Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

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<ul><li><p>ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 268, No. 2. February 1, pp. 605-616,1989 </p><p>Redox Cycling of Resorufin Catalyzed by Rat Liver Microsomal NADPH-Cytochrome P450 Reductase </p><p>DAVID R. DUTTON. GREGORY A. REED, AND ANDREW PARKINSON </p><p>Department of Pharmacology~ Toxicology. and Thera.peutics, lkiversity of Kansas hledical Center, Ka,nsas City, Kansas 66103 </p><p>Received August 12,1988, and in revised form September 30,1988 </p><p>The 0-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resoru- fin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7- alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the exten.t of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of re- duced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluo- rescent properties of resorufin were fully restored. These results indicate that the reduc- tion of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reac- tion. Stoichiometric measurements revealed a 1:l:l relationship between the amount of NADPH and O2 consumed and the amount of H202 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased -50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H202 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to HzOz. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing 0, to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxi- dation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-elec- tron reduction of resorufin (probably to the corresponding semiquinoneimine radical) </p><p> This research was supported by Grants ES 03765 and GM 37044 (awarded to A.P.), by Grant ES 04092 (awarded to G.A.R.) from the National Institutes of reer Development Award (ES 00166) from the Na- Health, and by BRSG SO7 RR05373. D.R.D. is sup- tional Institutes of Health. A preliminary account of ported by Training Grant ES 07079 from the National this work was presented in abstract form (D. R. Dut- Institutes of Health, and by Stauffer Chemical Co., ton and A. Parkinson (1987) Fed. Proc. 46,1957). Farmington, CT. A.P. is a recipient of a Research Ca- *To whom correspondence should be addressed. </p><p>605 0003-9861189 $3.00 Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. </p></li><li><p>606 DUTTON. REED, AND PARKINSON </p><p>which can either undergo a second, one-electron reduction (presumably to the corre- sponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion. The superoxide anion formed is then converted to hydrogen peroxide, either by a second, one-electron oxidation of reduced resorufin or by dismutation. In the accompanying paper, we show that the redox cycling of the 7-alkoxyresorufins by NADPH-cytochrome P450 reductase significantly affects their 0-dealkylation by puri- fied isozymes of rat liver microsomal cytochrome P450. ii 1989 i\cademic Press. Inc. </p><p>The 0-dealkylation of various 7-alkoxy- resorufins to the highly fluorescent com- pound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient method to study the induction of different forms of cytochrome P450 in liver micro- somes (l-7). In rats, for example, the O-de- alkylation of 7-ethoxyresorufin is cata- lyzed by cytochrome P45OcF whereas the 0-dealkylation of 7-pentoxy- and 7-benzyl- oxyresorufin is catalyzed by cytochrome P450b. The 30- to 60-fold induction of liver microsomal cytochrome P45Oc that results from treating rats with 3-methylcholan- threne (9, 16) is associated with a marked induction (&gt;30 fold) of the 0-dealkylation of 7-ethoxyresorufin (l-5). Similarly, the 30- to 60-fold induction of cytochrome P450b that results from treating rats with phenobarbital (9, 16) is associated with a marked induction (&gt;30 fold) of the O-deal- kylation of 7-pentoxy- and 7-benzyloxyre- sorufin (4-7). The direct fluorometric assav of 7-alk- </p><p>oxyresorufin 0-dealkylase acti;ity devel- oped for microsomal preparations must be modified for isolated rat hepatocytes and postmitochondrial supernatant (S9) frac- tions to avoid interference from DT-diaph- orase (NAD(P)H-quinone oxidoreductase, EC (17, 18). This cytosolic flavo- protein interferes with the 7-alkoxyreso- </p><p>3 Recently, a nomenclature system for cytochrome P450 isozymes was proposed based on the genes of the cytochrome P450 superfamily whose cDNA and/or amino acid sequences have been determined (8). We have included the new committee on Standardized Nomenclature conventions in boldface in this foot- note. The nomenclature system of Ryan et al. (S-11) is used throughout the manuscript. Cytochrome P450b (P450IIBl) is also known as P450 PB-B (12,13), P450 PB-4 (14), or P450 I-C (15); and cytochrome P45Oc (P450IAI) as P450 BNF-B (12.13). </p><p>rufin 0-dealkylase assay by reducing the liberated resorufin to a nonfluorescent product. This problem can be eliminated by incubating samples in the presence of the DT-diaphorase inhibitor, dicumarol, or by measuring fluorescence intensity after reactions are terminated by addition of or- ganic solvents, such as methanol or ace- tone (17-20). The latter method is based on the observation that reduced resorufin re- oxidizes after inactivation of DT-diapho- rase, with complete restoration of its fluo- rescent properties (17,18). More recently, a second problem with </p><p>the 7-alkoxyresorufin 0-dealkylation as- says has emerged from studies with puri- fied isozymes of rat liver cytochrome P450 (7, 21, 22j. As expected, purified cyto- chrome P45Oc, when reconstituted with NADPH-cytochrome P450 reductase and lipid, catalyzes the 0-dealkylation of 7- ethoxyresorufin at a rate which exceeds that catalyzed by liver microsomes from 3- methylcholanthrene-induced rats (23, 24). In contrast, purified cytochrome P450b is an unexpectedly poor catalyst of the O-de- alkylation of 7-pentoxy- and 7-benzyloxy- resort&amp; (7, 21, 22). Purified cytochrome P450b is, however, an effective catalyst of many other biotransformation reactions, including the 0-dealkylation of 7-ethoxy- coumarin (23, 25). Furthermore, antibody against cytochrome P450b effectively in- hibits the high rate of 7-pentoxy- and 7- benzyloxyresorufin 0-dealkylation cata- lyzed by liver microsomes from phenobar- bital-induced rats (6,7,22). We have undertaken studies to deter- </p><p>mine why purified cytochrome P450b, when reconstituted with NADPH-cyto- chrome P450 reductase and lipid, is an un- expectedly poor catalyst of the O-dealkyl- ation of 7-pentoxy- and 7-benzyloxyreso- </p></li><li><p>RAT LIVER MICROSOMAL NADPH-CYTOCHROME Pd50 REDUCTASE 607 </p><p>rufin. The studies described in this paper show that NADPH-cytochrome P450 re- ductase catalyzes the reduction of resoru- fin and the 7-alkoxvresorufins which, in the presence of 02, initiates a redox cycling re- action. Evidence is presented which sug- gests that the reosidation of reduced re- sorufin involves a one-electron reduction of molecular 0, to superoxide anion, which in turn forms hydrogen peroxide either by a second one-electron oxidation of reduced resorufin or by dismutation. The studies described in the accompanying paper (22) show that reduction of the 7-alkoxyresoru- fins by NADPH-cytochrome P450 reduc- tase impedes their 0-dealkylation by cyto- chrome P450b, but actually enhances their 0-dealkylation by cytochrome P45Oc. </p><p>EXPERIMENTAL PROCEDURES </p><p>CXenricn/s. Catalase (bovine liver), superoxide dis- mutase (bovine erythrocyte), horseradish peroxidase, cytochrome c, 3-(Ghydroxyphenyl)propionic acid, and diethylenetriaminepentaacetic acid (DETA- PAC? were purchased from Sigma Chemical Co. (St. Louis, MO). Resorufin (hgdroxyphenoxazone) and various 7-alkoxyresorufins were obtained from both Pierce Chemical Co. iRockford. IL) and Molecular Probes (Junction City, OR). Dicumarol [3.3-methy- lenebis(f-hydroxycoumarin)] was purchased from Aldrich Chemical Co. (Milwaukee, WI). Acetylated cytochrome c was prepared according to the method of Wada and Okunuki (26). Purijfcatio~ of ,~.~DPH-clltochrolrte P&amp;s~J reduc- </p><p>tase. NADPH-cytochrome P450 reductase was puri- fied from rat liver microsomes as described by Yasu- kochi and Masters (27). with modifications described previously (21). One nanomole of purified enzyme re- duced -3 rmol cytochrome c per minute at 22C in the presence of 330 mhl potassium phosphate buffer (pH 7.31, 1 mhf EDTA, 3 mM MgClz, 100 pM KCN, 50 pbr cytochrome c, and 100 pM NADPH. Reduction qi resorufi n end j-cllkoryresortili,cs. The </p><p>reduction of resorufin (final concentration 2.5 pM in 100 rnbl potassium phosphate buffer, pH 7-1) was mea- sured spectrophotometrically at room temperature (22C) as a decrease in absorbance at 570 nm (17,28). Similarly, the reduction of I-ethoxy-, 7-pentoxy-, and P-benzyloxgresorufin was monitored at 482, 433, and 138 nm, respectively. Spectra mere recorded on an SLM-Aminco DW-dC dual beam spectrophotometer. </p><p> Abbreviation used: DETAPAC, diethylenetri- aminepentaacetic acid. </p><p>For chemical reduction, a few grains of sodium dithi- onite were added to the contents of the sample cu- vette. For enzymatic reduction, NADPH-cytochrome P-150 reductase (0.1-1.0 nmol) was added to both the reference and sample cuvettes (final volume 1.0 ml) and reactions were initiated by addition of NADPH (final concentration 100 PM). The NADPH was added to the reference cuvette, so that a decrease in absor- bance due to reduction of resorufin in the reference cuvette was displayed as an apparent increase in ab- sorbance in the sample cuvette. N.lDPH and oqyen cowumpticm. NADPH and Oe </p><p>consumption were measured to determine the stoichi- ometry of resorufin reduction catalyzed by NADPH- cytochrome Pd50 reductase. Nt\DPH consumption was measured spectrophotometrically as a decrease in absorbance at 3-10 nm, under the same conditions used to measure the reduction of resorufin (see above). Oxygen consumption was measured with a Clark oxygen electrode under slightly different condi- tions. Under identical conditions to those used to measure resoruhn reduction or NADPH oxidation. the rate of Oz consumption was too slow to measure reliably. To increase the rate of O2 consumption, the concentration of resorufin was increased to 25 pM, and the temperature raised to 37C. The final concentra- tion of NADPH-cytochrome Pd50 reductase ranged from 0.1 to 1.0 phi in a final volume of 1.8 ml. Reactions were initiated by the addition of 180 nmol NADPH (final concentration 100 picl). The concentration of O;, was estimated to be 197 pht assuming 100% satura- tion at 37C in a 21; O4 atmosphere. Hydroyetr pemride J;wwo tiox Hydrogen peroxide </p><p>formation was measured by the fluorometric method of Zaitsu and Ohkuma (29). Reactions were carried out in 0.5-ml incubation mixtures at room tempera- ture, and contained 0.5 ~hl NADPH-cytochrome P150 reductase, 2.5 pM resorufln, and 50 PM NADPH. Reac- tions were terminated after 0. 5. 10, 15, or 20 min by the addition of 50 ~1 trichloroacetic acid (15% ). Each tube was treated with 200 ~1 of 7.5 mhl3-(l-hydroxy- phengl jpropionic acid, 2 ml of 150 mM Tris-HCI buffer, pH 8.5, and 100 ~1 of 2 units/ml horseradish peroxidase. Aftera lo-min incubation at 22C insolu- ble material was removed hy centrifugation (2000~ for 5 min). Fluorescence emission intensity was mea- sured at 404 ntn, with escitation at 320 nm. Standards for recovery and quantitative analysis were prepared from commercially available H,Op (Sigma Chemical Co.), the concentration of which was verified by titra- tion against potassium permanganate according to American Chemical Society specifications (30). Supero.ridc trniolr~h~,,lati0)2. Superoxide anion pro- </p><p>duction was determined spectrophotometrically from the rate of reduction of acetylated cytochrome c at 550 nm in the presence and absence of superoxide dis- mutase (final concentration 100 units/ml). The super- oxide dismutase-inhibitable rate of reduction of acet- </p></li><li><p>608 DUTTON, REED, AND PARKINSON </p><p>ylated cytochrome c was used to quantitate the amount of superoxide anion based on an extinction coefficient of 21 mM- em- for the reduced form of acetylated cytochrome c (31). It has been reported previously that acetylation of cytochrome c causes a much greater decrease in its rate of reduction by NADPH-cytochrome P450 reductase compared to its reactivity with superoxide anion, for which reason acetylated cytochrome c is preferentially reduced by superoxide anion (32). The acetylated eytochrome c prepared for these experiments was reduced by NADPH-cytochrome P450 reductase at 3-4s of the rate of reduction of native cytochrome c. Effects of cat&amp;use and superoxide dismutase. Where </p><p>indicated, reaction mixtures also contained 20 rg/ml catalase or superoxide dismutase (480 and 60 units/ ml, respectively) to determine the effects of these en- zymes on the rate of NADPH oxidation, Oa consump- tion, and resorufin reduction catalyzed by NADPH- cytochrome P450 reductase. </p><p>RESULTS </p><p>It has been reported previously that the oxidized and reduced form of resorulin can be distinguished spectrophotometrically and fluorometrically: Only the oxidized form of resorufin absorbs visible light (X,,, - 570 nm) and is fluorescent (X em,s1** - 585 nm, Lcitation - 530 nm) (17,28). As expected, addition of a few grains of the chemical reductant, sodium dithionite, to a solution of 2.5 ~.LM resorufin caused a com- plete loss of absorbance at 570 nm, as shown in Fig. 1. However, this loss of ab- sorbance was temporary, inasmuch as the absorbance at 570 nm was fully restored after 2-5 min (the time varied depending on how much solid sodium dithionite was added to the sample cuvette). The fluores- cent properties of resorufin, w...</p></li></ul>


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