Development of In Vitro Methodologies for the Investigation of Rat Liver Cytochrome P450 Gene Activation.

Sensitive, high-throughput, in vitro assays for the detection of potential rat CYP4A1 and rat CYP1A1 inducers have been developed using reporter gene technology. Large numbers of compounds can be rapidly screened with these assay systems, using only small quantities of test compound. A 102bp peroxisome-proliferator responsive element, isolated from the 5’-flanking region of CYP4A1 by Aldridge et al (1995), was shown to be responsive to several peroxisome proliferators and 9-cis retinoic acid in transiently transfected human hepatoma HepG2 cells. Co-transfection of an expression plasmid encoding mPPARa was found to be required for this response. Initial transfections were carried out using a chloramphenicol acetyl transferase (CAT) reporter gene construct containing the rabbit B-globin promoter upstream of the 102bp CYP4A1 element. In order to maximise sample throughput, subsequent transfections were performed using a secretory alkaline phosphatase (SEAP) reporter gene construct, which allowed reporter gene expression to be measured in small samples of cell culture media. The 102bp CYP4A1 element has been cloned into the SEAP reporter gene construct upstream of a SV40 early promoter. The response of the CYP4A1 element was not altered by the change of promoter and reporter gene. Upon exposure to a range of compounds, that included known peroxisome proliferators, PPAR activators and a number of cytochrome P450 inducers, the assay was able to differentiate between inducing compounds and negative controls. The first 1171bp of the rat CYP1A1 promoter was cloned upstream of the SV40 early promoter into a SEAP reporter gene construct. SEAP reporter gene expression was induced specifically by B-naphthoflavone in HepG2 cells that had been transiently transfected with the CYP1A1 reporter gene construct. Unidentified factors present in foetal calf serum were observed to reduce B-naphthoflavone induced reporter gene transcription. PD98059, a specific inhibitor of mitogen- activated protein kinase kinase (MEK) (Dudley et al. , 1995; Alessi et al. , 1995) was also found to modulate CYP1A1-reporter gene transcription. In conclusion, the CYP4A1 and CYP1A1 reporter gene assays that were developed have been found to be specifically responsive to their respective inducing compounds in a reproducible manner. These systems could potentially be used in the toxicological screening of novel compounds in the development of therapeutic drugs