2-Phenylphenanthridinone and Related Compounds: Aryl Hydrocarbon Receptor Agonists and Suicide Inactivators of P4501A1

Abstract

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 μM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a >80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.