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.     

Effects of ligand structure on the in vitro transformation of the rat cytosolic aryl hydrocarbon receptor.

Incubation of radiolabeled, 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) with rat hepatic cytosol for 2 h at 0 degrees C gave liganded aryl hydrocarbon (Ah) receptor complexes which were indistinguishable as determined by velocity sedimentation analysis and DNA-Sepharose column chromatography. Incubation of the cytosol plus the different radioligands for 2 h at 20 degrees C resulted in the formation of Ah receptor complexes which exhibited increased retention times on DNA-Sepharose columns. It was apparent that the amount of specifically bound Ah receptor complex or the levels of the transformed Ah receptor complex which eluted from the column with 0.2-0.3 M salt were dependent on the structure of the radioligand. For example, after incubation for 2 h at 20 degrees C the overall yields of the specifically bound transformed Ah receptor complex were 3.4, 2.0, 1.2, 1.9, 0.3, and 0.1%, respectively, using 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8-TCDF, and 2,3,7,8-TrCDD as radioligands. A more quantitative measure of the structure-dependent transformation of the liganded cytosolic Ah receptor complex was determined using a gel retardation assay with a consensus synthetic dioxin-responsive element (DRE) (26-mer, duplex). The EC50 values obtained for the concentration-dependent formation of the retarded DRE-Ah receptor complex using 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8-TCDF as ligands were 0.26, 0.35, 0.78, 1.75, 27.0, and 220 nM, respectively. The structure-dependent differences in these values were similar to their different potencies as Ah receptor agonists and these data suggest that the structure-dependent transformation of the liganded cytosolic Ah receptor may significantly contribute to the structure-activity relationships observed for 2,3,7,8-TCDD and related compounds.     

Synthesis and aryl hydrocarbon receptor binding properties of radiolabeled polychlorinated dibenzofuran congeners

Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.     

Differential ligand-dependent activation and a role for Y322 in aryl hydrocarbon receptor-mediated regulation of gene expression

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF). Non-traditional activators, including omeprazole (Omp), are thought to regulate AHR action through phosphorylation rather than binding to the receptor. In this study, we examined the ability of these compounds to induce AHR-dependent regulation of cytochrome P450 1A1 (CYP1A1) and CYP1B1 in T-47D human breast cancer cells. The role of Y322, a residue implicated in Omp-dependent activation of AHR was also investigated. All four compounds induced CYP1A1 and CYP1B1 mRNA expression, with Omp differing from the HAHs. Chromatin immunoprecipitation assays revealed ligand- and gene-selectivity in the recruitment patterns of AHR coactivators. We also found that residue Y322 of human AHR was important for maximum activation of AHR by 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF, but required for 2,3,7,8-TCDF and Omp in an AHR-deficient MCF-7 human breast cancer cell line. In summary, this study provides evidence for context- and ligand-selective differences in coactivator recruitment in AHR-regulated gene expression and reveal an important role of Y322 in AHR activation.     

Biomonitoring of polybrominated diphenyl ether (PBDE) pollution: a field study

Polybrominated diphenyl ethers (PBDEs) and cytochrome P450 enzyme activities were investigated in European eels (Anguilla anguilla) collected from seven sites in a coastal lagoon in the north-western Mediterranean Sea, Orbetello lagoon (Italy). Twelve PBDE congeners were measured in muscle and two CYP1A enzyme activities, 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BP(a)PMO), were investigated in liver microsomal fraction in order to obtain insights into the health of the lagoon environment. PBDE muscle levels were low and the most abundant congeners were 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5,5'-hexaBDE (BDE-153) and 2,2',4,5'-tetraBDE (BDE-49). EROD and B(a)PMO activities were also low and no differences were observed between eels from different sites. Multivariate analysis (PCA) did not indicate correlations between PBDEs and either P450 activities.     

Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Increasing effect of nori on the fecal excretion of dioxin by rats

The effects of nori (Porphyra yezoensis), a kind of red alga, on the gastrointestinal absorption and reabsorption of 17 types of dioxin were investigated in male Wistar rats. The rats were fed with 4 g of the control diet or 4 g of the nori diet containing a standard dioxin solution (233 ngTEQ/kg of body weight) for five consecutive days. In the group fed with the 10% nori diet, the fecal excretion of dioxin from days 1 to 5 was higher (p<0.01) than that of the control group by 5.5-fold for 2,3,7,8-TCDD, 6.6-fold for 1,2,3,7,8-pentaCDD, and 6.0-fold for 2,3,4,7,8-pentaCDF. In another experiment, the rats were fed with 4 g of the control diet containing a standard dioxin solution (2991 ngTEQ/kg of body weight) on the first day of the experiment and then given the control diet for 7 consecutive days, before being given either the control diet or the nori diet for 28 consecutive days more. In the group fed with the 10% Nori diet, the fecal excretion of dioxin during the period from days 8 to 35 was higher (p<0.01 or p<0.05) than that of the control group by 2.4-fold for 2,3,7,8-TCDD, 2.3-fold for 1,2,3,7,8-pentaCDD, and 2.4-fold for 2,3,4,7,8-pentaCDF. These results suggest that the administration of nori prevented dioxin from being efficiently absorbed and reabsorbed from the gastrointestinal tract, and might be useful for protecting humans exposed to dioxin from ill effects.     

Characterization of biphenyl dioxygenase of Pandoraea pnomenusa B-356 as a potent polychlorinated biphenyl-degrading enzyme

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Distribution of PCDDs/PCDFs and Co-PCBs in human maternal blood, cord blood, placenta, milk, and adipose tissue: dioxins showing high toxic equivalency factor accumulate in the placenta

To assess levels of dioxin background contamination and transfer of dioxins from mothers to unborn children and infants, concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar-polychlorinated biphenyls (Co-PCBs) were measured in human samples from expectant and nursing mothers living in Nara, Japan. The average toxic equivalency quantities (TEQs) of PCDDs/PCDFs and Co-PCBs from circulating maternal blood, cord blood, placenta, milk taken 3-10 d after delivery, milk taken one month after delivery, and adipose tissue were 26 and 9.3, 15 and 2.3, 31 and 1.2, 16 and 5.4, 18 and 8.8, and 16 and 7.7 pg-TEQ/g-fat, respectively. Among the various PCDD/PCDF congeners, 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF contributed most heavily to the TEQs of all maternal samples. Among the various Co-PCB congeners, 3,3',4,4',5-PeCB (#126), 2,3,3',4,4',5-HxCB (#156), and 2,3',4,4',5-PeCB (#118) contributed most heavily to the TEQs of all maternal samples. But, the concentrations and relative percentages of congeners differed among the various samples, suggesting that congeners showing high toxic equivalency factor accumulate in the placenta.     

Induction of 7-ethoxyresorufin-O-deethylase activity by planar chlorinated hydrocarbons and polycyclic aromatic hydrocarbons in cell lines from the rainbow trout pituitary

The induction of 7-ethoxyresorufin-o-deethylase (EROD) activity was examined in three rainbow trout pituitary cell lines: RTP-91E, RTP-91F and RTP-2. RTP-91E and RTP-91F were developed from the pituitary of a male and have epithelial-like and fibroblast-like morphologies, respectively. RTP-2, which was described previously, was developed from the pituitary of a female and has an epithelial-like shape. In all cell lines EROD activity was induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Immunoblotting with the polyclonal antibody, anti-trout CYP1A1(277-294)/KLH, confirmed induction of a 58-kDa polypeptide. Potential inhibitors of the aryl hydrocarbon receptor, geldanamycin and alpha-naphthoflavone, inhibited EROD induction by TCDD. Other compounds inducing EROD activity were 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3-methylcholanthrene (3MC). When judged by the concentration eliciting 50% of the maximal response (EC50), induction was similar in RTP-2 and RTP-91E, and less effective in RTP-91F. Regardless of the cell line, the rank order from most to least potent inducer on the basis of EC50 value was TCDD> or =PCDD>TCDF>PCB 126>3MC. When induction potencies were expressed relative to TCDD, the values obtained with the pituitary cell lines were similar to previously published values derived with a rainbow trout liver cell line.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

Damage to erythrocytes caused by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (in vitro)

The effects of the exposure of human erythrocytes to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin were studied. Particular attention was paid to lipid peroxidation, haemoglobin oxidation, and changes in the activity of catalase and glutathione peroxidase. Human erythrocytes at a 5% haematocrit were incubated with 2,3,7,8-TCDD at concentrations of 0.2 ppm to 1.6 ppm (ng-microg/ml erythrocytes) for 1 hour. The results obtained show that 2,3,7,8-TCDD induces the generation of lipid peroxides and the oxidation of Hb, and decreases the activity of catalase and glutathione peroxidase. This supports the thesis that TCDD causes oxidative stress in erythrocytes.     

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.     

Differentiation-dependent induction of CYP1A1 in cultured rat small intestinal epithelial cells,colonocytes, and human colon carcinoma cells: basement membrane-mediated apoptosis

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.     

Negative regulation of the murine cytosolic aldehyde dehydrogenase-3 (Aldh-3c) gene by functional CYP1A1 and CYP1A2 proteins.

We have examined enzyme activities and mRNA levels corresponding to aldehyde dehydrogenase-3 genes encoding cytosolic (ALDH3c) and microsomal (ALDH3m) forms. In contrast to negligible activities in the intact mouse liver, both ALDH3c and ALDH3m enzyme activities are inducible by benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mouse hepatoma Hepa-1c1c7 cell cultures. Constitutive mRNA levels of ALDH3c are virtually absent, whereas those of ALDH3m are substantial; using Hepa-1 mutant lines, we show that both ALDH3c and ALDH3m are TCDD-inducible by an Ah receptor-dependent mechanism. Basal mRNA levels of ALDH3c, but not those of ALDH3m, are strikingly elevated in untreated mutant cells lacking a functional CYP1A1 enzyme; low ALDH3c basal mRNA levels can be restored by introduction of a functional murine CYP1A1 or human CYP1A2 enzyme into these mutant cells. These data suggest that the TCDD induction process is distinct from the CYP1A1/CYP1A2 metabolism-dependent repression of constitutive gene expression; we suggest that this latter property classifies the Aldh-3c gene, but not the Aldh-3m gene, as a member of the murine [Ah] battery.