Increased endocrine activity of xenobiotic chemicals as mediated by metabolic activation.

The US EPA is faced with long lists of chemicals that require hazard assessment. This research is part of a larger effort to develop in vitro assays and QSARs applicable to untested chemicals on US EPA inventories through study of estrogen receptor (ER) binding and estrogen mediated gene expression in fish. The current effort investigates metabolic activation of chemicals resulting in increased estrogenicity. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10 and 10 M PLIN. Phenolphthalein (PLEIN), metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. PLEIN binds rtER with a relative binding affinity (RBA) to 17β-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg mRNA at 10 , 10 , and 10 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4'-methylenedianiline (MDA) was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10 , 10 , and 10 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, or azoxy-MDA were not observed. However, acetylated-MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays.