Evaluation of Estrogenic Activities of Hydroxylated Polycyclic Aromatic Hydrocarbons in Cigarette Smoke Condensate

Estrogenic activities of cigarette smoke condensates obtained from the extraction of particulate matters from mainstream and sidestream cigarette smoke with benzene/ethanol were evaluated by using a yeast two-hybrid assay system expressing human estrogen receptor alpha (hERalpha). To identify the constituents of the cigarette smoke condensate which are responsible for the estrogenic activity, the condensate was fractionated into eleven fractions by liquid-liquid extractions. Among these fractions, the neutral fractions of mainstream and sidestream smoke showed the strongest estrogen receptor-mediated activity by the yeast two-hybrid assay. Then the neutral fractions were fractionated by medium-pressure liquid chromatography with silica gel column. In the fractions that showed strong estrogenic activity, 2-hydroxyfluorene (2-OHFle), 2- and 3-hydroxyphenanthrene (2- and 3-OHPhe), 1-hydroxypyrene (1-OHPyr) and n-propyl-p-hydroxybenzoate (n-PHB) were identified by LC- and GC-MS and HPLC with fluorescence detection. 2-OHFle, 2-OHPhe and n-PHB exhibited estrogenic activity, whereas weak activity was observed with 3-OHPhe and 1-OHPyr. Several other hydroxylated polycyclic aromatic hydrocarbons having no activity were also identified. This is a first study to identify estrogenic hydroxylated PAHs in cigarette smoke condensate. The present findings points out the necessity for detailed investigation of exposure to aerosols containing apparently estrogenic compounds.