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Title: An investigation of host cell effects on the xenobiotic induction of cytochrome P450 3A
Author: Swales, Karen Elizabeth
ISNI:       0000 0001 3493 0546
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2002
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Species differences exist in the induction response of CYP3A genes to xenobiotics, and these are proposed to be due, in part, to host cell differences. These host cell effects were investigated functionally by trans-species transfections of alkaline phosphatase reporter genes containing ~1.5 kb of the CYP3A23 or CYP3A4 5' flanking regions into human HepG2 and rat FaO and H4IIEC3 hepatoma cells. HepG2 and FaO cells were demonstrated to support induction of the activity of both CYP3A constructs by 50 muM dexamethasone but H4IIEC3 cells could not. The receptor mRNA complement (CAR, GRa, HNF4a, PXR and RXRa) of the rat and human cell lines were characterised in comparison to rat and human liver using semi-quantitative RT-PCR to identify any differences. Principal component analysis (PCA) of the receptor mRNA levels was scattered indicating that rat liver does not resemble human liver and that hepatoma cell lines do not resemble their liver counterparts in terms of individual receptor mRNA levels. Increasing knowledge of nuclear receptor interactions with response elements led to consideration that response to xenobiotics may be due to relative receptor abundance in the cells, as opposed to individual receptor expression levels. PCA on comparison ratios showed clustering of human and HepG2 receptor ratios, supporting the observation that CYP3A reporter gene induction responses in HepG2 cells mimic those in vivo. From this data we hypothesise that relative receptor expression levels are key to determining CYP3A responsiveness to xenobiotics. However this hypothesis could not be further examined at the protein level, as Western blotting experiments were equivocal. The latter hypothesis was further examined through the use of TaqMan, expanding the receptor cohort to include COUPTFI and RXRs and gamma, plus CYP3A mRNA expression levels. In addition the effects of xenobiotics, dexamethasone, rifampicin, PCN and phenobarbital at a range of concentrations, were tested. The hepatoma cell lines did not have the same endogenous CYP3A mRNA expression or induction profiles as liver. The TaqMan results suggested that in human CYP3A regulation, the relative abundance of PXR heterodimerisation partners or accessory factors were more important than the level of PXR, whereas in rats, PXR was dominant in determining induction of CYP3A, indicating a further species difference. In conclusion, host cell effects on CYP3A regulation are dependent on receptor abundance and interactions, as well as differential receptor activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available