Abstract
In the present study, we evaluated the inducibility of cytochrome P-450 (CYP) CYP1A, CYP2B, CYP3A, and CYP4A by β-naphthoflavone, phenobarbital, dexamethasone, and clofibric acid, respectively, in primary hepatocyte cultures prepared from both fresh and cryopreserved rat hepatocytes. Rat hepatocytes were successfully thawed and cultured after cryopreservation in liquid nitrogen for up to 1 month. Percentage of total recovery, viable cell recovery, and final viability of the cells were 68%, 72%, and 85%, respectively. Regardless of whether they were cryopreserved or not, cultured hepatocytes exhibited near-normal morphology. Treatment of cryopreserved hepatocytes with β-naphthoflavone caused an 8-fold increase in 7-ethoxyresorufinO-dealkylase (CYP1A1/2) activity, with an EC50 of 1.5 μM; treatment with phenobarbital caused a 26-fold increase in 7-pentoxyresorufin O-dealkylase (CYP2B1/2) activity, with an EC50 of 10 μM; treatment with dexamethasone caused a 10-fold increase in testosterone 6β-hydroxylase (CYP3A1/2) activity, with an EC50 of 1.3 μM, whereas treatment with clofibric acid caused a 3-fold increase in lauric acid 12-hydroxylase (CYP4A1–3) activity, with an EC50 of 170 μM. The induction of CYP1A, CYP2B, CYP3A, and CYP4A enzymes by these inducers was confirmed by Western immunoblotting. The patterns of P-450 induction in cryopreserved rat hepatocytes, in terms of concentration response, reproducibility, magnitude, and specificity of response, were similar to those observed in freshly isolated hepatocytes. Additionally, the magnitude and specificity of induction was similar to that observed in vivo in rats. In conclusion, under the conditions examined, cryopreserved rat hepatocytes appear to be a suitable in vitro system for evaluating xenobiotics as inducers of P-450 enzymes.
Footnotes
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Send reprint requests to: Dr. Ajay Madan, XenoTech, LLC, 3800 Cambridge, Kansas City, KS 66103. E-mail: amadan{at}kumc.edu
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This work was funded in part by Grant ES03765 from the National Institutes of Health.
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↵2 The major dexamethasone-inducibleP-450 enzyme in rats was originally named cytochromeP-450p or P-450PCN, and was subsequently renamed CYP3A1 when the cDNA thought to encode this enzyme was isolated and sequenced (Gonzalez et al., 1985). However, it now appears that cytochrome P-450p is encoded byCYP3A23, and that CYP3A1 may be an allelic variant of this gene (Gonzalez et al., 1986; Ribeiro and Lechner, 1992; Kirata and Matsubara, 1993; Komori and Oda, 1994; Nagata et al., 1996; Mahnke et al., 1997; Wang and Strobel, 1997). For simplicity, the old nomenclature has been retained and the major PCN- and dexamethasone-inducible P-450 enzyme will be referred to as CYP3A1.
- Abbreviations used are::
- CYP
- cytochromeP-450
- DMEM
- Dulbecco’s modified Eagle’s medium
- DMSO
- dimethylsulfoxide
- EC50
- concentration effective in causing 50% of maximal induction
- EROD
- 7-ethoxyresorufinO-dealkylase
- FBS
- fetal bovine serum
- MCM
- modified Chee’s medium
- MEM
- modified Eagle’s medium
- PROD
- 7-pentoxyresorufinO-dealkylase
- Received July 20, 1998.
- Accepted November 24, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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