Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

Heme oxygenase-1 mRNA levels,metallothionein mRNA levels,lipid peroxidation and microsomal CYP1A activities in rats treated with 3,3-dichlorobenzidine and some other inducers of P450

The effect of 3,3-dichlorobenzidine (DCB), a potent inducer of CYP1A, on the levels of heme oxygenase-1 mRNA and metallothionein mRNAs was examined in the kidney, liver and lung of rats administered a single ip dose (157 μmol/kg) of the compound. DCB treatment increased heme oxygenase-I mRNA abundance in the kidney significantly from barely detectable levels in untreated animals; the maximum increase in the liver and lung was 24-fold and 4-fold, respectively. Hepatic microsomal heme oxygenase activity was also induced by DCB. In contrast with DCB, 2 other P450 inducers, β-naphthoflavone (β-NF) and phenobarbital did not elevate tissue HO-1 rnRNA levels. DCB pretreatment also elevated metallothionein mRNA levels in the kidney, liver and lung, with the effect in the lung being the least pronounced. In contrast with HO-1 mRNA, metallothionein mRNA was increased by the other P450 inducers examined. In vivo lipid peroxidation and in vitro NADPH-dependent microsomal lipid peroxidation were increased in the liver of DCB-treated rats but not in those of phenobarbital- or β-naphthoflavone-treated rats. Treatment with DCB or β-NF did not alter total hepatic microsomal P450 content, as measured spectrophotometrically, but induced the activity of CYP1A2. In contrast, the activity of CYP1A1 was induced to a lesser extent by DCB than by β-NF. The data show that DCB induces HO-1 as weD as P450 1A, confirm stimulation of lipid peroxidation by the compound, and suggest oxidative stress as a mechanism of HO-1 induction by the compound.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Acute sodium arsenite administration induces pulmonary CYP1A1 mRNA, protein and activity in the rat

Modulation of the cytochrome P450 (CYP) monooxygenase system (P450) by arsenite was investigated in male, adult Sprague-Dawley rats treated with a single dose (75 micromol/kg, sc) of sodium arsenite (As3+). Total CYP content and P450-dependent 7-pentoxyresorufin O-pentylation (PROD) and 7-ethoxyresorufin O-deethylation (EROD) activities of liver microsomes decreased maximally (33, 35, and 50% of control, respectively) 1 day after As3+ treatment. Maximum decreases of CYP content and P450 catalytic activities corresponded with maximum increases of microsomal heme oxygenase (HO) activity and with increased total plasma bilirubin concentrations. EROD activity increased maximally in lung (300%) 5 days after a single dose of As3+. Lung CYP1A1 mRNA and protein levels also increased maximally 5 days after treatment. A small but significant increase in EROD activity (65%) was observed in lung microsomes 24 h following a 1 h infusion of bilirubin (7.5 mg/kg) into rats. However, administration of bilirubin to the lung via intratracheal injection (0.25 and 2.5 mg/kg) did not increase CYP1A1 monooxygenase activity or mRNA. This study demonstrates that P450 is modulated in an isozyme (CYP1A1 vs CYP2B1/2) selective manner in rat lung after acute As3+ administration. Administration of bilirubin, a potential aryl hydrocarbon receptor (AHR) ligand, by infusion or intratracheal instillation did not upregulate pulmonary CYP1A1 at the mRNA level under our treatment conditions.     

Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta)

The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl)-1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose–response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 μg l⁻¹ propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.     

Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression.

Studies initiated to characterise the catalytic activity and expression of CYP1A1 in rat blood lymphocytes revealed significant activity of 7-ethoxyresorufin-O-deethylase (EROD) in rat blood lymphocytes. Pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (NF) resulted in significant induction in the activity of lymphocyte EROD suggesting that like the liver enzyme, EROD activity in lymphocytes is inducible and is mediated by the MC inducible isoenzymes of P450. The increase in the activity of EROD was associated with a significant increase in the apparent Vmax and affinity of the substrate towards EROD. That this increase in the activity of EROD could be primarily due to the increase in the expression of CYP1A1 isoenzymes was demonstrated by RT-PCR and western immunoblotting studies indicating an increase in the expression of CYP1A1 in blood lymphocytes after MC pretreatment. Significant inhibition in the EROD activity of MC induced lymphocyte by anti-CYP1A1/1A2 and alpha-naphthoflavone further provided evidence that the CYP1A1/1A2 isoenzymes are involved in the activity of EROD in blood lymphocytes. The data indicating similarities in the regulation of CYP1A1 in blood lymphocytes with the liver isoenzyme suggests that factors which may affect expression of CYP1A1 in liver may also affect expression in blood lymphocytes and that blood lymphocytes could be used as a surrogates for studying hepatic expression of the xenobiotic metabolising enzymes.     

A new method to purify hepatic CYP1A of Prochilodus scrofa, a Brazilian freshwater fish

Cytochromes P450 constitute a superfamily of the phase I enzymes whose primary task is the detoxification of both endogenous and xenobiotic compounds. Fish, among non-mammalian species, have received great interest because they are a direct food source for humans as well as conveyors of toxic chemicals to human beings. The aim of the present study was the purification of the hepatic isoform of CYP1A in Prochilodus scrofa (Prochilodontidae), a Brazilian fish, using only one chromatographic step. The purification of CYP1A was done by Reverse Phase HPLC on a C18 column. Purified CYP1A was characterized with respect to electrophoretic, immunochemical and biocatalyst properties. CYP1A fractions produced a single uniform band on SDS-PAGE with an apparent molecular mass of 58 kDa. Purified CYP1A of P. scrofa showed strong cross-reactivity with antibodies directed against CYP1A from trout. The fraction was also encapsulated in two different reconstituted systems; one composed of neutral lipids and another of negatively charged lipids. In both of them, we could detect EROD activity but not PROD activity, which confirms that the CYP1A was purified with all its enzyme activity. There was an increase of activity when CYP1A and NADPH cytochrome P450 (CYP) reductase were encapsulated in negatively charged lipids, which confirms that the charge of lipid is essential to CYP1A activity. All these characteristics strongly suggest that this new procedure is efficient for purifying hepatic CYP1A from P. scrofa, showing that the CYP1A isoform of this fish has a highly conserved protein region.     

Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.     

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.     

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist,Zanoterone

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6β-, 7α-, and 16α-hydroxytestosterone (6β-, 7α-, and 16α-OHT) and the de-alkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes form rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and β-naphthoflavone (β-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6β-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than that produced by PB or β-NF (⊆threefold increases). Z (high dose), PB, and β-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2–2.9-fold in females. β-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6β position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6β-OHT suggests that Z induced CYP 3A activity. Theses findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily. © 1997 John Wiley & Sons, Inc.     

Neurogenic-committed human pre-adipocytes express CYP1A isoforms

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of β-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants.     

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I(2)) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005 mg/kg Se, and I(2) deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T(4) (TT(4)), total T(3) (TT(3))], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I(2) deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I(2) and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I(2) status.     

Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.     

Modulation of drug-metabolizing enzymes by extracts of a herbal medicine Evodia rutaecarpa in C57BL/6J mice

Evodia rutaecarpa is a traditional Chinese medicine used for the treatment of gastrointestinal disorders and headache. To assess the possible drug interactions, effects of methanol and aqueous extracts of E. rutaecarpa on drug-metabolizing enzymes, cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. Treatment of mice with methanol extract by gastrogavage caused a dose-dependent increase of liver microsomal 7-ethoxyresorufin O-deethylation (EROD) activity. In liver, methanol extract at 2 g/kg caused 47%, 7-, 8-, 4-fold, 81% and 26% increases of benzo(a)pyrene hydroxylation (AHH), EROD, 7-methoxyresorufin O-demethylation (MROD), 7-ethoxycoumarin O-deethylation (ECOD), benzphetamine N-demethylation, and N-nitrosodimethylamine N-demethylation activities, respectively. Aqueous extract at 2 g/kg caused 68%, 2-fold, and 83% increases of EROD, MROD, and ECOD activities, respectively. For conjugation activities, methanol extract elevated UGT and GST activities. Aqueous extract elevated UGT activity without affecting GST activity. Immunoblot analyses showed that methanol extract increased the levels of CYP1A1, CYP1A2, CYP2B-, and GSTYb-immunoreactive proteins. Aqueous extract increased CYP1A2 protein level. In kidney, both extracts had no effects on AHH, ECOD, UGT, and GST activities. Three major bioactive alkaloids rutaecarpine, evodiamine, and dehydroevodiamine were present in both extracts. These alkaloids at 25 mg/kg increased hepatic EROD activity. These results demonstrated that E. rutaecarpa methanol and aqueous extracts could affect drug-metabolizing enzyme activities. Rutaecarpine, evodiamine, and dehydroevodiamine contributed at least in part to the increase of hepatic EROD activity by extracts of E. rutaecarpa. Thus, caution should be paid to the possible drug interactions of E. rutaecarpa and CYP substrates.     

Differential in vivo effects of α‐naphthoflavone and β‐naphthoflavone on CYP1A1 and CYP2E1 in rat liver,lung, heart,and kidney

Male Sprague–Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist β‐naphthoflavone (βNF), or the relatively weak AHR agonist α‐naphthoflavone (αNF). Animals treated with βNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with αNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by αNF treatment to levels comparable to that seen with βNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to βNF treatment, whereas αNF was without effect. Large increases of CYP1A1‐dependent 7‐ethoxyresorufin O‐deethylation (EROD) activity occurred with microsomes prepared from the tissues of βNF‐treated animals. Comparatively small changes were associated with αNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with βNF treatment. CYP2E1‐dependent p‐nitrophenol hydroxylase (PNP) activity was also increased by βNF treatment in microsomes prepared from kidney (3.1‐fold), whereas αNF was without effect. In contrast, αNF or βNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7‐pentoxyresorufin O‐deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that βNF and αNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR‐mediated toxicity. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 29–40, 1999     

Hepatic cytochrome P450 2A6 and 2E1 status in peri-tumor tissues of patients with Opisthorchis viverrini-associated cholangiocarcinoma

Endogenous nitrosation due to chronic inflammation is enhanced in opisthorchiasis and plays a crucial role in the development of cholangiocarcinoma (CCA). Hepatic cytochrome P450 (CYP) family enzymes, especially CYP2A6 and CYP2E1, are involved in the metabolism of procarcinogens; these two enzymes metabolize endogenous nitrosamines to carcinogenic N-dimethylnitrosamine (NDMA). CYP2A6 activity is increased in patients infected with Opisthorchis viverrini. Our aim was to determine whether the expression and function of CYP2A6 and 2E1 in the livers of patients with O. viverrini-associated cholangiocarcinoma (CCA) was altered compared to livers without CCA. Livers of CCA patients (n = 13 cases) showed increased enzyme activities, protein and mRNA levels of CYP2A6 whereas the enzyme activity and protein levels of CYP2E1 were markedly decreased (P < 0.05). CYP2E1 mRNA levels were not altered. Large numbers of inflammatory cells and increased iNOS expression was found in areas adjacent to the tumor. The data provide evidence to support the concept that enhanced CYP2A6 activity and diminished CYP2E1 activity probably involve to the progression of CCA.