Gebruiker:Conget/Kladblok
CYP1A1 bewerken
CYP1A1 is verantwoordelijk voor het omzetten van verschillende polycyclisch aromatisch koolwaterstof in electrofiel molecuul die covalente verbinding met DNA
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 west-ern 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 demon-strated 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 ~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,
~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-dimen-sional 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 TK1/–
cell line
expressing solely human CYP1A1 and differing from the
profile obtained using a polyclonal antibody directed
against CYP2E1 and cells expressing CYP2E1. Further-*Abbreviations: CYP, cytochrome P450s; EROD2, ethoxyresorufin O-deethylaase; IEF, isoelectric focusing; NEPHGE, non-equilibrium pH gradient
electrophoresis; PAHs, polycyclic aromatic hydrocarbons; PCDDs, polychlori-nated dibenzo-p-dioxins; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
©Oxford University Press 1361
more, mAb 1-12-3 recognized only one protein of identical
mobility on the two-dimensional blots from human liver
microsomes and AHH-1 TK1/–
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