Induction of CYP3A and associated terfenadineN-dealkylation in rat hepatocytes cocultured with 3T3 cells

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways:C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, andN-dealkylation to azacyclonol. In rat liver, only theN-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mol/L or 5 mol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.Abbreviations BSA bovine serum albumin - CYP cytochrome P450 - DMSO dimethyl sulfoxide - LDH lactate dehydrogenase - PCN pregnenolone-16-carbonitrile - SDS sodium dodecyl sulfate - SSC saline sodium citrate     

Modulation of cytochrome P450 gene expression in primary hepatocytes on various artificial extracellular matrices

We studied effect of artificial extracellular matrices (ECMs), such as collagen I, poly (N-p-vinylbenzyl-4-O-β-d-galactopyranosyl-d-gluconamide)(PVLA) and E-cadherin–IgG Fc (E-cad-Fc) on hepatic metabolism to identify the mechanism of in vivo hepatocellular functional and metabolic integrity. mRNA expression of liver function marker, cytochrome P450 (CYP) and transporter genes in hepatocytes were compared among used ECMs using real-time RT-PCR. mRNA expressions of Cyp2c29 and Cyp2d22 among CYP genes in hepatocytes on PVLA were recovered after 3 days due to enhanced liver-specific function by the spheroid formation of hepatocytes whereas mRNA expressions of CYP genes in hepatocytes on collagen and E-cad-Fc drastically decreased with time. mRNA expressions of the Cyp2c29 and Cyp2d22 in hepatocytes on PVLA were more recovered in the presence of epidermal growth factor (EGF) due to the more and bigger spheroid formation of hepatocytes. Multidrug resistance-associated protein 2 (Mrp2) protein was accumulated at intracellular lumen as similar to bile duct in hepatocyte spheroid formed on PVLA, indicating that spheroid formation of hepatocytes is very important for maintaining liver functions.     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

Prenatal liver stromal cells: Favorable feeder cells for long-term culture of hepatic progenitor cells

Clinical and pharmaceutical applications of primary hepatocytes (PHs) are limited due to inadequate number of donated livers and potential challenges in successful maintenance of PHs in culture. Freshly isolated hepatocytes lose their specific features and rapidly de-differentiate in culture. Bipotent hepatoblasts, as liver precursor cells that can differentiate into both hepatocytes and cholangiocytes (Alb- and Ck19-positive cells, respectively), could be used as an alternative and reliable cell source to produce enough PHs for drug discovery or possible clinical applications. In this study, growth factor-free coculture systems of prenatal or postnatal murine liver stromal cells (pre-LSCs or post-LSCs, respectively) were used as feeder cells to support freshly isolated mice hepatoblasts. DLK1-positive hepatoblasts were isolated from mouse fetuses (E14.5) and cocultured with feeder cells under adherent conditions. The hepatoblasts' bipotent features, proliferation rate, and colony formation capacity were assessed on day 5 and 7 post-seeding. Immunofluorescence staining showed that the hepatoblasts remained double positive for Alb and Ck19 on both Pre- and Post-LSCs, after 5 and 7 days of coculture. Moreover, application of pre-LSCs as feeder cells significantly increased the number of DLK1-positive cells and their proliferation rate (ie, increased the number of Ki-67 positive cells) on day 7, compared to Post-LSCs group. Finally, to address our ultimate goal, which was an extension of hepatoblasts ex vivo maintenance, 3D spheres of isolated hepatoblasts were, cultured in conditioned medium (CM) derived from pre-LSCs until day 30. It was observed that the CM derived from Pre-LSCs could successfully prolong the maintenance of hepatic progenitor cells (HPCs) in 3D suspension culture.     

Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases

We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3′:5′ cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca²⁺/calmodulin-dependent protein kinases II (independently of Ca²⁺) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Collagen type I gel cultures of adult rat hepatocytes as a screening induction model for cytochrome P450-dependent enzymes

Albumin secretion, expression of cytochrome P450 dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture time and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by beta-naphthoflavone (beta-NF), 3- methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5microM 3-MC or 25 microM beta-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2 mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16alpha-position (CYP2B1/2 and CYP2C11), the 7alpha-position (CYP2A1/2), and the 6beta-position (CYP3A1). DEX (10 microM) markedly increased testosterone 6beta- and 7alpha-hydroxylation. Expression and induction experiments of CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.     

Bromide alleviates fatty acid‐induced lipid accumulation in mouse primary hepatocytes through the activation of PPARα signals

Increased plasma free fatty acids (FFAs) and liver triglyceride (TG) accumulations have been implicated in the pathogenesis of hepatic steatosis. On the other hand, trace elements function as essential cofactors that are involved in various biochemical processes in mammals, including metabolic homeostasis. Notably, clinical and animal studies suggest that the plasma levels of bromide negatively correlate with those of TG, total cholesterol (TC) and high‐density lipoprotein‐cholesterol (HDL‐C). However, the effect of bromide on lipid accumulation and the direct molecular target responsible for its action remains unknown. Oil red O (ORO) and Nile red staining were used to detect the effect of bromide on lipid accumulation in mouse primary hepatocytes (PHs) treated with different doses of sodium bromide (NaBr) in the presence of FFAs (0.4 mM oleate/palmitic acid 1:1). Spectrophotometric and fluorometric analyses were performed to assess cellular TG concentrations and rates of fatty acid oxidation (FAO), respectively, in mouse PHs. We found that bromide decreased FFA‐induced lipid accumulation and increased FFA‐inhibited oxygen consumptions in mouse PHs in a dose‐dependent manner via activation of PPARα. Mechanical studies demonstrated that bromide decreased the phosphorylation levels of JNK. More importantly, the PPARα‐specific inhibitor GW6471 partially abolished the beneficial effects of bromide on mouse PHs. Bromide alleviates FFA‐induced excessive lipid storage and increases rates of FAO through the activation of PPARα/JNK signals in mouse PHs. Therefore, bromide may serve as a novel drug in the treatment of hepatic steatosis.     

Induction of DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (ICDD).

The compounds 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) are both inducers of the enzyme system aryl hydrocarbon hydroxylase. It has recently been reported that 3-MC is also an inducer of DT-diaphorase activity in rat liver. In this report the ability of TCDD to induce hepatic DT-diaphorase activity was examined. The results indicate that TCDD is approximately 17,000 times more potent as an inducer of DT-diaphorase activity than 3-MC.     

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Summary Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.     

Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber

We recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils. In this study, we first confirmed that the advantage of CVM chamber in comparison to the traditional culture chamber with porous polyethylene terephthalate membrane is to preserve a culture medium poured in its inside even though the under side is not a liquid phase but solid and gas phases. Subsequently, we designed three different culture systems to grow HepG2 cells in a culture medium (liquid phase) on the CVM which the under side is a culture medium, a plastic surface (solid phase) or 5 % CO₂ in air (gas phase) and aimed to develop a brief culture method useful for activating the liver-specific functions and analyzing the pharmacokinetics of fluorescein diacetate. HepG2 cells cultured for 2 days on the liquid–solid interface and subsequently for 1 day on the liquid–gas interface represented excellent cell viability and morphology in comparison to the others, and remarkably improved albumin secretion and urea synthesis to almost the same level of freshly isolated human hepatocytes and CYP3A4 activity to about half the level of differentiated HepaRG cells. Also, the cells rapidly absorbed fluorescein diacetate, distributed it in cytosol, metabolized it into fluorescein, and speedily excreted fluorescein into both bile canaliculus-like networks and extracellular solution. These data suggest that hepatic structure and functions of monolayered HepG2 cells can be induced within a day after the oxygenation from beneath the CVM.     

Collaborated regulation of female-specific murine Cyp3a41 gene expression by growth and glucocorticoid hormones

CYP3A41 is a female-specific major CYP3A in mouse livers. Adrenalectomy decreased expression of CYP3A41 as well as CYP3A11, another major CYP3A, and dexamethasone (DEX) restored the decreased expression. Hypophysectomy completely abolished CYP3A41 expression and growth hormone (GH) replacement only slightly restored the expression. Treatment with DEX alone did not induce expression of either CYP3A41 or CYP3A11 in hypophysectomized mice. However, combined treatment with GH and DEX strongly induced expression of CYP3A41 but not CYP3A11. In primary cultured mouse hepatocytes, DEX induced expression of both CYP3A41 and CYP3A11, and DEX-inducible expression of CYP3A41 was suppressed by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself enhanced basal expression of CYP3A11 mRNA, while it showed no inhibitory effect on DEX-inducible expression. These observations indicate that glucocorticoids may participate in the GH-dependent control of the Cyp3a41 gene expression, probably mediated via the glucocorticoid receptor, which may be different from that of the Cyp3a11 gene expression.     

Induction of cytochrome P-450-dependent hydroxylases in primary monolayer cultures of rat hepatocytes

Hydroxylation of androstenedione was studied in rat hepatocytes in primary monolayer culture following induction with phenobarbital. Six days after addition of phenobarbital and seven days after isolation of cells from liver, a maximal induction of total androstenedione hydroxylation of 5–6 times was seen at a phenobarbital concentration of 1·10⁻⁴ M. The 6β-, 7α- and 16α-hydroxylase activities showed different responses towards phenobarbital in agreement with the contension that different forms of cytochrome P-450 with different sensitivity towards phenobarbital participate in hepatic steroid hydroxylation. These results were obtained with cells supplemented with 1% (v/v) rat serum. The present cell culture system should be suitable for in vitro studies on mechanisms of induction of cytochrome P-450-dependent enzymes in normal liver cells.     

Enhancement of aldehyde dehydrogenase activity in human and rat hepatocyte cultures by 3-methylcholanthrene

Aldehyde dehydrogenase was measured in primary cultures of hepatocytes obtained with a two-step collagenase perfusion either from human hepatic tissue or from livers of Fisher rats. Basal enzyme activity declines gradually as a function of time in culture, but remains at all times higher when measured with propionaldehyde and NAD (P/NAD) than with benzaldehyde and NADP (B/NADP). Treatment of the cultures with 2 M of 3-methylcholanthrene for four days significantly increased the B-NADP activity of human and rat hepatocytes (tenfold and eightfold respectively). In human hepatocytes 3-methylcholanthrene increases also the P/NAD activity, but to a lesser extent (twofold), compared to the B/NADP activity. Due to the significant enhancement of B/NADP activity in cultures of human and rat hepatocytes after application of 3-methylcholanthrene, the initial difference in the basal activity levels between the P/NAD and B/NADP forms diminishes or, in the case of human hepatocytes, is even inverted. These results show for the first time that aldehyde dehydrogenase activity is increased in cultured human hepatocytes. This biochemical property is preserved in human and rat hepatocyte cultures, despite the rather quick loss of the basal aldehyde dehydrogenase activity.Abbreviations ALDH aldehyde dehydrogenase - B benzaldehyde - p-p-DDT 1,1,1,-trichlo-2,2,bis(p-chlorophenyl)ethane - DMSO dimethylsulfoxide - 3-MC 3-methylcholanthrene - MEM Minimal Essential Medium - P proprionaldehyde - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [¹⁴C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.