Metabolism of nitroxide spin labels in subcellular fraction of rat liver

Biochimica et Biophysica Acta, 1034 (1990) 285-289

285

Elsevier BBAGEN 23314

Metabolism of nitroxide spin labels in subcellular fraction of rat liver I. Reduction by microsomes A n n a I a n n o n e 1, A. Tomasi 2, V. Vannini 2 and Harold M. Swartz 1 z University of Illinois College of Medicine and ESR Research Center, Urbana, IL (U.S.A.) and 2 Istituto di Patologia Generale, Universit3 di Modena, Modena (Italy)

(Received 9 October 1989)

Key words: Nitroxide metabolism; Oxygen; Hydroxylamine; ESR; (Rat liver microsomes)

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding N A D P H , induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added N A D H or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of N A D P H cytochrome c reductase, cytochrome P-450, cytochrome b s and N A D H cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADHand NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondriai metabolism seems to play an even more important role in intact cells.

Introduction Recently, nitroxides have been used extensively in functional biological systems because of their new applications as contrast agents for M R I [1-3], including: the potential of reflecting metabolism [4,5]; as agents for ESR imaging and in vivo ESR [6-12]; as biochem-

Abbreviations: PDT, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl; Tempol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; Cat-l, trirnethylamino-2,2,6,6-tetramethylpiperidine-N-oxyl; 6-Tempamine, 4amino-2,2,6,6-tetramethylpiperidine-N-oxyl; 5-Tempamine, 3-amino2,2,5,5-tetramethylpyrrolidine-N-oxyl; PCA, 3-carboxyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl;5-doxyl stearate, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl; 10-doxyl stearate, 2-(8carboxyheptyl)-2-nonyl-4,4-dimethyl-3-oxazolidinyloxyl. Correspondence (permanent address): A. Iannone, Istituto di Patologia Generale, Via Campi 287, 41100 Modena, Italy.

ical probes of redox metabolism in cells [13-16]; as well as biophysical probes of motion, oxygen concentration, etc. [17-19]. In order for these uses to be accurate and fully utilized, there is a need for detailed knowledge of the metabolism of nitroxides in biological systems. Considerable information has already been obtained at the cellular level, including observations that nitroxides are reduced at different rates by different cell lines; that the principal products of their metabolism are the corresponding nonparamagnetic hydroxylamines [13]; that cells, in the presence of oxygen, can oxidize the hydroxylamines back to the nitroxides [13]; that the metabolism of nitroxides is affected by the ability to enter cells [14,16,21]; that the ring structure affects the rate of metabolism (six-membered tings are reduced faster than five-membered rings); and that the rates of reduction of nitroxides and oxidation of hydroxylamines are affected by the concentration of oxygen [4,14,20-22]. These studies at the cellular level, how-

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286 ever, are still incomplete and need to be supplemented by the more controlled studies that can be carried out in isolated subcellular fractions. This report presents results on isolated microsomes. It is part of an ongoing study of the three principal subcellular fractions (microsomal, mitochondrial and cytosolic). We previously reported on preliminary results from microsomes using a single type of nitroxide [29]. These studies are being done in freshly isolated hepatocytes because of the importance of the liver in redox metabolism• The use of freshly isolated cells also avoids some of the potentially atypical results that can occur with cells from long-term culture because of the loss of some functions during the period of culture• In this paper, we analyze in detail the reduction of a set of seven nitroxides in rat liver microsomes under different experimental conditions, including the effects of N A D P H and N A D H , the effect of induction of microsomal activity by phenobarbital and the effect of the presence of oxygen• Reduction by N A D P H - c y t o c h r o m e c reductase was studied specifically by using the purified enzyme• Materials and Methods

Chemicals [~sN]PDT was purchased from MSD Isotopes (St. Louis, MO); all the other nitroxides were purchased from Molecular Probes (Eugene, OR); the solutions of the aqueous soluble nitroxides were prepared in PBS (0.15 M N a C 1 / 3 mM K C 1 / 5 m M phosphate buffer (pH 7.4)), while the lipid soluble nitroxides were dissolved in ethanol. The concentration of nitroxides was determined by ESR. Seven nitroxides, chosen to reflect the range of important structural variations, were used for most of the experiments. The variables studied included the type of ring on which the nitroxide is located (6-membered pyperidine, 5-membered pyrrolidine or pyrroline, or doxyl ring), the charge of substituents and lipid solubility. N A D P H and N A D H were purchased from Sigma (St. Louis, MO) and dissolved in the same buffer used for the nitroxides. Phenobarbital also was purchased from Sigma and dissolved in drinking water (1 g/l). Potassium ferricyanide was purchased from Fisher Scientific (Fair Lawn, N J) and dissolved in PBS. Purified N A D P H - c y t o c h r o m e c reductase was a gift from Professor M. Ingelman-Sundberg of the Karolinska Institute (Stockholm, Sweden). Preparation of microsomes Male Sprague-Dawley rats of about 200-250 g body weight were kept on a standard laboratory diet and, when indicated, phenobarbital was added to the drinking water for 5 days. The rats were killed by decapitation after one night of starvation. The liver was removed, weighed and homogenized (10% w / v ) in ice-cold

0.15 M KC1, using a Teflon-glass homogenizer. Then the homogenate was centrifuged at 12000 x g for 10 rain at 2°C• The pellet was discarded and the supernatant was re-centrifuged at 12000 x g for 10 min. The pellet derived from this second centrifuging also was discarded and the supernatant was centrifuged at 105 000 × g for 40 min. The resulting pellet, containing microsomes, was washed with 0.15 M KC1 and recentrifuged at 105 000 × g for 40 rain. The pellets were stored at - 8 0 ° C and used within 3 weeks of preparation.

Biochemical assays The microsomal samples were not diluted until used. The individual enzymes were assayed as follows: (1) NADH-cytochrome c reductase. The microsomal pellets were suspended in 0.25 M sucrose immediately before being added to the cuvettes. The assay system contained 0.03 m M N A D H , 0.3 m M NaCN, 2.7% cytochrome c, 0.075 M Tris-HC1 buffer (pH 7.4) and about 6 m g / m l of microsomal proteins in a final volume of 3 ml [23]. The reduction of cytochrome c was followed at 550 nm. The rates were linear. The temperature was about 25 ° C. The extinction coefficient of 18.5 mM -1 cm 1 for reduced minus oxidized cytochrome c [24] was used to convert the values into nmol. of cytochrome c r e d u c e d / m i n per mg of proteins. (2) Cytochrome b5. The difference spectrum (reduced minus oxidized) was obtained by using two cuvettes containing the same fraction and 0.05 M phosphate buffer (pH 7.5), in a final volume of 3 ml [25]. Reduction was achieved by adding 30 m M Na2S204 to one of the cuvettes. The difference between the 424 nm maxim u m and the 410 nm minimum was converted into nmol. of cytochrome b s / m g protein using 112.1 m M - 1 • cm 1 as the molar extinction coefficient [26]. Preparation of samples The microsomal pellets were resuspended in a buffered solution containing 60 mM Tris-HC1 (pH 7.4), 60 m M KC1, 2 m M MgC12 and 5 m M nicotinamide, immediately before use. The microsomal suspensions were adjusted to contain 47.1 ___7.3 m g / m l of proteins. Protein concentration was estimated by the method of Lowry et al. [27]. The assay mixture always contained: 80/~1 of microsomal suspension, 1/~1 of aqueous soluble nitroxide (0.1 m M final concentration) and sufficient amounts of the buffered solution to bring the final volume to 0.1 ml. For the experiments in which the lipid soluble nitroxides were used, the assay mixture was drawn into a 0.5 ml plastic tube containing nitroxide that previously was added and dried on the walls of the tube. When the assay was performed in the presence of N A D P H or N A D H , the final concentration of each nucleotide was always 2 mM.

287 Purified N A D P H - c y t o c h r o m e c reductase was dissolved in PBS (10 U / l ) . The samples always contained: 1 /~1 of enzyme (100 U / l ) , 1 /zl of nitroxide (0.1 m M final concentration) and sufficient PBS buffer to bring the final volume to 0.1 ml. The samples containing microsomes and nitroxides were mixed and drawn into a gas-permeable Teflon tube (Zeus Industrial Products, Raritan, N J) and placed in a quartz ESR tube which was open at both ends, so that the gas (nitrogen or air as indicated) could be flowed around the sample in the ESR cavity. The temperature of the samples always was 37 ° C.

TABLE I

ESR measurements

5-Tempamine

A Varian E-109E ESR spectrometer with settings of 1 mW incident microwave power, modulation amplitude of 1 G and a time constant of 0.064 s was used to measure the rates of reduction of aqueous soluble nitroxides; for lipid soluble nitroxides the modulation amplitude was 2 G and the time constant was 0.128 s. A standard Varian flow dewar was used for temperature regulation in the ESR cavity. For monitoring the rates of reduction of nitroxides, the line height of the mid-field peak of the nitroxide spectrum was followed for a period of 15-30 min. First-order rates of reduction were calculated from the slope.

Measurements of hydroxylamines To determine whether the products of the metabolism of nitroxides were the corresponding hydroxylamines, after reduction of nitroxides in the presence of 2 m M N A D P H or N A D H and nitrogen, 1/.tl of potassium ferricyanide (1 m M final concentration), or 0.5 m M [15N]PDT, was added at the samples and the restoration of the ESR signal was recorded in the presence of air.

Rates of reduction of nitroxides by rnicrosomes in the presence of oxygen The final concentration of N A D P H and N A D H was always 2 raM. Data are expressed as reduction rate constants: means_+S.E. (No. experiments) in 10 4 m i n - 1/mg protein ( * * = P < 0.001 and * = P < 0.05 for difference from control). Nitroxide Cat-1 Tempol 6-Tempamine

PCA 5-Doxyl stearate 10-Doxyl stearate

Control

+ NADPH

+ NADH

2.1_+0.3

35-+4.5**

(8)

(5)

(5)

80-+3.4**

5.0-+0.6 (9) 3.3+_0.2

81_+17"* (9) 54_+8.2**

352_+55** (9) 68-+11"*

(7)

(6)

(6)

1.0 + 0.3 (7) 0.9-+0.2 (9) 0.6_+0.2 (8) 0.6 _+0.01 (4)

8.2 + 1.0 * * (5) 8.7-+1.6"* (5) 75_+28* (5) 30 + 10 * (6)

29 -+ 2.3 * * (4) 15-+2.3"* (6) 77_+17"* (4) 51 _+11" (7)

crease in the rate of reduction, with the largest relative rates of increase found with the lipophilic doxyl stearates and the m a x i m u m absolute rates occurring with 5-doxyl stearate and Tempol. The rates with N A D H were significantly greater than the rates with N A D P H for Cat-l, Tempol, 5-Tempamine and PCA. Table II summarizes the rates of reduction observed in the absence of oxygen. Without the addition of N A D P H or N A D H , the rates of reduction generally did not increase, in contrast with the situation in intact ceils [16]. In contrast to the results in the presence of oxygen, in the absence of oxygen the effects of N A D P H generally were greater than the effects with N A D H . T A B L E II

Statistical analyses Statistical significance was estimated by Student's t-test: the criterion for significance was P < 0.001 or P < 0.05. Results The results of the reduction of nitroxides by microsomes in the presence of oxygen are summarized in Table I with the data presented as first-order rates of reduction. In the absence of added N A D P H or N A D H , the rates of reduction were low. As occurs in most systems, nitroxides based on the 6-membered piperidine ring were reduced more rapidly than those on the 5-membered pyrrolidine rings [14,16,21]. The doxyl stearates had the slowest reduction rates, which contrasts with the usual situation in cellular systems [15]. The addition of either N A D H or N A D P H resulted in an 8- (for the pyrrolidine nitroxides) to 100-fold in-

Rates of reduction of nitroxides by rnicrosomes in the absence of oxygen The final concentration of N A D P H and N A D H was always 2 mM. Data are expressed as reduction rate constants: means_+S.E. (No. experiments) in 10 4 r a i n - l/rag protein (* = P < 0.05 and * * = P < 0.001 for difference from the corresponding groups in Table I). Nitroxide Cat-1 Tempol 6-Tempamine 5-Tempamine PCA 5-Doxyl stearate 10-Doxyl stearate

Control 2.7_+0.6

+ NADPH 573_+33**

+ NADH 209_+26**

(4)

(4)

(4)

8.7_+0.9* (5) 5.1_+0.7"

932_+100"* (6) 542_+112"*

750_+115" (6) 136_+16"*

(5)

(4)

(4)

3.7_+0.3**

418_+45"*

163+9.3"*

(4)

(4)

(4)

1.1_+0.1 (4) 3.5_+1.1"

56_+5.0** (4) 983_+21"*

41_+25"* (4) 493_+92**

(5)

(5)

(5)

1.7_+0.6"*

1295_+116"*

554_+133"*

(4)

(4)

(4)

288 TABLE III

TABLE V

Effect of intake of phenobarbital on the rates of reduction of nitroxides in microsomes

Oxidation of hydroxylamines back to nitroxides after reduction by NADPH cytochrome c reductase

Data are expressed as reduction rate constants: means±S.E. (No. experiments) in 10 -4 r a i n - l / r a g protein (* = P < 0.05 and ** = P < 0.001 for the difference from the corresponding groups in Table I).

0.1 mM nitroxides were reduced by 100 U / I of enzyme before adding 1 mM potassium ferricyanide. 0.5 mM [15N]PDT was used to reoxidize 5-Tempamine.

Nitroxide

Control

+ NADPH

+ NADH

Nitroxide

% Oxidized

Cat-1

2.9+0.6 (6) 7.3_+0.9* (5) 4.8+0.4* (8) 0.3+0.1" (6) 0.5±0.1 (7) 0.6+0.1 (5) 0.4+0.1 (4)

235___33** (9) 486±41"* (11) 278±46** (9) 268±31"* (6) 47±9.3* (7) 359_+53** (6) 373_+52** (6)

17+1.1"* (6) 128_+2.5" (5) 23±1.3"* (6) 7.8±0.6** (6) 7.1 +4.3* (7) 5.9+1.1"* (5) 6.6+1.0" (5)

Cat-1 Tempol 6-Tempamine 5-Tempamine PCA 5-Doxyl stearate 10-Doxyl stearate

98 61 100 93 87 85 83

Tempol 6-Tempamine 5-Tempamine PCA 5-Doxyl stearate 10-Doxyl stearate

To study the role of the various microsomal enzymes in the reduction of nitroxides, a group of rats was administered phenobarbital in the drinking water for 5 days. Phenobarbital is reported to increase N A D P H cytochrome c reductase activity and the content of cytochrome P-450 [28] and to diminish slightly the level of another microsomal flavoenzyme, N A D H - c y t o chrome c reductase, as well as cytochrome bs. Under our experimental conditions, we found that the N A D H - c y t o c h r o m e c reductase (184 + 49 n m o l / m i n per mg protein in control rats) decreased to 91 + 1.6 n m o l / m i n per mg protein in phenobarbital treated rats. The content of cytochrome b 5 (0.44 + 0.1 n m o l / m g protein in untreated rats) was significantly reduced to 0.19 + 0.08 n m o l / m g protein in phenobarbital treated rats. The rates of reduction of nitroxides in the microsomes from PB-treated rats (Table III), compared with those of microsomes from untreated animals (Table I),

showed the following differences: (1) in absence of any added substrate there were minimal changes in the rates of reduction; (2) in presence of N A D P H , the nitroxides were reduced significantly faster; (3) the NADH-dependent reduction of nitroxides was significantly decreased. This behaviour paralleled the measured levels of N A D P H - c y t o c h r o m e c reductase, cytochrome P-450 and N A D H - c y t o c h r o m e c reductase. In a previous paper, we demonstrated that the NADPH-dependent reduction of the six-membered ring Tempol correlates with activity of N A D P H - c y t o chrome c reductase and not with cytochrome P-450 [29]. In this study, we examined the reduction by purified N A D P H - c y t o c h r o m e c reductase of a set of seven nitroxides, both in the presence and absence of oxygen (Table IV). N A D P H was required for reduction of any of the nitroxides to occur, and in the presence of oxygen only the piperidine based nitroxides were reduced in this system. These results paralleled, in part, the results observed with N A D P H added to the crude microsomal fraction.

TABLE VI Oxidation of hydroxylamines back to nitroxides after reduction by microsomes

TABLE IV Rates of reduction of nitroxides by purified NADPH cytochrome c reductase Data are expressed as reduction rate constant in 10 -3 min-1. The reaction mixture contained 100 U / I NADPH-cytochrome c reductase, 2 mM N A D P H and 100 mM nitroxide.

The nitroxides (0.1 mM final concentration) were reduced by microsomes in nitrogen and in the presence of 2 mM N A D P H or NADH (values between brackets). The oxidation of the corresponding hydroxylamines back to the nitroxides was started by adding 1 mM potassium ferricyanide or 0.5 mM [15N]PDT, in the presence of air.

Nitroxide

Air

Nitrogen

Hydroxylamines (parent nitroxides)

Cat-I Tempol 6-Tempamine 5-Tempamine PCA 5-Doxyl stearate 10-Doxyl stearate

12 7 8 0 0 0 0

14 16 10 8 8 12 17

Cat-1 Tempol 6-Tempamine 5-Tempamine PCA 5-Doxyl stearate 10-Doxyl stearate

% Oxidized ferricyanide 100 (100) 100 (100) 100 (100)

[15N]PDT

83 (82) 90 (89) 78 (95) 86 (86)

289 To determine whether the products were hydroxylamines, we attempted to restore the nitroxides by using potassium ferricyanide or PDT, which selectively can oxidize hydroxylamines back to the corresponding nitroxides. The results of these experiments are shown in Tables V and VI and indicate that the products of the crude microsomal fraction and the purified NADPH-cytochrome c reductase primarily are the hydroxylamines. The efficiency of oxidation of the hydroxylamines was similar to that observed in other studies of hydroxylamines [13]. Discussion These results indicate that the rapid reduction of nitroxides by microsomes requires the presence of NADPH and NADH. The NADH-dependent reduction is consistent with a mechanism involving NADH-cytochrome c reductase and cytochrome b5. The role of cytochrome P-450 and the NADPH-dependent cytochrome c reductase in the reduction of nitroxides have already been studied in several papers [30-33]. We also had studied this reaction in detail [29] with the aqueous soluble nitroxide Tempol and with the purified NADPH-cytochrome c reductase. Those resuits indicated that the NADPH-cytochrome c reductase was active even in the absence of the cytochrome P-450. The results of the present paper extend this conclusion to other aqueous soluble nitroxides and also to lipid soluble nitroxides and, in general, are consistent with previous conclusions. The details of the effects on the rate of reduction, however, indicate that there may be roles of other NADPH-dependent enzyme systems as well. Although the products of the reduction by the microsomal fraction are similar to those observed in intact cells (i.e., the hydroxylamines) and there is an effect of oxygen on the rate of reduction, the reduction by microsomes shows a much greater oxygen effect for many of the nitroxides. The results of these studies suggest that the site of reduction observed in intact cells may not be due primarily to reduction by the microsomal fraction, hinting at an important role of other subcellular fractions. Acknowledgments This research was supported by NIH grant GM 35534 and also received the financial support of the Association for International Cancer Research (AICR, U.K.) and of the CNR (Italy) contract No. 88.0091244. It used the facilities of the University of Illinois ESR

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