Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes

Summary Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with β-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations > or = 200 microg/mL, whereas only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194, >200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.     

Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins

The cytochromes P450 (P450s) are a family of heme-containing monooxygenase enzymes involved in a variety of functions, including the metabolism of endogenous and exogenous substances in the human body. During lead optimization, and in drug development, many potential drug candidates are rejected because of the affinity they display for drug-metabolising P450s. Recently, crystal structures of human enzymes involved in drug metabolism have been determined, significantly augmenting the prospect of using structure-based design to modulate the binding and metabolizing properties of compounds against P450 proteins. An important step in the application of structure-based metabolic optimization is the accurate prediction of docking modes in heme binding proteins. In this paper we assess the performance of the docking program GOLD at predicting the binding mode of 45 heme-containing complexes. We achieved success rates of 64% and 57% for Chemscore and Goldscore respectively; these success rates are significantly lower than the value of 79% observed with both scoring functions for the full GOLD validation set. Re-parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the scoring functions resulted in a significant increase in the percentage of successful dockings against the heme binding proteins (Chemscore 73%, Goldscore 65%). The modified scoring functions will be useful in docking applications on P450 enzymes and other heme binding proteins.     

Renal localization, expression, and developmental regulation of P450 4F cytochromes in three substrains of spontaneously hypertensive rats

Cytochrome P450 4F isoforms have been shown to metabolize arachidonic acid to generate 20-hydroxyeicosatetraenoic acid (20-HETE), a potent eicosanoid that modulates vascular tone and renal tubular function. 20-HETE production in the kidney is implicated in the development of essential hypertension in the spontaneously hypertensive rat (SHR). In this study, we determined CYP4F mRNA localization and distribution in rat liver and kidney by in situ hybridization and real time quantitative PCR. CYP4Fs are regionally distributed in the kidney with CYP4F1, 4F4, and 4F5 being expressed more in the renal cortex than medulla while CYP4F6 shows higher medullary expression. We investigated developmental CYP4F gene expression in three different substrains of SHR. Distinct age-dependent patterns of expression were seen for individual CYP4F isoforms in Wistar-Kyoto (WKY) and three SHR substrains (B2, C, and A3). A steady increase in CYP4F1 expression with age was seen in each of the three substrains which correlate well with increased 20-HETE levels and elevated blood pressure seen in these animals. CYP4F4 expression increased significantly at 8 weeks followed by a precipitous fall in WKY and A3 strains at 12 weeks of age. In strains B2 and C, CYP4F4 levels started declining as early as 8 weeks of age. CYP4F5 and 4F6 levels fluctuated with age in a biphasic manner with a different profile for each sub-strain. Based on the expression profile and catalytic activity, CYP4F1 seems to be the most critical 4F isoform involved in the production of 20-HETE in the SHR kidney.     

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.     

The effect of dimethyl sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the co-expression with NADPH-cytochrome P450 reductase

HepG2 cells, a human hepatoma cell line, stably expressing NADPH-cytochrome P450 reductase (OR) and/or cytochrome P450 2D6 wild-type (CYP2D6-WT) or its variants (Pro34Ser, Gly42Arg, Arg296Cys and Ser486Thr) were established in the present study. The cultivation of HepG2 cells expressing CYP2D6-WT in the culture medium containing dimethyl sulfoxide (DMSO, 0.1% of final concentration) markedly increased the bufuralol (BF) 1'-hydroxylase activity compared with that of control cells when cultivated without DMSO. A similar effect was also observed in HepG2 cells stably expressing CYP2D6 and OR. The addition of hemin in place of DMSO to the culture medium resulted in no increase in the enzyme activity. Western blot analysis revealed that the levels of CYP2D6 protein were similar between DMSO-treated and non-treated HepG2 cells regardless of OR expression. Spectrophotometric analysis of reduced carbon monoxide-difference spectra of HepG2 cells expressing CYP2D6-WT and/or OR demonstrated that the addition of DMSO increased the peak height of functional CYP2D6 at 450 nm. These results suggest that the increase in CYP2D6 activity is attributable to the radical-scavenging effect of DMSO. The HepG2 cell lines stably expressing OR and CYP2D6 or its variants in combination with DMSO treatment may be useful for screening the cytotoxicity of chemical compounds which undergo oxidation by CYP2D6.     

Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts

Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.     

Effect of silybin and its glycosides on the expression of cytochromes P450 1A2 and 3A4 in primary cultures of human hepatocytes

Four beta-glycosides of flavonoligan silybin, i.e. silybin beta-galactoside, silybin beta-glucoside, silybin beta-maltoside, silybin beta-lactoside were synthesized in order to improve silybin water solubility and bioavailability (Kren et al., J Chem Soc, Perkin Trans 1, 2467-2474, 1997). The presented paper deals with the effect of silybin and its synthetic beta-glycosides on the expression of two major cytochrome P450 isoforms, CYP1A2 and CYP3A4. Primary cultures of human hepatocytes were the model of choice. mRNAs were analyzed using Northern blot and P-radiolabelled probes. CYP protein content was determined by immunoblotting using specific antibodies. Silybin and its beta-glycosides do not induce expression of CYP1A2 and CYP3A4. Tested compounds did not affect inducible expression of CYP1A2 and CYP3A4 by dioxin and rifampicin, respectively, as evaluated at the level of mRNAs and proteins. Silybin and its beta-glycosides do not interfere with the expression of CYP1A2 and CYP3A4, are not likely to produce drug-drug interactions in terms of the inducibility of two important cytochromes P450.     

Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-depen-dent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver :body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate ω- and ω-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-de-alkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.     

Alcohol-mediated effects on the level of cytochrome P-450 and heme biosynthesis in cultured rat hepatocytes

Interaction of alcohol and drugs in the liver appears to involve common microsomal oxidative enzymes which utilize cytochrome P-450. Since alcohol augments the toxicity of a variety of drugs, the regulation of the P-450 hemoprotein, a primary component in hepatic drug metabolizing systems, may play a vital role in this phenomenon. We utilize an adult rat liver culture system as a model to explore the action of levels of alcohol below that which is necessary to produce intoxication in humans. The addition of 16 mM ethanol (70 mg/dl) to these hepatocytes results in a 49.5% decrease in cytochrome P-450 activity after 24 h, and a 3-fold increase in the activity of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic heme biosynthesis. Furthermore, ethanol treatment also causes a transient decrease in the level of intracellular heme. However, the diminished level of total heme does not appear to act as a repressor for δ-aminolevulinate synthase, since it occurs after the initial stimulation of the enzyme by ethanol.     

Developmental changes of serum steroids produced by cytochrome P450c17 in rat

Serum levels of 17-hydroxypregnenolone, dehydroepiandrosterone, 17-hydroxyprogesterone, and androstenedione were measured during the postnatal development of rats 1-14 weeks of age. A significant decrease in the serum levels of these steroids with increasing age was observed, using multiple regression analysis: 17-hydroxypregnenolone (beta= -1.56, S.E.= 0.25, P < 0.00001), dehydroepiandrosterone (beta= -0.43, S.E.= 0.07, P < 0.00001), 17-hydroxyprogesterone (beta= -2.51, S.E.= 0.45, P < 0.00001), and androstenedione (beta= -1.63, S.E.= 0.33, P < 0.00001). A sex-related difference was not found. The observed decline in the serum levels of the steroids was directly proportional to the previously reported decrease in mRNA expression and enzyme activity of cytochrome P450c17 in the rat liver. Yet, despite this decrease to undetectable levels in liver after 7-8 weeks, significant amounts of 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione were still observed in the rat serum. This may partly be due to the mRNA expression of cytochrome P450c17 in tissues other than the liver, such as the testis and/or duodenum, after 4 weeks of age. Serum levels of pregnenolone, progesterone, and corticosterone in the developing rats were also examined.     

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of ¹³C- and ¹⁵N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis¹³C,¹⁵N-labeled His₄CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His₄CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.