P-nitrophenol Hydroxylation Research Articles

Effects of Water Miscible Organic Co-solvents on Enzyme Kinetic Parameters of Drug Metabolizing Enzymes in Rat Liver Microsomes

We have investigated the effects of methanol, acetonitrile, dimethyl sulphoxide or dioxane on enzyme kinetic parameters (Km and Vmax) of p-nitrophenol hydroxylation, phenacetin deethylation and metoprolol metabolism in rat liver microsomes, at different solvent concentrations. In the case of p-nitrophenol hydroxylation, methanol (0.1 %-0.75 % v/v) and dimethyl sulphoxide (0.01 %-0.075 % v/v) showed 1.3 and 2.5-fold increase respectively, in the Km with a less marked effect on the Vmax. In contrast, dimethyl sulphoxide (0.1 %-0.75% v/v) showed a 1.96-fold decrease in the Vmax. Unlike, methanol and dimethyl sulphoxide, acetonitrile showed activation of the p-nitrophenol hydroxylation activity as indicated by a 0.7-fold decrease in the Km. In the case of metoprolol metabolism, dioxane resulted in a 1.5 to 3.1-fold increase in the Km of all the three metabolite formation pathways in concentration dependent manner, with less marked effect on Vmax of the reaction. Interestingly, while acetonitrile did not affect the Km for metabolite 1 and 2 formation, the Km of metabolite 3 formation was decreased in a concentration dependent manner again with little effect on all the three Vmax values. Further, methanol had little to no effect on the Vmax of metabolite 1 and 2 formation while metabolite 3 formation was increased by 2-fold. The Km of all three metabolite formations was found to be decreased in presence of methanol. The phenacetin deethylation activity in rat liver microsomes followed atypical kinetics. Methanol and dimethyl sulphoxide did not affect the auto activation kinetics at concentration range studied. Dimethyl sulphoxide and dioxane appeared to be unsuitable for characterizing the Cytochrome450 mediated reactions because they showed a very significant effect on the Vmax/Km ratio, starting at concentrations of 0.025 % v/v and 0.25 % v/v, respectively. Methanol and acetonitrile at concentration <0.5 % v/v appeared to be acceptable solvents for solubilization of substrates metabolized by Cytochrome450s.

Effects of water-soluble organic co-solvents on the metabolic kinetics of drugs in rat liver microsomes

The aim of this research was to explore how different water-miscible organic co-solvents, such as methanol, acetonitrile, dimethyl sulphoxide, and dioxane, influence the kinetic characteristics of enzymes in rat liver microsomes that metabolize drugs. Our study revealed that the impact of these co-solvents on enzyme activity was highly dependent on the specific metabolic route involved and the concentration of the co-solvent used. Methanol and dimethyl sulphoxide showed a notable increase in the Km of p-nitrophenol hydroxylation, while acetonitrile showed activation of this metabolic pathway. Interestingly, dioxane resulted in a significant increase in the Km of metoprolol metabolism, while acetonitrile decreased the Km of metabolite 3 formation. In addition, our study findings revealed that the Vmax/Km ratio was significantly affected by dimethyl sulphoxide and dioxane, suggesting their unsuitability for characterizing Cytochrome450 mediated reactions. On the other hand, methanol and acetonitrile were found to be suitable solvents for solubilizing substances that Cytochrome 450s metabolise, as long as their concentration remained below 0.5% v/v. Overall, our study sheds light on the potential effects of chemical co-solvents that are water soluble on enzyme kinetics and highlights the importance of carefully selecting appropriate solvents to study the in vitro metabolism of drugs.

In vitro inhibition of human CYP2E1 and CYP3A by quercetin and myricetin in hepatic microsomes is not gender dependent.

This is the first in vitro study to investigate gender-related differences in the regulation of human cytochrome P450 by the flavonoids. Activities of CYP2E1 and CYP3A were measured in the presence of quercetin, myricetin, or isorhamnetin in hepatic microsomal pools from male and female donors. Hydroxylation of p-nitrophenol (PNPH) was measured to determine CYP2E1 activity, and O-dealkylation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was measured to determine CYP3A activity. Quercetin, but not myricetin or isorhamnetin, competitively inhibited PNPH activity in human recombinant cDNA-expressed CYP2E1 with the Ki=52.1±6.31μM. In the human microsomes, slight inhibition of PNPH activity by quercetin was not considered as physiologically relevant. Quercetin inhibited BFC activity in human recombinant cDNA-expressed CYP3A4 competitively with the Ki=15.4±1.52μM, and myricetin - noncompetitively with the Ki=74.6±7.99μM. The degree of inhibition by quercetin was similar between genders. Myricetin showed somewhat stronger inhibition in female pools, but the Ki values were higher than physiologically relevant concentrations. Isorhamnetin did not affect either PNPH or BFC activity. We concluded that observed inhibition of CYP2E1 and CYP3A by some flavonols were not gender-dependent.

In Silico Approach to Support that p-Nitrophenol Monooxygenase from Arthrobacter sp. Strain JS443 Catalyzes the Initial Two Sequential Monooxygenations.

p-Nitrophenol (PNP), used primarily for manufacturing pesticides and dyes, has been recognized as a priority environmental pollutant. It is therefore important to reduce the input of this toxicant into the environment and to establish approaches for its removal from the contaminated sites. PNP monooxygenase, a novel enzyme from Gram-positive bacteria like Arthrobacter sp. and Bacillus sp., that comprises two components, a flavoprotein reductase and an oxygenase, catalyzes the initial two sequential monooxygenations to convert PNP to trihydroxybenzene. Accurate and reliable prediction of this enzyme-substrate interactions and binding affinity are of vital importance in understanding these catalytic mechanisms of the two sequential reactions. As crystal structure of the enzyme has not yet been published, we built a homology model for PNP monooxygenase using crystallized chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100 (3HWC) as the template. The model was assessed for its reliability using PROCHECK, ERRAT and ProSA. Molecular docking of the physiological substrates, PNP and 4-nitrocatechol (4-NC), was carried out using Glide v5.7 implemented in Maestro v9.2, and the binding energies were calculated to substantiate the prediction. Docking complexes formed by molecular level interactions of PNP monooxygenase-PNP/4-NC without or with the cofactors, FAD and NADH, showed good correlation with the established experimental evidence that the two-component PNP monooxygenase catalyzes both the hydroxylation of PNP and the oxidative release of nitrite from 4-NC in B. sphaericus JS905. Furthermore, molecular dynamics simulations performed for docking complexes using Desmond v3.0 showed stable nature of the interactions as well.

In silico approach to support that p-nitrophenol monooxygenase from Arthrobacter sp. strain JS443 catalyzes the initial two sequential monooxygenations.

p-Nitrophenol (PNP), used primarily for manufacturing pesticides and dyes, has been recognized as a priority environmental pollutant. It is therefore important to reduce the input of this toxicant into the environment and to establish approaches for its removal from the contaminated sites. PNP monooxygenase, a novel enzyme from Gram-positive bacteria like Arthrobacter sp. and Bacillus sp., that comprises two components, a flavoprotein reductase and an oxygenase, catalyzes the initial two sequential monooxygenations to convert PNP to trihydroxybenzene. Accurate and reliable prediction of this enzyme-substrate interactions and binding affinity are of vital importance in understanding these catalytic mechanisms of the two sequential reactions. As crystal structure of the enzyme has not yet been published, we built a homology model for PNP monooxygenase using crystallized chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100 (3HWC) as the template. The model was assessed for its reliability using PROCHECK, ERRAT, WHATCHECK and ProSA. Molecular docking of the physiological substrates, PNP and 4-nitrocatechol (4-NC), was carried out using Glide v5.7 implemented in Maestro v9.2, and the binding energies were calculated to substantiate the prediction. Docking complexes formed by molecular level interactions of PNP monooxygenase-PNP/4-NC without or with the cofactors, FAD and NADH, showed good correlation with the established experimental evidence that the two-component PNP monooxygenase catalyzes both the hydroxylation of PNP and the oxidative release of nitrite from 4-NC in B. sphaericus JS905. Furthermore, molecular dynamics simulations performed for docking complexes using Desmond v3.0 showed stable nature of the interactions as well.

Interactions between CYP2E1 and CYP2B4: Effects on Affinity for NADPH-Cytochrome P450 Reductase and Substrate Metabolism

Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent K(M) and k(cat) of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR increased up to 23-fold with virtually no change in the k(cat) for the reaction, however, at higher concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the k(cat) also decreased by 11-fold. Additionally, CYP2E1 increased the apparent K(M) of CYP2B4 for BNZ by 8-fold and the apparent K(M) did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent K(M) values of CYP2B4 and CYP2E1 for CPR are similar, the apparent K(M) of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR.

Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevanceKaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer. Aim of the studyIn order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated. Materials and methodsThe impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively. ResultsIn vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of Vmax (15.276±0.206nmol/min) and lowest of Ki value (0.008±0.002μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low Km value (1.599±0.147pmol). For in vivo studies, mice were orally treated with 250mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity. ConclusionsKaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.

Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270μgl−1) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Modulation of cytochrome P450 and induction of DNA damage in Cyprinus carpio exposed in situ to surface water treated with chlorine or alternative disinfectants in different seasons

Epidemiological studies have shown an association between consumption of disinfected drinking water and adverse health outcomes. The chemicals used to disinfect water react with occurring organic matter and anthropogenic contaminants in the source water, resulting in the formation of disinfection by-products (DBPs). The observations that some DBPs are carcinogenic in animal models have raised public concern over the possible adverse health effects for humans. Here, the modulation of liver cytochrome P450-linked monooxygenases (MFO) and the genotoxic effects in erythrocytes of Cyprinus carpio fish exposed in situ to surface drinking water in the presence of disinfectants, such as sodium hypochlorite (NaClO), chlorine dioxide (ClO2) and peracetic acid (PAA), were investigated in winter and summer. A complex induction/suppression pattern of CYP-associated MFOs in winter was observed for all disinfectants. For example, a 3.4- to 15-fold increase was recorded of the CYP2B1/2-linked dealkylation of penthoxyresorufin with NaClO (10 days) and PAA (20 days). In contrast, ClO2 generated the most notable inactivation, the CYP2E1-supported hydroxylation of p-nitrophenol being decreased up to 71% after 10 days’ treatment. In summer, the degree of modulation was modest, with the exception of CYP3A1/2 and CYP1A1 supported MFOs (62% loss after 20 days PAA).The micronucleus (MN) induction in fish circulating erythrocytes was also analysed as an endpoint of genotoxic potential in the same fish population. Significant increases of MN induction were detected at the latest sampling time on fish exposed to surface water treated with chlorinate-disinfectants, both in winter (NaClO) and summer (NaClO and ClO2), while no effect was observed in fish exposed to PAA-treated water.These results show that water disinfection may be responsible for harmful outcomes in terms of MFO perturbation and DNA damage; if extrapolated to humans, they ultimately offer a possible rationale for the increased urinary cancer risk recorded in regular drinking water consumers.

Para-nitrophenol hydroxylation by fish liver microsomes: kinetics and effect of selective cytochrome P450 inhibitors

The study investigated the kinetics of p-nitrophenol hydroxylase (PNPH) in hepatic microsomes obtained from Atlantic salmon (Salmo salar). The selective inhibitors for some major mammalian cytochrome P450 (CYP450) were used to investigate the potential inhibitory effect on enzymes involved in p-nitrophenol hydroxylation. The following inhibitors were used: α-naphtoflavone (CYP1A), ellipticine (CYP1A1), furafylline (CYP1A2), 8-methoxypsoralen (8MOP, CYP2A6), 4-methylpyrazole (4MP, CYP2A6/2E1), diallyl sulfide (DAS, CYP2E1), and ketoconazole (CYP3A4). Additionally, the natural steroids 17-beta-oestraiol (E2) and testosterone were investigated as potential inhibitors of PNPH activity. It was found that formation of 4-nitrocatechol from p-nitrophenol followed monophasic kinetics with K(m) = 0.17 ± 0.03 mM and V(max) = 21.8 ± 1.05 pmol/min/mg. PNPH activity was competitively inhibited by diallyle sulfide with the K(i) value of 285.1 ± 94.2 μM μM and uncompetitively by ellipticine with K(i) value of 65.7 ± 7.8 μM. Moreover, E2 showed an ability to reduce PNPH activity through the mechanism-based inhibition mode. Our results suggest that hepatic microsomes from Atlantic salmon possess CYP2E1-like activity. However, specific isoform-mediated PNPH activity should be identified.

Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+

Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

Aim Some carotenoids such as canthaxantin, astaxanthin and beta apo-8'€™-carotenal were reported to have modulatory effect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1. Methods Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation) were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test. Results Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase) was significantly reduced by repeated administration of 100mg/ kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot). Conclusion The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase) in the rat. (Med J Indones 2009; 18: 233-8)

Effect of taurine supplementation on cytochrome P450 2E1 and oxidative stress in the liver and kidneys of rats with streptozotocin-induced diabetes

To investigate whether diabetes-induced alterations of CYP2E1 and oxidative stress can be modulated by dietary taurine supplementation, male Wistar rats were divided into non-diabetic, diabetic, and diabetic taurine-supplemented groups (administered at 2% in the drinking water). Increased levels of CYP2E1-catalyzed p-nitrophenol hydroxylation were found in liver and kidney microsomes of rats with STZ-induced diabetes compared to those of non-diabetic control rats. Immunoblot and RT-PCR analyses of CYP2E1 protein and mRNA levels in the liver and kidneys showed the same trend as with enzyme activities. Taurine supplementation significantly decreased the enzyme activity and expression (protein and mRNA) of CYP2E1 in diabetic rat kidneys. Plasma β-hydroxybutyrate concentration was significantly reduced in taurine-treated diabetic rats. The induction of heme oxygenase-1 mRNA was suppressed by taurine treatment in diabetic rat kidneys. An increase in reduced glutathione (GSH) and a higher ratio of reduced to oxidized glutathione (GSH/GSSG) together with lower values of thiobarbituric acid-reactive substances (TBARS) were observed in the kidneys of taurine-treated diabetic rats. However, taurine supplementation caused only a slight or insignificant effect on these alternations in the liver of diabetic rats. Our results show dietary taurine may reduce CYP2E1 expression and activity, and oxidative stress in kidneys of diabetic rats.

Differential inhibition of rat and human hepatic cytochrome P450 by Andrographis paniculata extract and andrographolide

The inhibitory effect of Andrographis paniculata extract (APE) and andrographolide (AND), the most medicinally active phytochemical in the extract, on hepatic cytochrome P450s (CYPs) activities was examined using rat and human liver microsomes. For this purpose, CYP1A2-dependent ethoxyresorufin- O-deethylation, CYP2B1-dependent benzyloxyresorufin- O-dealkylation, CYP2B6-dependent bupropion hydroxylation, CYP2C-dependent tolbutamide hydroxylation, CYP2E1-dependent p-nitrophenol hydroxylation and CYP3A-dependent testosterone 6β-hydroxylation activities, were determined in the presence and absence of APE or AND (0–200 μM). APE inhibited ethoxyresorufin- O-deethylation activity in rat and human liver microsomes, with apparent K i values of 8.85 and 24.46 μM, respectively. In each case, the mode of inhibition was noncompetitive. APE also inhibited tolbutamide hydroxylation both in rat and human microsomes with apparent K i values of 8.21 and 7.51 μM, respectively and the mode of inhibition was mixed type. In addition, APE showed a competitive inhibition only on CYP3A4 in human microsomes with K i of 25.43 μM. AND was found to be a weak inhibitor of rat CYP2E1 with a K i of 61.1 μM but did not affect human CYP2E1. In conclusion, it cannot be excluded from the present study that APE could cause drug–drug interactions in humans through CYP3A and 2C9 inhibition.

The inhibitory effect of tannic acid on cytochrome P450 enzymes and NADPH-CYP reductase in rat and human liver microsomes

Tannic acid has been shown to decrease mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. The purpose of this study was to evaluate the effect of tannic acid on cytochrome P450 (CYP)-catalyzed oxidations using rat liver microsomes (RLM) and human liver microsomes (HLM) as the enzyme sources. In RLM, tannic acid showed a non-selective inhibitory effect on 7-methoxyresorufin O-demethylation (MROD), 7-ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation, p-nitrophenol hydroxylation and testosterone 6β-hydroxylation activities with IC 50 values ranged from 14.9 to 27.4 μM. In HLM, tannic acid inhibited EROD, MROD and phenacetin O-deethylation activities with IC 50 values ranged from 5.1 to 7.5 μM, and diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and testosterone 6β-hydroxylation with IC 50 values ranged from 20 to 77 μM. In baculovirus-insect cell-expressed human CYP 1A1 and 1A2, the IC 50 values of tannic acid for CYP 1A1- and 1A2-catalyzed EROD activities were 23.1 and 2.3 μM, respectively, indicating that tannic acid preferably inhibited the activity of CYP1A2. Tannic acid inhibited human CYP1A2 non-competitively with a Ki value of 4.8 μM. Tannic acid was also found to inhibit NADPH-CYP reductase in RLM and HLM with IC 50 values of 11.8 and 17.4 μM, respectively. These results suggested that the inhibition of CYP enzyme activities by tannic acid may be partially attributed to its inhibition of NADPH-CYP reductase activity.

Is Bupropion a More Specific Substrate for Porcine CYP2E than Chlorzoxazone and p‐Nitrophenol?

Porcine microsomes are able to hydroxylate chlorzoxazone and p-nitrophenol, the most commonly used human test substrates for CYP2E1. However, in pigs, CYP2E appears not to be the only enzyme involved in the hydroxylation of chlorzoxazone and p-nitrophenol, as the enzyme capacity and immunochemical level of the apoprotein do not correlate. The present study shows that the hydroxylation of chlorzoxazone and p-nitrophenol is inhibited 50-65% by anti-human CYP2A6, suggesting that these substrates are metabolized almost equally well by CYP2A and CYP2E in pigs. To find an alternative probe to porcine CYP2E, bupropion, another human substrate, was examined. Incubation with bupropion concentrations ranging from 0.05 to 20 mM and with various inhibitors revealed that this substrate is metabolized by both CYP2A and CYP2E. At the high substrate concentration (5 mM), however, the CYP2A6 inhibition decreased compared to inhibition percentages found using the low substrate concentration (0.5 mM). The opposite was found for CYP2E, as inhibition studies with antibodies and diethyldithiocarbamate indicate that it catalysed a negligible part of the reaction at the low substrate concentration and up to 84% at the high concentration. Thus, hydroxylation of bupropion follows the same pattern in pigs as in human beings and the activity measured in pigs is comparable with the human counterpart. Furthermore, bupropion is a more specific substrate for CYP2E than chlorzoxazone and p-nitrophenol although not perfect.

Protective effects of isothiocyanates alone or in combination with vitamin C towards N‐nitrosodibutylamine or N‐nitrosopiperidine‐induced oxidative DNA damage in the single‐cell gel electrophoresis (SCGE)/HepG2 assay

The aim of this study was to investigate the protective effect of isothiocyanates alone or in combination with vitamin C towards N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP)-induced oxidative DNA damage in the single cell gel electrophoresis (SCGE)/HepG2 assay. Phenethyl isothiocyanate (PEITC) and indole-3-carbinol (I3C) alone showed a weak protective effect towards NDBA (0.1 microm, 26-27%, respectively) or NPIP (1 microm, 26-28%, respectively)-induced oxidative DNA damage. Allyl isothiocyanate (AITC) alone did not attenuate the genotoxic effect provoked by NDBA or NPIP. In contrast, HepG2 cells simultaneously treated with PEITC, I3C and AITC in combination with vitamin C showed a stronger inhibition of oxidative DNA-damage induced by NDBA (0.1 microm, 67%, 42%, 32%, respectively) or NPIP (1 microm, 50%, 73%, 63%, respectively) than isothiocyanates (ITCs) alone. One feasible mechanism by which ITCs alone or in combination with vitamin C exert their protective effects towards N-nitrosamine-induced oxidative DNA damage could be by the inhibition of their cytochrome P450 dependent bioactivation. PEITC and I3C strongly inhibited the p-nitrophenol hydroxylation (CYP2E1) activity (0.1 microm, 66-50%, respectively), while the coumarin hydroxylase (CYP2A6) activity was slightly reduced (0.1 microm, 25-37%, respectively). However, the ethoxyresorufin O-deethylation (CYP1A1) activity was only inhibited by PEITC (1 microm, 55%). The results indicate that PEITC and I3C alone or PEITC, I3C and AITC in combination with vitamin C protects human-derived cells against the oxidative DNA damaging effects of NDBA and NPIP, two food carcinogenic compounds.

Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119-131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography-mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The KM values calculated for these compounds were in the millimolar range: 1.21+/-0.07 mM for chlorzoxazone, 2.52 +/- 0.08 mM for aniline, 0.81+/-0.04 mM for propranolol. The values of vmax for chlorzoxazone and propranolol were 46.0+/-9.0 and 7.6+/-3.4 nmol min-1 nmol-1, respectively. These values are higher then those measured for the human enzymes. The vmax value for aniline was 9.4+/-1.3 nmol min-1 nmol-1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1–2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO 2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2β-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO 2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO 2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.

Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes

The testicular steroids androstenone (A), 17β-oestradiol (E2) and testosterone (T) were tested for their ability to alter CYP2E1 and CYP2A activity in porcine liver microsomes from male and female pigs. This is the first in vitro study indicating that sex steroids have a potential to modify microsomal CYP2E1 activity, the main skatole-metabolising enzyme. A and E2 exerted an inhibitory effect on CYP2E1 mediated hydroxylation of p-nitrophenol to p-nitrocatechol although the mechanism of this inhibition differed for these steroids. The inhibitory effect of A on CYP2E1, as determined by kinetic analysis, might be due to the competitive binding of A and p-nitrophenol to the same site of CYP2E1. Including E2 into the incubations resulted in decreased activities of CYP2E1 in male microsomes through a mixed mode of inhibition. Including pre-incubation steps eliminated this inhibition in male microsomes, and resulted in increased CYP2E1 activities in the microsomes from female pigs. Testosterone was ineffective as an inhibitor of either CYP2E1 or CYP2A activities. Overall, our findings indicate that A and E2 have the potential to modify the catalytic activities of porcine CYP2E1 in vitro. However, the significance of this modification for skatole metabolism in vivo is questionable.