Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.     

Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes

The aim of this study was to test suitable culture conditions for maintaining normal cellular cytoarchitecture and inducibility of P450 enzymes in primary cultures of human hepatocytes by prototypical inducers. The selectivity and sensitivity of a sandwich culture system were determined by treating cultures with a number of clinically relevant drugs that are known to be inducers of either rodent and/or human P450 enzymes. The results showed that considerable induction of CYP3A4 activity is observed at DMSO concentrations greater than 0.1% (v/v). No differences in P450 induction response were observed between cultures maintained under different matrix conditions. However, the matrix condition considered to be optimal for maintaining cellular integrity, protein yields, and P450 enzyme induction was a sandwich configuration in combination with modified Chee's medium containing insulin (6.25 microg/mL) and dexamethasone (< or =0.1 microM). Under these conditions, induction of CYP3A4 occurred at clinically relevant drug concentrations, and maximal activities were achieved after 3 days of exposure. Overall, the response of human hepatocyte cultures to treatment with both positive and negative modulators was found to reflect that observed in vivo with respect to both enzyme specificity and potency of enzyme induction, although considerable sample-to-sample variability was observed in the magnitude of induction.     

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.     

Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2?

While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Effects of pretreatment with pyrazole and inducers of mixed function oxidases on DNA repair elicited by dimethylnitrosamine in rat hepatocytes in vivo and in vitro

Experiments were performed to investigate the relationship between the rate of oxidative metabolism of dimethylnitrosamine (DMN) by rat liver microsomes (i.e., DMN demethylase activity, DMNd) and its genotoxicity in liver, as assessed by the in vitro and in vivo/in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assays. Pretreatment of rats with pyrazole (PYR) resulted in a 4-fold increase in DMNd and a 3-fold greater DNA repair response to in vivo administration of 5 mg DMN/kg body weight. Pretreatment with phenobarbital (PB), dichlorodiphenyltrichloroethane (DDT), 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF) or Aroclor 1254 (ARO) produced a variable degree of inhibition of DMNd and had no significant effects on the response to DMN in the in vivo/in vitro HPC/DR assay. DNA repair elicited by DMN in vitro was decreased in hepatocytes from rats pretreated with 3-MC, while PB, DDT, beta-NF and ARO pretreatments had little effect on the response. In contrast, PYR pretreatment produced a 4.5-6.7-fold increase in the in vitro DNA repair response to DMN, and extended detection of positive responses to lower concentrations. Most of the inducers had no effect on DNA repair elicited by the direct acting alkylator, methyl methanesulfonate (MMS). Thus, the pretreatment-related changes in DMN-induced DNA repair were probably due to alterations in DMNd rather than to effects on the DNA repair capacity of the hepatocytes.     

Hepatic mixed function oxidase system and effect of phenobarbital, 3-methylcholanthrene and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane in developing chickens.

Contents of hepatic microsomal protein, aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, hydrogen peroxide formation, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 were examined in control, phenobarbital (PB), 3-methylcholanthrene (3-MC) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) treated group of 1-28 days old chickens. Increase in aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 was noticed at all stages of development during administration of PB and 3-MC. But these enzyme activities were not always paralleled by increase in age. Aminopyrine N-demethylase was increased in early stages only during DDT administration, which indicates that the form of cytochrome P-450, responsible for aminopyrine N-demethylation is present in early stages only. However, acetanilide hydroxylase was decreased in all stages of development, in postnatal development the basal activities of the enzymes for various substrates do not exhibit identical pattern, the degree of inducibility by inducers varied in relation to age of animal. Hydrogen peroxide formation increased in all stages of developing chickens due to the administration of PB and DDT. It however decreased due to 3-MC administration which may be due to induction of high spin cytochrome P-450.     

A test using cultured cells with induced mixed-function oxygenase in the unscheduled DNA synthesis assay for detecting promutagens/procarcinogens

The human FL cell line contains very low levels of constitutive AHH activity, but it could be greatly induced by NE, beta-NF and 3-MC, and induced slightly by PB. When two different types of inducer, for example, 3-MC and PB or 3-MC and NE were given in combination, an additive inductive effect was not observed. Both the constitutive and induced AHH in FL cells have characteristics of MFO, namely, NADPH-dependence and CO-sensitivity. The fact that the constitutive and induced AHH in FL cells could be inhibited by a known hydroxylase inhibitor 7,8-BF indicated that the AHH in FL cells belongs to the cytochrome P-448 dependent MFO type. After removal of inducer from the medium, the induced AHH activity remained at a high level for at least 24-36 h. By using AHH-induced FL cells in the UDS assay system for the detection of promutagens/procarcinogens, we found that AFB1 and 3-MC did not induce a UDS reaction in uninduced FL cells, while in beta-NF induced cells, 10(-6)-10(-4) M AFB1 and 10(-7)-10(-6) M 3-MC elicited a very significant UDS reaction, which was concordant with the results obtained in the UDS assay system using HeLa cells or FL cells supplemented with liver microsomes or using primary cultured hepatocytes as indicator cells. B(a)P elicited the UDS reaction at concentrations of 10(-6)-10(-3) M in beta-NF induced cells, whereas 10(-4)-10(-3) M was required in uninduced cells. The results above indicate that this new design is feasible, but further study is needed to assure its accuracy.     

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.     

Regulation of cytochrome P450 gene expression in the olfactory mucosa

The mammalian olfactory mucosa (OM) is unique among extrahepatic tissues in having high levels, and tissue-selective forms, of cytochrome P450 (CYP) enzymes. These enzymes may have important toxicological implications, as well as biological functions, in this chemosensory organ. In addition to a tissue-selective, abundant expression of CYP1A2, CYP2A, and CYP2G1, some of the OM CYPs are also known to have an early developmental expression, a resistance to xenobiotic inducers, and a lack of responsiveness to circadian rhythm. Efforts to fully characterize the regulation of CYP expression in the OM, and to identify the underlying mechanisms, are important for our understanding of the physiological functions and toxicological significance of these biotransformation enzymes, and may also shed unique light on the general mechanisms of CYP regulation. The aim of this mini-review is to provide a summary of current knowledge of the various modes of regulation of CYPs expressed in the OM, an update on our mechanistic studies on tissue-selective CYP expression, and a review of the literature on xenobiotic inducibility of OM CYPs. Our goal is to stimulate further studies in this exciting research area, which is of considerable importance, in view of the constant exposure of the human nasal tissues to inhaled, as well as systemically derived, chemicals, the prevalence of olfactory system damage in individuals with neurodegenerative diseases, and the current uncertainty in risk assessments for potential olfactory toxicants.     

Biotransformation of tamoxifen in a human endometrial explant culture model

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.     

Evaluation of the effect of culture matrices on induction of CYP3A isoforms in cultured porcine hepatocytes

Several bioartificial liver devices have been developed as temporary therapy for patients suffering from fulminant hepatic failure. Some of these devices contain porcine hepatocytes entrapped in collagen matrices. In order to improve the function of these BAL devices, there exists a need to optimize metabolic function of cultured hepatocytes. The goal of these investigations was to evaluate the effect of altering culture conditions on rifampin-mediated induction of CYP3A isoforms in cultured porcine hepatocytes. Midazolam metabolism was compared in porcine hepatocytes cultured in a monolayer configuration on collagen gels, in a sandwich configuration between collagen gels and a Matrigel overlay, and in spheroidal cultures. The effect of culture conditions was evaluated, by measuring CYP3A-mediated metabolism of midazolam and by immunoblotting to detect CYP3A proteins, in control cultures and in rifampin-treated cultures. Results obtained by normalizing the metabolism rate data to cell numbers (based on DNA content) present at the end of the culture experiment, showed that there was no difference between the different culture conditions tested. Our results suggest that culturing porcine hepatocytes as spheroids or in a sandwich configuration between collagen and Matrigel, offers no advantage in terms of CYP3A-mediated metabolic function on a per cell basis compared to culturing on collagen gels.     

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.     

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.     

Effect of enzyme inducers on metabolism of 1-nitropyrene in human hepatoma cell line HepG2.

We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.     

Oltipraz inhibits 3-methylcholanthrene induction of CYP1A1 by CCAAT/enhancer-binding protein activation

Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding proteinbeta (C/EBPbeta) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBPbeta for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 microM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10-20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 microM oltipraz treatment. Oltipraz (10 microM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBPbeta and partly C/EBPdelta. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBPbeta overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBPbeta by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.     

Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alteration of the CYP apoprotein profile that after 24 h of culture is quite different from that found in the liver of uninduced rats. This process is reverted by the early and efficient inhibition of NO synthesis, which allows for (1) maintenance of total P450 holoenzyme content, (2) preservation of the initial constitutive CYP pattern in culture and (3) the early expression of the normal inducibility in response to model inducers.     

Scoparone O-demethylase assay is not useful to differentiate the effects of model inducers of cytochrome P-450 in rabbit and guinea pig liver

Coumarin derivative, scoparone (6,7-dimethoxycoumarin), is regioselectively O-demethylated into isoscopoletin (I) and scopoletin (S). This oxidation is inversely influenced by cytochrome P-450 inducers in the rat such as 3-methylcholantrene (3-MC) and phenobarbital (PB). The I/S ratio is higher than 1.5 with 3-MC treatment whereas it is lower than 0.5 with PB treatment. With regards to this contrasting effect, it gas been suggested that the I/S ratio should be useful to differemtiate between the effects of these types of inducers. We studied the consequences of in vivo PB and 3-MC treatment on scoparone biotransformation in guinea pig and rabbit. In these two species, at the basal state, scoparone biotransformation was enhanced in comparison to the rat. Moreover, in these untreated animals, two other metabolites were formed. After 3-MC or PB treatment, scoparone metabolism is, in contrast to the rat, inappropriate to differentiate between the P-450 profile of other animals.