The proposed study will examine in rodents and man the metabolism and toxicity of methoxychlor and chlorotrianisene (TACE), both present in pesticidal preparations of methoxychlor. The importance of studying these compounds is: a) they are potentially toxic environmental estrogens, b) they represent structural analogs of several classes of pesticidal and nonpesticidal aromatic xenobiotics and c) they provide useful models for investigating mechanisms controlling alternate routes of metabolism. The knowledge of TACE metabolism is particularly important because TACE is a therapeutic estrogen and is a structural analog of triphenylethylene antiestrogens and tumor-imaging agents. The metabolism of methoxychlor and TACE by two pathways, leading to estrogenic products and to reactive intermediates (RIs), will be investigated. The covalent binding of methoxychlor and TACE to proteins is dramatically stimulated by treatment of rats with phenobarbital and methylcholanthrene, respectively, indicating that different complements of cytochrome P-450s catalyze the activation of the two compounds. It is planned to identify the cytochrome P-450s catalyzing the two pathways and determine how sexual maturation affects the level of these P-450s and the metabolism of these compounds. Antibodies to isoforms of P-450 will be used to define the P-450s catalyzing the formation of metabolites and RIs. Catalytic activity of reconstituted cytochrome P-450s in the two pathways will be examined. The metabolites will be identified by HPLC and mass spectrometry and their estrogenic activity determined. To establish whether the in vitro activation of the estrogen receptor increases gene expression, a integrated assay will be developed. This method will identify proestrogens and estrogens and determine whether the apparent decrease of estrogen receptor by TACE is due to receptor inactivation or by masking of the estrogen binding site. Also, proestrogenic activity of other pollutants, e.g. PCBs, will be determined by that procedure. It is planned to identify the RIs of methoxychlor (M*) and of TACE (T*) by formation of adducts with N-acetylcysteine (NAC) and with small peptides. The amino acid(s) in the P-450 or albumin bound to M* and T* and their location in the polypeptide will be identified by proteolysis and isolation of radiolabeled M*-and T*-linked peptides. The cause for the high covalent binding of T* to albumin and whether albumin could trap T* in vivo will be explored. Cellular toxicity of M* and T* and of their NAC and GSH adducts will be examined in hepatocytes.
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