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P. U. Stephensen et al.
mechanism for induction of CYP1A1 protein and activity.
Gillner et al. [29] previously demonstrated that the Ah
receptor binding affinity of ASG, measured as IC50 values of
the inhibition of specific [3H]TCDD binding in rat liver
cytosol, was more than 400 times lower than the corre-
sponding value for ICZ and in the same range as for I3C.
Since ASG has a very low affinity for the Ah receptor, it is
likely that ASG is transformed into a more potent ligand
and CYP1A1 inducer. ICZ, or a compound with an iden-
tical retention time, was detected in the ASG media at 24
and 72 hr. The possibility that other inducers are formed
from ASG cannot be excluded. We therefore suggest that
the induction of CYP1A1 protein by ASG is caused by
transformation to the potent inducer ICZ and/or other
potent products. Furthermore, it cannot be excluded from
the present experiments that ASG may interact with other
regulatory factors as well.
Preobrazhenskaya et al. [30] have previously shown that
ASG incubated with gastric juice for 5 hr at 37° give rise to
a very complex mixture containing ICZ, and that the
concentration of ICZ in gastric juice was approximately 20
times higher after incubation under these conditions with
ASG than after incubation with I3C. Our experiments
indicate that ICZ may also be formed from ASG under
physiological conditions at neutral pH. In contrast, ASG
dissolved in PBS caused an EROD induction with the same
efficiency at 8 hr as found for the positive control at 24 hr.
This induction pattern corresponds to the ICZ-induced
EROD activity shown by Chen et al. [19]. One can
therefore speculate that ASG is transformed to ICZ in the
aqueous buffer before addition to the medium. However,
ICZ was not detected using HPLC in the ASG–PBS
solution when incubated at 37° for up to 24 hr, whereas
another fluorescent, more polar compound was detected.
The identity of this compound has not been established but
the ASG oligomers do not exhibit fluorescence, and the
unidentified compound is likely to be a polycyclic polar
product. The early response in EROD activity observed
when using PBS as ASG solvent is therefore not caused by
the presence of ICZ in the initial PBS solution. Further-
more, the low level of ICZ detected in the media cannot by
itself account for the entire CYP1A1 induction observed.
Degradation of ASG is likely to result in the release of AA,
and the effect of AA on induction of CYP enzymes is not
well characterised. It cannot be excluded that AA in vitro
may enhance the inducing potential of ASG/ICZ. The
observed EROD kinetic is a combination of a fast induction
by ICZ or an ICZ-like compound, ASG, and other poten-
tial inducers formed from ASG and a slow disappearance of
the inhibitor ASG from the medium over time. The
half-life of ICZ is about 10 min [19], and the effect
presented here is likely caused by a continuous formation of
ICZ from an excess amount of ASG. Induction of CAT is
observed above 10 M ASG, but because of a significant
ASG-mediated inhibition in whole cells above 1 M, a
significant induction of EROD is only observed above 50
M ASG.
In conclusion, our results show that exposure to ASG
induces CYP1A1 expression in Hepa 1c1c7 cells. ASG
treatment induces Ah receptor-driven CAT activity and
therefore presumably induces CYP1A1 via activation of the
Ah receptor. According to the literature, ASG acts as a
weak Ah receptor agonist compared to ICZ or an ICZ-like
substance, but the induction may in part be explained by
the transformation of ASG to the more potent inducer ICZ.
In addition to inducing EROD activity, ASG also inhibits
this activity. As ASG is the main transformation product
from glucobrassicin in the presence of AA, the observation
that ICZ, or an ICZ-like substance, may also be formed at
neutral pH is of great interest.
We thank Charlene Schaldach (University of California, Berkeley,
CA) for undertaking the CAT assay, Dr. Simon Bolvig (University of
California, Berkeley, CA) for the NMR analysis, and Dr. Niels
Agerbirk (The Royal Veterinary and Agricultural University, Den-
mark) for helpful discussions on ASG synthesis. Valuable comments on
the manuscript from Dr. Hanne-Cathrine Bisgaard (Roskilde Univer-
sity) are appreciated. This work was supported by the Danish Cancer
Society (P. U. S.), the Wedell-Wedellsborg Foundation (P. U. S.,
C. B., O. V.), The Foundation for Disease Treatment without use of
Animal Experiments (O. V.), the US Department of Defense, Army
Breast Cancer Research Program Grant DAMD17-96-1-6149
(L. F. B.) and NIH Grant CA 69056 (L. F. B.).
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