Carcinogenesis, Vol 19, 1361-1368, Copyright © 1998 by Oxford University Press
AT Drahushuk, BP McGarrigle, KE Larsen, JJ Stegeman and JR Olson
Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of
numerous polycyclic aromatic hydrocarbons into electrophilic species
capable of binding covalently to DNA and has therefore been postulated to
be involved in the initiation of carcinogenesis. The expression of CYP1A1
protein appears not to be constitutive, but is readily inducible by aryl
hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental
animals, especially the liver. To date, there is conflicting evidence for
the expression or inducibility of CYP1A1 protein in human liver. In this
present study, we report the detection of CYP1A1 in all 20 human liver
microsomal samples tested by standard western immunoblotting with
chemiluminescent detection using a specific monoclonal antibody (mAb
1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3
has been shown previously to specifically recognize CYP1A1 in mammals. This
system consistently demonstrated a detection sensitivity as low as
0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels
were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg
microsomal protein. Additionally, the inducibility of CYP1A1 protein was
demonstrated by incubating precision-cut human liver slices in dynamic
organ culture for up to 96 h in the presence of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3
was tested using several purified human and rat cytochrome P450s to ensure
that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react
with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize
CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with
human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to
confirm CYP1A1 as the immunoreactive protein detected in human liver,
microsomal samples were subjected to two-dimensional electrophoresis
involving isoelectric focusing followed by SDS-PAGE and immunoblotting.
Utilizing mAb 1-12-3, the human liver microsomal samples displayed an
immunoblotting profile matching that obtained from a microsomal preparation
from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing
from the profile obtained using a polyclonal antibody directed against
CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1- 12-3 recognized
only one protein of identical mobility on the two- dimensional blots from
human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while
displaying no reaction to cells expressing only CYP2E1. In conclusion,
CYP1A1 appears to be expressed in human liver at low levels and is
inducible upon exposure to TCDD.
ARTICLES
Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture
Department of Pharmacology and Toxicology, SUNY, Buffalo, NY 14214, USA.
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