Expression of cytochrome P‐450 enzymes in cultured human hepatocytes

Abstract

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIEl decreased to 25 -50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin 0-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIEl was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn.These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.The cytochrome P-450 (P-450) monooxygenase system (EC 1.14.14.1; for nomenclature see [I] and [2]) consists of a large family of enzymes responsible for the oxidation of drugs and environmental pollutants, as well as many endogenous compounds. More than 20 distinct forms of P-450 have been purified from rat liver [3, 41 (and references therein). These distinct enzymes are encoded by different genes and several forms are inducible with agents such as phenobarbital, 3-methylcholanthrene, polyhalogenated biphenyl mixtures, or pregnenolone 16a-carbonitrile (PCN) [5]. While much information has been gathered concerning P-450 in experimental animals, the application of this knowledge to human beings has lagged [l, 6,7]. It is now well established that P-450 forms may contribute to inter-individual variations among humans in biological responses to drugs and other chemicals [8]. Genetic polymorphisms have been recognized in drug oxidations in humans [9] and possible relationships to cancers and other lesions have been proposed [lo-131. Furthermore, induction of P-450 may be clinically important since in some c...

Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduced cells

“…The results obtained in our experiments indicate that the activities so far examined, representative of cytochromes recombinant virus was prepared from homologous recombination in 293 cells of two plasmids pJM17 24 and 1A2, 2A1, 2B6 and 3A3/5, [28][29][30] were not substantially altered after adenovirus-mediated gene transduction pACCMV␤gal. After infecting human hepatocytes with the recombinant virus, changes in liver-specific ( Figure 7).…”

“…The former is inducible by methylchol-4-chloro-3-indolyl-␤-d-galactopyranoside (B-4252), ␣-glucoamilase (A-3042; specific activity 5000 U/ml) (Hoechst antrene, via a cytosolic receptor that is translocated to the nucleus where it activates gene transcription. 30,31 Fibrino-33258 (B-2883)) and ␣-glucosidase (G-3651, specific activity 50 U/mg) were from Sigma (St Louis, MO, USA). gen synthesis increases in the course of the systemic inflammatory response and is induced by interleukin 6.…”

Liver cell models in in vitro toxicology.

“…About 50% of the initial CYP content is still found after 1 week of culture (29,73), and all major CYPs (i.e., CYPiA, CYP2C, CYP2E, and CYP3A) remain well expressed and responsive to specific inducers (72). Specific inductions were demonstrated with rifampicin (72,74), 3-methylcholanthrene (72), omeprazole (75), and dihydralazine (76).…”

“…Phenotypic changes with time in culture also include overexpression in adult rat hepatocytes of P-glycoprotein, the product of multidrug resistance gene(s), (29,(71)(72)(73). About 50% of the initial CYP content is still found after 1 week of culture (29,73), and all major CYPs (i.e., CYPiA, CYP2C, CYP2E, and CYP3A) remain well expressed and responsive to specific inducers (72).…”

Transcriptional Activation of Cytochrome P450 CYP2C45 by Drugs Is Mediated by the Chicken Xenobiotic Receptor (CXR) Interacting with a Phenobarbital Response Enhancer Unit

“…Diazepam (4), ibuprofen (5), phenytoin (6), sildenafil (7), and warfarin (8) are some examples of clinically used drugs, whose metabolism involves enzymes of the CYP2C subfamily. CYP2C genes show a variety of regulation patterns including sex-dependent regulation (9), constitutive expression, or transcriptional activation by classical P450 inducers such as phenobarbital (PB), dexamethasone (DEX), and rifampicine (10).…”