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1.
J Chromatogr B Analyt Technol Biomed Life Sci ; 852(1-2): 631-4, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17321806

ABSTRACT

An LC-MS/MS method has been developed for the quantitative determination of a protein drug (Tenecteplase; M(W) 58,777 Da) in rat plasma. The protein was digested with trypsin without prior clean-up of the plasma sample, without the use of a label nor internal standard. A limited validation was performed to assess the linearity, the sensitivity and the specificity of the method. In addition, the developed method was applied to the quantitative analysis of Tenecteplase in rat plasma samples originating from a single-dose study in rats.


Subject(s)
Chromatography, Liquid/methods , Fibrinolytic Agents/blood , Tandem Mass Spectrometry/methods , Tissue Plasminogen Activator/blood , Animals , Enzyme-Linked Immunosorbent Assay , Rats , Sensitivity and Specificity , Tenecteplase
2.
J Nutr ; 131(6): 1714-22, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11385058

ABSTRACT

The potential benefits of a high fruit and vegetable intake on the antioxidant status and on relevant biomarkers of oxidative damage to lipids, proteins and DNA and on (functional) markers of oxidative stress were evaluated. A randomized, free living, open placebo-controlled cross-over trial of 3 wk, with a 2-wk washout period between treatments, was performed in a group of 22 male smokers with a relatively low vegetable and fruit intake using a vegetable burger and fruit drink. The vegetable burger and fruit drink increased serum levels of vitamin C, alpha-carotene, beta-carotene, beta-cryptoxanthin and zeaxanthin and plasma total antioxidant capacity. However, no effects were demonstrated on any marker of oxidative damage to lipids (malondialdehyde F(2)-isoprostane) proteins (carbonyls) and DNA (Comet assay) and (functional) markers of oxidative stress (reduced/oxidized glutathione ratio, glutathione-S-transferase alpha, glutathione-S-transferase pi and nuclear transcription factor-kappaB). Apparently, these increased levels of antioxidants in serum were not sufficiently high to show beneficial changes with the selected biomarkers. Alternatively, oxidative stress in male smokers with a relatively low fruit and vegetable intake might have been still too low to demonstrate a beneficial effect of antioxidants.


Subject(s)
Antioxidants/administration & dosage , Fruit , Smoking/physiopathology , Vegetables , Adult , Blood Pressure , Body Weight , Carotenoids/blood , Cholesterol/blood , Cholesterol, HDL/blood , Cross-Over Studies , DNA Damage , Humans , Male , Malondialdehyde/blood , Middle Aged , Monocytes/ultrastructure , Oxidative Stress , Time Factors , Triglycerides/blood , Vitamin A/blood , Vitamin E/blood
4.
Environ Toxicol Pharmacol ; 10(4): 141-52, 2001 Sep.
Article in English | MEDLINE | ID: mdl-21782570

ABSTRACT

This review summarizes current knowledge on glutathione (GSH) associated cellular processes that play a central role in defense against oxidative stress. GSH itself is a critical factor in maintaining the cellular redox balance and has been demonstrated to be involved in regulation of cell signalling and repair pathways. Enhanced expression of various enzymes involved in GSH metabolism, including glutathione peroxidases, γ-glutamyl cysteinyl synthetase (γ-GCS), glutathione S-transferases (GST) and membrane proteins belonging to the ATP-binding cassette family, such as the multidrug resistance associated protein, have all been demonstrated to play a prominent role in cellular resistance towards oxidative stress. This review stresses the fact that aco-ordinateinterplay between these systems is essential for efficient protection against oxidative stress.

5.
Toxicol Sci ; 58(1): 15-22, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11053536

ABSTRACT

In vitro and in vivo skin absorption of the pesticide propoxur (2-isopropoxyphenyl N-methyl carbamate, commercially Baygon(TM) and Unden (TM); log Po/w 1.56, MW 209.2) was investigated. In vivo studies were performed in rats and human volunteers, applying the test compound to the dorsal skin and the volar aspect of the forearm, respectively. In vitro experiments were carried out in static diffusion cells using viable full-thickness skin membranes (rat and human), non-viable epidermal membranes (rat and human) and a perfused-pig-ear model. Percutaneous penetration of propoxur in human volunteers was measured by analysis of its metabolite (2-isopropoxyphenol) in blood and urine; in all other studies radiolabeled propoxur ([ring-U-(14)C]propoxur) was used. In order to allow for direct comparison, experimental conditions were standardized with respect to dose (150 microg propoxur per cm(2)), vehicle (60% aqueous ethanol) and exposure time (4 h). In human volunteers, it was found that approximately 6% of the applied dose was excreted via the urine after 24 h, while the potential absorbed dose (amount applied minus amount washed off) was 23 microg/cm(2). In rats these values were 21% and 88 microg/cm(2), respectively. Data obtained in vitro were almost always higher than those obtained in human volunteers. The most accurate in vitro prediction of the human in vivo percutaneous absorption of propoxur was obtained on the basis of the potential absorbed dose. The absorbed dose and the maximal flux in viable full-thickness skin membranes correlated reasonably well with the human in vivo situation (maximal overestimation by a factor of 3). Epidermal membranes overestimated the human in vivo data up to a factor of 8, but the species-differences observed in vivo were reflected correctly in this model. The data generated in the perfused-pig-ear model were generally intermediate between viable skin membranes and epidermal membranes.


Subject(s)
Epidermis/metabolism , Insecticides/pharmacokinetics , Propoxur/pharmacokinetics , Skin Absorption , Animal Testing Alternatives , Animals , Ear, External/blood supply , Ear, External/metabolism , Humans , In Vitro Techniques , Male , Models, Animal , Perfusion , Rats , Rats, Wistar , Species Specificity , Swine
6.
Biochem Pharmacol ; 57(12): 1383-90, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10353259

ABSTRACT

The cyclopentenone prostaglandin A2 (PGA2) is known to inhibit cell proliferation, and metabolism of this compound thus might be important in controlling its ultimate function. The glutathione-related metabolism of PGA2 was therefore investigated both with purified glutathione S-transferase P1-1 (GSTP1-1) and with IGR-39 human melanoma cells. Firstly, the irreversible inhibition of human GSTP1-1 and its mutants C47S, C101S, and C47S/C101S was studied. PGA2 appeared to inhibit GSTP1-1 mainly by binding to the cysteine 47 moiety of the enzyme. This binding was reversed by a molar excess of GSH, indicating that retro-Michael cleavage occurs. Secondly, after exposing IGR-39 human melanoma cells to PGA2, both diastereoisomers of the PGA2-glutathione conjugate are excreted into the medium, although with a clear excess of the S-form, due to its preferential formation by the GSTP1-1 present in the cells. Thirdly, the effect of PGA2 on intracellular GST activity was determined by quantification of the excreted glutathione conjugate S-(2,4-dinitrophenyl)glutathione (DNPSG) after exposure to 1-chloro-2,4-dinitrobenzene. DNPSG excretion was inhibited after incubation with 10 or 20 microM PGA2 for 1 or 4 hr, as a result of glutathione depletion, reversible GST inhibition, and covalent modification of intracellular GST. Furthermore, PGA2 also inhibited transport of DNPSG by the multidrug resistance-associated protein, an effect that was reversible and competitive. In conclusion, PGA2 modulates all three aspects of the glutathione-mediated biotransformation system, i.e. GSH levels, GSTP1-1 activity, and transport of GSH conjugates. A role for GSTP1-1 as a specific transport protein inside the cell is indicated.


Subject(s)
Glutathione/metabolism , Prostaglandins A/metabolism , Biotransformation , Glutathione S-Transferase pi , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Melanoma , Tumor Cells, Cultured
7.
Cancer Res ; 58(20): 4616-23, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9788613

ABSTRACT

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Breast Neoplasms/metabolism , Glutathione Transferase/physiology , Isoenzymes/physiology , Thiotepa/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , ATP-Binding Cassette Transporters/physiology , Biological Transport , Biotransformation , Cell Division/drug effects , Female , Glutathione S-Transferase pi , Humans , Multidrug Resistance-Associated Proteins , Thiotepa/pharmacology
8.
Xenobiotica ; 27(8): 801-17, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9293617

ABSTRACT

1. The metabolic fate of the insecticide teflubenzuron, orally dosed to the male Wistar rat, was investigated. Particular attention was paid to the metabolic fate of the benzoyl and aniline moiety after hydrolysis of the urea bridge. 2. The 0-48-h urinary and faecal metabolic patterns and recoveries showed that for a dose range of 4-53 mumol (1.5-20 mg) teflubenzuron, 90% of the dose was excreted in the faeces mainly in unmodified form, approximately 4.6% was absorbed from the lumen and excreted in the urine, and 5.4% was retained in the body. Metabolites excreted in the urine could be identified as benzoate and aniline derivatives originating from the two aromatic rings of teflubenzuron liberated from the parent molecule by hydrolysis of the urea bridge. 3. The amount of urinary benzoate-type metabolites was about eight times the amount of aniline-type metabolites, indicating significant differences in efficiency of urinary excretion of the benzoate moiety as compared with the aniline ring. 4. To investigate further the possible reason underlying this difference in urinary excretion efficiency between the two aromatic derivatives formed from teflubenzuron, dose-recovery studies of these aniline- and benzoate-type metabolites were performed. These studies confirmed the discrepancy observed between the urinary recovery of the benzoyl and the aniline moiety of teflubenzuron. 5. Additional results of the present study indicate that the above discrepancy can be explained by the fact that the benzoate derivative is excreted mainly in its unmetabolized form, whereas the aniline derivative needs additional phase I and II modifications before it can be excreted from the body, the former being a relatively slow reaction. Furthermore, conversion of the halogenated aniline derivative in phase I metabolism might result in a reactive benzoquinone-type or N-oxidized primary metabolite, which can be retained in the body due to reaction with cellular macromolecules.


Subject(s)
Benzamides/metabolism , Benzoates , Insecticides/metabolism , Anilides/urine , Aniline Compounds/urine , Animals , Benzamides/urine , Benzoates/urine , Feces/chemistry , Hydrolysis , Insecticides/urine , Kinetics , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/metabolism , Rats , Rats, Wistar
9.
FEBS Lett ; 419(1): 112-6, 1997 Dec 08.
Article in English | MEDLINE | ID: mdl-9426231

ABSTRACT

The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Glutathione/analogs & derivatives , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Adenosine Triphosphate/physiology , Animals , Biological Transport , Cell Line , Cell Polarity , Cyclic AMP/pharmacology , Dogs , Erythrocytes , Ethacrynic Acid/metabolism , Glutathione/metabolism , Humans , Kidney/cytology , Methotrexate/pharmacology , Mice , Mice, Mutant Strains , Microsomes/metabolism , Prostaglandins A, Synthetic/metabolism , Stereoisomerism
10.
Toxicol Appl Pharmacol ; 141(2): 403-15, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8975765

ABSTRACT

The in vitro and in vivo metabolic profiles of a series of C4-substituted 2-fluoroanilines were determined and compared to their capacity to induce methemoglobinemia and nephrotoxicity in male Wistar rats. Qualitative and quantitative relationships between the biotransformation and the toxic endpoint of the halogenated anilines were defined. The rate of in vitro N-hydroxylation of the aniline derivatives correlated with the capacity of the compounds to induce methemoglobinemia (r = 0.96). In the experiments on the nephrotoxicity, attention was focused on the relative importance of the C4- and C6-hydroxylated metabolites of the C4-substituted 2-fluoroanilines. In vivo, the formation of 4-aminophenol metabolites was demonstrated to vary in the opposite order as the formation of the 6-aminophenol metabolites. 1H-NMR urinalysis and characterization of a set of conventional biochemical urinary parameters revealed the occurrence of nephrotoxicity upon exposure to the aniline derivatives and were most consistent with damage at the proximal tubular site. Comparison of the extent of nephrotoxicity to the extent of formation of the 4-aminophenol and/or 6-aminophenol metabolites, respectively, indicates a predominant role for the C4-hydroxylation route, not the C6-hydroxylation route, in the induction of nephrotoxic effects. Thus, a qualitative relationship is observed for the extent of C4-hydroxylation of the aniline derivatives and the extent of their in vivo nephrotoxicity. In addition, comparison of the extent of 4-aminophenol formation and nephrotoxicity of both 2-fluoroaniline and 2,4-difluoroaniline pointed at a possible role for a bioactivation pathway through oxidative dehalogenation, resulting in direct formation of a 1,4-benzoquinoneimine as the primary metabolite in the case of 2,4-difluoroaniline. Altogether, it is concluded that a decrease in C4-hydroxylation in the series of aniline derivatives results in a metabolic switch to C6- and N-hydroxylation and, consequently, a shift in the type of toxic endpoint observed, i.e., from nephrotoxicity to methemoglobinemia.


Subject(s)
Aniline Compounds/toxicity , Aniline Compounds/metabolism , Animals , Hydroxylation , Kidney/drug effects , Male , Methemoglobinemia/chemically induced , Rats , Rats, Wistar , Structure-Activity Relationship
11.
FEBS Lett ; 391(1-2): 126-30, 1996 Aug 05.
Article in English | MEDLINE | ID: mdl-8706899

ABSTRACT

The multidrug resistance protein MRP has been shown to mediate the transport of glutathione S-conjugates across membranes. In this study we demonstrate that the glutathione S-conjugate of the diuretic drug ethacrynic acid, which is an efficient inhibitor of glutathione S-transferases, is a high-affinity substrate and inhibitor of the glutathione S-conjugate pump associated with MRP. This implies that ethacrynic acid may modulate drug resistance of tumor cells not only by inhibiting glutathione S-transferase activity, but also by inhibiting the export of drug conjugates from the cell by MRP.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Ethacrynic Acid/metabolism , Glutathione/metabolism , Adenosine Monophosphate/pharmacology , Biological Transport/drug effects , Carcinoma, Non-Small-Cell Lung , Cell Membrane/metabolism , Drug Resistance, Multiple , Glutathione/pharmacology , Humans , Kinetics , Lung Neoplasms , Multidrug Resistance-Associated Proteins , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sulfinpyrazone/pharmacology , Transfection , Tumor Cells, Cultured
12.
Toxicol Appl Pharmacol ; 139(1): 71-83, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8685911

ABSTRACT

In the present study both the biotransformation patterns and the capacity to induce methemoglobinemia of a series of fluoronitrobenzenes were investigated. This was done to investigate to what extent variation in the number and position of the halogen substituents influence the metabolic fate of the fluoronitrobenzenes, thereby influencing their capacity to induce methemoglobinemia. The results obtained were compared to the effect of the fluorine substituent patterns on the calculated electronic characteristics and, thus, on the chemical reactivity of the fluoronitrobenzenes. Analysis of the in vivo metabolic profiles demonstrates a dependence of the extent of nitroreduction, of glutathione conjugation, and of aromatic hydroxylation with the pattern of halogen substitution. With an increasing number of fluorine substituents at electrophilic carbon centers, 24-hr urine recovery values decreased and fluoride anion elimination increased, due to increased reactivity of the fluoronitrobenzenes with cellular nucleophiles. In vitro studies even demonstrated a clear correlation between calculated parameters for the electrophilicity of the fluoronitrobenzenes and the natural logarithm of their rate of reaction with glutathione or with bovine serum albumin, taken as a model for cellular nucleophiles (r = 0.97 and r = 0.98, respectively). Increased possibilities for the conjugation of the fluoronitrobenzenes to cellular nucleophiles were accompanied by decreased contributions of nitroreduction and aromatic hydroxylation to the overall in vivo metabolite patterns, as well as by a decreased capacity of the fluoronitrobenzenes to induce methemoglobinemia. In vitro studies on the rates of nitroreduction of the various fluoronitrobenzenes by cecal microflora and rat liver microsomes revealed that the changes in the capacity of the fluoronitrobenzenes to induce methemoglobinemia were not due to differences in their intrinsic reactivity in the pathway of nitroreduction, leading to methemoglobinemia-inducing metabolites. Thus, the results of the present study clearly demonstrate that the number and position of fluorine substituents in the fluoronitrobenzenes influence the capacity of the fluoronitrobenzenes to induce methemoglobinemia, not because their intrinsic chemical reactivity for entering the nitroreduction pathway is influenced. The different methemoglobinemic capacity must rather result from differences in the inherent direct methemoglobinemic capacity and/or reactivity of the various toxic metabolites and/or from the fact that the halogen substituent pattern influences the electrophilic reactivity, thereby changing the possibilities for reactions of the nitrobenzenes with glutathione and, especially, other cellular nucleophiles. When the number of fluorine substituents increases, the electrophilicity of the fluoronitrobenzenes can become so high that glutathione conjugation is no longer able to compete efficiently with covalent binding of the fluoronitrobenzenes to cellular macromolecules. As a consequence, it can be suggested that with an increasing number of fluorine substituents at electrophilic carbon centers in a nitrobenzene derivative, a toxic end point of the nitrobenzene other than formation of methemoglobinemia can be foreseen.


Subject(s)
Fluorine/chemistry , Methemoglobinemia/chemically induced , Nitrobenzenes/chemistry , Nitrobenzenes/toxicity , Acetylcysteine/chemistry , Acetylcysteine/toxicity , Administration, Oral , Animals , Biotransformation , Cattle , Cecum/drug effects , Cecum/metabolism , Chromatography, High Pressure Liquid , Fluorine/urine , Glutathione/metabolism , Hydroxylation , Liver/drug effects , Liver/metabolism , Magnetic Resonance Spectroscopy , Male , Nitrobenzenes/administration & dosage , Oxidation-Reduction , Rats , Rats, Wistar , Serum Albumin, Bovine/metabolism , Structure-Activity Relationship
13.
Chem Biol Interact ; 100(2): 187-201, 1996 Mar 25.
Article in English | MEDLINE | ID: mdl-8646791

ABSTRACT

In the present study it is demonstrated that MO-QSARs (quantitative structure activity relationships based on calculated molecular orbital substrate characteristics) of cytochrome P450-catalysed biotransformation of benzene derivatives obtained in previous studies for Wistar rats, can be extrapolated to other species, including man. First, it was demonstrated that the regioselectivity of the in vivo aromatic hydroxylation of two fluorobenzene derivatives can be quantitatively predicted, on the basis of the calculated density distribution of the reactive pi-electrons in the aromatic ring of the fluorobenzene derivative, for all experimental animal species tested. Second, it was investigated whether the preferential site for in vitro aromatic hydroxylation of 3-fluoroaniline could be predicted on the basis of the same calculated parameter. This was done because extrapolation to human systems requires in vitro instead of in vivo experiments. The results obtained indicated that the variation in the regioselectivity of the aromatic hydroxylation of 3-fluoroaniline by liver microsomes from different species, including man, was only a few percent, and was mainly directed by calculated chemical reactivity characteristics of the 3-fluoraniline substrate. Finally, possibilities for the extrapolation from rat to other species, of the MO-QSAR for the rate of in vitro C4 hydroxylation of a series of aniline derivatives converted in an iodosobenzene-supported microsomal cytochrome P450 system, were investigated. Experiments with liver microsomes from rats, mice, rabbit and man resulted in clear MO-QSARs with correlation coefficients for the relationship between the 1n k(cat) and the E(HOMO) of the aniline substrates that were > or = to 0.97 in all cases. Thus, the results of the present study clearly demonstrate that MO-QSARs previously described for Wistar rats can be extrapolated to mice, rabbit, guinea pig and even to man. Regioselectivities obtained and QSAR lines for the rate of conversion plotted against calculated E(HOMO) values of the aniline derivatives are similar for the various species investigated. Altogether, these results strongly support the conclusion that the conversion of the relatively small benzene derivatives in the relatively large and aspecific active sites of the mammalian cytochromes P450, even when derived from various species, are mainly dependent on chemical reactivity parameters of the substrates. Therefore, the results of the present study support the hypothesis that MO-based QSARs obtained in rat for the cytochrome P450 catalysed aromatic hydroxylation of benzene derivatives can provide a basis for prediction of biotransformation pathways in different species, including man.


Subject(s)
Aniline Compounds/metabolism , Benzene Derivatives/metabolism , Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/metabolism , Structure-Activity Relationship , Aged , Animals , Arylsulfatases/metabolism , Benzene Derivatives/urine , Biotransformation , Female , Glucuronidase/metabolism , Humans , Hydroxylation , Kinetics , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/enzymology , Middle Aged , Rabbits , Rats , Rats, Wistar , Rodentia
14.
Biochem Pharmacol ; 49(9): 1235-48, 1995 May 11.
Article in English | MEDLINE | ID: mdl-7763304

ABSTRACT

The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent Vmax for the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed that the observed decrease in 4-aminophenol formation was accompanied by a metabolic switch to 2-aminophenols and N-hydroxyanilines, while products resulting from NIH-type mechanisms were not observed. For a C4-chloro-, bromo-, or iodo-substituted 2-fluoroaniline the Vmax for the oxidative dehalogenation was reduced by the additional electron withdrawing fluorine substituent at the C2 position in a similar way.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Aminophenols/metabolism , Aniline Compounds/chemistry , Aniline Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Halogens/chemistry , Animals , Kinetics , Male , Microsomes, Liver/metabolism , Oxidation-Reduction , Rats , Rats, Wistar , Safrole
15.
Chem Biol Interact ; 94(1): 49-72, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7820880

ABSTRACT

The in vivo metabolite patterns of 2,5-difluoroaminobenzene and of its nitrobenzene analogue, 2,5-difluoronitrobenzene, were determined using 19F NMR analysis of urine samples. Results obtained demonstrate significant differences between the biotransformation patterns of these two analogues. For the aminobenzene, cytochrome P450 catalysed aromatic hydroxylation presents the main metabolic pathway. 2,5-Difluoronitrobenzene was predominantly metabolised through glutathione conjugation leading to excretion of 5-fluoro-2-(N-acetylcysteinyl)-nitrobenzene and fluoride anions, and, to a minor extent, through cytochrome P450 catalysed hydroxylation and nitroreduction. Pretreatment of the rats with various inducers of cytochrome P450 enzymes, known also to influence glutathione S-transferase enzyme patterns, followed by exposure to the 2,5-difluoroamino- or 2,5-difluoronitrobenzene, generally resulted in metabolite patterns that varied only to a small (< or = 12%) extent. Based on these results it was concluded that the biotransformation enzyme pattern is not the predominant factor in determining the metabolic route of these two model compounds. Additional in vitro microsomal and cytosolic incubations with 2,5-difluoroaminobenzene and 2,5-difluoronitrobenzene qualitatively confirmed the in vivo results. NADPH/oxygen supported microsomal cytochrome P450 catalysed hydroxylation was observed only for 2,5-difluoroaminobenzene whereas cytosolic GSH conjugation occurred only in incubations with 2,5-difluoronitrobenzene as the substrate. Outcomes from molecular orbital calculations provided a working hypothesis that can explain the difference in metabolic pathways of the nitro- and aminobenzene derivative on the basis of their chemical characteristics. This hypothesis states that the chances for a nitro- or aminobenzene derivative to enter either a cytochrome P450 or a glutathione conjugation pathway are determined by the relative energy levels of the frontier orbitals of the compounds. The aminobenzene derivative has relatively high energy molecular orbitals leading to an efficient reaction of its highest occupied molecular orbital (HOMO) with the singly occupied molecular orbital of the cytochrome P450 (FeO)3+ intermediate, but a low reactivity of its lowest unoccupied molecular orbital (LUMO) with the HOMO of glutathione. The nitrobenzene, on the other hand, has molecular orbitals of relatively low energy, explaining the efficient interaction, and, thus, reaction between its LUMO and the HOMO electrons of glutathione, but resulting in low reactivity with the SOMO electron of the cytochrome P450 (FeO)3+ reaction intermediate.


Subject(s)
Aniline Compounds/chemistry , Aniline Compounds/pharmacokinetics , Nitrobenzenes/chemistry , Nitrobenzenes/pharmacokinetics , Xenobiotics , Aniline Compounds/urine , Animals , Biotransformation , Chemical Phenomena , Chemistry, Physical , Cytochrome P-450 Enzyme System/metabolism , Cytosol/enzymology , Enzyme Induction/drug effects , Glutathione/metabolism , Glutathione Transferase/metabolism , Hydroxylation , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/enzymology , Nitrobenzenes/urine , Rats , Rats, Wistar
16.
Chem Res Toxicol ; 7(5): 590-8, 1994.
Article in English | MEDLINE | ID: mdl-7841336

ABSTRACT

The cytochrome P450 (P450) catalyzed 4-hydroxylation of halogenated anilines was investigated with special emphasis on possible relationships between kinetic parameters and physicochemical and electronic characteristics of the substrates. The most important observation of the present study was a correlation (r = 0.96) between the natural logarithm of the apparent maximum reaction rate kcats for 4-hydroxylation of the aniline substrates in a iodosobenzene-supported microsomal cytochrome P450-catalyzed reaction and the energy of the highest molecular orbital [E(HOMO)] of the anilines. This result is in accordance with a mechanism that proceeds by an initial electrophilic attack of the P450 (FeO)3+ intermediate on the frontier pi electrons of the aniline substrates. In the iodosobenzene-supported aniline 4-hydroxylation this electrophilic attack is the rate-limiting step. In the NADPH/oxygen-supported cytochrome P450-catalyzed 4-hydroxylation of the anilines a correlation of the natural logarithm of kcats with E(HOMO) was not observed and the kcats values were lower than observed in the iodosobenzene-supported reaction. From this result it is concluded that, although the NADPH/oxygen-supported microsomal 4-hydroxylation of the halogenated anilines proceeds by the same cytochrome P450 (FeO)3+ intermediate and, thus, by a similar electrophilic attack of the (FeO)3+ on the pi electrons of the substrate, this attack is no longer the rate-limiting step of the reaction. Additional results of the present study demonstrate that the apparent Michaelis constant Kms of the NADPH/oxygen-supported 4-hydroxylation of the anilines decreases with increasing hydrophobicity of the aniline derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Aniline Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Aminophenols/metabolism , Aniline Compounds/chemistry , Animals , Chemical Phenomena , Chemistry, Physical , Hydroxylation , Kinetics , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/metabolism , Molecular Conformation , NADP/metabolism , Rats , Rats, Wistar , Structure-Activity Relationship
17.
Anal Biochem ; 220(1): 165-71, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7978241

ABSTRACT

A sensitive and efficient spectrophotometric assay is described for the determination of 2-aminophenols in biological samples. Using ferric ions as a metal catalyst, 2-aminophenol dimerizes in an acidic aqueous environment to 2-hydroxyisophenoxazin-3-one, an intensively colored dye. The newly developed assay is suitable for the detection of 2-aminophenols in the micromolar range. The paper demonstrates that this chemical assay is also applicable for the determination of 2-aminophenols substituted with a halogen at the ortho, meta, or para position of the aromatic ring with respect to the amino moiety, lambda max, and the molar extinction coefficient varying with the substituent pattern. Results obtained for fluorinated 2-aminophenol detection in biological samples by either this new method or 19F NMR are similar. This observation corroborates that the newly developed assay is suitable for detection of 2-aminophenols in biological samples.


Subject(s)
Aminophenols/analysis , Mass Spectrometry , Aminophenols/urine , Aniline Compounds/chemistry , Animals , Calibration , Coloring Agents/analysis , Cytochrome P-450 Enzyme System/chemistry , Hydrocarbons, Halogenated/chemistry , In Vitro Techniques , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/chemistry , Molecular Structure , Oxazines/analysis , Rats , Rats, Wistar , Reproducibility of Results , Spectroscopy, Fourier Transform Infrared
18.
Drug Metab Dispos ; 21(2): 218-30, 1993.
Article in English | MEDLINE | ID: mdl-8097689

ABSTRACT

In vivo and in vitro biotransformation of secondary aromatic amines was investigated using 4-fluoro-N-methylaniline as the model compound. Attention was focused on the role of cytochromes P-450 and the flavin-containing monooxygenase in formation of the various metabolic products. In vitro studies using microsomal preparations, purified reconstituted cytochromes P-450 IA1 and IIB1 and purified flavin-containing monooxygenase, demonstrated that N-demethylation, N-hydroxylation, and 4-hydroxylation accompanied by dehalogenation were all catalyzed by both the cytochrome P-450 system and the flavin-containing monooxygenase. The turnover rates of the two monooxygenase systems for the various metabolite formations and the reaction pathways involved, were shown to vary significantly. This study provides direct experimental support for the conclusion that the aromatic ring hydroxylation of secondary N-methylamines can be a consequence of flavin-containing monooxygenase catalyzed N-hydroxylation rather than of direct aromatic ring hydroxylation. The results obtained in vitro were compared with data from urine analysis of rats exposed to 4-fluoro-N-methylaniline. In this way it could be demonstrated that the major phase I biotransformation reactions for formation of urine excretable metabolites are (i) the cytochrome P-450-catalyzed N-demethylation followed by aromatic ring hydroxylation of the 4-fluoroaniline formed, and (ii) flavin-containing monooxygenase and cytochrome P-450-dependent formation of defluorinated 4-hydroxy-N-methylaniline.


Subject(s)
Aniline Compounds/pharmacokinetics , Cytochrome P-450 Enzyme System/metabolism , Flavins/metabolism , Mixed Function Oxygenases/metabolism , Aniline Compounds/metabolism , Animals , Biotransformation , Chromatography, High Pressure Liquid , Dealkylation , Fluorine Radioisotopes , Hydroxylation , In Vitro Techniques , Magnetic Resonance Spectroscopy , Male , Microsomes, Liver/enzymology , Mixed Function Oxygenases/isolation & purification , NADPH-Ferrihemoprotein Reductase/metabolism , Rats , Rats, Wistar
19.
Chem Biol Interact ; 85(2-3): 151-72, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1493607

ABSTRACT

The in vitro and in vivo metabolism of monofluoroanilines was investigated. Special attention was focused on the regioselectivity of the aromatic hydroxylation by cytochromes P-450 and the mechanism by which this reaction might proceed. The results clearly demonstrate that the in vitro and in vivo regioselectivity of the aromatic hydroxylation by cytochromes P-450 is dependent on the fluoro-substituent pattern of the aromatic aniline-ring. Results from experiments with liver microsomes from differently pretreated rats demonstrate that the observed regioselectivity for the aromatic hydroxylation is not predominantly determined by the active site of the cytochromes P-450. To investigate the underlying reason for the observed regioselectivity, semi-empirical molecular orbital calculations were performed. Outcomes of these calculations show that neither the frontier orbital densities of the LUMO/LUMO + 1 (lowest unoccupied molecular orbital) of the monofluoroanilines nor the spin-densities in their NH. radicals can explain the observed regioselectivities. The frontier orbital densities of the HOMO/HOMO - 1 (highest occupied molecular orbital) of the monofluoroanilines however, qualitatively correlate with the regioselectivity of the aromatic hydroxylation. Based on these results it is concluded that the cytochrome P-450 dependent aromatic hydroxylation of monofluoroanilines does not proceed by hydrogen or electron abstraction from the aniline substrate to give an aniline-NH. radical. The results rather suggest that cytochrome P-450 catalyzed aromatic hydroxylation of monofluoroanilines proceeds by an electrophilic attack of the (FeO)3+ species of cytochrome P-450 on a specific carbon atom of the aromatic aniline-ring.


Subject(s)
Aniline Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Aniline Compounds/pharmacokinetics , Aniline Compounds/urine , Animals , Biotransformation , Fluorine , Fluorobenzenes/metabolism , Fluorobenzenes/pharmacokinetics , Fluorobenzenes/urine , Hydroxylation , Magnetic Resonance Spectroscopy/methods , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Models, Chemical , Rats , Rats, Wistar , Stereoisomerism
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