Investigating the mechanisms of regulation of drug disposition is important in understanding pharmacokinetic (PK) variability and rationalising drug-drug interactions. Ultimately, drug absorption in the intestine and drug elimination from the liver are the major determinants of PK. Key proteins involved in these processes are cytochrome P450 enzymes (CYPs) and ABC-transporters. These proteins are transcriptionally regulated by a complex network of nuclear receptor (NR) type transcription factors and many of these NRs are activated by endogenous or exogenous chemicals. The study of these mechanisms in rodent . models has been complicated by the reported species differences in activation of NRs. The aim of this thesis was to assess the spectrum of induction of CYPs, transporters and NRs in response to paradigm NR-activators in a battery of human and rat in-vitro systems. The aim of chapter two was to validate robust methodology for quantitatively assessing mRNA and protein expression of CYP2B6, CYP3A4, ABCB1, ABCC1, ABCC2, CAR, FXR, PXR and their rat homologues. In addition, since there are many conflicting reports in the literature, the impact of using dexamethasone (OEX) as a media supplement was assessed. Previous studies have illustrated species differences with pregnenalone-16a-carbonitrile (PCN) activating rodent but not human PXR, and rifampicin (RIF) activating human but not rodent PXR. The data presented in this chapter illustrate that OEX induces human PXR, CYP3A4, ABCB1 and rat orthologues. Furthermore, in the absence of OEX, RIF and PCN increased expression of human CYP3A4, ABCB1 and rat orthologues to a similar extent. These data suggest that there is a maximal induction and the addition of OEX effectively increases the baseline. However, since the maximum is fixed a reduction in the observed effect is evident. Therefore, although there are species differences in activation of PXR, the impact on target gene expression appears to be similar. For this reason, OEX was not incorporated into the culture media in SUbsequent chapters. The aim of chapter three was to investigate the impact of Phenobarbital (PB; CAR activator), PCN (rodent PXR activator), chenodeoxycholic acid (COCA; FXR activator), RIF (human PXR activator) and 9-cis-retinoic acid (9-cisRA; RXR activator) on expression of CYPs, transporters and NRs in human (Caco-2) and rat (iec-6) intestinal cell lines. Significant induction of transcripts in both human and rat intestinal cell lines were observed. However, some differences were seen in the induction profiles in Caco-2 versus lec-6 cells. In general, logarithmic correlations between the changes in mRNA and protein were also observed. Lower changes in mRNA elicited linear increases in protein but there appeared to be a threshold beyond which no additional increase in protein was observed. In chapter four, these experiments were repeated in the human (HepG2) and rat (H411e) hepatic cell lines. Again, induction of transcripts in both human and rat hepatic cell lines was observed. In hepatic and intestinal cell lines concentration-dependent responses were observed in most cases. However, i~ some cases, bell shaped concentration-response profiles were observed. In most cases these were related to toxicity but in some cases sub-maximal induction was observed at concentrations that were non-toxic. These data indicate that using a single concentration may result in gross underestimation of induction and may also explain some of the discrepancies in the literature. Logarithmic correlations between the changes in mRNA and protein in HepG2 and H411 e cells were also observed. Similar to intestinal cell lines, lower changes in mRNA elicited linear increases in protein. This is most likely due to the time which analysis was conducted; mRNA induction being more rapid than that of protein. The aim of chapter five was to investigate these observations in human (cryopreserved from two donors) and rat (fresh) hepatocYtes. In these studies, a sub·toxic concentration (1IJM) causing maximal induction of most transcripts in cell lines was chosen and a time course was conducted to circumvent problems associated with a decrease in viability over time. Protein was not investigated because insufficient material was observed after incubation with typical NR activators. Generally, maximal induction was observed at 6 hours in human and at 4 hours in rat hepatocytes, and in most instances time-dependent induction was observed. Interestingly, there was variation in the observed induction between the human donors. These data were in broad agreement with data generated in cell lines, and where overlap existed were also in agreement with previous studies in the literature. The aim of chapter six was to investigate activation of a range of human NRsby the activators utilised in previous chapters. This was particularly important given that similar induction of target genes was observed, even where species differences have been documented. In order to probe the mechanisms that underpin these observations, mammalian 1 hybrid ligand binding assays were employed for PXR, VOR, PPARy, LXRa, FXRa and RXRa in Caco-2 and CHO-K1 cells. All NRs showed some degree of promiscuity, with LXRa appearing the most promiscuous. PPARy was activated by PCN, which is particularly interesting in light of previously reported PPAR response elements within the PXR proximal promoter. COCA significantly activated FXRa and LXRa in CHO-K1 cells. In addition, LXRa, FXRa and VOR were all activated by COCA in Caco-2 cells. In summary, variation in induction was observed between model systems for both tissue and species. However, in the absence of OEX, both rat and human PXR and PXR target genes were activated by PCN and RIF to a similar extent. In addition, a number of novel observations were made. For example upregulation of NRs by their purported activators was observed, which raises the possibility of self regulation. Further studies, which utilise reporter constructs from the target gene promoters coupled with NR expression vectors, are now required to investigate which NRs are involved. Work has now also begun to utilise targeted knockdown of NRs (with SiRNA), in order to determine the relative contribution of individual NRs in the reported induction.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:502298 |
Date | January 2008 |
Creators | Martin, Philip John |
Publisher | University of Liverpool |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
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