Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture

Abstract

enzymes are known to be controlled by many different stimThe influence of cell density and epidermal growth uli, including physiopathological, environmental, and genetic factor (EGF) on the expression and inducibility of cytofactors, so that changes in the battery of CYP enzymes are chrome P450 (CYP) genes of the CYP3A and CYP1A famiexpected to occur not only from one individual to another, lies in adult human hepatocytes in primary culture has but also in the same individual as a function of time, for been evaluated. Only when cultured at subconfluence example. These changes may have critical consequences in and in the presence of EGF did hepatocytes exhibit a pharmacology and toxicology, especially in those individuals proliferative response, assessed by measuring DNA synwho are significantly exposed to xenobiotics. thesis and cyclin A accumulation. In the absence of EGF,The CYP3A and CYP1A families have been investigated the accumulation of CYP3A4 and CYP1A2 messenger extensively because they play a critical part both in pharmaRNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in cology and toxicology. 3,4 In humans, the CYP3A family has subconfluent culture with respect to confluent cultures. three functional genes, including CYP3A4, CYP3A5, and The presence of EGF only slightly decreased the accu-CYP3A7. 1,2,5 CYP3A4 is the major CYP protein expressed in mulation of these mRNAs in both confluent and subcon-the adult liver, where it may represent up to 60% of the fluent cultures. The accumulation of CYP2D6 and total amount of CYP proteins. 6 This CYP is involved in the CYP2E1 proteins, which are constitutively expressed in oxidative metabolism of the majority of drugs in current use, confluent cultures, and the production of fibrinogen and as well as in the activation of many procarcinogens, including apolipoprotein (Apo) B100 exhibited similar behavior, polycyclic aromatic hydrocarbons and aflatoxins. 5,7 CYP3A4 while nicotinamide adenine dinucleotide phosphate cy-is inducible by many structurally unrelated compounds such tochrome c reductase activity was affected neither by as rifampicin (RIF), glucocorticoids, imidazole-derivatives, cell density nor by EGF. In contrast, the accumulation and phenobarbital. 8-10 CYP3A7 is the major form of CYP exof CYP1A1 mRNA in response to dioxin was similar in pressed in the human fetal liver. 11,12 Shortly before birth, confluent and subconfluent cultures, irrespective of the however, the accumulation of CYP3A7 messenger RNA presence of EGF. Interestingly, CYP3A7, a gene that is (mRNA) and protein decreases to a low or undetectable level preferentially expressed in the fetal liver, was expressed in the newborn and adult liver. In contrast, the level of constitutively neither in confluent nor in subconfluent CYP3A4 mRNA and protein rapidly increases during the cultures, irrespective of the presence of EGF. It is con-perinatal period. 13 CYP3A5, which is expressed in a polymorcluded that the loss of cell-cell contacts ...

Repression of Cytochrome P450 Activity in Human Hepatocytes in Vitro by a Novel Hepatotrophic Factor, Augmenter of Liver Regeneration

“…In addition, the mitogenic effect of hepatotrophic growth factors such as HGF, EGF, and transforming growth factor ␣ on human hepatocytes and their influence on hepatic drug metabolism such as P450 enzyme modulation have been discovered and characterized by several authors (Greuet et al, 1997;Donato et al, 1998;De Smet et al, 2001;Tinel et al, 2003). To our knowledge, the present study provides the first evidence of a modulating effect of the hepatotrophic factor ALR on the hepatic P450-metabolizing enzyme activities.…”

“…Pathological disorders of the liver including tissue injury, inflammation, cirrhosis, and tumor growth were shown to be associated with an impairment of hepatic drug metabolism, particularly the one mediated by P450 (Tygstrup et al, 2002;Shibuya et al, 2003;Villeneuve and Pichette, 2004). Factors of the so-called acute phase mediators, e.g., IL-6, IL-1, and TNF-␣ as well as growth factors like HGF and EGF, were identified to down-regulate P450 expression (Muntane-Relat et al, 1995;Greuet et al, 1997;Donato et al, 1998;De Smet et al, 2001;Tinel et al, 2003).…”

Dexamethasone Induces Pregnane X Receptor and Retinoid X Receptor-α Expression in Human Hepatocytes: Synergistic Increase of CYP3A4 Induction by Pregnane X Receptor Activators

“…One hundred micrograms of total proteins or 50 g of nuclear extracts from 10 7 hepatocytes were separated by SDS-polyacrylamide gel electrophoresis (10%), then electroblotted onto Imobilon P (Millipore, Bedford, MA). Membranes were incubated with specific antibodies against multidrug resistance glycoprotein (MDR; Ab-1; Oncogene Research Products, Cambridge, MA), RXR␣ (⌬N 197; Santa Cruz Biotechnology, Santa Cruz, CA), or CYP3A6 (Greuet et al, 1997) and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia, Cardiff, England).…”

Colchicine Down-Regulates Cytochrome P450 2B6, 2C8, 2C9, and 3A4 in Human Hepatocytes by Affecting Their Glucocorticoid Receptor-Mediated Regulation

“…Levels of CYP3A4 and tyrosine aminotransferase (TAT) mRNAs were determined by Northern blot using [␣-32 P]dCTP-labeled cDNA probes (INSERM U128, Montpellier, France) and mouse TAT cDNA (gift from Dr. T. Grange, Institute J. Monod, Paris, France), respectively. To determine CYP2B6, CYP2C8, CYP2C9, PXR, CAR, and GR mRNAs, the total RNA was analyzed by ribonuclease protection assay as described previously (Greuet et al, 1997). Preparation of riboprobe plasmids for CYP2B6, CYP2C8, CYP2C9, PXR, CAR, and GR were described elsewhere (Pascussi et al, 2000b).…”