QUANTIFICATION OF CYP1A1 AND 2B1/2 IN RAT HEPATOCYTES CULTURED IN MICROWELLS BY IMMUNOLOGICAL METHODS

The quantification of P-450 enzymes (CYP proteins) is of great relevance for drug metabolism and toxicity studies. Their measurement in cultured cells requires a sufficient amount of cellular protein to isolate a minimal amount of functional microsomes. The difficulties in obtaining microsomes from a small number of cultured hepatocytes make it necessary to use more sensitive methods. Immunological methods can overcome such drawbacks. However, their use is prevented by the fact that P-450 enzymes are membrane proteins that require the use of detergents for adequate solubilization which, in turn, can interfere with the antigen-antibody recognition. A sensitive immunoassay (competitive indirect ELISA) was developed which allowed quantification of CYP1A1 and CYP2B1/2 in small culture plates with a reduced number of cells. The main advantages of this ELISA were: (1) precise measurement of both proteins over a wide range of antigen concentration (10-10,000 fmol CYP proteins); (2) minimal sample handling allowing reproducible titrations in small number of cells; (3) minimization of interference by detergents; (4) an excellent correlation (r = 0.96-0.98) between the immunologically determined CYP1A1 and CYP2B1/2, and the activities of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD), respectively. This assay allowed a precise monitoring of the expression of CYP proteins in cultured cells. Upon incubation of hepatocytes with 3-methylcholanthrene the levels of immunoreactive CYP1A1 and CYP2B1/2 were paralleled by an increase of the enzymatic activities of EROD and PROD, respectively. In cultured rat hepatocytes treated with phenobarbital, significant EROD activity was detected in the absence of immunoreactive CYP1A1 induction. Analogously, there was a dissimilarity between enzymatic activity and protein levels in ageing cultures: the decrease of PROD activity preceded by several hours that of the immunoreactive CYP2B1/2, which emphasizes the need to determine individual CYPs, in addition to their enzymatic activities.