Tomcod i.p. injected with a single dose of 2,3,7,8-TCDD exhibited a profile of prolonged CYP1A mRNA induction of at least 25 days. Levels of CYP1A mRNA in tomcod caged at the mill site and transferred to clean water remained significantly induced for at least 3 days post-transfer. In combination, these results suggest that CYP1A mRNA was induced in caged tomcod by constituents of the mill's effluents and that the rates of clearance of CYP1A mRNA may provide additional information concerning the identity of environmental inducers.

Differences in induction by xenobiotics in murine tissues and the Hepa 1c1c7 cell line of mRNAs encoding glutathione transferase, quinone reductase, and CYP1A P450s

Levels of mRNAs encoding class-alpha glutathione transferases, class-mu glutathione transferases, quinone reductase, and cytochrome P450 1A were measured after xenobiotic induction in murine tissues and in the Hepa1c1c7 murine hepatoma cell line. RNA levels in liver and intestinal mucosa were determined after induction with phenobarbital, butylated hydroxyanisole, beta-naphthoflavone, isosafrole, or combinations of these compounds. The tissue culture cells were presented with combinations of butylated hydroxyanisole, tert-butyl-hydroquinone, and beta-naphthoflavone. In murine liver and intestinal mucosa, the greatest induction (5-15-fold) of glutathione transferases and quinone reductase was seen with butylated hydroxyanisole. Administration of phenobarbital or beta-naphthoflavone has only a modest effect (2-3-fold). In contrast, cytochrome P450 1A mRNA levels increase only slightly after BHA induction but are induced dramatically by beta-naphthoflavone. The pattern of induction is different in Hepa1c1c7 cells; there the greatest induction of all mRNAs occurred with beta-naphthoflavone. Administration of antioxidants with other xenobiotics increases mRNA levels only slightly over the levels obtained with BHA in murine tissues, or with beta-naphthoflavone in Hepa1c1c7 cells. mGSTM1 (GT8.7, Yb1), the most abundant glutathione transferase mRNA in murine liver, is also the most abundant glutathione transferase mRNA in both normal and induced Hepa1c1c7 cells. Our results suggest that BHA induction in murine liver and intestinal mucosa of class-mu and class-alpha glutathione transferases may involve regulatory elements and mediators that function poorly in Hepa1c1c7 cells.

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.

The mechanism of action of α-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced CYP1A1 gene expression

Treatment of rat hepatoma H-4-II E cells with α-naphthoflavone (αNF) (10 −8, 10 −7, 10 −6 m) resulted in only minimum induction of ethoxyresorufin O-deethylase (EROD) activity and cytochrome P4501A1 mRNA levels only at 10 −6 m. In contrast, 2,3,7,8-tetrachlorodi-benzo- p-dioxin (TCDD) caused maximum or near maximum induction responses at 10 −8 and 10 −9 m. In a timecourse study with TCDD (10 −9 m), and TCDD plus αNF (cotreated), αNF significantly inhibited the induction of EROD activity and cytochrome P4501A1 mRNA levels by TCDD for 6–24 h after initial exposure of the cells to the chemicals. In addition, treatment of the cells with 10 −9 m TCDD in the presence or absence of 10 −8, 10 −7, and 10 −9 m αNF showed that the latter compound inhibited the induction effects by TCDD in a concentration-dependent manner and these inhibitory effects could be overcome, in part, by a higher concentration of TCDD (10 −8 m). Treatment of the rat hepatoma H-4-II E cells with [ 3H]TCDD showed that within 60 min, there was an initial rapid increase in nuclear [ 3H]TCDD receptor complex levels (38 fmol/mg protein) which decreased to less than 10 fmol/mg protein within 4 h and remained relatively constant for up to 24 h. However, in cells treated with [ 3H]TCDD (10 −9 m) plus αNF (10 −6 m) the levels of the nuclear [ 3H]TCDD receptor complex were <5 fmol/mg protein throughout the 24-h time course. These data, coupled with the results which indicate that the αNF competitively inhibits the binding of [ 3H]-TCDD to the cytosolic aryl hydrocarbon (Ah) receptor, suggest that αNF inhibits the TCDD-mediated induction of CYP1A1 gene transcription and translation by direct competition for cytosolic Ah receptor binding sites.