Down-regulation of murine Cyp1a-1 in mouse hepatoma Hepa-1c1c7 cells by bisphenol A

Cultured mouse hepatoma Hepa-1c1c7 cells were treated with either bisphenol A or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of bisphenol A in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as determined by analysis of 7-ethoxyresorufin O-deethylase (EROD) activities. Bisphenol A alone did not affect the activity of Cyp1a-1-specific EROD; in contrast, TCDD-induced EROD activities were markedly reduced in the concomitant treatment of TCDD and bisphenol A in a dose-dependent manner. Treatment with tamoxifen, an antiestrogen that acts through the estrogen receptor, did not affect the suppressive effects of bisphenol A on TCDD-induced EROD activity. TCDD-induced Cyp1a-1 mRNA levels were markedly suppressed in the concomitant treatment of TCDD and bisphenol A consistent with their effects on EROD activity. Transient transfection assay using dioxin-response element (DRE)-linked luciferase revealed that bisphenol A reduced transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter of the Cyp1a-1 gene. These results suggest the down-regulation of the Cyp1a-1 gene expression by bisphenol A in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear Ah receptor but not mediated through estradiol receptor.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta VIII. Effect of Mevinolin on Photoinduced Carotenogenesis

Mevinolin, which is a highly specific competitive inhibitorof 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase,was used in a search for photoinducible enzyme(s) other thanHMG-CoA reductase in the pathway of carotenoid biosynthesisin Rhodotorula minuta. The photoinduced production of carotenoids was competitivelyinhibited by mevinolin. The concentration of mevinolin thatis required to inhibit completely the production of carotenoidsdepends on the light dose given to the cells. However, the relationshipbetween the inhibition ratio and the concentration of mevinolinwas almost identical regardless of the light dose. These resultssuggest that the activity of enzymes involved in the formationof HMG-CoA may not be affected by light. When an adequate amount of mevalonate was added to the growthmedium that contained sufficient mevinolin for the completeinhibition of the photoinduction of the production of carotenoids,the same quantity of carotenoids was produced as in the absenceof mevinolin. Moreover, the production of carotenoids in thepresence of both mevinolin and mevalonate was inhibited by cycloheximide. It appears from these results that one or more photoinducibleenzymes, such as HMG-CoA reductase, may be present in the carotenogenicpathway beyond mevalonate. (Received April 12, 1989; Accepted January 16, 1990)     

Isoprenoid synthesis in isolated embryonic Drosophila cells. Sterol-independent regulatory signal molecule is distal to isopentenyl 1-pyrophosphates

Embryonic Drosophila cells (Kc cells) were used to further characterize sterol-independent modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 3-Methyl-3-5-dihydroxyvalerate (mevalonate), 3-fluoromethyl-3,5-dihydroxyvalerate (fluoromevalonate), and 3-ethyl-3,5-dihydroxyvalerate (homomevalonate) were tested as modulators. Although mevalonate caused a rapid, reversible suppression of reductase activity, fluoro- and homomevalonate increased activity; fluoromevalonate was more effective than homomevalonate. Mevalonate, added simultaneously with fluoromevalonate, blocked the analogue's effect on Kc cell reductase activity. However, mevalonate did not suppress an established fluoromevalonate increase in HMG-CoA reductase activity. Fluoromevalonate blocked [1-14C, 5-3H]mevalonate conversion to 14CO2- and 3H-labeled lipids and [3H] mevalonate 5-pyrophosphate accumulated. Neither protein nor RNA synthesis were required for mevalonate-mediated suppression of reductase activity. However, fluoromevalonate's effect on reductase activity required protein synthesis. Furthermore, in the absence of protein synthesis, fluoromevalonate-stabilized Kc cell HMG-CoA reductase activity. We have concluded that mevalonate, fluoromevalonate, homomevalonate, and compactin (mevinolin) modulated HMG-CoA reductase activity because they altered isoprenoid carbon flow to a post-isopentenyl 1-pyrophosphate regulatory, signal molecule.     

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.     

Mevinolin-resistant mutations identify a promoter and the gene for a eukaryote-like 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the archaebacterium Haloferax volcanii.

Both eukaryotes and archaebacteria use 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaebacteria need for the isoprenoid side chains of their unique and characteristic lipids. The drug mevinolin inhibits HMG-CoA reductase in eukaryotes and in the halophilic archaebacteria, and we have used a spontaneous mutation to mevinolin resistance in the construction of a selectable shuttle vector for Haloferax volcanii. Sequence analysis shows that this resistance determinant encodes an HMG-CoA reductase very like its eukaryotic homologs, but sharing with the one sequenced eubacterial HMG-CoA reductase (that of Pseudomonas mevalonii) few residues other than those common to all HMG-CoA reductases. Characterization of several spontaneous mevinolin-resistant mutants reveals that they are of two sorts: amplifications of the HMG-CoA reductase gene with varying amounts of flanking sequence, and point mutants upstream of the HMG-CoA reductase coding region. We compared sequence and expression of a mutant gene of the latter class to those of the wild-type gene. The point mutation found affects the TATA box-like "distal promoter element," results (like gene amplification) in resistance through the synthesis of excess gene product, and provides the first true genetic definition of an archaebacterial promoter.     

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Inhibition by mevinolin of plant growth, sterol formation and pigment accumulation

To obtain information on the importance of a functional mevalonate synthesis for plant growth and development, we investigated the effect of mevinolin, a highly specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase (the mevalonate-producing enzyme) on growth, sterol accumulation and pigment formation of radish seedlings (Raphanus sativus L. cv. Saxa Treib) and in part also wheat seedlings (Triticum aestivum L. cv. Kolibri). Mevinolin applied during germination inhibits root elongation and development of lateral roots in etiolated and light-grown radish seedlings. This effect cannot be overcome by exogenous GA₃, but by addition of mevalonic acid, the product of the internally inhibited reaction. This emphazises the specifity of the mevinolin effect and indicates that the biosynthesis of mevalonic acid is a mandatory requirement for root growth. In light-grown radish seedlings mevinolin also affects hypocotyl length-growth and inhibits sterol accumulation, but has little effect on the chlorophyll and carotenoid accumulation in the chloroplasts of the cotyledons. This indicates the possible presence of an independent mevalonate synthesizing pathway within the plastids and suggests a low transport rate of mevinolin from the radish roots to the cotyledons. When mevinolin is directly applied to the leaves at higher concentrations, it also reduces the light-induced chlorophyll and carotenoid accumulation as has been shown with etiolated primary leaves of wheat. This inhibition is age-dependent and proceeds to a higher extent in older than in younger etiolated leaf tissue. From our results we conclude that plastids possess an independent HMG-CoA reductase. In the cotyledons of radish, mevinolin seems to induce a senescence retardation and sun-type growth response, as has been evaluated by measuring the fast and slow chlorophyll fluorescence induction kinetics (Kautsky effect). These responses may be due to inhibitor-induced changes in the intracellular phytohormone balance.     

Mechanism of benzo[a]pyrene-induced Cyp1a-1 gene expression in mouse Hepa 1c1c7 cells: role of the nuclear 6 s and 4 s proteins.

Treatment of wild-type (wt) aryl hydrocarbon (Ah)-responsive mouse Hepa 1c1c7 cells with benzo[a]pyrene (B[a]P) caused a concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast, B[a]P was inactive as an inducer in Ah nonresponsive class 1 and class 2 mutant cell lines. In parallel experiments, the nuclear fractions from wt cells treated with 10(-7) M [3H]B[a]P contained both the 4 s carcinogen binding protein and the 6 s (Ah receptor) complexes, whereas only the 4 s complex was present in the nuclear fraction of the class 2 mutant cells. The results obtained from cotreatment of wt Hepa 1c1c7 cells with 10(-6) or 10(-7) M B[a]P and 5 x 10(-7) or 10(-7) M 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) showed that MCDF inhibited the induction of EROD activity and Cyp1a-1 mRNA levels by B[a]P. Moreover, using 10(-7) M [3H]B[a]P and unlabeled MCDF, it was shown that MCDF not only inhibited the induction response but also caused a concentration-dependent decrease in levels of the nuclear 6 s complex but not the 4 s complex. In contrast, in situ competition studies with unlabeled 10(-6) M benzo[ghi]-perylene (B[ghi]P) resulted in the elimination of the nuclear [3H]B[a]P 4 s complex (but not the 6 s complex); however, the EROD activity and Cyp1a-1 mRNA levels in cells treated with 10(-7) M B[a]P in the presence or absence of 10(-6) M B[ghi]P were not significantly different. These results indicate that the 4 s binding protein is not required for the induction of Cyp1a-1 gene expression in Hepa 1c1c7 cells and suggest that B[a]P and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce Cyp1a-1 gene expression via a common mechanism which involves binding to the Ah receptor.     

Isoprenoid synthesis in Halobacterium halobium. Modulation of 3-hydroxy-3-methylglutaryl coenzyme a concentration in response to mevalonate availability

Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.     

Requirement for mevalonate in cycling cells: quantitative and temporal aspects

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.