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Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices
1Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Germany.
Abstract:
Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.
Insights
Precision-cut liver slices maintain key functions like protein synthesis and drug metabolism for up to 48 hours in vitro. These slices are valuable for studying hepatic functions and drug metabolism induction.
Area of Science:
- Hepatology
- In vitro toxicology
- Drug metabolism research
Background:
- Investigating the viability and functionality of precision-cut liver slices is crucial for their application in toxicological and pharmacological studies.
- Understanding the stability of hepatic functions over time in vitro is essential for designing reliable experimental protocols.
Purpose of the Study:
- To evaluate the stability of various hepatic functions and drug metabolism in precision-cut rat liver slices over a 48-hour incubation period.
- To assess the utility of precision-cut liver slices as a model for studying drug-induced changes in hepatic gene expression.
Main Methods:
- Precision-cut rat liver slices were prepared using a KRUMDIECK slicer (200-250 microm thickness).
- Slices were incubated in modified William's medium E at 37°C for 2, 24, and 48 hours.
- Assessed parameters included protein, DNA, potassium, glutathione, lactate dehydrogenase (LDH) leakage, thiobarbituric acid-reactive substances, albumin synthesis, and cytochrome P450 (CYP)-dependent enzyme activities (7-ethoxycoumarin O-deethylation and testosterone hydroxylation).
- CYP1A1-mRNA expression was measured by competitive RT-PCR after beta-naphthoflavone exposure.
Main Results:
- Key cellular components (protein, DNA, potassium, glutathione) remained stable over 48 hours.
- Lactate dehydrogenase (LDH) and thiobarbituric acid-reactive substance leakage were highest in the initial 2 hours, then decreased.
- Albumin synthesis decreased to approximately 70% after 48 hours.
- CYP-dependent 7-ethoxycoumarin O-deethylation was stable, while testosterone hydroxylation decreased.
- Beta-naphthoflavone induced CYP1A1-mRNA expression by over 1000-fold within 6 hours.
Conclusions:
- Precision-cut liver slices demonstrate remarkable stability in core cellular components and several metabolic functions for up to 48 hours.
- These slices are a valuable in vitro tool for studying diverse hepatic functions, drug metabolism, and induction of drug-metabolizing enzymes.
- The model is suitable for investigating drug effects and gene expression changes in liver tissue.