Cytochrome P450 1A1 Gene Expression Research Articles

Induction of cytochrome P4501A1 genes and metallothionein gene expression in tilapia exposed to coastal sediments in Hong Kong

Sediment samples were collected from different sites including Tolo Harbour, Victoria Harbour, Deep Bay and the south of Lantau Island (a reference site) to determine the existing impacts of sediment-associated contaminants on fish. Many biomonitors have been used to study water pollution in Hong Kong, however, the use of molecular studies on the sublethal physiological effects of coastal sediment exposure in fish is limited. The use of tilapia to detect cytochrome P4501A1 (CYP1A1) and metallothionein (MT) gene expression in liver tissues using RT-PCR was studied in the present project. The fish were exposed for 7 days to coastal sediments. Three control groups were also used; one ‘negative’ control group was maintained in seawater only, while the second and third ‘positive’ control groups were injected i.p. with β-napthoflavone (40 μg/g body weight) and cadmium chloride (10 μg/g body weight), respectively. The results showed that exposure of tilapia to the sediments induced MT and/or CYP1A1 levels in the liver tissues. This enables us to devise highly sensitive biomarkers for monitoring metal and trace organic contamination in local waters.

Quercetin Inhibits Benzo[a]Pyrene-Induced DNA Adducts in Human Hep G2 Cells by Altering Cytochrome P-450 1A1 Gene Expression

Quercetin is one of the most abundant of the naturally occurring flavonoids. It has been estimated that about 25-50 mg of quercetin are consumed from the daily diet. The chemopreventive effect of quercetin on dietary carcinogen has been intensely studied in animal models; however, knowledge regarding the molecular mechanism is still limited. In this study, the human hepatoma Hep G2 cell line was used to investigate how quercetin prevents benzo[a]pyrene (B[a]P)-induced DNA adducts. The Hep G2 cells were treated with 10 μM B[a]P for 18 hours in the presence or absence of quercetin. The DNA adduct levels, evaluated by 32 P postlabeling, decreased in a dose-dependent manner after treatment with quercetin. Cytochrome P-450 1A1 (CYP1A1) and glutathione S-transferase involvement have been well demonstrated in the modulation of B[a]P-induced DNA damage. From the assays of both enzyme activities, quercetin inhibits CYP1A1-linked ethoxyresorufin O-deal-kylase activity more effectively than glutathione S-transferase activity. To elucidate the molecular mechanisms, reverse transcriptase-polymerase chain reaction and Western blot were used to evaluate whether the decrease in CYP1A1 enzyme activity by quercetin is mediated because of alterations of CYP1A1 transcription or mRNA stability. The results indicated that quercetin significantly inhibits B[a]P-induced CYP1A1 mRNA and protein expression. From these findings, we conclude that quercetin suppresses B[a]P-induced DNA damage in human Hep G2 cells by altering CYP1A1 gene expression. Thus we suggest that dietary quercetin may have a long-term preventive effect on chemical carcinogenesis, especially in people who eat a diet rich in fruits and vegetables.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

Regulation of Cytochrome P4501A Metabolism by Glutathione

Gene expression of cytochrome P4501A (CYP1A) in the rainbow trout Oncorhynchus mykiss is dependent on aromatic hydrocarbon receptor signal transduction, and is markedly sensitive to tissue thiol status. Tissue glutathione (GSH) status was manipulated by exogenous GSH, L-buthionine-[S,R]-sulfoximine (BSO), lipoate or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Tissue GSH contents were significantly elevated in GSH- and lipoate-supplemented trout. Hepatic, renal and plasma GSH levels were markedly arrested in BSO-treated trout. Oxidized glutathione (oxidized GSH) levels were significantly elevated in the BCNU-supplemented group. Both BCNU treatment and BSO-induced GSH deficiency increased steady-state levels of hepatic CYPIA mRNA. Additional exposure to 0.1 mg/kg 3,3',4,4'-tetrachlorobiphenyl marginally suppressed the tetrachlorobiphenyl-dependent CYP1A induction in BSO-treated livers compared with the respective thiol treatment groups. Tetrachlorobiphenyl exposures altered efficiencies of thiol treatments and increased oxidized GSH content in all but the BSO-treated groups. However, exposure to 5 mg/kg tetrachlorobiphenyl altered effects of thiol treatments on CYP1A mRNA to a small extent, but catalytic activity of CYP1A was many times suppressed in BSO-treated and lipoate-supplemented fish. These results suggest that thiol status interferes with CYPIA metabolism in a two-way mode of action and provide further evidence for a cross-talk between cytochrome P4501A and glutathione.

UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan

Induction of cytochrome P-4501A1 (CYP1A1) activity by UV has been observed earlier in animal studies via a mechanism that has not yet been resolved. Our previous data have indicated that formylated indolocarbazoles which are formed by UV irradiation of tryptophan solutions are very potent Ah-receptor agonists. To evaluate the effect of UV light on cytochrome P4501A1 gene expression, we studied the induction of CYP1A1 mRNA by UV irradiation of cultured human keratinocytes (HaCaT cell line), primary human blood lymphocytes and mouse Hepa-1 cells. The cells were exposed to UV light delivered by a bank of 6 Philips TL20/12RS sun lamps emitting primarily in the UVB range in the absence and presence of tryptophan. A semiquantitative reverse transcriptase-linked polymerase chain reaction (RT-PCR) was used for analysis of gene expression in the treated cells. The results show that the CYP1A1 mRNA level induced by UV in the presence of tryptophan was higher than that induced by UV alone in both HaCaT cells and lymphocytes after 3 h of incubation post-UV irradiation. To find out if the induction by UV light is caused by the formation of an Ah receptor ligand, Hepa-1 wild-type and Ah receptor deficient c12 cell lines were applied. Wild-type (wt) cells were inducible either by the tryptophan photoproduct 6-formylindolo[3,2- b]carbazole (FICZ) or by UV-irradiation but very low or undetectable levels were observed in the c12 cells. This shows that the induction of gene expression by FICZ and UV is Ah receptor dependent. Together, these results indicate that UV-induced CYP1A1 gene expression in mammalian cells is mediated by an Ah receptor ligand formed from tryptophan. Thus, the photoproducts of tryptophan are suggested to be mediators of light via binding to the Ah receptor and as such also could have a role in light-regulated biological rhythms.

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture.

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells.

Strain Differences in Cytochrome P4501A1 Gene Expression Caused by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat Liver: Role of the Aryl Hydrocarbon Receptor and Its Nuclear Translocator

Rat strain variation in hepatic cytochrome P4501A1 (CYP1A1) gene expression caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated along with possible underlying mechanism. TCDD at a single oral dose of 13.5 ng/kg body weight significantly increased hepatic CYP1A1 mRNA expression in DRH, Long–Evans Cinamon (LEC), Long–Evans (LE), and Holtzman (HO) rats, but not in Sprague–Dawley (SD), Wistar–Imamichi (WI), Lewis (LEW), and Fisher-344 (F344) strains. All showed significant induction of CYP1A1 mRNA at a dose of 40 ng/kg, the relative levels decreasing in the order DRH, LEC, HO, LE, F344, WI, LEW, and SD. A more than 35-fold difference in the induction of CYP1A1 RNA was evident between the DRH and SD strains. Based on CYP1A1 induction, classification into two distinctly separate groups was possible, high responders (DRH, LEC, HO, and LE) and low responders (SD, LEW, WI, and F344). The expression levels closely correlated with the steady-state aryl hydrocarbon receptor (AhR) mRNA expression, this being approximately four-fold higher in the high than in the low responder group. Analysis of the aryl hydrocarbon receptor nuclear translocator (ARNT) showed the presence of a wild type as well as an alternately spliced variant in all strains, with a 45-bp deletion whose sequence corresponded to part of 5′ end of the basic region of the basic helix–loop–helix domain. Expressed levels of both products were almost equal in all the strains except DRH, LEC and HO, where the wild form predominated. The results suggest that differential expression of both AhR and ARNT are responsible for rat strain-specific differences in TCDD induced CYP1A1 expression.

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.

Biomarker responses in whitefish (Coregonus lavaretus L. s.l.) experimentally exposed in a large lake receiving effluents from pulp and paper industry.

Physiological and biochemical biomarker responses were studied in juvenile whitefish (Coregonus lavaretus L. s.l.) exposed experimentally to effluent from the forest industry. The large study area (609 km2), Southern Lake Saimaa, in Southeast Finland, receives 330,000 m3 d-1 of biologically and 55,000 m3 d-1 of chemically treated effluents, discharged from two integrated elementary chlorine free (ECF) bleached kraft pulp and paper mills, from one ECF pulp mill, and from one mill producing unbleached pulp and cardboard. The assessment of exposure to effluent discharged from the mills was based on lake water chlorophenolics (CPs) and resin acids (RAs) measured in samples collected from the 22 experimental sites along the area. Despite the low levels of effluent constituents in the lake, they were still accumulated in detectable levels in fish bile, indicating an exposure to the bioactive compounds of effluents. In comparison to the reference area, a two- to four-fold increase in ethoxyresorufin O-deethylase (EROD) activity was observed in whitefish exposed in the vicinity (1-6 km) of all the mills. However, cytochrome P450 1A1 (CYP1A1) gene expression was increased in only one of the receiving areas, indicating higher sensitivity of the EROD activity in the present study. There were no statistically significant correlations between EROD activity and the ambient water concentrations of the CPs, the RAs, or effluent dilution expressed by water sodium concentration. Neither bile chlorophenolics nor bile resin acids showed a significant correlation with EROD. No significant changes in circulating reproductive steroids, 17beta-estradiol and testosterone, in juvenile whitefish were observed. The vitellogenin gene was expressed in the vicinity of the pulp mill discharging the most wood-derived compounds, i.e. resin acids and wood-sterols, including beta-sitosterol. No differences were observed in plasma immunoglobulin M, glucose, or lactate concentrations between the effluent sources.

Transport of Rat Liver Glycine N-Methyltransferase into Rat Liver Nuclei

Rat liver cytosolic glycine N-methyltransferase (GNMT) catalyzes the S-adenosylmethionine-dependent methylation of glycine to sarcosine. It is comprised of four identical 292-amino acid residue subunits. Recently, evidence has been provided to show that GNMT is identical to the cytosolic receptor for benzo[a]pyrene, which induces cytochrome P450 1A1 gene expression. In the present study we show that chemical modification of purified rat liver GNMT with fluorescein isothiocyanate (FITC) resulted in dissociation of the tetrameric enzyme and was accompanied by loss of enzyme activity. Amino acid sequence analysis of the FITC-labeled peptides obtained by hydrolysis of the modified protein with Staphylococcus aureus V8 protease revealed that lysines 45, 89, 92, 96, 122, and 147 were modified. Lys-122 and Lys-147 were derivatized in tetrameric, dimeric, and monomeric forms of the enzyme. Lysines 45, 89, 92, and 96 were derivatized only in monomeric GNMT, suggesting that modification of these residues resulted in GNMT dissociation. The modified monomeric GNMT was quickly transported into isolated rat liver nuclei. This transport was specific for the GNMT monomer, since neither tetramer nor dimer was able to enter the nuclei. Bovine carbonic anhydrase, similar in size to the GNMT monomer, was labeled with FITC to a similar extent but was not transported into the nuclei. Disruption of the nuclei containing fluorescein-labeled GNMT and subsequent extraction of the nuclear lysate with both high and low salt buffers recovered FITC-GNMT only in the chromatin pellet. Our study supports the suggestion of an additional function for GNMT, probably connected with regulation of cytochrome P450 1A1 gene expression.

The homodimeric form of glycine N-methyltransferase acts as a polycyclic aromatic hydrocarbon-binding receptor.

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein, which was recently identified as glycine N-methyltransferase [Raha, A., Wagner, C., MacDonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 268, 5750-5756]. Glycine N-methyltransferase (GNMT) is one of those unique proteins which exhibit diversity in function. Different subunit configurations are involved in its enzymatic role as a methyltransferase and as PAH-binding receptor. Here we report a systematic study of the oligomeric state of GNMT in the presence of benzo[a]pyrene (B[a]P) in vivo and in vitro. We have used chemical cross-linking and denaturing polyacrylamide gel electrophoresis to show that the B[a]P-binding unit of GNMT is a homodimer. We recently reported that phosphorylation is involved in the interaction of B[a]P with the 4S PAH-binding protein [Bhat, R., Weaver, J. A., Wagner, C., Bodwell, J. E., and Bresnick, E. (1996) J. Biol. Chem. 271, 32551-32556]. In the present study, this observation has been amplified by using bacterially expressed GNMT, which was post-translationally modified by a reticulocyte lysate and ATP-generating system. This modification was also accompanied by the formation of homodimers in the presence of B[a]P. These results indicate that post-translational modification is involved in determining the final configuration, i.e., dimeric form, of GNMT which then acts as a PAH-binding receptor.

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.

Regulation of 3,3′,4,4′-Tetrachlorobiphenyl Induced Cytochrome P450 Metabolism by Thiols in Tissues of Rainbow Trout*

We observed that glutathione (GSH) status regulates the Ah receptor inducible cytochrome P4501A (CYP1A) gene expression and catalytic activity in 3,3′,4,4′-tetrachlorobiphenyl (TCB) exposed rainbow trout. Tissue GSH status of TCB (1 mg/kg body weight, in corn oil) injected fish was manipulated by a) injecting (i.p.) GSH (0.25 g/kg), b) arresting GSH synthesis by l-buthionine-[S,R]-sulfoximine (BSO; 6 mmol/kg) injection for 3 and 6 days. Our attempt to manipulate GSH levels by lipoate supplementation (16 mg/kg) was not productive. Both BSO- and lipoate-supplemented fish maintained a low tissue redox (GSSG/GSH) ratio. Activities of glutathione peroxidase and glutathione reductase were elevated following 3 days of GSH supplementation in GSH rich tissues. Low activities of these enzymes were observed in BSO treated GSH deficient tissues. TCB injection markedly induced hepatic and renal CYP1A catalytic (ethoxyresorufin O-deethylase [EROD]) activities. This effect was further potentiated (3-fold) in GSH-supplemented fish tissues. In contrast, EROD induction by TCB was markedly suppressed in GSH deficient (BSO-treated) and lipoate-supplemented fish. The suppression of CYP1A catalytic activities in GSH deficient and lipoate-supplemented fish was consistently associated with a suppression of TCB induced CYP1A mRNA and protein expressions in these groups. In glutathione-supplemented fish, TCB induced CYP1A protein expression was markedly higher following 3 days of GSH supplementation. Results of our study suggest that tissue thiol status modulates cytochrome P450 CYP1A gene expression and catalytic activity.

Rat Liver 4S-Benzo[a]pyrene-Binding Protein Is Distinct from GlycineN-Methyltransferase

Rat liver 4S-benzo[a]pyrene-binding protein (BAP) was reported to be identical to the subunit (Mr32,500) of the tetrameric glycineN-methyltransferase (GNMT). We have reevaluated this study. When a liver cytosol was subjected to Sephacryl S-200 gel permeation chromatography, enzyme activity as well as cross-reactivity with anti-GNMT antibody were found only at the elution position of the purified GNMT. Chromatograph of the cytosol pretreated with [3H]benzo[a]pyrene showed two peaks in the void volume and at the position of an approximateMrof 40,000. The latter, corresponding to the 4 S BAP, did not cross-react with the anti-GNMT antibody. An extract of nuclei in which GNMT was proposed to act as a mediator of cytochrome P4501A1 gene expression contained the tetrameric GNMT but no binding activity. The lung, in which no GNMT mRNA occurred, had the 4 S BAP. Extracts ofEscherichia coliand COS-7 cells expressing large amounts of GNMT showed no 4 S BAP. These findings suggest that the 4 S BAP is distinct from the subunit of GNMT.

ATP Depletion Affects the Phosphorylation State, Ligand Binding, and Nuclear Transport of the 4 S Polycyclic Aromatic Hydrocarbon-binding Protein in Rat Hepatoma Cells

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. Phosphorylation and dephosphorylation have been suggested to influence the function of many cytosolic receptors and transcription factors. The ATP level within H4IIE rat hepatoma cells could be depleted by treatment with sodium azide or 2,4-dinitrophenol; restoration of the original ATP levels occurred with addition of glucose to the cell culture. ATP depletion reduced the phosphate content of the 4 S protein by approximately 25-30%, which lowered the binding of benzo[a]pyrene (B[a]P) to the 4 S protein by >60%. This effect could not be reversed by the addition of ATP to the binding reaction mixtures. Alkaline phosphatase treatment of the purified 4 S protein in a cell-free system also reduced the B[a]P binding to the protein. Cells treated with a protein phosphatase inhibitor, okadaic acid, and a protein kinase inhibitor, staurosporin, affected the B[a]P binding of the 4 S protein positively and negatively, respectively. These data suggested that phosphorylation is involved in the interaction of the 4 S protein with the PAH. The nuclear translocation of the predominantly cytosolic binding protein has been investigated after ligand binding. Western blots with the immunopurified 4 S PAH-binding protein from cytosolic and nuclear lysates showed significant differences in the distribution of the 4 S receptor between cytosolic and nuclear compartments in control and ATP-depleted cells. Ligand binding stimulated the movement of the receptor into the nucleus, which was completely blocked by reducing the intracellular ATP concentration. These findings provide new information on the role of ATP and phosphorylation on the interaction of B[a]P with 4 S PAH-binding protein and its nuclear translocation.

Phenobarbital Induction of CYP1A1 Gene Expression in a Primary Culture of Rainbow Trout Hepatocytes

In mammals, phenobarbital (PB) is an in vivo inducer of the cytochrome P4502B (CYP2B) family, whereas in teleosts PB induction of cytochrome P450 is unclear. We show that teleost cytochrome P4502K1 (CYP2K1) protein levels and 7-pentoxyresorufin-O-deethylase activity were not induced by exposure of primary cultures of rainbow trout hepatocytes to PB. Instead, cytochrome P4501A1 (CYP1A1) gene expression was strongly induced by PB, based upon observations of marked increases in CYP1A1 mRNA, CYP1A1 protein, and 7-ethoxyresorufin-O-deethylase activity. In accordance with these data we provide a temporal study employing antibodies for the aromatic hydrocarbon (Ah) receptor that showed an increase in Ah receptor in nuclear extracts prepared from cells exposed to PB. Employment of the electrophoretic mobility shift assay (EMSA) showed PB to cause activation or "transformation" of the Ah receptor in nuclear extracts. Studies employing actinomycin D and cycloheximide indicated that PB induction of CYP1A1 was regulated at both the transcriptional and post-transcriptional levels. Nuclear run-off experiments confirm that PB causes an increase in CYP1A1 transcription. Inhibition of protein synthesis led to the superinduction of CYP1A1 mRNA, suggesting the regulation of teleost CYP1A1 may involve a labile repressor protein. These findings suggest that PB induction of the CYP1A1 gene involves the Ah receptor and is via transcriptional activation.

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.