Abstract
To evaluate the theory that within precision-cut liver slices intercellular transport occurs in parallel with cellular metabolism and to illustrate the constraints this places on clearance predictions, the kinetics of ethoxycoumarin O-deethylation have been determined under varying conditions of hepatic cytochrome P-450 activity. Liver slices, isolated hepatocytes, and microsomes were obtained from rats treated with the inducers phenobarbital (PB) and β-naphthoflavone (βNF) and the inhibitor aminobenzotriazole (ABT). In hepatocytes and microsomes, a two-site kinetic model with a high-affinity, low-capacity site and an unsaturated low-affinity, high-capacity site described the hydroxycoumarin formation data. There were marked increases in Vmax (2- to 5-fold and 50- to 70-fold for PB and βNF, respectively) in both systems and in CLint (3- and 9-fold for PB and βNF, respectively) in hepatocytes and substantial decreases in both parameters (3–8 and 12–23% of control, respectively) in ABT hepatocytes and microsomes. A qualitatively similar response was evident in slices obtained from livers of rats treated with phenobarbital and ABT, but although slices from βNF livers produced high metabolic rates (comparable to slices obtained from livers of rats treated with phenobarbital), these showed a linear increase with substrate concentration without indication of a high-affinity site. The intrinsic clearance parameters were scaled to full liver capacity using hepatocellularities and microsomal recovery indices to allow direct comparison of these responses. The slice system consistently underestimated the effects of the modifiers. When compared with hepatocytes, estimates of 30, 15, and 1% for ABT, PB, and βNF, respectively, were observed and the degree of underestimation was dependent on the magnitude of intrinsic clearance and was consistent with the above theory.
Footnotes
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Send reprint requests to: Dr. J. B. Houston, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, U.K. E-mail: BHOUSTON{at}fs1.pa.man.ac.uk
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Financial support was received for part of this work from European Centre for the Validation of Alternative Methods (ECVAM) (European Commission, JRC Ispra, Italy) under the terms on contract 12356-96-11.
- Abbreviations used are::
- CLint
- intrinsic clearance
- CYP
- cytochrome P-450
- EC
- 7-ethoxycoumarin
- PB
- phenobarbital
- βNF
- β-naphthoflavone
- ABT
- 1-aminobenzotriazole
- HC
- 7-hydroxycoumarin
- UT
- untreated
- SRW
- standard rat weight of 250g
- Received July 22, 1998.
- Accepted December 21, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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