Gebruiker:Conget/Kladblok

CYP1A1

CYP1A1 is verantwoordelijk voor het omzetten van verschillende polycyclisch aromatisch koolwaterstof in electrofiel molecuul die covalente verbinding met DNA

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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