4-nitrocatechol and 4-nitrophenol

4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and

Topics: Bacterial Proteins; Biotransformation; Burkholderia; Catechols; Dioxygenases; Hydroquinones; Nitrophenols; Pseudomonas

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.

Topics: Arthrobacter; Bacterial Proteins; Binding Sites; Biodegradation, Environmental; Catalysis; Catalytic Domain; Catechols; Environmental Pollutants; Molecular Docking Simulation; Molecular Dynamics Simulation; Nitrophenols; Oxygenases; Protein Binding; Protein Conformation; Substrate Specificity

The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ± 0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ± 1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5-12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8 ± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0 ± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1.

Topics: Aldehydes; Catechols; Cytochrome P-450 CYP2A6; Cytochrome P-450 CYP2E1; Enzyme Inhibitors; Humans; Kinetics; Nitrophenols; Oxidation-Reduction; Structure-Activity Relationship

4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.

Topics: Bacterial Proteins; Benzoquinones; Catechols; Cloning, Molecular; Escherichia coli; Genes, Bacterial; Hydroquinones; Multigene Family; Nitrophenols; Oxidation-Reduction; Oxygenases; Rhodococcus; Sequence Analysis, DNA; Substrate Specificity

Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.

Topics: Biodegradation, Environmental; Blotting, Southern; Burkholderia; Catechols; Chromatography, Gas; Cloning, Molecular; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Mass Spectrometry; Molecular Weight; Nitrophenols; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA

A 9.2-kb DNA fragment encoding the enzymes of a p-nitrophenol (PNP) catabolic pathway from Pseudomonas putida DLL-E4 was cloned and sequenced. Ten open reading frames (ORFs) were found and five ORFs were functionally verified. The pnpA and pnpC gene products were purified to homogeneity by Ni-NTA chromatography. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase which converts p-nitrophenol to para-benzoquinone in the presence of NADH and FAD. PnpC is a 1,2,4-trihydroxybenzene (BT) 1,2-dioxygenase which converts BT to maleylacetate. The hydroquinone (HQ) dioxygenase (PnpC1C2) multi-component protein complex was expressed in Escherichia coli via plasmid pET-pnpC1C2 containing pnpC1 and pnpC2. This complex converts HQ to gamma-hydroxymuconic semialdehyde. pnpR is a lysR-type regulator gene. PnpR is a positive regulator involved in HQ degradation in pnp gene cluster. These results demonstrate that a pathway encoded by the pnp gene cluster is involved in degradation of HQ and BT in P. putida DLL-E4.

Topics: Biodegradation, Environmental; Catechols; Chromatography, High Pressure Liquid; Cloning, Molecular; Dioxygenases; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Mass Spectrometry; Molecular Sequence Data; Multigene Family; Nitrophenols; Pseudomonas putida; Sequence Analysis, DNA; Substrate Specificity; Transcription, Genetic

Liquid-phase decomposition of 4-nitrophenol (4-NP) and formation of hydrogen peroxide (H(2)O(2)) induced by contact glow discharge electrolysis (CGDE) were investigated. Experimental results showed that the decays of 4-NP and total organic carbon (TOC) obeyed the first-order and pseudo-first-order reaction kinetics, respectively. The major intermediate products were 4-nitrocatechol, hydroquinone, benzoquinone, hydroxyhydroquinone, organic acids and nitrite ion. The final products were carbon dioxide and nitrate ion. The initial formation rate of H(2)O(2) decreased linearly with increasing initial concentration of 4-NP. Addition of iron ions, especially ferric ion, to the solution significantly enhanced the 4-NP removal due to the additional hydroxyl radical formation through Fenton's reaction. A reaction pathway is proposed based on the degradation kinetics and the distribution of intermediate products.

Topics: Carbon Dioxide; Catechols; Electrolysis; Environmental Restoration and Remediation; Hydrogen Peroxide; Kinetics; Nitrates; Nitrophenols; Quinones; Water Pollutants, Chemical

Experiments have been performed with a semicontinuous batch reactor to compare the degradation efficiency of nitrobenzene in aqueous solution by the ultrasonic processes of single field, opposite dual fields, and orthogonal dual fields. Ultrasound with dual fields can improve the degradation efficiency of nitrobenzene compared to that of single field, and the improvement phenomenon is even more pronounced in the orthogonal dual-field system. The degradation reactions of nitrobenzene in the three processes all follow the pseudofirst-order kinetic model. The mechanism investigation indicates the degradation proceeds via hydroxyl radical (*OH) oxidation. The enhancement efficiency of orthogonal dual fields is attributed to an obvious synergetic effect, which accelerates the *OH initiation from 0.28 micromol L(-1) min(-1) for a single field to 0.98 micromol L(-1) min(-1) compared with 0.42 micromol L(-1) min(-1) for opposite dual fields, resulting in rapid formation of an increased diversity of byproducts and an advanced degree of mineralization of total organic carbon (TOC). The introduction of an ultrasonic field placed in the different spatial position causes a variable kinetic order during the removal of TOC. The degradation byproducts are identified by gas chromatography mass spectrometry and ion chromatography, including p-, m-nitrophenol, malonic acid, nitrate ion, 4-nitrocatechol, phenol, maleic acid, oxalic acid, hydroquinone, 1,2,3-trihydroxy-5-nitrobenzene, and acetic acid.

Topics: Acetic Acid; Carbon; Catechols; Chromatography, Gas; Hydroquinones; Hydroxyl Radical; Ions; Kinetics; Maleates; Malonates; Nitrates; Nitrobenzenes; Nitrophenols; Oxalic Acid; Ultrasonics; Water

Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.

Topics: Adsorption; Aluminum Oxide; Animals; Bioreactors; Catechols; Cytochrome P-450 CYP2E1; Enzymes, Immobilized; Gold; Hydroxylation; Male; Membranes, Artificial; Microsomes, Liver; Nitrophenols; Rats; Rats, Sprague-Dawley; Time Factors

Interference of antiviral agent adefovir, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) with microsomal drug metabolizing system was investigated in rats. The content of total liver cytochrome P450 (CYP) was lowered while that of its denaturated form, P420, was elevated in animals intraperitoneally treated with PMEA (25 mg/kg). Similar effect was observed after treatment with a prodrug of adevofir, adefovir dipivoxil (bisPOM-PMEA). The CYP2E1-dependent formation of 4-nitrocatechol from p-nitrophenol was diminished, though the specific activity of p-nitrophenol hydroxylase remained unchanged. PMEA had no influence on expression of CYP2E1 protein and mRNA and mRNAs of other P450 isoenzymes (1A1, 1A2, 2C11, 3A1, 3A2, and 4A1). It may be concluded that repeated systemic administration of higher doses of PMEA results in a partial degradation of rat CYP protein to inactive P420.

Topics: Adenine; Animals; Antiviral Agents; Catechols; Cytochrome P-450 Enzyme System; Female; Microsomes, Liver; Nitrophenols; Organophosphonates; Rats; Rats, Inbred Lew; RNA, Messenger

Arthrobacter protophormiae strain RKJ100 is capable of utilizing p-nitrophenol (PNP) as well as 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy. The degradation of PNP and NC by this microorganism takes place through an oxidative route, as stoichiometry of nitrite molecules was observed when the strain was grown on PNP or NC as sole carbon and energy sources. The degradative pathways of PNP and NC were elucidated on the basis of enzyme assays and chemical characterization of the intermediates by TLC, GC, (1)H NMR, GC-MS, UV spectroscopy, and HPLC analyses. Our studies clearly indicate that the degradation of PNP proceeds with the formation of p-benzoquinone (BQ) and hydroquinone (HQ) and is further degraded via the beta-ketoadipate pathway. Degradation of NC involved initial oxidation to generate 1,2,4-benzenetriol (BT) and 2-hydroxy-1,4-benzoquinone; the latter intermediate is then reductively dehydroxylated, forming BQ and HQ, and is further cleaved via beta-ketoadipate to TCA intermediates. It is likely, therefore, that the same set of genes encode the further metabolism of HQ in PNP and NC degradation. A plasmid of approximately 65 kb was found to be responsible for harboring genes for PNP and NC degradation in this strain. This was based on the fact that PNP(-) NC(-) derivatives were devoid of the plasmid and had simultaneously lost their capability to grow at the expense of these nitroaromatic compounds.

Topics: Arthrobacter; Biodegradation, Environmental; Catechols; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Kinetics; Magnetic Resonance Spectroscopy; Nitrites; Nitrophenols; Oxidation-Reduction; Plasmids

Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.

Topics: Biodegradation, Environmental; Catechols; Cloning, Molecular; Genes, Bacterial; Gram-Negative Aerobic Rods and Cocci; Mixed Function Oxygenases; Molecular Sequence Data; Nitrophenols; Sequence Analysis, DNA; Soil Microbiology

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O2 pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O2 pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1, 2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO2-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of 14CO2 from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled 14C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source.

Topics: Aerobiosis; Biodegradation, Environmental; Catechols; Light; Models, Chemical; Nitrites; Nitrogenase; Nitrophenols; Rhodobacter capsulatus

4-Nitrocatechol was identified as a product of transformation of 4-nitrophenol by bacterial strain Corynebacterium sp.8/3 using direct acetylation of biodegradation samples by acetic anhydride followed by GC-MS analysis. The identity of 4-nitrocatechol, in the form of diacetate, was confirmed by electron-impact spectra and spectra recorded under chemical ionization conditions (positive and negative modes). Negative-ion chemical ionization was used for quantification of 4-nitrocatechol in biodegradation samples in a concentration range of 1-25 mg/l.

Topics: Biodegradation, Environmental; Catechols; Corynebacterium; Gas Chromatography-Mass Spectrometry; Nitrophenols

Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.

Topics: Bacillus; Catechols; Hydroxylation; Nitrites; Nitrophenols; Oxidation-Reduction; Oxygenases

We have isolated two soil bacteria (identified as Arthrobacter aurescens TW17 and Nocardia sp. strain TW2) capable of degrading p-nitrophenol (PNP) and numerous other phenolic compounds. A. aurescens TW17 contains a large plasmid which correlated with the PNP degradation phenotype. Degradation of PNP by A. aurescens TW17 was induced by preexposure to PNP, 4-nitrocatechol, 3-methyl-4-nitrophenol, or m-nitrophenol, whereas PNP degradation by Nocardia sp. strain TW2 was induced by PNP, 4-nitrocatechol, phenol, p-cresol, or m-nitrophenol. A. aurescens TW17 initially degraded PNP to hydroquinone and nitrite. Nocardia sp. strain TW2 initially converted PNP to hydroquinone or 4-nitrocatechol, depending upon the inducing compound.

Topics: Arthrobacter; Biodegradation, Environmental; Catechols; Cresols; Gene Expression Regulation, Bacterial; Nitrophenols; Nocardia; Phenol; Phenols; Soil Microbiology; Soil Pollutants

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.

Topics: Base Sequence; Catechols; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; DNA, Complementary; Dose-Response Relationship, Drug; Humans; Kinetics; Microsomes, Liver; Mixed Function Oxygenases; Molecular Sequence Data; Nitrophenols; Oxidoreductases, N-Demethylating; Xenobiotics

A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 microM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes.

Topics: Catechols; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Glycerol; Humans; Hydroxylation; In Vitro Techniques; Microsomes, Liver; Nitrophenols; Oxidoreductases, N-Demethylating; Quality Control; Solvents; Spectrophotometry, Ultraviolet

Carbon tetrachloride (CCl4) is a classical pericentral hepatotoxicant; however, precise details of its mechanism of action remain unknown. One possibility is that Kupffer cells participant in this mechanism since CCl4 elevates calcium, and the release of toxic eicosanoids and cytokines by Kupffer cells is calcium-dependent. Therefore, these studies were designed to evaluate the role of Kupffer cells in CCl4 toxicity in the rat in vivo. Kupffer cells were destroyed selectively with gadolinium chloride treatment (10 mg/kg GdCl3 iv) 1 day prior to administration of CCl4 (4 g/kg ig). Twenty-four hours after CCl4 treatment, rats were anesthetized, blood samples were drawn for aspartate aminotransferase (AST) determination, which is indicative of parenchymal cell damage, and trypan blue was infused into the liver to stain the nuclei of dead hepatocytes. AST levels were in the normal range and trypan blue staining was negligible in livers from vehicle- or GdCl3-treated rats. As expected, CCl4 treatment alone elevated AST levels to values over 4000 U/liter and caused massive cell death (60-90 trypan blue-positive cells/pericentral field). In dramatic contrast, the elevation in AST and cell death due to CCl4 were almost completely prevented by GdCl3 treatment. In attempts to understand this phenomenon, metabolic and detoxification pathways were assessed. CCl4 is metabolized via cytochrome P450 II.E.1; however, GdCl3 treatment did not alter this pathway as assessed from p-nitrocatechol formation from the selective substrate, p-nitrophenol. GdCl3 treatment also had no effect on hepatic glutathione levels. On the other hand, GdCl3 treatment significantly reduced infiltration of neutrophils resulting from exposure to CCl4. These data clearly support the hypothesis that Kupffer cells participate in the mechanism of toxicity of CCl4 in vivo, possibly by release of chemoattractants for neutrophils.

Topics: Animals; Aspartate Aminotransferases; Carbon Tetrachloride; Catechols; Cell Death; Female; Gadolinium; Kupffer Cells; Liver; Nitrophenols; Rats; Rats, Sprague-Dawley; Receptors, Leukocyte-Adhesion

In perfused livers from fed and fasted beta-naphthoflavone-treated C57BL/6J mice, maximal rates of p-nitroanisole O-demethylation were 30-40 mu moles/g/hr and 15-20 mu moles/g/hr respectively. The detergent saponin, at concentrations ranging from 0.001 to 0.005%, was infused between 2 and 30 min to establish optimal conditions to permeabilize plasma membranes. Permeabilization was assessed by release of lactate dehydrogenase and stimulation of p-nitroanisole O-demethylation by citrate. Saponin (0.005% for 5 min) alone had little effect on the rates of p-nitroanisole O-demethylation or conjugation of p-nitrophenol by perfused livers. Further, dicarboxylates or NADPH had no effect on rates of monooxygenation by perfused mouse liver in the absence of saponin. In saponin-treated livers from fasted mice, however, rates of monooxygenation were increased rapidly by infusion of dicarboxylates (10 mM malate, citrate, or isocitrate) or an NADPH-generating system (60 and 110% respectively), over a 6-8 min period. During this time period, cellular energetics were not comprised as reflected by normal rates of glucuronidation of p-nitrophenol. Thus, non-permeable metabolites can enter saponin-permeabilized cells in the perfused liver. Rates of monooxygenation were increased 40-60% in livers from fed mice by citrate, NADPH (200 microM) or an NADPH-generating system. In contrast, saponin decreased mixed-function oxidation assayed in isolated microsomes incubated with an NADPH-generating system. Taken together, these data support the hypothesis that maximal rates of monooxygenation in intact hepatocytes from fed as well as fasted mice is limited by the availability of NADPH.

Topics: Animals; Benzoflavones; beta-Naphthoflavone; Catechols; Cell Membrane Permeability; Citrates; Citric Acid; Liver; Male; Mice; Mice, Inbred C57BL; Mixed Function Oxygenases; NADP; Nitrophenols; Oxidoreductases, O-Demethylating; Perfusion; Saponins

Topics: Aminophenols; Animals; Catechols; Nitrobenzenes; Nitrophenols; Rabbits