Conjugates of estrogenic chemicals, endogenous as well
as xenobiotic, are mainly excreted via bile into the intestine.
Therefore, measurement of estrogenic activity in bile
yields useful information about an organism's internal
exposure to (xeno-)estrogens. Although previous studies
in The Netherlands have reported estrogenic activity in male
fish bile, the contribution of natural hormones and
xenobiotic substances to this activity is unknown. To
identify compounds responsible for estrogenic activity in
fish bile, we developed a bioassay-directed fractionation
method for estrogenic chemicals. In this approach, the in
vitro reporter gene assay ER-CALUX (Estrogen Responsive
Chemical Activated Luciferase Gene Expression) was
used to assess estrogenic activity in deconjugated bile
samples and to direct RP-HPLC fractionation and chemical
analysis (by GC−MS) of estrogenic compounds. The
method was applied to bile from male breams (Abramis
brama) collected at three locations in The Netherlands. At
one of these locations, the River Dommel, extremely
high levels of plasma vitellogenin and a high incidence of
intersex gonads in these male breams have previously
been observed, indicating the exposure to estrogens. In
this study, the natural hormones 17β-estradiol, estrone, and
estriol accounted for the majority of estrogenic activity
in male bream bile. At the River Dommel, the synthetic
contraceptive pill component ethynylestradiol was found in
effective concentrations as well. The detected natural
and synthetic hormones may be responsible for the estrogenic
effects observed in wild bream from this location.
Furthermore, a large number of xenobiotic chemicals was
detected at relatively high levels in bile, including triclosan,
chloroxylenol, and clorophene. Although chloroxylenol was
shown for the first time to be weakly estrogenic, these
compounds did not contribute significantly to the estrogenic
activity observed.