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Chem. Res. Toxicol. 2009, 22, 1342–1350
Oxidative and Reductive Metabolism of Tris(p-carboxyltetrathiaaryl)methyl Radicals by Liver Microsomes Christophe Decroos, Yun Li, Gildas Bertho, Yves Frapart, Daniel Mansuy, and Jean-Luc Boucher* Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, UniVersite´ Paris Descartes, 45 rue des Saints Pe`res, 75270 Paris Cedex 06, France ReceiVed April 9, 2009
Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals are particularly stable carbon-centered free radicals that are used as contrast agents in NMR imaging and as probes for in vivo oximetry by electron paramagnetic resonance (EPR) imaging. However, nothing is known so far on the metabolism of these persistent radicals in mammals. This article describes the metabolism of two TAM radicals by rat, human, and pig liver microsomes. It shows that microsomal transformation of these free radicals leads to two major metabolites resulting from an oxidation or a reduction of the present compounds. The structures of these metabolites were completely established by 1H and 13C NMR spectroscopy, mass spectrometry, and comparison with authentic compounds. Under aerobic conditions, liver microsomes catalyzed the oxidative decarboxylation of TAM radicals by NADPH and O2 with formation of the corresponding quinone-methide products. This reaction was dependent on cytochromes P450 and cytochrome P450 reductase and greatly implied the involvement of superoxide. Under anaerobic conditions, these enzymes catalyzed the reduction of TAM radicals to the corresponding triarylmethanes. This reduction was strongly inhibited by O2. These metabolic transformations should be considered when using such TAM radicals for pO2 measurement by EPR imaging, especially in tissues in which fast oxidative (inflammation sites) or reductive (hypoxic tissues) metabolism could occur. Introduction The dioxygen partial pressure (pO2)1 is an important parameter in the metabolic processes of cells (1), and measurement of tissue oxygenation in vitro and in vivo is of crucial importance in the study of both physiological and pathological processes (2). Various methods have been developed to detect and monitor pO2 (3). They include invasive optical (4-6) and electrochemical techniques (7, 8). An increasing number of new noninvasive imaging techniques used to assess quantitative tissue O2 mapping are now currently under development (9, 10). Among these methods, electron paramagnetic resonance imaging (EPRI) measures the O2-dependent line widths (3) of exogenous paramagnetic contrast agents such as nitroxides and triarylmethyl (TAM) radicals, as well as particulate-based probes such as chars and lithium phthalocyanines (10). Spin-spin interaction between the paramagnetic spin probes and the dioxygen molecule, which is also paramagnetic, influences the relaxation time of the probe and its peak to peak line width (11). For these imaging modalities, a useful spin probe should display one single sharp line on its EPR spectrum with a highly O2-dependent line width (3). Furthermore, it must possess a good stability in the presence of biological systems, a low toxicity, and a good water solubility. * To whom correspondence should be addressed. Tel: +33 1 42 86 21 91. Fax: + 33 1 42 86 83 87. E-mail:
[email protected]. 1 Abbreviations: ESI, electrospray ionization; DEX, dexamethasone; DPI, diphenyliodonium chloride; EPRI, electron paramagnetic resonance imaging; HMBC, heteronuclear multiple bond correlation; HRMS, high-resolution mass spectrometry; HSQC, heteronuclear single quantum correlation; PB, phenobarbital; pO2, dioxygen partial pressure; QM, quinone-methide; RPHPLC, reversed-phase HPLC; SOD, superoxide dismutase; SKF 525A, N,Ndiethylaminoethyl 2,2-diphenylvalerate hydrochloride; TAM, triarylmethyl; tBuOOH, tert-butylhydroperoxide; TOF, time-of-flight; X, xanthine; XO, xanthine oxidase.
Figure 1. Oxidation of TAMs 1a,b into the corresponding QMs 2a,b by superoxide.
The requirements for the probes also include a uniform concentration in the samples under comparison, this concentration being kept at levels where the self-broadening is minimal but high enough to provide an adequate signal-to-noise ratio. TAM radicals 1a,b (Figure 1) developed by Nycomed are particularly stable carbon-centered radicals that have been developed for use as contrast agents in NMR imaging (12-15) and as probes for EPRI (11, 16-18). They have been demonstrated to be very effective spin probes in measuring pO2 in vivo and in vitro through line-broadening analysis because their EPR spectrum displays one narrow single line, the line width of which linearly depends upon the O2 concentration (11, 16-18). Oxo63, 1b, is a compound highly soluble in water and weakly toxic (LD50, 8 mmol kg-1) (15). It is stable in the presence of many oxidoreductants such as ascorbate, glutathione, NADPH, hydrogen peroxide, and hydroxyl radical (19, 20). In addition, it has been reported that Oxo63 may be used for the specific measurement of superoxide, O2•-, in cell-free and cellular systems (19, 20). Recently, we have shown that TAMs 1a,b very efficiently react with superoxide with the formation of the
10.1021/tx9001379 CCC: $40.75 2009 American Chemical Society Published on Web 06/22/2009
Microsomal Metabolism of TAM Radicals
corresponding diamagnetic quinone-methides (QMs) 2a,b (Figure 1) that result from an oxidative decarboxylation of the starting TAM radicals (21). The pharmacokinetics of Oxo63 in mice has been followed by EPR spectroscopy and imaging (22). However, nothing is known presently on the in vivo or in vitro metabolism of stable free radicals such as 1a,b (23). This article describes for the first time the metabolism of TAMs 1a,b, by rat, human, and pig liver fractions. It shows that hepatic microsomes that contain the main enzymatic systems involved in the oxidative and reductive metabolism of drugs and xenobiotics efficiently catalyze the reduction of 1a,b into the corresponding triarylmethane derivatives by NADPH, as well as their oxidation into the corresponding QMs by NADPH and O2. This study fully identifies the predominant metabolites formed by these reactions and shows that cytochromes P450 and cytochrome P450 reductases are the main enzymatic systems involved.
Experimental Procedures Chemicals. Tris-(8-carboxyl-2,2,6,6-tetramethylbenzo-[1,2-d;4,5d′]bis[1,3]dithiol-4-yl)methyl sodium salt (1a) was synthesized according to a previously described method (13, 24). Oxo63 (1b) was a generous gift from Dr M.C. Krishna (NIH, Bethesda, MD). NADPH and NADH were purchased from Boehringer (Mannheim, Germany), and xanthine (X), xanthine oxidase (XO), superoxide dismutase (SOD), cytochrome c, catalase, EDTA, diphenyliodonium chloride (DPI), clotrimazole, N,N-diethylaminoethyl 2,2-diphenylvalerate hydrochloride (SKF 525A), metyrapone, and tertbutylhydroperoxide (tBuOOH) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). All solutions were prepared in phosphate buffer (0.1 M, pH 7.4) in the presence of EDTA (100 µM). All other chemicals and solvents were of the highest grade commercially available. Spectroscopic Methods. All NMR experiments [1H, 13C, heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC)] were carried out at room temperature on a Bruker Biospin Avance II 500 MHz spectrometer, and chemical shifts are reported in ppm (δ) relative to tetramethylsilane; s is used for singlet. Infrared spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrophotometer. UV-visible spectra were recorded on a Uvikon 942 spectrometer (Kontron Biotech). HPLC-MS studies were performed on a Surveyor HPLC instrument coupled to a LCQ Advantage ion trap spectrometer (Thermo, Les Ulis, France). Mass spectra were obtained by electrospray ionization (ESI) in both positive (ESI+) and negative (ESI-) ionization detection modes under the following conditions: sheath gas, 40; auxiliary gas, 10; spray voltage, 5 kV; capillary temperature, 300 °C; capillary voltage, 7 V; and scanning in full scan mode (m/z from 200 to 1500). High-resolution mass spectra (HRMS) were obtained by ESI in time-of-flight (TOF) detection mode on a LCT (Waters-Micromass) spectrometer at ICSN (Gif sur Yvette, France). EPR spectra were recorded at 20 °C using a Bruker Elexsys 500 EPR spectrometer operating at X-band (9.85 GHz) with a TM 110 cavity and an AquaX quartz cell, under the following conditions: modulation frequency, 100 kHz; modulation amplitude, 0.03 G; time constant, 40.96 ms; conversion time, 40.96 ms; and microwave power, 1 mW. Data acquisition, processing, and double-integration were performed using Bruker software. Synthesis of QM, 2a. QM 2a was obtained by reaction of 1a with superoxide radical generated by the X/XO system as previously described (21). Compound 1a (10.0 mg, 9.4 µmol) was dissolved in 100 mL of 0.1 M phosphate buffer (pH 7.4), containing 100 µM EDTA, 1 mM X, and 0.04 U mL-1 XO. The solution was kept at 37 °C for 90 min with a slow bubbling of dioxygen. The organic products were extracted (3 × 100 mL) with a diethyl ether/
Chem. Res. Toxicol., Vol. 22, No. 7, 2009 1343 acetonitrile (1/1, v/v) mixture, and the solvents were evaporated under vacuum. The crude product was purified by reversed-phase (RP)-flash chromatography over a prepacked C18 column (AIT, Houilles, France) using a gradient from 5/95 to 20/80 of acetonitrile/ water mixture to afford 9.0 mg (quantitative yield) of pure compound 2a as a purple solid. 1H NMR (CD3OD): 1.65 (s, 6H), 1.72 (s, 6H), 1.74 (s, 6H), 1.76 (s, 6H), 1.82 (s, 6H), 1.83 (s, 6H). 13 C NMR (CD3OD): 29.41, 31.05, 32.22, 33.28, 34.57, 62.90, 63.83, 63.97, 128.89, 130.76, 132.37, 138.87, 139.95, 141.08, 141.20, 144.35, 145.31, 158.49, 173.17, 174.07. UV-vis (H2O) λmax (nm) (ε in M-1 cm-1): 276 (34500), 360 (8000), and 546 (12000). MS (ESI+) m/z ) 970.9 ([M + 3H]+). HRMS (TOF-ESI-) calcd for C39H37O5S12 ([M + H]-), 968.9290; found, 968.9277. IR (acid form, neat, cm-1): 3191, 2960, 1702, 1607. Synthesis of Tris-(8-carboxyl-2,2,6,6-tetramethylbenzo-[1,2d;4,5-d′]bis[1,3]dithiol-4-yl)methane Sodium Salt, 3a. Compound 1a (10.0 mg, 9.4 µmol) was dissolved in 1 mL of degassed water, and Na2S2O4 (4.0 mg, 23.0 µmol) was added at once. The solution immediately turned dark blue and then slowly light yellow. After 15 min at room temperature, the resulting solution was lyophilized. The crude product was dissolved in 1 mL of methanol and filtered to remove salts. The solution was concentrated in vacuo to afford 10 mg of compound 3a in quantitative yield. 1H NMR (CD3OD): 1.62 (s, 9H), 1.72 (s, 9H), 1.73 (s, 9H), 1.76 (s, 9H), 5.47 (s, 1H). 13 C NMR (CD3OD): 28.62, 28.78, 34.00, 35.15, 62.39, 62.80, 63.33, 129.68, 129.77, 138.89, 139.76, 140.50, 140.79, 174.23. UV-vis (H2O) λmax (nm) (ε in M-1 cm-1): 272 (36100) and 376 (8000). MS (ESI+) m/z ) 1000.9 ([M + 4H]+). HRMS (TOF-ESI-) calcd for C40H39O6S12 ([M + 2H]-), 998.9395; found, 998.9410. IR (acid form, neat, cm-1): 3195, 2979, 1689. Preparation of Liver Microsomes and Cytosol Fractions. Male Sprague-Dawley rats (200-250 g) were provided laboratory chow and water ad libitum. After 7 days of adaptation, animals were treated either with phenobarbital (PB) (0.1% in drinking water for 5 days) or dexamethasone (DEX) (50 mg kg-1, in corn oil, i.p. for 4 days). Control animals were treated with corn oil (0.5 mL, i.p., for 4 days). Liver cytosols and microsomes were prepared by differential centrifugation as previously reported and stored at -80 °C until use (25). Microsomes from pig liver (obtained from a slaughterhouse) were obtained following an identical protocol. Human liver microsomes were obtained from BD-Gentest (Le Pont de Claix, France). Protein concentrations were determined by the Bradford assay with bovine serum albumin as standard (26). Cytochrome P450 contents were determined by the method of Omura and Sato (27). The cytochrome c reductase activity of the various microsomes was measured according to a previously described procedure (28) and were 160 ( 19, 306 ( 35, 601 ( 71, 120 ( 13, and 132 ( 15 nmol reduced cyt c min-1 mg prot-1, for liver microsomes from untreated, PB-treated, and DEX-treated rats and from humans and pigs, respectively. Microsomal dealkylation of 7-ethoxycoumarin activities (29) were 70 ( 8, 303 ( 22, and 156 ( 18 pmol produced 7-hydroxycoumarin min-1 mg prot-1 for liver microsomes from untreated, PB-treated, and DEX-treated rats, respectively, in agreement with previous literature data (30). Incubations of 1a in the Presence of Liver Microsomes. Anaerobic incubations were performed at 37 °C in 1 cm path length quartz cuvettes previously purged with argon and stopped with a rubber septum. Liver microsomes were gently degassed by argon flowing for 5 min at 4 °C at the surface of the sample. In parallel, the buffer was degassed by argon bubbling for at least 30 min at 4 °C and added to degassed liver microsomes. Typical anaerobic incubation mixtures (final volume, 200 µL) contained 100 µM 1a in 0.1 M phosphate buffer (pH 7.4), 100 µM EDTA, and 0.5-1.8 mg mL-1 microsomal proteins. After equilibration for 5 min at 37 °C, the reactions were started by addition of NADPH (1 mM final concentration) and monitored by UV-vis spectroscopy at 37 °C either by repetitive scanning between 380 and 880 nm or by following the decrease in absorbance at 469 nm as a function of time. An aliquot of the reaction mixture was quenched by addition of cold acetonitrile (same volume) containing 4-chlorobenzamide
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Figure 2. EPR characteristics of TAM 1a and EPR analysis of reaction of 1a with liver microsomes and NADPH. EPR spectra of a 100 µM solution of 1a in phosphate buffer, pH 7.4, under (A) aerobic (about 260 µM O2) and (B) anaerobic conditions. (C) Time-dependent variation of 1a concentration (deduced from the 1a EPR signal intensity) (0) and line width of the 1a EPR signal (b) during incubation of 1a with PB-treated rat liver microsomes and NADPH in an AquaX cell (incubation at 20 °C in 0.1 M phosphate buffer, pH 7.4, containing 100 µM EDTA, 100 µM 1a, 1 µM P450, and 1 mM NADPH) as described in the Experimental Procedures.
(internal standard; final concentration, 100 µM), centrifuged for 10 min at 13000 rpm, and analyzed by HPLC-MS. Identical incubation procedures were followed for experiments performed under aerobic conditions except for the use of 2-7 mg mL-1 of microsomal proteins and of dioxygen-saturated buffer after O2 bubbling for 10 min. Analysis of the incubates by UV-vis and HPLC-MS was performed as described above. To quantitate the possible formation of 3a by HPLC, tBuOOH (80 mM) was added at the end of the incubation, and the reaction mixture was reincubated for 10 min at 37 °C. Under these conditions, a complete oxidation of the remaining 1a to 2a was observed, whereas compound 3a was unaltered. Transformations of 1b by PB-treated rat liver microsomes were performed under identical conditions. Reversed-Phase (RP)-HPLC Analysis. Analyses of 1a metabolites were performed at room temperature on a 150 mm × 3.9 mm Novapak C18 column (Waters, St. Quentin en Yvelines, France) using a Spectra Physics HPLC system. The mobile phase was a mixture of solvent A (water + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid) with the following gradient: 0-2 min, isocratic elution with 95% A; 2-32 min, linear increase from 5 to 95% B; 32-35 min, isocratic elution with 95% B; 35-38 min, linear decrease from 95 to 5% B; and 38-45 min, reequilibration at 5% B. The flow rate was 1 mL min-1. The absorbance was monitored at 270 and 240 nm and recorded using the Borwin data acquisition system. Under these conditions, the retention times for 1a, 2a, and the internal standard were 20.1, 23.0, and 9.0 min, respectively, whereas compound 3a coeluted with 1a. Metabolites were further identified by LC-MS under the MS conditions indicated above. Data were recorded and analyzed with the XCalibur acquisition system. Separation and identification of 1b metabolites were performed at room temperature on a 250 mm × 4.6 mm Polarity C18 column (Waters) using the same LC-MS system. The mobile phase and the gradient were identical to those used for TAM 1a except that the flow rate was 500 µL min-1. The absorbance was monitored at 270 nm. Under these conditions, the retention times for 1b and 2b were 15.0 and 15.6 min, respectively, whereas compound 3b coeluted with 1b.
Results Transformation of TAM 1a by Rat Liver Microsomes Followed by EPR and UV-Visible Spectroscopy. The EPR spectrum, at 20 °C, of a solution of 1a in 0.1 M phosphate buffer, pH 7.4, in an Aqua X cell, shows a single peak with a g value of 2.0030 and a line width of 180 mG, as expected for an aerated solution of 1a in water (24) (Figure 2A). The addition of either liver microsomes from PB-pretreated rats or 1 mM NADPH alone did not change its EPR signal intensity and line
width, indicating that 1a was stable under these conditions (data not shown). By contrast, a time-dependent decrease of the EPR signal intensity and of the line width was observed when 1a was mixed with PB-treated rat liver microsomes and NADPH. Figure 2C shows the variation of the 1a concentration that was measured by double integration of the EPR signal of 1a using the adapted Bruker software. The observed decrease of the intensity of the EPR signal indicated that 1a was transformed into diamagnetic product(s). Figure 2C also shows a decrease of the line width of the EPR signal of 1a, indicating that the dioxygen concentration in the AquaX cell (quartz tubes isolated from the atmosphere) decreased as a function of time. It is noteworthy that the EPR signal line width is proportional to the O2 concentration, whereas it does not depend on the concentration of 1a (at least for 1a concentrations lower than ∼3 mM). The data of Figure 2C indicated that two phases were involved in the consumption of 1a. The first one mainly occurred during the first 15 min and was concomitant to dioxygen consumption, whereas the second one mainly occurred later and under low dioxygen concentrations (line width around 90 mG very close to that observed for 1a under anaerobic conditions, Figure 2B). The UV-visible absorption spectrum of 1a (in 0.1 M phosphate buffer) shows a characteristic peak at 469 nm (ε ) 16000 M-1 cm-1) (see Figure S1 of the Supporting Information). This UV-vis spectrum was unaffected by the addition of either PB-treated rat liver microsomes or NADPH alone (data not shown). However, time-dependent changes were observed when 1a was mixed with microsomes and NADPH. At 37 °C, during the first 6 min of the experiment, a decrease in absorbance at 469 nm was observed with a concomitant increase in absorbance at 532 nm (Figure 3A). Thereafter, the absorbance at 469 nm further decreased without any increase of the absorbance in the 532 nm region (Figure 3B). Replot of the absorbance at 469 nm as a function of time clearly identified two distinct slopes (Figure 3D). The data of Figure 3 (A, B, and D for 0 e t e 12 min) confirmed the EPR results showing that consumption of 1a in the presence of liver microsomes and NADPH, under conditions of limited access of dioxygen in EPR Aqua X cell or in UV-vis microcuvette, occurred in two phases. During the first phase, dioxygen consumption occurred and 1a was mainly transformed into a species absorbing at 532 nm, whereas in the second phase, 1a was transformed into another diamagnetic species showing no clear visible absorption between 420 and 700 nm. Because of extensive consumption of O2 during
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Figure 4. Main reaction occurring during anaerobic reduction of TAMs 1a,b by rat liver microsomes and NADPH.
Figure 3. UV-vis study of the reaction of 1a with PB-treated rat liver microsomes and NADPH. Time-dependent changes of the UV-vis difference spectrum (relative to the UV-vis spectrum of the incubate at t ) 0, before NADPH addition) of an incubation of 1a with PBtreated rat liver microsomes (conditions as indicated in Figure 2 except for temperature, 37 °C). (A) Aerobic phase (from 0 to 6 min), (B) anaerobic phase (from 7 to 12 min), (C) after reoxygenation of the solution (from 13 to 17 min) by O2 bubbling at 12 min, and (D) plot of the absorption at 469 nm as a function of time during the A, B, and C phases of the reaction.
the first phase, the reaction in the second phase occurred under quasi-anaerobic conditions (Figure 2B and 3B). Accordingly, after reintroduction of O2 after 12 min (Figure 3C, D for t g 12 min), the aerobic formation of the 532 nm absorbing species restarted with a rate almost identical to that of the first phase (Figure 3D, compare Figure 3D, 0 e t e 6 min and t g 12 min). To more clearly identify the role of dioxygen in the transformation of 1a in the presence of rat liver microsomes and NADPH, we performed the following reactions either in deoxygenated buffer (anaerobic conditions) or in buffer previously saturated with O2 (aerobic conditions). Metabolism of TAM 1a under Anaerobic Conditions. As mentioned above (Figure 2B), the EPR spectrum of 1a in a 0.1 M phosphate buffer (pH 7.4) that has been extensively deoxygenated by pumping under vacuum and bubbling of argon for
at least 30 min displays a line width of 90 mG. This line width did not further decrease after an additional bubbling of argon, indicating that the solution was reasonably anaerobic. Careful addition, under argon, of concentrated rat liver microsomes that have been gently degassed by argon flowing at the surface of the suspension did not lead to any significant change of the EPR signal of the solution. After the addition of NADPH, one observed a time-dependent decrease of the EPR signal intensity without any change in its line width, suggesting that 1a was transformed into diamagnetic compound(s) and that the solution remained anaerobic during the reaction (data not shown). Following the same reaction by UV-vis spectroscopy showed a linear time-dependent decrease in the absorbance at 469 nm of 1a without the appearance of any new peak in the visible region (data not shown). To identify the new diamagnetic species formed during this reaction, the incubation mixtures were analyzed by RP-HPLC using UV-vis and MS detection. HPLC analyses showed the presence of only one new species having the same retention time (20.1 min) as 1a but a distinct UV-vis spectrum (λmax at 272 and 376 nm) (see Figure S1 of the Supporting Information) and a MS (ESI+) molecular ion peak at m/z ) 1000.9 corresponding to that of 1a + one mass unit as expected for [3a + 4H]+ (see Table S1 of the Supporting Information). These results suggested that reduction of 1a into the diamagnetic triarylmethane compound 3a has occurred (Figure 4). Complete identification of this metabolite as triarylmethane 3a was obtained by comparison of its HPLC retention time and of its UV-vis and MS spectra with those of an authentic compound prepared by reduction of 1a with excess sodium dithionite. The structure of triarylmethane 3a was completely confirmed by a detailed analysis of its 1H and 13C NMR spectra, including 1H-13C correlations using HSQC and HMBC techniques (see Figures S2-S6 of the Supporting Information). The 1 H (five singlets between 1.62 and 5.47 ppm) and 13C (14 peaks between 28.62 and 174.23 ppm) NMR signals of 3a were assigned thanks to the detection of many C-H correlations between 13C and 1H nuclei through up to five bonds. Thus, its 1 H NMR spectrum exhibited a singlet at 5.47 ppm (1 H) as expected for a Ar3CH benzylic proton (12). This NMR analysis indicated that the aryl rings were not coplanar because of the steric repulsion between their bulky substituents. The molecule has a C3 symmetry axis, explaining the number of singlets found for the methyl protons (four for the aryl rings). The molecular ion observed in HRMS (TOF-ESI-) at m/z ) 998.9410, corresponding to [3a + 2H]- (calculated value, 998.9395), was also in agreement with structure 3a (see Table S1 of the Supporting Information). The anaerobic reduction of 1a into triarylmethane 3a by NADPH in the presence of rat liver microsomes absolutely required active microsomal proteins as heat-inactivated microsomes were inactive (Table 1). The rat liver cytosolic fraction was 100 times less active than the microsomal fraction. NADPH
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Table 1. Effects of Incubation Conditions on the Anaerobic Reduction of 1a into Triarylmethane 3a and on the Aerobic Oxidation of 1a into QM 2a by PB-Treated Rat Liver Microsomes anaerobic incubations
aerobic incubations
conditions
formation of triarylmethane 3aa
formation of QM 2ab
CS - microsomes denaturated microsomesd - microsomes + cytosole - NADPH - NADPH + NADHf + NADH + FAD + FMNg + clotrimazole (100 µM) + metyrapone (100 µM) + SKF 525 A (100 µM) + DPI (100 µM)h + SOD (1000 U mL-1) + catalase (1000 U mL-1) + SOD + catalase
22.3 ( 1.1
