Changes in estrogen receptor, DNA ploidy, and estrogen metabolism in rat hepatocytes during a two-stage model for hepatocarcinogenesis using 17 alpha-ethinylestradiol as the promoting agent.

17 alpha-Ethylestradiol (EE2) was administered chronically to diethylnitrosamine (DEN)-initiated (200/mg/kg, i.p.) adult ovariectomized Sprague-Dawley rats, by means of Silastic implants at an estimated dose of 90 micrograms/kg/day. Isolated hepatocytes from DEN/EE2-treated animals exhibited a 2- to 3-fold increase in nuclear estrogen receptor (ER) levels throughout the promotion period. Furthermore, approximately 30-40% of the receptor was occupied when quantified by an exchange assay. For all groups the ER had a sedimentation coefficient of approximately 8S for unoccupied ER and a binding affinity for 17 beta-estradiol of 0.25 nM. An ER of lower affinity for estradiol was present in animals initiated with DEN and/or promoted with EE2. The increase in hepatocyte ER was associated with a 5.2-fold increase in gamma-glutamyl transpeptidase and 2.5-fold decrease in glucose-6-phosphatase activity at 20 weeks. EE2 treatment caused a 50% increase in the maximal binding capacity (Bmax) of hepatic epidermal growth factor receptors, but the equilibrium binding constant (Kd) did not change. Modulation of mitotic activity of hepatocyte subpopulations by EE2 treatment was indicated by an increase in the proportion of diploid hepatocytes and an increase in the number of hepatocytes undergoing DNA synthesis. In general, effects on ER, epidermal growth factor receptor, gamma-glutamyl transpeptidase and glucose-6-phosphatase were greater in DEN/EE2-treated animals than in rats receiving only EE2. Modification of receptor pathways associated with hepatocyte growth control, ER and epidermal growth factor receptor, may be contributing factors in the clonal expansion of preneoplastic cells during EE2 promotion of hepatocarcinogenesis.

Relationships among benzo(a)pyrene metabolism, benzo(a)pyrene-diol-epoxide:DNA adduct formation, and sister chromatid exchanges in human lymphocytes from smokers and nonsmokers.

In the present study, benzo(a)pyrene (BP) metabolism, DNA adduct formation, ethoxyresorufin-O-deethylase activity, and sister chromatid exchange induction by BP were compared in human lymphocytes prepared from whole blood of smokers and nonsmokers following an in vitro incubation with BP. There was an approximate 7- to 10-fold variation in all parameters measured. To determine the source of this variation, participants were resampled, the assays were repeated, and all the data were analyzed to assess (a) smoking-related effects, (b) differences in multiple samples from the same individual, and (c) intraindividual, experimental, and interindividual variation. No smoking-related effects were observed except for baseline sister chromatid exchange frequency. The variation observed for BP-related DNA adducts and ethoxyresorufin-O-deethylase activity was primarily due to interindividual variation. For example, in vitro formation of DNA adducts did not change when samples were obtained at different times from the same individual and were not influenced significantly by culture conditions. No significant correlation existed between DNA adduct formation and BP metabolism [correlation coefficient (r) = 0.27] for either the total population or when segregated based on smoking status. Furthermore, no correlation was seen between DNA adducts and sister chromatid exchange induction by BP. Our studies have compared a number of commonly used lymphocyte markers and conclude that it is difficult to predict changes in one marker based on changes in another. However, in vitro formation, of PB-derived DNA adducts is consistent over time for individuals.

Dose-response relationships for chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a rat tumor promotion model: quantification and immunolocalization of CYP1A1 and CYP1A2 in the liver.

The mechanisms responsible for the braod spectrum of effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are not entirely clear but seem to involve an initial interaction with the Ah receptor. A major uncertainty in risk assessment for TCDD is the lack of adequate dose-response relationships following chronic exposure to TCDD. Induction of cytochrome P-450 enzymes (CYP1A1 and CYP1A2) is one of the most sensitive responses to TCDD and its structural analogues. We have used a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats to evaluate dose-response relationships for induction of CYP1A1 and CYP1A2 in diethylnitrosamine-initiated as well as in noninitiated rats. After initiation with a single dose of diethylnitrosamine, TCDD was administered biweekly by p.o. gavage at doses equivalent to 3.5, 10.7, 35.7, and 125 ng/kg/day for 30 weeks. CYP1A1 and CYP1A2 concentrations were quantified in hepatic microsomes by radioimmunoassay and localized in hepatic tissue slices by immunohistochemical techniques. Radioimmunoassay data revealed a maximum induction of 200-fold for CYP1A1 and 10-fold for CYP1A2 and there were no statistically significant differences between initiated and noninitiated rats. Induction at the lowest dose (3.5 ng/kg/day) was 20-fold for CYP1A1 and 3-fold for CYP1A2. Mathematical analysis indicates that the best fit of the induction data are inconsistent with a threshold for this response. There was a linear relationship between administered dose and TCDD liver concentration over the entire dose range of the study. This indicates that induction of CYP1A2 does not significantly alter the distribution of TCDD in our chronic dosing regimen. Immunolocalization of CYP1A1 and CYP1A2 revealed the same localization and induction pattern for both isozymes in the cytoplasm of hepatocytes. However, the hepatic distribution pattern was not uniform with the most intense staining observed around central veins. These studies help to clarify dose-response relationships for dioxin-mediated effects and demonstrate different sensitivity of hepatocytes to the effects of TCDD.

Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat.

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.

Ingestion of soil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters hepatic enzyme activities in rats.

Female rats were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in either corn oil or contaminated soil from the Minker site in Missouri. Eight doses ranging from 0.015 to 5 micrograms TCDD/kg were used in the corn oil group; the range was 0.015 to 5.5 micrograms TCDD/kg in the TCDD-contaminated soil group. Rats in a third group were given equal amounts of soil uncontaminated with TCDD. No acute toxicity or effects on body weight gain were observed at these doses. In general, equivalent doses of TCDD in corn oil or TCDD in soil produced similar increases in hepatic aryl hydrocarbon hydroxylase activity (AHH) and UDP glucuronyltransferase activity although effects were slightly greater in the TCDD-corn oil groups. In the corn oil groups, the induction of AHH ranged from about 30-fold at the highest dose to twofold at the lowest dose studied. TCDD also caused an increase in cytochrome P-450 concentration and a shift in spectral peak from 450 to 448 nm. There was no effect of TCDD on ethylmorphine N-demethylase, consistent with previous reports. Liver concentrations of TCDD (mean +/- SD) in the 5-micrograms/kg groups were 40.8 +/- 6.3 ppb in the TCDD corn oil group and 20.3 +/- 12.9 ppb in the TCDD-contaminated soil group. Our results suggest that the bioavailability of TCDD in soil in rats is approximately 50%. Therefore, ingestional exposure to TCDD-contaminated soil may constitute a significant health hazard in view of its extremely high toxicity and relatively high bioavailability.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated decreases in dexamethasone binding to rat hepatic cytosolic glucocorticoid receptor.

An investigation of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the liver cytosolic glucocorticoid receptor (GRc) in intact and adrenalectomized (ADX) rats, using equilibrium binding analysis, sucrose gradient sedimentation, and affinity labeling experiments, clearly demonstrated that TCDD significantly reduced the binding capacity (Bmax) of the hepatic GRc but did not alter the apparent equilibrium dissociation constant (Kd). This effect was maximal after 24 hr and was still present 22 days after treatment. Western blot analysis revealed that TCDD treatment did not cause a comparable decrease in the levels of immunodetectable receptor protein, which suggests that the steroid-binding properties of the hepatic GRc are altered, rather than the absolute concentration of receptor protein. Studies of TCDD effects on the uptake of GRc by nuclei indicated that TCDD treatment did not alter the ability of the steroid-GRc complex to be taken up by nuclei; however, TCDD treatment did increase the total capacity of liver nuclei to bind steroid-GRc complexes. TCDD dose-response studies that compared the hepatic GRc steroid binding of ADX and intact rats indicated that adrenalectomy markedly enhanced the response to TCDD. Significant effects on the GRc binding in ADX animals were induced at TCDD doses that were 10,000 times lower than those required for a response in intact rats. Analysis of two other biochemical markers demonstrated that ADX rats were 10-fold more sensitive to the induction of microsomal benzo[a]pyrene hydroxylase but of similar sensitivity to reduction of epidermal growth factor receptor binding, when compared with the responses of intact animals. These data indicate that adrenal status may be important in modulating the responses of the animals to TCDD and that the alteration of the hepatic GRc pathway may have a role in some of the actions of TCDD.

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.