O-demethylation Of P-nitroanisole Research Articles

Heterologous Co-expression of CYP6B7 and NADPH-Dependent Cytochrome P450 Reductase From Helicoverpa armigera (Lepidoptera: Noctuidae) in Pichia pastoris.

As important metabolic enzymes, the function of cytochrome P450 monooxygenases (CYPs) has been demonstrated repeatedly through various means, including heterologous expression systems. Unfortunately, most model systems typically lack expression of a conspecific NADPH-dependent cytochrome P450 reductase (CPR), which is the electron transfer partner of CYPs. As a result, the activities of heterologously expressed insect CYPs may not accurately reflect detoxification activities in vivo. Previously, CYP6B7 from Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) (HaCYP6B7) has been expressed in the Pichia pastoris GS115 strain and shown to detoxify bifenthrin, fenvalerate and chlorpyrifos. However, it remains to be determined if co-expression of HaCYP6B7 with HaCPR will enhance the detoxification ability of the expression system. In the present study, HaCYP6B7 and HaCPR genes were co-expressed in P. pastoris using a reconstituted expression vector, pPICZA-HaCYP6B7-HaCPR. Protein expression was confirmed by Western blot, and the detoxification activities of microsomal fractions to p-nitroanisole O-demethylation (PNOD), 7-ethoxycoumarin O-deethylation (ECOD), fenvalerate and chlorpyrifos were measured. Co-expression of HaCYP6B7 with HaCPR resulted in PNOD and ECOD activities of 1.90 nmol/min/mg·protein and 12.39 pmol/min/mg·protein, which were 1.6- and 1.5-fold of that catalyzed by HaCYP6B7 expressed alone, respectively. Furthermore, microsomes of pPICZA-HaCYP6B7-HaCPR-GS115 had higher detoxification activity than that of pPICZA-HaCYP6B7-GS115 to fenvalerate, but not chlorpyrifos. The results indicated that co-expression of HaCYP6B7 with conspecific CPR could enhance the detoxification activities to some substrates comparing with expression of HaCYP6B7 alone.

Heterologous expression of Helicoverpa armigera cytochrome P450 CYP6B7 in Pichia pastoris and interactions of CYP6B7 with insecticides.

Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot. Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively. The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry.

Melatonin protects the cytochrome P450 system through a novel antioxidant mechanism

Melatonin, an endogenous hormone, is used as an antioxidant drug in doses quite higher than the endogenous circulating levels of this hormone. Hepatic endoplasmic reticulum contains the cytochrome P450 (CYP450) system, which catalyzes one biotransformation pathway of melatonin; this organelle is also one of the main sources of reactive oxygen species in cells. Therefore, we proposed that the antioxidant activity of this hormone may have a biological relevance in the organelle where it is biotransformed. To evaluate this postulate, we used Fe(3+)/ascorbate, an oxygen free radical generating system that leads to lipid peroxidation, loss of protein-thiol content, and activation of UDP-glucuronyltransferase in rat liver microsomes. We found that mM concentrations of melatonin prevented all these oxidative phenomena. We also found that Fe(3+)/ascorbate leads to structural alterations in the CYP450 monooxygenase, the enzyme that binds the substrate in the CYP450 system catalytic cycle, probably through direct oxidation of the protein, and also inhibited p-nitroanisole O-demethylation, a reaction catalyzed by the CYP450 system. Notably, melatonin prevented both phenomena at microM concentrations. We provide evidence suggesting that melatonin may be oxidized by oxygen free radicals. Thus, we postulate that melatonin may be acting as an oxygen free radical scavenger, and Fe(3+)/ascorbate-modified melatonin would be directly protecting the CYP450 system through an additional specific mechanism. Pharmacological relevance of this phenomenon is discussed.

High Performance Liquid Chromatography Assay for p-Nitroanisole o-Demethylation by Cytochrome P450 Enzymes in Musca domestica L.

Abstract A novel sensitive method was developed for measuring p -nitroanisole o -demethylation (PNOD) catalyzed by cytochrome P450 enzymes in crude homogenates of housefly, Musca domestica L., using high performance liquid chromatography (HPLC) with diode array detector. This method is based on the use of liquid-liquid extraction and HPLC with reversed-phase C 18 column and gradient ultraviolet detection at 316 nm. The detection limit ( S/N of 3/1) of p -nitrophenol ( p NP) was 0.1 ng. The relative standard deviation (RSD) of peak area response of the HPLC analysis system was 1.0%. The average recoveries of 0.351, 1.755 and 8.755 μg of p NP added to incubation mixtures ranged from 92.3% to 87.6%, and the RSD of recoveries of three added levels ranged 2.64%–5.90%. The relationships between peak areas and concentration were linear in the range of 9.72–486 ng with good coefficient of determination ( r 2 = 0.9995). This method is sensitive, precise, and reproducible and is also applicable to the assay of p -nitroanisole o -demethylases, which have low catalytic activities, present in other insects or organisms. With this method, the comparison of P450 o -demethylation activity of an imidacloprid-resistant strain with a susceptible strain of M. domestica (L.) was conducted to identify the roles of P450 enzymes involved in housefly resistance to imidacloprid. The results indicated that the resistance mechanism of housefly to imidacloprid was intensely assisted with elevated P450 activities.

Essential Role of Singlet Oxygen Species in Cytochrome P450-dependent Substrate Oxygenation by Rat Liver Microsomes

Previously, we reported that singlet oxygen (1O2) was involved in rat liver microsomal P450-dependent substrate oxygenations in such reactions as p-hydroxylation of aniline, O-deethylation of 7-ethoxycoumarin, omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine. In order to confirm the generality of 1O2 involvement, we have further investigated which kinds of reactive oxygen species (ROS) are formed during P450-dependent substrate oxygenation in microsomes. We examined CYP2E1-dependent hydroxylation of p-nitrophenol in rat liver microsomes in the presence of some ROS scavengers, because CYP2E1 has been reported to predominantly generate ROS in the hepatic microsomes and to relate with the oxidative stress in the body. The addition of 1O2 quenchers, beta-carotene, suppressed the hydroxylation of p-nitrophenol. Furthermore, a nonspecific P450 inhibitor, SKF525A, and a ferric chelator, deferoxamine, both suppressed the hydroxylation. No other ROS scavengers such as superoxide dismutase (SOD), catalase, or mannitol altered the reaction. 1O2 was detectable during the reaction in the microsomes as measured by an electron spin resonance (ESR) spin-trapping method when 2,2,6,6-tetramethyl-4-piperidone (TMPD) was used as a spin-trapping reagent. The 1O2 was quenched by additions of beta-carotene, p-nitrophenol, and SKF525A. The reactivity of p-nitrophenol and 1O2 correlated linearly with its hydroxylation rate in the microsomes. On the basis of these results, we conclude that 1O2 contributes to the p-nitrophenol hydroxylation in rat liver microsomes, by adding a new example of 1O2 involvement in the CYP2E1-dependent substrate oxygenations.

Liver microsomal biotransformation of nitro‐aryl drugs: mechanism for potential oxidative stress induction

Toxic effects of several nitro-aryl drugs are attributed to the nitro-reduction that may be suffered in vivo, a reaction that may be catalysed by different reductases. One of these enzymes is NADPH-cytochrome P450 reductase, which belongs to the cytochrome P450 oxidative system mainly localized in the endoplasmic reticulum of the hepatic cell. This system is responsible for the biotransformation of oxidative lipophilic compounds, so that oxidative and reductive metabolic pathways of lipophilic nitro-aryl drugs can take place simultaneously. Because of the affinity of nitro-aryl drugs (xenobiotics) for the endoplasmic reticulum, we propose this subcellular organelle as a good biological system for investigating the toxicity induced by the biotransformation of these or another compounds. In this work we used rat liver microsomes to assess the oxidative stress induced by nitro-aryl drug biotransformation. Incubation of microsomes of rat liver with nifurtimox and nitrofurantoin in the presence of NADPH induced lipoperoxidation, UDP-glucuronyltransferase activation and an increase in the basal microsomal oxygen consumption. Nitro-aryl-1,4-dihydropyridines did not elicit these prooxidant effects; furthermore, they inhibited lipoperoxidation and oxygen consumption induced by Fe3+/ascorbate. Nifurtimox and nitrofurantoin modified the maximum absorption of cytochrome P450 oxidase and inhibited p-nitroanisole O-demethylation, an oxidative reaction catalysed by the cytochrome P450 system, signifying that oxidation may proceed in a similar way to that described for nitro-aryl-1,4-dihydropyridines. Thus the balance between lipophilic nitro-aryl drug oxidation and reduction may be involved in the potential oxidative stress induced by biotransformation.

Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera: Noctuidae): tissue difference and induction.

Differences in the microsomal P450 monooxygenase system and its inducibility by pentamethylbenzene (PMB) and naphathalene (NA) were investigated in midgut and fatbody tissues of the cotton bollworm, Helicoverpa armigera (Boddie), larvae. Orthogonal array design was used to establish the optimal conditions for measuring Aldrin epoxidation (AE). The optima for AE were similar for the midgut and the fatbody at a temperature of 30 degrees C, pH 7.4, and a time of 10 min. In comparison to fatbody, the midgut had higher levels of total cytochrome P450s, p-nitroanisole O-demethylation (ODM) and AE. In vivo administration of 0.2% PMB or 0.2% NA resulted in higher microsomal protein content and levels of total cytochrome P450 as well as the two examined monooxygenase activities. Total cytochrome P450 and ODM activity were induced to a greater degree in the fatbody. In the midgut, PMB was significantly more effective on ODM than NA. Differences existed in SDS-PAGE profiles between the midgut and the fatbody. The induction of the midgut with PMB and of the fatbody with NA and PMB resulted in marked intensification of the protein bands with molecular masses of 59,100, 53,400, 50,400 Da.

Evidence for Singlet Oxygen Involvement in Rat and Human Cytochrome P450-dependent Substrate Oxidations

Recently, we proposed that singlet oxygen ((1)O(2)) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of (1)O(2) is general in the P450-dependent substrate oxidations. In the present study, we examined omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of beta-carotene and NaN(3) significantly suppressed these reactions in a concentration-dependent manner, and (1)O(2) during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a (1)O(2)-spin trapping reagent, where the addition of (1)O(2) quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-(1)O(2) adduct. Next, we examined the effect of (1)O(2) quenchers on P450-dependent reactions in the human liver microsomes, and (1)O(2) was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that (1)O(2) is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.

Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism

Three inhibitory monoclonal antibodies specific to cytochrome P450 3A4/5 (CYP3A4/5), CYP2C8/9/19 and CYP2E1, respectively, were used to assess the contribution of the P450s to the metabolism of seven substrates in liver microsomes from 18 human donors, as measured by monoclonal antibody inhibition phenotyping of the substrate conversion to product(s). Metabolism of seven substrates by recombinant cytochromes P450 and human liver microsomes was performed in the presence of monoclonal antibodies and their metabolites were analyzed by high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrophotometry (GC-MS) to measure the magnitude of inhibition. Our results showed that CYP3A4/5 contributes to testosterone 6β-hydroxylation, taxol phenol formation, diazepam 3-hydroxylation, diazepam N-demethylation, and aflatoxin B1 3-hydroxylation in human liver by 79.2%, 81.5%, 73.2%, 34.5% and 80%, respectively. CYP2E1 contributes to chlorzoxazone 6-hydroxylation, p-nitroanisole O-demethylation, and toluene hydroxylation by 45.8%, 27.7% and 44.2% respectively, and CYP2C8/9/19 contribute to diazepam N-demethylation by 30.6%. The additive contribution (75.3%) of human CYP3A and CYP2C to diazepam N-demethylation was also observed in the presence of both anti-CYP3A4/5 and anti-CYP2C8/9/19 monoclonal antibodies. The contribution of individual P450s to the specific metabolic reaction in human liver varies greatly in the individual donors and the substrates examined. Thus, inhibitory monoclonal antibodies could play a unique role in defining the single or subfamily of cytochrome P450 that is responsible for the metabolism of specific drugs.

Autonomous regulation of prostaglandin production in placenta and fetal membranes

Recently, we proposed that singlet oxygen (1O2) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of 1O2 is general in the P450-dependent substrate oxidations. In the present study, we examined ω- and (ω-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of β-carotene and NaN3 significantly suppressed these reactions in a concentration-dependent manner, and 1O2 during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a 1O2-spin trapping reagent, where the addition of 1O2 quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-1O2 adduct. Next, we examined the effect of 1O2 quenchers on P450-dependent reactions in the human liver microsomes, and 1O2 was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that 1O2 is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.

Food Restriction Stimulates Conjugation of P-Nitrophenol in Perfused Rat Liver

Rates of conjugation of p-nitrophenol were studied in livers from normal and food-restricted rats perfused with either p-nitroanisole or p-nitrophenol. Female Sprague-Dawley rats had ad libitum access to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a nonrecirculating system. Maximal rates of conjugation of p-nitrophenol, generated either from the O-demethylation of p-nitroanisole (200 μM) or from the infusion of p-nitrophenol (70 μM), were elevated significantly nearly twofold by food restriction. Thus, food restriction stimulates conjugation in the intact liver cell. Specifically, rates of conjugation were increased from 2.1 ± 0.2 to 3.7 ± 0.4 and from 3.3 ± 0.6 to 5.8 ± 0.5 μmol/g/h when 200 μM p-nitroanisole or 70 μM p-nitrophenol were infused, respectively. On the other hand, rates of conjugation were not affected by food restriction when low concentrations of p-nitroanisole (50 μM) or p-nitrophenol (20 μM) were infused. Further, food restriction did not alter rates of conjugation in isolated microsomes supplemented with excess UDPGA. Interestingly, both UDP-glucose and UDP-glucuronic acid were increased significantly in liver extracts from food-restricted rats when livers were perfused with high but not low concentrations of p-nitrophenol. Under these conditions, the increase in UDP-glucuronic acid was threefold. Moreover, food restriction increased carbohydrate release from the liver about twofold. Glycogen content was also increased significantly in liver extracts from 8.4 ± 1.9 to 60.4 ± 13.8 mmol/kg wet weight by food restriction. Taken together, these data support the hypothesis that food restriction stimulates conjugation of p-nitrophenol concentrations by increasing the supply of the pivotal cofactor UDP-glucuronic acid from carbohydrate reserves (e.g., glycogen).

Inhibition of Insect Cytochromes P450 by Furanocoumarins

Coumarin, benzofuran, and 16 furanocoumarins were evaluated as inhibitors of Manduca sexta midgut cytochrome P450-catalyzed O-demethylation of p-nitroanisole. 8- O-Alkylpsoralens with nonpolar alkyl groups of five or fewer carbons had the lowest I 50 values. I 50 values of 5- O-alkylpsoralens increased with the size of the O-alkyl group and were greater than psoralen. Psoralens with polar O-alkyl groups such as alcohols had high I 50 values. Psoralens with substituents on the furan ring were not inhibitory. Coumarin, which lacks a furan ring, was a reversible inhibitor with an I 50 value two orders of magnitude higher than psoralen. Benzofuran which lacks the pyrone ring was a weak mechanism-based inhibitor. The angular furanocoumarin angelicin had an I 50 value comparable to 8- O-alkylpsoralens. All of the inhibitory furanocoumarins tested were mechanism-based irreversible inhibitors. The structure-activity results are consistent with a proposed mechanism of inhibition whereby the furanocoumarin is oxidized by cytochrome P450 at the double bond of the furan ring forming an unstable epoxide that can react with cytochrome P450. [ 3H]Xanthotoxin labels numerous proteins when incubated with microsomes in the presence of NADPH. Although a P450-containing protein fraction separated by chromatography has the highest tritium/protein ratio, the lack of specificity limits the usefulness of radiolabeling with furanocoumarins for identification of cytochromes P450.

Food restriction and stimulation of monooxygenation of p-nitroanisole in perfused rat liver

This study assessed the effects of food restriction on the metabolism of model monooxygenase substrates in the perfused rat liver. Female Sprague-Dawley rats had access ad lib. to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a non-recirculating system, and the rates of monooxygenation of p-nitroanisole and 7-ethoxycoumarin were measured. The results indicate that food restriction stimulated p-nitroanisole O-demethylation from 2.9 ± 0.2 to 4.6 ± 0.5 μmol/(g.hr) when saturating concentrations of p-nitroanisole were infused. Concomitantly, the ratio of β-hydroxybutyrate to acetoacetate (B/A) and the rates of ketogenesis (B + A) were increased significantly by food restriction. Further, p-nitroanisole (200 μmnol/L) increased hepatic malate concentration nearly 3-fold in liver extracts from food-restricted rats. However, infusion of either a low concentration of p-nitroanisole (50 μmol/L) or 7-ethoxycoumarin (200 μmol/L) did not alter these parameters. On the other hand, food restriction did not alter rates of monooxygenation in isolated microsomes supplemented with excess NADPH. Taken together, these data support the hypothesis that high concentrations of p-nitroanisole increased monooxygenation in food-restricted rats by stimulating fatty acid oxidation, which elevates the mitochondrial NADH/NAD + ratio. This, in turn, increases the availability of reducing equivalents in the form of NADPH by a malate-pyruvate exchange system, leading to increased drug metabolism.

Polysubstrate Monooxygenases and Other Xenobiotic-Metabolizing Enzymes in Susceptible and Resistant Colorado Potato Beetle

The activities of key defensive enzymes against xenobiotics were studied in two azinphosmethyl-resistant and one susceptible strain of Colorado potato beetle ( Lepinotarsa decemlineata Say) with emphasis on the microsomal polysubstrate monooxygenase system. Monooxygenase activity comparable to that in other coleopterans was observed in the oxidation of NADPH, O-demethylation of p-nitroanisole, N-demethylation,of aminopyrine, and epoxidation of aldrin. Aniline hydroxylase activity was not detected. Monooxygenase activity against aminopyrine was largely confined to the microsomal fraction of homogenates and was more active in the larval gut than in the fat body. Microsomal NADPH oxidation was inhibited strongly in vitro by SKF 525-A and in vivo by prior treatment of insects with piperonyl butoxide. The activity of the monooxygenase system was approximately twice as high in the resistant strains as that in the susceptible strain. Glutathione transferase activity was also elevated in the resistant strains, but no elevation in carboxyesterase activity was seen. Piperonyl butoxide pretreatment increased the toxicity of azinphosmethyl several-fold more strongly in the resistant than susceptible strain, indicating a significant role for monooxygenase activity in resistance. However, in both strains, resistance to azinphosmethyl is multifactorial and enhanced monooxygenase activity is only one contributing factor.

Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes.

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Ethylbenzene-mediated induction of cytochrome P450 isozymes in male and female rats

Male and female Holtzman rats were exposed to ethylbenzene, and the effect on liver microsomal activities was studied. Hydrocarbon- and sex-dependent effects on P450-dependent metabolism of drugs and aromatic hydrocarbons were investigated. Hydrocarbon treatment produced two patterns of induction in cytochrome P450-dependent activities: (1) induction common to both sexes; and (2) induction exclusively in females. Benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, p-nitroanisole O-demethylation and aromatic hydroxylation of toluene were induced in both sexes after rats were exposed to ethylbenzene. The rate of benzphetamine N-demethylation increased 4-fold in females and nearly doubled in males. The increase in O-deethylation of 7-ethoxy-coumarin was 3-fold in females and doubled in males, while p-nitroanisole O-demethylation increased 4-fold in both sexes after exposure to ethylbenzene. Ethylbenzene had its greatest effect upon the formation of aromatic hydroxylated metabolites of toluene. Ethylbenzene exposure increased the rate of o-cresol formation by 4- and 9-fold in female and male rats, respectively. The formation rate of p-cresol was undetectable in either sex prior to hydrocarbon exposure; however, after the rats were given ethylbenzene, rates increased to 0.4 nmol/min/mg protein in females and to 0.9 nmol/min/mg protein in the males. Ethylbenzene exposure selectively induced aminopyrine demethylation, aniline hydroxylation, N, N-dimethylnitrosamine N-demethylation (DMNA) and aliphatic hydroxylation of toluene in females. Rates for aminopyrine, aniline, and DMNA were increased 50% over controls, while formation of benzyl alcohol from toluene was enhanced to 260% of control. Western immunoblotting indicated that ethylbenzene treatment induced cytochrome P450 2B1/2B2 to a greater extent in male rats and cytochrome P450 2E1 only in females. Ethylbenzene exposure did not affect significantly the level of cytochrome P450 1A1.

Separation of cytochrome P-450 containing vesicles from the midgut microsomal fraction of Manduca sexta

1. Microsomes prepared from midgut tissue of Manduca sexta by differential centrifugation are a heterogeneous population of vesicles. 2. Upon centrifugation of microsomes in a sucrose gradient of 30%, 15% and 10%, specific activity of cytochrome P-450 catalyzed O-demethylation of p-nitroanisole (a marker enzyme for endoplasmic reticulum) was increased 2.5 to 3.5-fold in the 15% fraction. 3. Specific activities of alkaline phosphatase and leucine aminopeptidase (marker enzymes for brush border membranes) were increased 3.1 to 5.7-fold in the pellet. 4. Differential centrifugation is a useful step in the purification of cytochrome P-450 enzymes.

Dietary Magnesium Depletion: p-Nitroanisole Metabolism and Glucuronidation in Rat Hepatocytes and Hepatic Microsomal Membranes

Male Sprague-Dawley rats were pair fed either a control magnesium diet (480 mg/kg) or a magnesium-deficient diet (30 mg/kg) for 10 days to produce magnesium depletion. Serum magnesium concentration declined 60%, but tissue magnesium deficiency was not apparent. Hepatic parenchymal cells and microsomal membranes were isolated to evaluate in vitro metabolism of p-nitroanisole and conjugation of its metabolite, p-nitrophenol (pNP). O-Demethylation of p-nitroanisole was decreased slightly in both magnesium depletion hepatocytes and microsomal membranes. However, pNP glucuronide formation was specifically decreased 50% in the magnesium depletion cells at all p-nitroanisole concentrations. Similar results were found in the microsomal reaction system measuring pNP conjugation. Thus, early stages of magnesium depletion may result in a specific decrease in the pNP UDP-glucuronyl transferase activity.

Inhibition of hepatic microsomal cytochrome P450 by cannabidiol in adult male rats.

The mechanism of inhibitory effect of cannabidiol (CBD) on the hepatic drug-metabolizing enzyme system was studied in adult male rats in vivo. Time course studies revealed that microsomal d-benzphetamine N-demethylation and testosterone 2 alpha-, 16 alpha- and 17-oxidation were markedly suppressed 6 to 48 h after the single administration of CBD (10 mg/kg, intraperitoneally). Decreases in activities of aniline hydroxylation and p-nitroanisole O-demethylation and in content of total cytochrome P450 were intermittent and moderate. On the other hand, no change was observed in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase activity or cytochrome b5 content in the hepatic microsomes of the CBD-treated rats. Western blotting analysis showed a marked decrease in the male-specific cytochrome P450 UT-2 in the hepatic microsomes, especially 24 to 48 h after pretreatment with CBD. It is possible that CBD given 6 to 12 h before the sacrifice might interact with cytochrome P450 as a substrate, resulting in inhibition of the drug-metabolizing enzyme activities in the earlier stages. In the later stages from 24 to 48 h after CBD treatment, the reduction in content of the male-specific cytochrome P450 UT-2 may play a major role in the inhibitory effect of CBD on the hepatic drug-metabolizing enzyme system in the adult male rat in vivo.

Metabolism of fusarin C by rat liver microsomes: Role of esterase and cytochrome P-450 enzymes with respect to the mutagenicity of fusarin C in Salmonella typhimurium

Fusarin C (FC) is a potent mutagen present on Fusarium moniliforme contaminated corn. This compound requires metabolic activation for which microsomes from phenobarbital-induced rats are most effective. Inhibition of the simultaneously induced esterase activity, which produced a less mutagenic metabolite, doubled the mutagenicity of FC. Carbon monoxide inhibited the mutagenicity of FC, suggesting the involvement of a heme containing enzyme. However, monoclonal antibodies specific for the phenobarbital-induced cytochrome P-450 enzymes PB-4 and PB-5, while inhibiting O-demethylation of p-nitroanisole and aflatoxin B 1 mutagenicity, had no effect on FC mutagenicity. This implies that either these enzymes are not involved in the activation of FC or FC competes well with the antibodies for binding to the cytochrome P-450 enzymes. Two additional metabolites of FC were detected. One had an ultraviolet spectrum similar to FC; the other had a λ max at 326 nm, and its retention time on reverse phase HPLC was very sensitive to changes in pH.