Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion ofnumerous polycyclic aromatic hydrocarbons into electrophilic speciescapable of binding covalently to DNA and has therefore been postulated tobe involved in the initiation of carcinogenesis. The expression of CYP1A1protein appears not to be constitutive, but is readily inducible by arylhydrocarbon (Ah) receptor ligands in a majority of tissues of experimentalanimals, especially the liver. To date, there is conflicting evidence forthe expression or inducibility of CYP1A1 protein in human liver. In thispresent study, we report the detection of CYP1A1 in all 20 human livermicrosomal samples tested by standard western immunoblotting withchemiluminescent detection using a specific monoclonal antibody (mAb1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3has been shown previously to specifically recognize CYP1A1 in mammals. Thissystem consistently demonstrated a detection sensitivity as low as0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levelswere quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mgmicrosomal protein. Additionally, the inducibility of CYP1A1 protein wasdemonstrated by incubating precision-cut human liver slices in dynamicorgan culture for up to 96 h in the presence of2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3was tested using several purified human and rat cytochrome P450s to ensurethat the protein being detected was CYP1A1. mAb 1-12-3 did not cross-reactwith human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognizeCYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 withhuman CYP2E1, approximately 75-fold less compared with CYP1A1. In order toconfirm CYP1A1 as the immunoreactive protein detected in human liver,microsomal samples were subjected to two-dimensional electrophoresisinvolving isoelectric focusing followed by SDS-PAGE and immunoblotting.Utilizing mAb 1-12-3, the human liver microsomal samples displayed animmunoblotting profile matching that obtained from a microsomal preparationfrom a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differingfrom the profile obtained using a polyclonal antibody directed againstCYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1- 12-3 recognizedonly one protein of identical mobility on the two- dimensional blots fromhuman liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, whiledisplaying no reaction to cells expressing only CYP2E1. In conclusion,CYP1A1 appears to be expressed in human liver at low levels and isinducible upon exposure to TCDD.