Induction Of Aryl Hydrocarbon Hydroxylase Activity Research Articles

Disruption of glomerular cell-cell and cell-matrix interactions in hydrocarbon nephropathy

Environmental chemicals play an etiological role in greater than 50% of idiopathic glomerular diseases. The present studies were conducted to define mechanisms of renal cell-specific hydrocarbon injury. Female rats were given 10 mg/kg benzo(a)pyrene (BaP) once a week for 16 wk. Progressive elevations in total urinary protein, protein/creatinine ratios, and microalbuminuria were observed in rats treated with BaP for up to 16 wk. The nephropathic response involved early reductions in mesangial cell numbers and fibronectin levels by 8 wk, coupled to transient increases in podocyte cellularity. Changes in podocyte numbers subsided by 16 wk and correlated with rebound increases in mesangial cell numbers and fibronectin levels, along with increased alpha-smooth muscle actin and Cu/Zn superoxide dismutase and fusion of podocyte foot processes. In culture, mesangial cells were more sensitive than podocytes to hydrocarbon injury and expressed higher levels of inducible aryl hydrocarbon hydroxylase activity. Naïve mesangial cells exerted a strong inhibitory influence on podocyte proliferation under both direct and indirect coculture conditions, and this response involved a mesangial cell-derived matrix that selectively inhibited podocyte proliferation. These findings indicate that hydrocarbon nephropathy in rats involves disruption of glomerular cell-cell and cell-matrix interactions mediated by deposition of a mesangial cell-derived growth-inhibitory matrix that regulates podocyte proliferation.

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the 32P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 108 nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min-1 10-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.

Environmental Factors and Aryl Hydrocarbon Hydroxylase Activity (CYP1A1 Phenotype) in Human Lymphocytes

Since aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene (BP) and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine AHH activity in the determination of susceptibility to lung cancer. Lymphocytes from healthy male adults (239) of non-smokers and smokers were cultured in vitro and assayed for non-induced and 3-methylcholanthrene (MC)-induced AHH activity and AHH inducibility (MC-induced AHH activity/non-induced AHH activity). A day-to-day variation in AHH activity was not observed while a seasonal variation was apparent. Very wide differences in non-induced AHH and MC-induced AHH activities were observed. The association of some selected environmental factors and AHH activity was studied. Age was related to non-induced AHH activity (Spearman's rank correlation coefficients (r), r = 0.185, p < 0.005) and AHH inducibility (r = -0.329, p < 0.001). Coffee consumption was associated with non-induced (age-adjusted r = 0.138, p < 0.05) and MC-induced AHH activity (age-adjusted r = 0.173, p < 0.01). Cigarette smoking was correlated with non-induced AHH activity (age-adjusted r = 0.191, p < 0.005) and AHH inducibility (age-adjusted r = -0.191, p < 0.005). No significant association was observed for any other selected factors, including alcohol intake or broiled food consumption. In conclusion, AHH activity might be affected by cigarette smoking and coffee consumption, and was dependent on the age of the donor. Day-to-day and seasonal variation analyses showed that this assay method was reproducible and reliable and AHH inducibility might be a useful biomarker in cancer epidemiology. As those factors may affect the AHH activity, a careful control of those factors to AHH activity is necessary in epidemiological studies on the association between AHH inducibility in human lymphocytes and lung cancer.

The relationship between aryl hydrocarbon hydroxylase and polymorphisms of the CYP1A1 gene.

We examined the relationship between aryl hydrocarbon hydroxylase (AHH) and the frequency of a Msp I mutation in the 3′‐flanking region of cytochrome P450 (CYP) 1A1 (Mspl polymorphism) and another mutation in exon 7 (Ile‐Val polymorphism) in 84 healthy male subjects in Fukuoka, Japan. AHH inducibility (3‐methylcholanthrene (MC)‐induced AHH activity/non‐induced AHH activity) was correlated with the MspI polymorphism (P<0.0001) and age class (P=0.015), whereas no correlation was found for the Ile‐Val polymorphism (P=0.509). Age‐adjusted AHH inducibility (mean±SE) of the predominant homozygote (genotype A), the heterozygote (genotype B) and a homozygote rare allele (genotype C) genotypes was 4.89±0.36, 4.82±0.29 and 13.61±1.44, respectively. The genotype C showed much higher AHH inducibility than genotypes A and B (P<0.001), while no significant difference was observed between genotypes A and B. Non‐induced AHH activity was also correlated with these polymorphisms. The AHH activity of a homozygous mutant Val/Val genotype (0.076±0.010 pmol/min/106 cells) was significantly higher (P<0.05) than that of the wild‐type homozygous Ile/Ile (0.044±0.004 pmol/min/106 cells) and heterozygous Ile/Val (0.047±0.007 pmol/min/106 cells) genotypes. Our study suggests that the genotypes C and Val/Val, which are more frequent in smoking‐related lung cancer, are closely related with high AHH inducibility and high non‐induced AHH activity, respectively. Thus, the positive relationship between AHH inducibility and lung cancer is supported by our study. If our results are confirmed and the assessment of genotype becomes feasible on a population basis, identification of smokers who have genetically high susceptibility to lung cancer (genotype C or Val/Val) may become important for the prevention of lung cancer.

Effects of polychlorinated dibenzo- p-dioxin and dibenzofuran congeners in human lymphoblastoid cells on aryl hydrocarbon hydroxylase activity

The separate and concurrent effects of polychlorinated dibenzo- p-dioxin (PCDD) and dibenzofuran (PCDF) congeners on aryl hydrocarbon hydroxylase (AHH) activity in human lymphoblastoid cells were examined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD) induced AHH activity about 9-fold acetone-treated AHH activity, while octachlorodibenzo- p-dioxin (octaCDD) did not affect the AHH activity and 2,4,6,8-tetrachlorodibenzofuran (2,4,6,8-tetraCDF) reduced the AHH activity by 3470. The concurrent effect of 2,3,7,8-tetraCDD with 1,3,6,8-tetraCDD on AHH inducibility was much smaller than expected. The concurrent effect of 2,3,7,8-tetraCDD with 2,4,6,8-tetraCDF, octaCDD or 3,3′,4,4′,5,5′-hexaCB was as much as the effect of 2,3,7,8-tetraCDD alone. There was a difference between 3-methylcholanthrene (MC) and 2,3,7,8-tetraCDD in the concurrent effects of 3-methylsulfone-3′,4,4′,5-tetraCB (3-MSF-3′,4,4′,5-tetraCB) on AHH activity. The concurrent effect of MC with 3-MSF-3′,4,4′,5-tetraCB on AHH activity was equal to the effect of MC alone while that of 2,3,7,8-tetraCDD and 3-MSF-3′,4,4′,5-tetraCB on AHH activity was inhibitory. These results imply that congeners of PCDD and PCDF are considered to be more or less effective and isozymes of cytochrome P450, key enzymes for the AHH, induced by them are differently inhibited.

Nitroreduction of 2,4-dinitrotoluene [formula omitted] by cytochrome P-450 induced H4IIE cells

Conditions have been established for H4IIE rat hepatoma cell cultures in which effects of cytochrome P-450 induction on the metabolism of a munitions wastestream pollutant can be studied. Under these conditions, the polychlorinated hydrocarbon 2,3,4,7,8-pentachlorodibenzfuran (PCDBF) induced cytochrome P-450 (1A1) aryl hydrocarbon hydroxylase (AHH) activity over a wide range of concentrations without significant cytotoxic effects. The munition pollutant 2,4-dinitrotoluene (2,4-DNT) did not induce AHH activity itself, but its metabolism was considerably altered when applied to PCDBF induced cultures. Production of amino nitrotoluene isomers was greatly enhanced in induced cultures as compared to uninduced controls, as was the conversion of radiolabeled 2,4-DNT to relatively more polar metabolites. To some extent, the results with H4IIE cells parallel those reported for animals exposed to 2,4-DNT after induction of cytochrome P-450 AHH activity. The preliminary findings suggest that with further development and validation, H4IIE cultures could be of use in characterizing metabolites that result from exposure to chemical mixtures involving a P-450 (1A1) inducer.

Proline Is Required for Transcriptional Control of the Aromatic Hydrocarbon‐inducible P1450 Gene in C57BL/6 Mouse Monolayer‐cultured Hepatocytes

Expression of aryl hydrocarbon hydroxylase (AHH) and the corresponding gene, CyplA1 or P1450 in mice, in C57BL/6 mouse hepatocytes in primary culture was investigated after exposure to benz[a]‐anthracene with respect to proline‐related metabolic regulation. When the cells were cultivated in complete Waymouth MB752/1 (Way), prominent induction of AHH by benz[a]anthracene was observed, whereas the induction was inefficient in the same but proline‐deflcient medium [Way‐(‐pro)]. Constitutive AHH activities decreased with increasing culture period. P1450 gene transcripts were slightly expressed when the medium was changed, independently of whether the cells were cultivated in either Way or Way(‐pro), followed by decrease within 24 h and no apparent induction of AHH. However, treatment with Δ1‐pyrroline‐S‐carboxylic acid (P5C), a biosynthetic precursor for proline, dose‐dependently increased basal AHH activities in the cells cultivated in Way‐(‐pro). Benz[a]anthracene induction of AHH in cells cultivated in Way(‐pro) was additively increased in the presence of P5C as much as with proline. Treatment with o‐aminobenzaldehyde, which inactivates P5C, drastically reduced the induced AHH activities in hepatocytes cultivated in either P5C‐added Way (‐pro) or Way medium. Benz[a]anthracene induced both P1450 and P3450 mRNAs. Neither proline nor P5C increased the induced transcripts within 12 h after the start of benz[a]‐anthracene treatment, but the two compounds increased the amounts of P1450 mRNA found at later time points. After treatment with actinomycin D, the half‐life of the induced P1450 mRNA was approximately 12 h, being independent of the presence of either proline or P5C. Our observations suggest that induction of AHH after treatment with polycyclic aromatic hydrocarbon is dependent on proline‐related metabolism which influences the transcriptional process of P1450 gene expression.

Interrelationship of cellular fluorescein formation ability, an indication of cell viability, with environmental factors and with aryl hydrocarbon hydroxylase activity

Fifty-eight lymphoblastoid cell lines were determined both for their cellular fluorescein formation ability (CFFA) and aryl hydrocarbon hydroxylase (AHH) activities, while each one was interviewed for several factors of his (her) living environment. Simple correlation coefficients of CFFA were found to be correlated positively with the daily use of oil stoves and negatively with AHH inducibility. Multivariate analysis performed to obtain partial regression coefficient indicated that CFFA was correlated negatively with the 3-methylcholanthrene (MC)-induced AHH activity. These results suggest the presence of a negative relationship between CFFA and the drug metabolizing ability, AHH activity, in the human lymphoblastoid cells.

The Ah receptor, cytochrome P450IA1 mRNA induction, and aryl hydrocarbon hydroxylase in a human lymphoblastoid cell line

The immunosuppressive and carcinogenic effects of aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and 3-methylcholanthrene (MC) on B lymphocytes of adult rodents and the induction of cytochrome P450IA1 and aryl hydrocarbon hydroxylase (AHH) in human mitogenactivated lymphocytes and B-lymphoblastoid cell lines are believed to be mediated by the Ah receptor. However, there has not been a direct demonstration or characterization of the Ah receptor in denned populations of any of these cells. We report here the detection and characterization of an abundant, high-affinity B lymphocyte Ah receptor in the AHH-inducible human B lymphoblastoid cell line BCR-5. Our results represent the first characterization of a human lymphocyte receptor in a well-defined lymphocyte population. Sucrose density gradient analysis of BCR-5 cytosols incubated with [ 3H]TCDD revealed a characteristic 9 S specific binding peak. The maximum concentration of Ah receptor was about 200 fmol/mg protein. Specific binding to the Ah receptor was also detected with [ 3H]MC and, to a lesser extent, with [ 3H]benzolalpyrene. The apparent binding affinity ( K d ) for [ 3H]TCDD (determined by saturation analyses) was about 5 nM. A specific [ 3 H] TCDD-Ah receptor complex which sedimented at 5 S was extracted from nuclei of BCR-5 cells incubated at 37° with [ 3H]TCDD. The Ah receptor of BCR-5 cells is thus similar in characteristics to that identified in other cell lines. When BCR-5 cells were exposed in culture for 24 hr to increasing concentrations of benz[ a]anthracene there was a concentrationdependent increase in induction and a good correlation ( r = 0.98) between the level of induced AHH activity and the relative abundance of cytochrome P450IA1 mRNA. The human B lymphoblastoid cell line BCR-5, therefore, has a complete regulatory mechanism for Ah receptor-mediated induction of cytochrome P450IA1 that is essentially the same as that which has been well established in many rodent species. The accessibility of human blood lymphocytes and the ease of establishment of B lymphoblastoid cell lines from any donor provide a source of pure cultures of human B lymphocytes which can be grown continuously in vitro for the study of mechanisms related to Ah receptor-mediated cytochrome P450IA1 induction, immunosuppression and carcinogenesis.

Regulation of mouse P1450 gene expression in monolayer-cultured hepatocytes from responsive and non-responsive strains

Regulation of P(1)450 gene expression in mouse hepatocytes from responsive (C57BL/6) and non-responsive (DBA/2) strains in primary culture was investigated with respect to aryl hydrocarbon hydroxylase (AHH) activity and P450 transcript levels. Although significant induction of AHH activity in C57BL/6 mouse hepatocytes after exposure to benz[aanthracene (BA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was observed 24 h after the beginning of cultivation, the response was more prominent after longer periods. AHH induction in DBA/2 mouse hepatocytes by TCDD was also evident after 24 h treatment, but that by BA was delayed, only becoming significant after 3 days. Limited treatment with cycloheximide (CHI) for the initial 8 h affected AHH activity measured after 24 h; BA-induced AHH activity was decreased if the treatment started day 1 after seeding of the cells from either strain, whereas if started at day 3 the enzyme activities in hepatocytes from C57BL/6 strain were approximately doubled and those from DBA/2 increased to 130%. Treatment with dibutyryl cAMP or forskolin, a specific activator for adenyl cyclase, increased BA-induced AHH activities. 3-Methoxybenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, significantly increased both basal and BA-induced AHH activities of hepatocytes from both strains at days 3 and 5, reduction of P(1)450 transcripts also being evident in the latter case. The observations indicate qualitatively similar but quantitatively different regulation of AHH induction in both responsive and non-responsive mouse strains. Furthermore the regulation changed with increasing cultivation period. Previously described regulation mechanisms in cultured cells were observed to operate a few days after seeding, possibly after adaptation of hepatocytes to the culture conditions.

Increase of CYP1A1 mRNA and AHH activity by inhibitors of either protein or RNA synthesis in mouse hepatocytes in primary culture

Regulation of CYP1A1 gene expression in mouse hepatocytes from C57/BL6 strain in primary culture was investigated with respect to aryl hydrocarbon hydroxylase (AHH) activity and mRNA levels. Small amounts of the CYP1A1 gene transcripts were detected without the presence of any known AHH inducers but after medium change. Maximal level of expression was approximately 6-9 h after the procedure, followed by a decrease to an undetectable level 24 h later. After temporary treatment of hepatocytes for 10 h with cycloheximide, an inhibitor of protein synthesis, AHH activity measured 14 h later was at a normal low level, although medium-change-associated CYP1A1 mRNA were increased by cycloheximide treatment. However, if cells were treated with either actinomycin D, alpha-amanitin or cordycepin, which are inhibitors of RNA synthesis, after exposure to cycloheximide, prominent induction of AHH activity was observed, the levels being almost equal to those for hepatocytes treated with benz[a]anthracene, a potent AHH inducer. With the same experimental protocol benz[a]anthracene-induced AHH activity was enhanced approximately 4-fold. After washing out cycloheximide and/or benz[a]anthracene, the decrease in the mRNA amounts was delayed in the presence of actinomycin D and half amounts were found even 12 h later; while without actinomycin D they reduced with a half-life of 3-4 h. The observations indicate that CYP1A1 gene expression might be regulated at the post-transcriptional level with compensatory mRNA stabilization under conditions of blocked further production, resulting in elevation of AHH activity.

A comparative analysis of the electrostatic potentials of some structural analogues of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and of related aromatic systems

AbstractWe have carried out an ab initio STO‐5G computational analysis of the electrostatic potentials of four structural analogues of the highly toxic 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and four related aromatic systems benzo[a]pyrene, benz[a]anthracene, and two isomeric benzoflavones. These systems, to varying degrees, induce aryl hydrocarbon hydroxylase activity and are believed to interact with the same cytosolic receptor in initiating their biochemical responses. Our present results for the TCDD analogues support and have allowed us to further qualify our early observations regarding factors which are linked to high biological activities in the dibenzo‐p‐dioxins and structurally similar systems. We find that a high degree of activity appears to require nonoverlapping negative potentials above all or most of the lateral regions, with an observed optimum range of magnitudes. In systems with central oxygens, it is required that the negative oxygen potentials be small and weak; however, oxygen negative regions in the molecule are not necessary for high activity. The observed differences between the potential patterns of the four aromatic systems and those of TCDD and its active analogues may reflect an inherent dissimilarity in the nature of their interactions with the cytosolic receptor. That the carbonyl oxygen negative potential of 7,8‐benzoflavone is significantly more negative than its isomer's supports earlier speculation concerning the former's more effective inhibiting effects on metabolic oxidation.

The Association of Age with the Induction of Drug-metabolizing Enzymes in Human Monocytes

In vivo studies in man suggest that the enzyme-inducing effect of environmental influences such as drugs or smoking may be reduced in elderly people. We have investigated the basal activity and response to induction of the oxidative enzyme, aryl hydrocarbon hydroxylase (AHH) in human monocytes. Three groups were studied: ten fit young, ten fit elderly and eight frail elderly subjects. Significant induction of AHH activity in response to the hydrocarbon benz(a)anthracene was achieved in all three groups. No impairment of basal AHH activity or in the synthesis of new enzyme protein was noted with age or frailty. There is still no direct evidence of an age-associated fall in drug metabolizing enzyme activities in man.

Use of Human Epidermal Cells in the Study of Carcinogenesis.

Because of the importance of human cells, particularly human epithelial cells, in cancer research, we have studied certain phases or events of carcinogenesis using human epidermal cells in primary culture. 1) We found that human epidermal cells are capable of metabolizing benzo[a]pyrene. Large inter-individual variations are found in the basal and induced arylhydrocarbon-hydroxylase activities. 2) UV-induced unscheduled DNA synthesis was demonstrated in human epidermal cells on autoradiographs. We also found that DNA repair is defective in epidermal cells isolated from xeroderma pigmentosum by a new explant-outgrowth culture. 3) Human epidermal cells are unique in that there is a large number of binding sites to phorbol esters compared with mouse epidermal cells, but there is no down-regulation. Further, human epidermal cells show essentially negative responses to tumor promoters, i.e., no stimulation of DNA synthesis, sugar uptake, and no induction of ornithine decarboxylase activity. 4) Human epidermal cells contain 1.5 x 10(5) binding sites per cell for epidermal growth factor (EGF), whereas squamous cell carcinomas of skin and oral cavity have larger amounts of EGF receptors in the order of 10(6) per cell. 5) Based on the above results, we attempted to transform human epidermal cells by the treatment with chemical carcinogens, but until now no transformation was obtained.

Use of Human Epidermal Cells in the Study of Carcinogenesis

Because of the importance of human cells, particularly human epithelial cells, in cancer research, we have studied certain phases or events of carcinogenesis using human epidermal cells in primary culture. 1) We found that human epidermal cells are capable of metabolizing benzo[a]pyrene. Large inter-individual variations are found in the basal and induced arylhydrocarbon-hydroxylase activities. 2) UV-in-duced unscheduled DNA synthesis was demonstrated in human epidermal cells on autoradiographs. We also found that DNA repair is defective in epidermal cells isolated from xeroderma pigmentosum by a new explant-outgrowth culture. 3) Human epidermal cells are unique in that there is a large number of binding sites to phorbol esters compared with mouse epidermal cells, but there is no down-regulation. Further, human epidermal cells show essentially negative responses to tumor promoters, i.e., no stimulation of DNA synthesis, sugar uptake, and no induction of ornithine decarboxylase activity. 4) Human epidermal cells contain 1.5 × 10 5 binding sites per cell for epidermal growth factor (EGF), whereas squamous cell carcinomas of skin and oral cavity have larger amounts of EGF receptors in the order of 10 6 per cell. 5) Based on the above results, we attempted to transform human epidermal cells by the treatment with chemical carcinogens, but until now no transformation was obtained.

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals: Phenobarbital and TCDD induce the same form of cytochrome P450

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different “classes”. Both 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P 1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducerreceptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD ( Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.

2‐(4′‐Chlorophenyl)benzothiazole is a potent inducer of cytochrome P450IA1 in a human and a mouse cell line

Two benzothiazole derivatives, 2-(4'-chlorophenyl)benzothiazole (CPBT) and 2-(4'-formylphenyl)benzothiazole (FPBT) were studied for their ability to induce aryl hydrocarbon hydroxylase activity in a mouse and a human cell line. In both the mouse hepatoma cell line, Hepa-1, and the human choriocarcinoma cell line, JEG-3, a high aryl hydrocarbon hydroxylase activity was observed after treatment with CPBT. In contrast, FPBT had a very weak inducing capacity in both cell lines. The maximal induction by CPBT was several times (e.g. in Hepa-1 about fourfold on average) greater than that observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific cDNA probe for mouse cytochrome P4501A1 gene was used to quantify mRNA levels in Hepa-1 cells. CPBT increased cytochrome P450IA1 mRNA to a level of 88% of that induced by TCDD. Immunoblot analysis with monoclonal antibody 1-7-1, directed against rat liver cytochrome P450IA1 and P450IA2, showed that the amount of P450IA1 is substantially increased in Hepa-1 cells after treatment with CPBT. The observation that CPBT competed TCDD off its specific cytosolic binding site suggests a receptor-mediated induction of cytochrome P450IA1 mRNA. An in vitro activation effect did not explain the exceptionally high hydroxylase activity. The results show that CPBT is a more efficient inducer of aryl hydrocarbon hydroxylase than TCDD in Hepa-1 and JEG-3 cells and that the induction is supported by P450IA1. The discordant effect of CPBT on mRNA and aryl hydrocarbon hydroxylase activity suggests that post-translational modifications of P450IA1 account for a major part of the increased enzyme activity.

Relation between Cytochrome P450IA1 Expression and Estrogen Receptor Content of Human Breast Cancer Cells

Multidrug resistance (MDR) in an MCF-7 human breast cancer cell line (MCF7/Adr) is associated with decreased drug accumulation and overexpression of P-glycoprotein as well as alterations in the levels of specific drug-metabolizing enzymes, including decreased activity of the phase I drug-metabolizing enzyme aryl hydrocarbon hydroxylase (AHH) and increased expression of the anionic form of the phase II drug-metabolizing enzyme glutathione S-transferase. Since the development of MDR in this MCF-7 cell line is also associated with a loss of estrogen receptors (ER), we have examined the expression of cytochrome P450IA 1, the gene encoding AHH activity, in other breast cancer cell lines not selected for drug resistance but expressing various levels of ER. These studies show that a relationship exists between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AHH activity and the ER content in a series of breast cancer cell lines. In these cell lines expression of AHH activity is regulated, at least in part, at the level of P450IA 1 RNA. While TCDD-specific binding proteins (Ah receptors) were found in each of the breast cancer cell lines, there was no apparent relation between the level of nuclear TCDD-binding proteins and the level of TCDD-inducible P450IA 1 expression. Previous studies from our laboratory have described an inverse relationship between levels of the anionic form of glutathione S-transferase and ER in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells.

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p less than 0.005), two had approximately the same, and two had significantly higher (p less than 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 +/- 0.09 for clone 1 (Hs-1) and 0.94 +/- 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p less than 0.005), two had approximately the same, and one had significantly higher levels (p less than 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).

TCDD receptor ligands present in extracts of urban air particulate matter induce aryl hydrocarbon hydroxylase activity and cytochrome P-450c gene expression in rat hepatoma cells

In previous studies we have shown that substances associated with particulates collected from urban air and automobile exhaust bind with high affinity to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor present in rat liver cytosol. In this study we used a rat hepatoma cell line, H4IIE, to investigate the effect of such substances on an enzyme system, aryl hydrocarbon hydroxylase (AHH), which is regulated via the TCDD receptor. The results demonstrate that AHH activity in the H4IIE cell line can be induced by extracts of particulates collected from urban air and automobile exhausts in a dose-dependent manner, and that the AHH activity is inducible by five polycyclic aromatic hydrocarbons (PAHs) including 1-/3-nitrobenzo[a]pyrene and 6-chlorochrysene, all present in extracts of particulates from urban air and automobile exhausts. The induction of AHH activity is correlated to apparent TCDD receptor affinity for investigated PAHs (r = 0.85) and particulate extracts. Biochemically, treatment of the cells with 5,6-benzoflavone significantly increased the level of cytochrome P-450c but not P-450d as shown by immunoblotting and analysis of mRNA levels. The data indicate that substances present in extracts of urban air particulates can interact with the TCDD receptor in intact cells and cause an accumulation of cytochrome P-450c mRNA leading to an increased synthesis of the gene product and thus an increase in enzyme activity.