Determinants of polycyclic aromatic hydrocarbon-DNA adducts in human placenta.

To determine the relative contributions of tobacco smoking and P-450 metabolism (cytochrome P-450IAI) in the formation of benzo(a)pyrenediol-epoxide and other polycyclic aromatic hydrocarbon-DNA adducts in vivo, 16 human placentas were assayed for aryl hydrocarbon hydroxylase activity and (+/-)r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-DN A adduct levels. Immunoaffinity chromatography columns, conjugated with monoclonal antibodies raised against benzo(a)-pyrene-diol-epoxide-deoxyguanosine, were used to concentrate polycyclic aromatic hydrocarbon-DNA adducted nucleotides, and synchronous fluorescence spectroscopy was used specifically to detect r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP-tetrol) extracted from acid hydrolysates of immunoconcentrated materials. Data were analyzed for associations with maternal dietary and smoking habits, umbilical cord blood cotinine levels, and placental aryl hydrocarbon hydroxylase levels. Complex mixtures of fluorescent materials were present in organic solvent extracts of acid hydrolysates of immunoconcentrated nucleotide-adducts from all placentas with patterns of fluorescence that may be associated with tobacco smoking determined by generation of spectral fluorescence excitation-emission matrices. BP-tetrols were detected in extracts from 8 placentas: 5 of 7 from smokers and 3 of 9 from nonsmokers. Placental aryl hydrocarbon hydroxylase activity was significantly higher in placentas from which BP-tetrols were extracted [3.9 +/- 2.4 [corrected] (mean +/- SE) pmol 3-hydroxybenzo(a)pyrene mg protein-1 min-1], than among placentas from which BP-tetrols were not extracted (0.4 +/- 0.2 [corrected] pmol 3-hydroxybenzo(a)pyrene mg protein-1 min-1) (P = 0.03, Student's t test). This association was independent of maternal smoking or umbilical cord blood cotinine levels. These results indicate that while maternal tobacco smoking is associated with the accumulation of putative, but as yet unidentified, polycyclic aromatic hydrocarbon-DNA adducts in placenta, metabolic capacity appears to be the principal determinant for the (+/-)r-7,t-8-dihydroxy-c-9,10 epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-DNA adduct levels detected.     

The effect of cigarette smoke on aryl hydrocarbon hydroxylase activity and cytochrome P450 content in rat liver and lung microsomes.

Levels of activity of the enzyme aryl hydrocarbon hydroxylase and cytochrome P450 have been estimated in lung and liver of rats exposed to graded doses of cigarette smoke and in human bronchial mucosa of smokers, non-smokers and patients with lung cancer. Exposure of rats to smoke of four cigarettes increased both hepatic and pulmonary aryl hydrocarbon hydroxylase activity. Exposure to smoke of four cigarettes daily for seven and 14 days did not result in higher aryl hydrocarbon hydroxylase levels in the liver than one day's exposure, but in the lung longer exposures caused greater increases in enzyme activity. Injection of benzo(a)pyrene at two dose levels caused a much greater increase in both liver and lung aryl hydrocarbon hydroxylase than smoking. The lower dose maximally stimulated the enzyme in both organs. The changes in aryl hydrocarbon hydroxylase activity were accompanied by significant increases in both liver weight and the cytochrome P450 content of liver microsomes but the increase in cytochrome P450 did not parallel those in aryl hydrocarbon hydroxylase activity. Of 40 surgical and autopsy specimens of human lung and tracheal mucosa from smokers, non-smokers and cancer patients, only one was found to have detectable aryl hydrocarbon hydroxylase activity. The relationship between aryl hydrocarbon hydroxylase induction by cigarette smoke in human tissues and the development of bronchogenic carcinoma in smokers remains unclear.     

Strain-dependent differences in the metabolism of 3-methylcholanthrene by maternal, placental, and fetal tissues of C57BL/6J and DBA/2J mice

C57BL/6J (B6) and DBA/2J (D2) mice have different susceptibilities to developmental toxicity and transplacental carcinogenesis induced by in utero exposure to polycyclic aromatic hydrocarbons, which has been associated with polycyclic aromatic hydrocarbon metabolism and inducibility at the Ah locus. The distribution of total 3-methylcholanthrene (3-MC)-associated radioactivity in maternal, placental, and fetal tissues of beta-naphthoflavone-pretreated pregnant B6 and D2 mice was determined up to 12 h after p.o. exposure to [6-14C]-3-MC (63 mg/kg, 20 mu Ci) on gestational day 17. 3-MC-associated radioactivity in maternal plasma was not significantly different in the two strains. However, D2 tissue homogenates had consistently higher levels of 3-MC-associated radioactivity, which included both bound and free parent compound and metabolites. Increased metabolism of 3-MC by B6 maternal liver was suggested by the induced levels of aryl hydrocarbon hydroxylase activity in that tissue and by the observation that levels of total radioactivity decreased more rapidly in B6 tissues than in D2 tissues. The D2 fetal lung, the target tissue for 3-MC-induced transplacental carcinogenesis, appeared to accumulate 3-MC-associated radioactivity for a longer period of time than either the D2 fetal liver or the B6 fetal tissues. This study suggests that the genetic differences in fetal susceptibility to the developmental toxicity and transplacental carcinogenesis of 3-MC may be related to the presystemic elimination of the compound from both maternal and fetal tissues.     

Development of an assay for aryl hydrocarbon (benzo(a)pyrene) hydroxylase in human peripheral blood monocytes.

An assay has been developed and measurements of aryl hydrocarbon [benzo(a)pyrene] hydroxylase have been made in peripheral blood monocytes from a human population. Treatment with benz(a)anthracene in cell culture increased aryl hydrocarbon hydroxylase activity from 6.5 to 37-fold in monocytes from each of 25 apparently healthy donors. A weak correlation (r = 0.38) was observed between the induction ratios obtained with monocytes and lymphocytes from the same donors. Reproducibilities of the monocyte and lymphocyte assays were comparable. In monocytes, the measurement of basal and induced aryl hydrocarbon hydroxylases activity does not require pretreatment with mitogens as is the case with lymphocytes. Monocytes also exhibit a much wider range of induction ratios than do lymphocytes.     

Induction of aryl hydrocarbon hydroxylase activity and pulmonary carcinoma.

Aryl hydrocarbon hydroxylase activity in lymphoblasts from normal Finnish adults and from patients with pulmonary carcinomas and other types of malignancy has been studied by a modification of previously used techniques. High absolute induced aryl hydrocarbon hydroxylase activity was found in 39% of patients with untreated lung cancer but only in 15% of normal people. No increased frequency was found in the control group comprising other malignancies. The diagnosis of pulmonary carcinoma was made at a lower mean age (4.9 years younger) in the individuals with high aryl hydrocarbon hydroxylase activity than in those with low activity. High absolute aryl hydrocarbon hydroxylase activity was dominantly inherited in normal individuals, and the frequency of athe Ahb gene in the Finnish population was 8%.     

Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes

Serially cultivated keratinocytes of human and rat epidermis and esophagus were compared with respect to their sensitivity to toxic effects of 3-methylcholanthrene and ability to metabolize benzo(a)pyrene. 3-Methylcholanthrene was highly toxic to the human keratinocytes and to early-passage rat epidermal keratinocytes, as evidenced by markedly reduced growth upon continuous exposure or reduced colony-forming ability after 1-day exposure to concentrations of 0.4 to 40 microM in the culture medium. Rat esophageal and late-passage rat epidermal cells appeared insensitive to 3-methylcholanthrene by these criteria. All the cell types except late-passage rat epidermal cells metabolized benzo(a)pyrene at comparable rates, and expressed aryl hydrocarbon hydroxylase maximally inducible by similar concentrations of 3-methylcholanthrene. Biotransformation in the human cells was greatly inhibited by alpha-naphthoflavone, a specific inhibitor of aryl hydrocarbon hydroxylase. The lack of toxicity of 3-methylcholanthrene toward late-passage rat epidermal cells can be attributed to the low constitutive rate of biotransformation these cells exhibit. The insensitivity of rat esophageal cells despite substantial metabolic activity reflects the importance of intrinsic differences among keratinocytes derived from different epithelia and species in determining toxic response. Human cervical and monkey esophageal keratinocyte cultures also actively metabolized benzo(a)pyrene, illustrating further the utility of the culture system for exploring differences among species and epithelial cell types.     

Population distribution of placental benzo(a)pyrene metabolism in smokers

Human placental microsomes isolated from term placentas derived from nonsmoking women and women smoking 1 to 40 cigarettes a day were analyzed for the metabolism of benzo(a)pyrene measured as various metabolites by HPLC and/or as aryl hydrocarbon hydroxylase (AHH)6 activity. In accordance with other reports, AHH activity was several times higher in smokers than in nonsmokers. Regression analysis on 13 different placental tissues from women smoking from 1 to 40 cigarettes demonstrated a high correlation (r = 0.8 to 0.9) between AHH activity (or the formation of benzo(a)pyrene phenols resolved by HPLC) versus the formation of the procarcinogenic benzo(a)pyrene-7,8-diol. Subsequent studies on placentas derived from 67 women who smoked 10 to 40 cigarettes per day demonstrated a definite dose-response relationship between AHH activity and the number of cigarettes smoked/day. The dose-response curve was sigmoidal in shape; however, when the data were plotted on a semi-log scale the curve assumed a linear shape, reaching saturation of AHH induction beyond 20 to 25 cigarettes/day. While mean AHH activity was dependent upon the number of cigarettes smoked/day, considerable interindividual variability in AHH (ranging more than 1,000-fold in some cases) was observed among individuals with comparable smoking histories, i.e. smoking the same number of cigarettes. Population distribution suggested clustering of the population in the low-AHH-activity region while cord-blood thiocyanate analysis and twin studies suggested that genetic factors contributed to a major portion of the inter-individual variability in AHH activity observed among smokers.     

Effect of 17-beta-estradiol and testosterone on aryl hydrocarbon hydroxylase activity in mouse tissues in vivo and in cell culture

Initiation of skin tumors by 7,12-dimethylbenz[a]anthracene is more effective during diestrus than during estrus and thus may be inhibited by estrogens. We therefore studied the effect of sex hormones on aryl hydrocarbon hydroxylase, an enzyme system which may be a common pathway in metabolizing both polycyclic hydrocarbons and steroids. 1. The hydroxylase system is present in mouse skin and liver and is inducible by the administration of polycyclic hydrocarbons such as 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene. The control and inducible enzyme activities are slightly lower in castrated male mice which have received subcutaneous implants of 17-β-estradiol. 2. In mouse fetal cell cultures, 17-β-estradiol in the growth medium prevents hydroxylase induction only at concentrations 6 to 60 times greater than the level of the polycyclic hydrocarbon inducer in the medium. 3. In a cell-free in vitro system, both 17-β-estradiol and testosterone competitively inhibit polycyclic hydrocarbon hydroxylation, even at levels of steroid that are lower than that of the polycyclic hydrocarbon substrate. With the hydroxylase system from mouse liver, lung, kidney, adrenal and skin in the cell-free system, 17-β-estradiol is generally more inhibitory than testosterone. This difference in inhibition between an estrogen and an androgen may be related to sex differences in the metabolism of drugs and in the susceptibility to carcinogenesis by polycyclic hydrocarbons.     

Large interindividual variations in metabolism of benzo(alpha)pyrene by peripheral lung tissue from lung cancer patients.

A very large variation (44-fold) was observed in the ability of short-term organ cultures of peripheral lung tissue from lung-cancer patients to metabolize the environmental carcinogen benzo(alpha)pyrene to organic solvent-soluble metabolites. The amounts of benzo(alpha)pyrene (2 microM) metabolized ranged from little (1%) to almost total (96.2%) metabolism within 24 h of culture. Previous work by Kellerman et al. (1973) has suggested a relationship between susceptibility to lung cancer and the indicibility of aryl hydrocarbon hydroxylase activity in cultured human lymphocytes. The metabolic fate of carcinogenic polycyclic aromatic hydrocarbons in the respiratory tract in vivo is undoubtedly more closely mimicked by short-term organ culture of human lung than by cultured lymphocytes. Thus the very wide interindividual variation observed in pulmonary metabolism of benzo(alpha)pyrene in this study and the large variations in covalent binding to human bronchial DNA observed by Harris et al. (1976) strongly suggest that there may be little basis for screening humans for variations in lymphocyte aryl hydrocarbon hydroxylase activity as a means of assessing their susceptibility to lung cancer.     

Polycyclic aromatic hydrocarbon-DNA adducts in spontaneously aborted fetal tissue

Fetal tissue and placentas from 15 human spontaneous abortions were evaluated for DNA adducts of polycyclic aromatic hydrocarbons (PAHs), using a competitive enzyme-linked immunosorbent assay (ELISA) with fluorescent end-point detection. PAH-derived adducts were found in 43% of placentas, 27% of fetal liver samples and 42% of fetal lung specimens, thus confirming that the human fetus is a target for DNA damage. As there was only 60% concordance between placenta and fetal lung or liver on the presence or absence of detectable PAH adducts, the placenta was not a good surrogate for adduct formation in other fetal organs. PAH-derived adducts in fetal liver and lung presumably form as a result of transplacental exposure to environmental stimuli. Since none of the positive fetal samples were from women who reported smoking during pregnancy, cigarette smoke is, in this case, an unlikely candidate and the adducts detected must be due to some other common source(s) of hydrocarbon exposure. The high frequency of positive samples in our small series casts some doubt on whether fetal PAH-DNA adducts identify a population at increased risk for transplacental carcinogenesis.     

Polycyclic hydrocarbon-produced toxicity, transformation, and chromosomal aberrations as a function of aryl hydrocarbon hydroxylase activity in cell cultures

The cytotoxic effect of benzo[a]pyrene alone in fetal rat hepatocytes in culture is prevented by phenobarbital. Benzo[a]pyrene is not toxic to HTC, A9, or HeLa cells in which aryl hydrocarbon hydroxylase activity is either absent or very low; however, benzo[a]pyrene is cytotoxic to each of these three established cell lines grown together with the liver cells in the presence of phenobarbital. Polycyclic hydrocarbon-produced cytotoxicity is associated with chromatid breaks, whereas aneuploidy is more closely correlated with malignant transformation of hamster secondary cultures. Benz[a]anthracene or a-naphthoflavone competitively inhibits the hydroxylation of other polycyclic hydrocarbons such as the carcinogens 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene. The exposure of fetal hamster secondary cells to excessive amounts of benz[a]anthracene prior to, during, and following treatment with 7,12-dimethylbenz[a]anthracene prevents malignant cell transformation from occurring.     

Benzo(alpha)pyrene effects on mouse epithelial cells in culture.

The effect of benzo (a) pyrene on the growth in culture of 5 mouse epithelial cell strains was examined. These epithelial cells are highly sensitive to the cytotoxic action of benzo (a)-pyrene. In addition, the activity of the benzol (a) pyrene-metabolizing system, aryl hydrocarbon hydroxylase, is low but highly iducible by the carcinogen. As the sensitivity of a cell strain to the cytotoxic action of benjo (a) pyrene decreased, the inudcibility of the hydroxylase also decreased,. However, a strong correlation could not be found between cytotoxicity and the level of uninduced or induced hydroxylase when the values from different cell strains were compared. These experiments suggest that thehydroxylase is important in determining the sensitivity of epithelial cells to the cytotoxic action of benzo (a) pyrene, but other factors may also modulate this sensitivity.     

Aryl hydrocarbon hydroxylase activity and microsomal cytochrome content of human fetal tissues

Aryl hydrocarbon hydroxylase (AHH) and microsomal cytochromes were measured in tissues of human features aborted by prostaglandins. Cytochrome P-450 concentrations and AHH activity were about 4 times higher in adrenal glands than in liver. AHH was present in testes, ovaries, and vagina and uterus at levels equal to or greater than those in the liver. In lung, kidney, and intestines it was present at levels lower than those in the liver. Mean hepatic AHH and hepatic and adrenal cytochromes P-450 and b5 were not significantly different in prostaglandin and hysterotomy abortuses; mean adrenal AHH was significantly lower in prostaglandin abortuses, but the ranges in both groups were overlapping. Neither fetal sex nor maternal cigarette smoking affected hepatic or adrenal AHH activity or microsomal cytochrome concentrations or difference spectra. Hepatic and adrenal AHH activities were concentrated in microsomes and were correlated with microsomal P-450 content. These findings are constant with P-450 mediation of AHH in human fetal tissues. The data demonstrate the utility of prostaglandin abortuses for studies of fetal tissue mixed-function oxidase activity and point to the endocrine glands and target tissues in addition to the liver as potential sites for activating chemical carcinogens and cytotoxins in the human fetus.     

The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase.

Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.     

Cytochrome P-450-dependent xenobiotic metabolizing activity in Zymbal's gland, a specialized sebaceous gland of rodents

Homogenates of Zymbal's glands from beta-naphthoflavone-treated rats and mice have 7-ethoxycoumarin O-deethylase activity, while those from rats also have aryl hydrocarbon hydroxylase activity. Measured concentrations of cytochrome P-450 in microsomes from Zymbal's glands of beta-naphthoflavone-treated rats are not higher than those from untreated rats. Studies of inhibitors of 7-ethoxycoumarin O-deethylation and aryl hydrocarbon hydroxylation in homogenates of Zymbal's glands from beta-naphthoflavone-treated rats suggest that these enzyme activities are catalyzed by cytochrome P-450. These findings indicate that reactive metabolites of chemical carcinogens may be formed in Zymbal's gland, a target organ for chemical carcinogenesis.     

Histological, histochemical and electron microscopic changes of the placenta induced by maternal exposure to hyperthermia in the rat.

Both clinical and experimental investigations have shown that maternal hyperthermia during critical stages of embryo development can induce malformations in the offspring. Studies of the effect of heat stress on the placental functions are limited to the ewes, but that on microscopic structure is unknown. In the present study, rats were exposed to 41 or 42 degrees C for 1 h on gestation day (GD) 9. The controls were sham treated. Fetuses and placentas were collected on GD 20. Intrauterine growth retardation (IUGR) and several craniofacial malformations were observed in the fetuses of the heat-treated group. The placentas of the 42 degrees C group were significantly lighter in weight than those of the control. Light microscopy (LM) revealed thickening, hyalinization and occasional lymphocytic infiltration of the decidua basalis. Giant cells were prominent and glycogen cells had degenerated, leaving behind large cysts in the basal (spongy) zone. Best's carmine stain with or without diastase indicated the reduction in number and degeneration of glycogen cells and cyst formation. The labyrinthine zone was relatively thin in comparison to that of the controls. Perivascular fibrosis and paucity of vascularization were other features of the placentas of the hyperthermia group. Electron microscopy (EM) revealed lipid droplet accumulation in the trophoblast, the presence of myelin bodies and an increased production of collagen in the basal zone. Perivascular fibrosis appeared to have contributed to placental barrier thickening. EM also revealed accumulation of glycogen and lipid droplets in the trophoblasts and fibrin secretion into the extracellular space of the labyrinthine zone. These data suggest that placental pathology possibly contributes to fetal growth retardation in maternally heat-stressed rat fetuses.     

A relationship between cord blood and maternal blood lymphocytes and term placenta in the induction of aryl hydrocarbon hydroxylase activity

Correlations between lymphocyte aryl hydrocarbon hydroxylase (AHH) inducibility in cord blood and maternal lymphocytes and placental AHH activity were studied in 15 smokers and 11 non-smokers. Placental AHH activity was extremely low in the non-smokers regardless of the lymphocyte AHH induction ratio, but was elevated to a variable extent in the smokers, in whom it showed a statistically significant correlation (r = 0.75, P < 0.01) with cord blood lymphocyte AHH inducibility. The correlation between maternal lymphocyte AHH inducibility and placental AHH activity was poor (r = 0.04, not significant). These findings suggest that AHH induction in man may be ‘systemically’ regulated and that the genetic background will determine the extent of induction at a given level of exposure to polycyclic aromatic hydrocarbons.     

The effect of modifiers of microsomal enzymes on chemical oncogenesis in cultures of C3H mouse cell lines.

Two cell lines, both derived from the C3H mouse and each having different responses (oncogenic and cytotoxic) to polycyclic aromatic hydrocarbon oncogens, were studied with respect to their drug-metabolizing enzymes. The 10T1/2CL8 cells (a C3H mouse embryo fibroblastic cell line) were much more effective in converting 3-methylcholanthrene (3-MC) to 3-MC water-soluble metabolites, 3-MC phenols, and 3-MC-bound cellular macromolecules than were CVP3SC6 cells (a new line of C3H mouse adult ventral prostate fibroblasts). Basal aryl hydrocarbon hydroxylase activity was higher in 10T1/2CL8 cells than in CVP3SC6 cells, while the reverse was found for epoxide hydrase activity (using 3-methylcholanthrene-11, 12-oxide as substrate. 3-MC or benz(a)anthracene induced epoxide hydrase activity in both cell lines to about the same extent. 3-MC did not induce aryl hydrocarbon hydroxylase activity in CVP3SC6 cells. Aryl hydrocarbon hydroxylase activity was markedly induced in both cell lines by benz(a)anthracene and was slightly induced in 10T1/2CL8 cells by 3-MC. In a chemical oncogenesis cell culture system, transformation of 10T1/2CL8 cells mediated by 3-MC could be increased two- to threefold by treating the cell cultures with: either benz(a)anthracene, styrene oxide, cyclohexene oxide, or 1,2,3,4-tetrahydrona=phthalene-1,2-oxide; or with cyclohexene or 1,2-dihydrona-phthalene, alkene precursors of cyclohexene oxide and 1,2,3,4-tetrahydronaphthalene-1,2-oxide, respectively. When 10T1/2CL8 cells were treated with a combination of benz(a)anthracene and cyclohexene, 3-MC-mediated transformation was increased 7.8-fold. CVP3SC6 cells that were not transformed by 3-MC or other hydrocarbon oncogens were transformed by a combined treatment with benz(a)anthracene, 1,2-dihydronaphthalene, and 3-MC.     

The widely-distributed tumour protein, oncomodulin, is a normal constituent of human and rodent placentas

Oncomodulin, first found in tumours, turns out to be a highly conserved oncodevelopmental protein in human and rodent placentas. The human and rat placental oncomodulins were visualised immunohistochemically. The placental oncomodulins are identical to each other, and to tumour oncomodulin with respect to amino acid composition, chromatographic elution and the pattern of the peptides released by trypsin action.     

Structure-dependent induction of aryl hydrocarbon hydroxylase in human breast cancer cell lines and characterization of the Ah receptor

The structure-dependent induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined in the MCF-7, T47-D, and MDA-MB-231 human breast cancer cell lines. Both the MCF-7 and T47-D cells were responsive to the induction effects of the halogenated aryl hydrocarbons and the structure-induction relationships were comparable to the reported structure-activity (induction, receptor binding, and toxicity) relationships observed in rodents and rodent cells in culture. The induction of ethoxyresorufin O-deethylase in the T47-D cells was the most sensitive aryl hydrocarbon (Ah) receptor-mediated response in both cell lines and this enzyme activity was more inducible than aryl hydrocarbon hydroxylase. In contrast, the three congeners were inactive as monooxygenase enzyme inducers in the MDA-MB-231 cells. Despite the differential Ah responsiveness of the cell lines, incubation of the cells with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin followed by extraction of the nuclei with high salt and velocity sedimentation analysis of the extracts showed that specifically bound nuclear Ah receptor complexes were present in the three cell lines. The sedimentation coefficients (and levels) for the nuclear receptors were 6.6 S (32.1 fmol/mg protein/mg DNA), 6.9 S (61.6 fmol/mg protein/mg DNA), and 7.4 S (38.2 fmol/mg protein/mg DNA) in the T47-D, MCF-7, and MDA-MB-231 cell lines, respectively. Cytosolic receptor was also detected in the MCF-7 and MDA-MB-231 cells. Thus, despite the differences in Ah responsiveness of the T47-D and MDA-MB-231 cells, comparable levels of nuclear receptor were detected in both cell lines. Furthermore, the elution profiles of the nuclear receptors from DNA-Sepharose columns by using a salt gradient were similar and this suggested that defects in the DNA-binding activity of MDA-MB-231 nuclear receptor complexes were not major factors associated with their failure to respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds.