1. The induction of CYP3A enzymes was investigated using a range of structurally unrelated drugs using in vivo and in vitro models. Hepatic microsomal testosterone 6(3-hydroxylation, anti-CYP3A immunoblot analysis, and molecular biology approaches were utilised in the investigation. 2. Using the rat as an in vivo model, potent induction of CYP3A enzymes was observed after administration of the synthetic glucocorticoid dexamethasone (at 150mg.kg.day for 4 days) and pregnenolone 16?-carbonitrile (at 150mg.kg-1.day-1 for four days). However, no induction was observed after administration of rifampicin (at 50 g.kg-1.day-1 for 4 days, a dose which causes potent induction in the rabbit). 3. Investigations into the effects of drug exposure on testosterone 6?- hydroxylation in cultured female rat hepatocytes revealed a positive in vivo/in vitro correlation. Cultured cells were treated with the same drugs (at 50 M concentration) for 72hrs. Dexamethasone was shown to be more potent than PCN, and rifampicin again had no effect. Dexamethasone-mediated induction of testosterone 6?- hydroxylation was dose-dependent and was shown to be maximal after 72hrs exposure. 4. The presence of the differentiating agent dimethylsulphoxide at 2% (v/v) in the cultiure medium enhanced CYP3A induction by the synthetic steroids by approximately 100% (p 0.05). 5. The potent glucococorticoid antagonist RU 486 induced testosterone 60- hydroxylation 5-fold when administered at 50mg.kg-1.day-1 for 4 days. Induction of the CYP3A protein was confirmed by immunoblot analysis of liver microsomes. Administration of RU 486 at 50mg.kg-1.day-1 did not antagonise the induction of testosterone 6?-hydroxylatiomn by dexamethasone at 150mg.kg-1.day-1. 6. Dexamethasone (0.1 to 10 M) -mediated induction of testosterone 6(3- hydroxylation in cultured rat hepatocytes was attenuated in the presence of RU 486. It is not known whether this was due to effects on CYP3 A gene expression or inhibition of enzyme mediated activity at the active site of the enzyme. 7. The lipid lowering drug SK F 98016 (150mg.kg-1.day-1) induced testosterone 6?-hydroxylation 10-fold when administered at 150mg.kg-1.day-1 for 4 days. This was confirmed by immunoblot analysis. Co-administration of RU 486 with SK F 98016 attenuated induction of CYP3A-mediated enzyme activity. The mechanism of induction of the CYP3A genes by SK F 98016 may therefore involve 'steroidal' compounds, the action of which is antagonised by RU 486. The dexamethasone- mediated increase in spectrally determined cytochrome P450 levels was also attenuated after co-administration with RU 486. As CYP3A induction was not affected by co-administration of dexamethasone with the anti-glucocorticoid RU 486, this result suggests that the glucocorticoid receptor may be involved in the induction of other P450 genes. 8. Treatment of rat hepatocytes with SK F 98016 (50 M) for 72 hours did not result in an increase in testosterone 6?-hydroxylation. In fact testosterone 6?-, 16?- and 17-oxidation activities were reduced to 50% of the activities measured in untreated hepatocytes. This pointed to some P450 inhibitory potential of SK F 98016. Investigation of the inhibitory potential of SK F 98016 on testosterone 60- hydroxylation in hepatic microsomes from PCN-treated rats showed an inhibitory effect with an IC50 of 50 M. The inhibitory effect seen in hepatocytes is similar to the effects of exposure to clotrimazole (50 .M) for 72 hours where testosterone metabolism at the 60 and 17 positions were inhibited by >90%. 9. To investigate whether the lack of inducing effect of SK F 98016 was due to the very high lipophilicity and extensive partitioning into the cultured hepatocyte, therefore resulting in a non-physiological state, cultured hepatocytes were exposed to the same drugs with albumin (from bovine serum, at the concentration present in human blood-36g/litre) in the medium in attempt to encourage an equilibrium of drug concentration between the medium and the inside of the hepatocyte. No significant induction of testosterone 60-hydroxylation was observed in the presence of albumin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:312674 |
| Date | January 1995 |
| Creators | Williams, J. Andrew |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602279 |
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