2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in wild type and variant mouse hepatoma cells. Nuclear location and strength of nuclear binding

The volume of aqueous solvent present during subcellular fractionation of mouse hepatoma (Hepa 1c1c7) cells influences the distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptors between the nuclear and cytosolic fractions. When the effects of dilution are minimized, at least 80% of the receptors associate with nuclei. The receptors bind relatively strongly to nuclei, as measured by their release by KCl. TCDD-receptor complexes bind more strongly to nuclei than do unoccupied receptors. A temperature-dependent event further enhances the binding of TCDD-receptor complexes to nuclei. A class of variant cells contains receptors which bind relatively weakly to nuclei; this defect accounts for the variant phenotype. We conclude that, in the intact cell, TCDD receptors are located within the nucleus and that the temperature-dependent event in the induction of cytochrome P1-450 gene expression is one which strengthens the binding of the TCDD-receptor complex to chromatin.     

Suppression of aryl hydrocarbon hydroxylase activity in human primary lung carcinoma x mouse hepatoma somatic cell hybrids

Variants of the mouse hepatoma cell clone inducible for aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) (EC 1. 14. 14.1) activity and deficient in hypoxanthine guanine phosphoribosyl-transferase (EC 2.4.2.8), and human primary lung carcinoma cell clone noninducible for AHH activity and deficient in thymidine kinase (EC 2.7.1.21) were isolated. The variant lines characterized for AHH inducibility and drug resistant phenotype were utilized to study somatic cell hybrids for the expression of AHH induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In two hybrids AHH activity was not expressed. In view of these results we conclude that aryl hydrocarbon hydroxylase activity is suppressed in AHH noninducible human lung carcinoma x AHH inducible mouse hepatoma cell hybrids.     

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.     

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin induces NADPH cytochrome c (P-450) reductase in rat hepatoma cells in culture

The lack of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) (EC 1.14.14.1) induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a clone of rat hepatoma (HTC cl-1) cells is not caused by the lack of nuclear Ah receptor or by a deficiency in the activity of NADPH-cytochrome c (P-450) reductase. Treatment of HTC cl-1 cell line with TCDD for 18 h in culture resulted in a reproducible 500-600% increase in reductase activity without concomitant expression in AHH activity. These data suggests that TCDD induces cytochrome c reductase activity and that the lack of inducible AHH activity in rat hepatoma cells could reflect a defect in the structural gene (s) encoding for cytochrome P1-450, or an Ah receptor with a faulty DNA binding domain.     

Identification of the Ah receptor in selected mammalian species and induction of aryl hydrocarbon hydroxylase

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea-pig by means of sucrose density centrifugation analysis and hydroxyapatite binding assays. Using 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74-691 fmol [3H]TCDD/mg cytosolic protein and apparent dissociation constants ranged over 0.30-7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3-methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a]pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor.     

Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols. 7-Hydroxyellipticine as a competitive antagonist of cytochrome P-450 induction

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague-Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The binding characteristics of Ah receptors, before and after separation, were assessed by competition of various chemicals as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene, benzo[a]pyrene, beta-naphthoflavone and ellipticines with [3H]3-methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin as radioligands. The rationale of this approach is supported by the results obtained and the major conclusions are as follows. 1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light-density component and 4-S carcinogen-binding protein). 2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9-S binding peak with [3H]benzo[a]pyrene as a radioligand. 3. The intrinsic binding characteristics of the separated Ah receptors of Sprague-Dawley rats and C57BL/6 mice were similar if not identical. 4. A good correlation exists between the competitive potency (IC50) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7-hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50 approximately 5-10 microM) without inducing aryl hydrocarbon hydroxylase. 5. When coadministered with various inducers, 7-hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.     

A third genetic locus affecting the Ah (dioxin) receptor

We have isolated a new benzo(a)pyrene-resistant clone, c35, of the mouse hepatoma line, Hepa-1. Cytochrome P1-450 mRNA and P1-450-dependent aryl hydrocarbon hydroxylase (AHH) activity are no longer inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in c35. The phenotype of c35 is unstable in long-term culture. A subclone, c35-1, having partially restored AHH inducibility, was studied in detail. The concentration of dioxin required to give half-maximal induction of AHH activity was 16-fold greater in c35-1 than in Hepa-1. Scatchard analysis showed that c35-1 contains reduced levels of the Ah (dioxin) receptor, which mediates induction of P1-450, but that the affinity of the receptor for dioxin is unaltered. In vivo assays confirmed that c35-1 possesses reduced levels of receptor but showed that it is even more severely affected in nuclear translocation of the receptor. Somatic cell hybridization experiments demonstrated that c35 is recessive and belongs to a new, third complementation group of mutants defective in Ah receptor activity. We propose that c35 is mutated either in the ligand-binding Ah receptor polypeptide or in another polypeptide required for receptor function and that in c35-1 partial reversion has occurred to generate a polypeptide which is still impaired in its role in promoting nuclear translocation and/or DNA binding.     

Intestinal organic anion transport, glutathione transferase and aryl hydrocarbon hydroxylase activity: effect of dioxin.

Benzyl penicillin, p-aminohippurate and phlorizin are passively transported across the intestinal wall of rat everted gut sacs. The mid-intestine takes up more organic anion than the duodenum or terminal ileum and also moves significantly more anion into the serosal fluid than the more proximal or distal segments of intestine. About the same amount (40%) of the anion taken up by the various regions of the intestine remains in the wall of the gut sac after incubation. This pattern of organic anion transport down the intestine is different than the aboral gradient of glutathione S-transferase activity found in the intestine, suggesting that the two mechanisms are not directly related in the intestine. Oral and intraperitoneal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases iron transport and aryl hydrocarbon hydroxylase activity in the intestine and aryl hydrocarbon hydroxylase and glutathione S-transferase activity in the liver. By contrast, intestinal glutathione S-transferase and organic anion transport are not affected by TCDD treatment. Intestinal transport and enzymatic mechanisms apparently respond differently to TCDD than those previously described in the liver and kidney.     

Curcumin suppresses the transformation of an aryl hydrocarbon receptor through its phosphorylation

Halogenated and polycyclic aromatic hydrocarbons induce diverse biochemical responses through the transformation of a cytosolic aryl hydrocarbon receptor (AhR). In mouse hepatoma Hepa-1c1c7 cells, curcumin, a yellow pigment of Curcuma longa, did not inhibit the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced translocation of the AhR into the nucleus, but rather accelerated it. In the nucleus, curcumin inhibited the TCDD-induced heterodimerization of the AhR with an AhR nuclear translocator (Arnt), an essential partner for the transformation, and also dose-dependently inhibited the TCDD-evoked phosphorylation of both the AhR and Arnt. Moreover, curcumin significantly inhibited the TCDD-induced activation of protein kinase C (PKC), which is involved in the transformation, decreased the TCDD-induced DNA-binding activity of the AhR/Arnt heterodimer, and downregulated CYP1A1 expression. In a cell-free system, curcumin inhibited the binding of 3-methylcholanthrene, an AhR agonist, to the receptor. These results indicate that curcumin is able to bind to the AhR as a ligand, but suppresses its transformation by inhibiting the phosphorylation of AhR and Arnt, probably by PKC.     

Induction of cytochrome P450-dependent monooxygenase activities in rat hepatoma H-4-IIE cells in culture by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds: mechanistic studies using radiolabeled congeners

Treatment of rat hepatoma H-4-IIE cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) resulted in the structure-dependent induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities. The induction potencies followed the order 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,7,8-PeCDD approximately 1,2,3,7,8-PeCDF greater than 1,2,7,8-TCDF greater than 2,3,7-TrCDD and were comparable to structure-toxicity relationships which have previously been reported. In contrast, many of the properties of these compounds were structure-independent. For example, using tritiated congeners of high specific activity (greater than 30 Ci/mmol) the sedimentation coefficients (S) for the nuclear and cytosolic aryl hydrocarbon (Ah) receptor complexes were 5-6 and 9-10 S, respectively, for all the radioligands. Moreover, examination of the processing of nuclear Ah receptor complexes for the radiolabeled congeners showed that after 6 h, the rates of nuclear processing were very low and varied between 0.006 and 0.0385 fmol degraded/mg protein/mg total DNA. These results were consistent with the reported stability and persistence of the nuclear Ah receptor complexes and in addition, there were no apparent structure-dependent differences in the processing rates. Inspection of the nuclear receptor levels and the corresponding induced enzyme activities for the congeners showed that there was a linear correlation between average nuclear receptor complex levels (18-42 h) and induced enzyme activities (32-42 h) for all six radioligands; these data indicated that the rates of cytochrome P450-dependent gene expression correlated with the levels of nuclear Ah receptor complex. In contrast, the accumulation of occupied nuclear receptor complexes in rat hepatoma H-4-IIE cells was structure-dependent and appeared to be one of the factors which governed the observed structure-induction and the previously reported structure-toxicity relationships for 2,3,7,8-TCDD and related halogenated aryl hydrocarbons.     

Variation in aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity in heteroploid and predominantly diploid rat liver cells in culture

Aryl hydrocarbon (benzo(a)pyrene) hydroxylase is present and inducible in Buffalo rat liver cells in culture. There is substantial variation in both basal and inducible hydroxylase activities among heteroploid subclones isolated from a heteroploid parent population, and among diploid subclones isolated from a diploid parent population. This variation is not related to differences in the growth characteristics of the subclones, or to differences in their chromosome number. The results indicate that substantial heterogeneity in both basal and induced hydroxylase activity develops during the growth of both heteroploid and diploid cell strains in culture. These findings indicate that diploid cell populations are not necessarily homogeneous with respect to aryl hydrocarbon hydroxylas activity. This observation may complicate the interpretation of experiments involving somatic cell hybridization or polycyclic hydrocarbon-induced transformation and/or cytotoxicity. This heterogeneity in hydroxylase activity develops rather rapidly (2-3 mo of culture), in the absence of any apparent mutational stress.     

Ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver, lung, and thymus

The ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in Sprague-Dawley rats by quantitation of the receptor in hepatic, lung, and thymic cytosol. Concentrations of hepatic and lung cytosolic receptors increased rapidly after birth and remained at the highest levels from days 2 to 21. After this time, receptor levels in these tissues slowly declined with age. In the thymus, cytosolic receptor concentrations remained high from days 2 to 42 following birth. In these tissues and at all times examined, the receptors demonstrated very high affinities for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. From days 15 to 42 following birth, no consistent sex related differences in receptor content or affinity were observed in any of these tissues.