Kun Chae

Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8 tetrachlorodibenzofuran in chicks. I. Relationship of chemical parameters.

Abstract Five hexachlorobiphenyl (HCB) isomers and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were fed to chicks for 21 days. Liver weights increased, with the smallest increase produced by 2,3,6,2′,3′,6′-HCB and the largest by 2,4,6,2′,4′,6′-HCB. Weight gain was decreased by 2,3,4,2′,3′,4′-, 2,3,6,2′,3′,6′-, and 2,4,5,2′,4′,5′-HCB, while 1μg/kg of TCDF did not affect body weight or liver weight. Pathological changes in the liver were greatest in the 2,4,6,2′,4′,6′-HCB group, moderate in the 2,3,4,2′,3′,4′- and 2,3,6,2′,3′,6′-HCB groups, and mildest in the 3,4,5,3′,4′,5′-, 2,4,5,2′,4′,5′-HCB, and 5μg/kg TCDF groups, with no significant effect for the 1μg/kg TCDF group. Thymic involution and edema were observed with 3,4,5,3′,4′,5′ HCB and TCDF. 3,4,5,3′,4′,5′-HCB and 5μg/kg of TCDF were lethal. HCB retention indices from gas chromatography correlated well with adipose-tissue concentration, with the exception of the 2,4,6,2′,4′,6′-isomer, which had the smallest retention index but relatively high tissue accumulation. Extraction p -values correlated poorly, but the 2,3,6,2′,3′,6′-HCB gave the smallest p -value, whereas the 2,4,6,2′,4′,6′-HCB p -value was next to the highest. The HCB retention indices generally correlated with their overall biological response and were highest in isomers with 3,4-substitution.

Estrogen receptor stereochemistry: ligand binding and hormonal responsiveness

Estrogen stimulation of the uterus produces a spectrum of biochemical responses that are customarily linked together. This report is an overview of a series of studies by our laboratory investigating the role of different ligand structures in eliciting hormonal responses. Diethylstilbestrol (DES) and certain structural analogs, indenestrol A (IA), indenestrol B (IB), and pseudo-DES, were used as probes to segregate various genomic responses previously considered interrelated, most notably the events of specific protein synthesis and DNA synthesis. These compounds have weak uterotrophic activity; however, they interact with high affinity specifically with mouse uterine estrogen receptors (ERs). All of them produce stoichiometrically similar amounts of ER complex in the nucleus. Indenestrol A and IB possess a single chiral carbon atom and exist as a mixture of enantiomers (ENTs). Competitive binding assays of pure ENTs and cytosolic ERs demonstrated a stereochemical chiral preference for the IA isomer but not IB. This preference was also evident from nuclear ER occupancy experiments. Biologic activity of the IA ENTs also demonstrated differences as seen by receptor binding. Ornithine decarboxylase (ODC) activity was stimulated 600% by DES and partially by IA (rac). All of the ODC activity produced by IA (rac) was due to the IA(C3)-S ENT. Uterine DNA synthesis was measured after treatment with the IA compounds. Indenestrol A (rac) increased DNA synthesis to 40% of the level seen with DES. The weak ENTs showed no activity and the active ENTs were weaker than the IA racemic. These compounds should be useful probes for studying the individual responses involved in estrogen-induced uterine growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse Steroid Sulfotransferases: Substrate Specificity and Preliminary X-Ray Crystallographic Analysis

Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones. The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively. In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester. These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol. Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol. The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A.

Toxicity of selected symmetrical hexachlorobiphenyl isomers in the mouse

Abstract Five-week-old male mice were given: 0.3, 1, 3, 10, 30, 100, or 300 ppm of 3,4,5,3′,4′,5′-hexachlorobiphenyl (HCB); 10, 30, 100 or 300 ppm of 2,4,5,2′,4′,5′-HCB, 2,3,6,2′,3′,6′-HCB, or 2,4,6,2′,4′,6′-HCB in feed daily for 28 days. HCB residue levels were determined in adipose tissue and liver. There were marked differences in dose response and severity of pathologic effects among the isomers. 3,4,5,3′,4′,5′-HCB was the most toxic isomer causing mortality, and body and organ weight effects at all dose levels, and was the only isomer which produced excess porphyrin accumulation. It was also the most concentrated isomer in fat and liver. 3,4,5,3′,4′,5′-HCB caused subcutaneous edema, enlargement of liver with accentuated hepatic lobular markings, fatty infiltration, hepatocellular swelling and necrosis, and atrophy of the thymus. 2,4,6,2′,4′,6′-HCB and 2,4,5,2′,4′,5′-HCB caused the same lesions but to a lesser degree. Slight cardiomyopathy was observed with 2,4,6,2′,4′,6′-HCB. The decreasing order of overall toxicity was 3,4,5-HCB >> 2,4,6-HCB > 2,4,5-HCB > 2,3,6-HCB.

Production and characterization of antisera specific for chlorinated biphenyl species: initiation of a radioimmunoassay for Aroclors.

Abstract Antisera to 4-amino-4′-monochlorobiphenyl, 2-amino-4,5,3′,4′-, and 3-amino-2,6,2′,6′-tetrachlorobiphenyl have been produced in rabbits by conjugating these compounds to proteins using the mixed anhydride method. Extensive characterization studies indicated the antisera were fairly specific to their nonaminated biphenyl analogs. Values for these isomers in Aroclors by radioimmunoassay and gas chromatography correlated well for several of the antisera. Suggestive evidence is presented indicating the feasibility of employing radioimmunoassays for determining the Aroclor product number and concentration in environmental samples. A feature of the assay is the use of nonionic detergents to solubilize the extremely hydrophobic chlorinated biphenyls in a manner permitting their binding to antibodies.

Multiple estrogen binding sites in the uterus : stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.

Separation of indenestrol A and B isomers and enantiomers by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) methods have been developed for the separation of substituted indenestrol A and B isomers on different columns. The isomers were separated by normal-phase liquid chromatography with a silica gel column. Enantiomers of these compounds were separated by chiral HPLC and the most successful separations were achieved with a Chiralcel OJ column.

A new synthesis of tetrachlorofluorodibenzo-p-dioxin.

The 1,2,3,4-tetrachloro-7-fluorodibenzo-p-dioxin has been synthesized via condensation of 4-fluorocatechol and pentachloronitrobenzene. This compound could be used as an internal standard for the analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin by chromatographic methods.