NADPH-induced microsomal chemiluminescence without accompanying lipid peroxidation as measured by the thiobarbituric acid assay

112

Biochimica et Biophysica Acta, 5 8 6 ( 1 9 7 9 ) 1 1 2 - - 1 2 2 © Elsevier/North-Holland

Biomedical Press

BBA 28964

NADPH-INDUCED MICROSOMAL CHEMILUMINESCENCE WITHOUT ACCOMPANYING LIPID P E R O X I D A T I O N AS M E A S U R E D BY THE T H I O B A R B I T U R I C ACID ASSAY *

SANDRA

L. G U T H A N S ,

W I L L I A M H. B A R I C O S * * a n d R I C H A R D

H. S T E E L E

Department o f Biochemistry, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, LA 70112 (U.S.A.) (Received October 18th, 1978) (Revised manuscript received February 19th, 1979)

Key words: Chemiluminescence; Peroxidation; Thiobarbituric acid assay; Redox chain; NADPH; (Microsome)

Summary Pyrophosphate added to rat liver microsomes blocks both NADPH-induced chemiluminescence and lipid peroxidation, whether NADPH is added in bulk or generated with an NADPH-regeneration system. Addition of KCN to these systems released the pyrophosphate suppression of NADPH-induced chemiluminescence without restoring lipid peroxidation. A hexose or hexose derivative is required for the display o f KCN-released chemiluminescence with galactose 6-phosphate and glucose 6-phosphate being most effective. The integral KCNreleased chemiluminescence for pyrophosphate inhibited systems is proportional to microsomal protein, NADPH, and glucose-6-phosphate dehydrogenase concentrations. Pyrophosphate also blocks ascorbic acid induced chemiluminescence. Additionally ascorbic acid inhibits the KCN release of NADPHinduced chemiluminescence. This work establishes a point a divergence in the microsomal redox chain between NADPH-induced chemiluminescence and lipid peroxidation as measured by the thiobarbituric acid assay.

Introduction

For many years this laboratory has been investigating the possible functionality o f high energy electronic excitation states in mammalian systems * The work described in this paper was carried o u t by Sandra L. G u t h a n s in partial fulfillment of the requirements for the Doctor o f Philosophy Degree. * * To w h o m correspondence concerning this paper should be addressed.

113 other than vision. Our experimental approach has been to look for light emission in biological systems. Such luminescent systems potentially mediate their reactions (dark photochemistry, [1]) through high energy states. Several laboratories, including our own, have reported that microsomal chemiluminescence correlates closely with lipid peroxidation when induced by either NADPH or ascorbic acid [2--5]. Howes and Steele [2] presented data, however, which suggested that the NADPH-induced chemiluminescence could not have originated solely from the breakdown of lipid peroxides. These workers reported subsequently [6] that both NADPH-induced chemiluminescence and lipid peroxidation were suppressed in the presence of hydroxylatable substrates such as polycyclic aromatic hydrocarbons. They. suggested that the NADPHinduced microsomal chemiluminescence, lipid peroxidation, and substrate hydroxylations are mutually competitive processes mediated by a common excited state intermediate. These studies were extended by Shoaf and Steele [3] who showed that the addition of KCN to NADPH- or ascorbic acid-induced peroxidized microsomes or their chloroform/methanol extracts produced a marked instantaneous burst of chemiluminescence followed by a rapid decline (the cyanide flash). Furthermore, the addition of KCN to either the peroxidized microsomes or a chloroform/methanol extract of peroxidized microsomes gave rise to gas evolution, reduction of methylene blue or nitroblue tetrazolium, hydroxylation of aniline, and O-demethylation of p-nitroanisole. These reactions are compelling evidence for the involvement of 1-hydroxy hydroperoxides, and experiments with chemically prepared compounds supported this suggestion. These workers postulated that substrate oxidations were effected by ~ -~ lr* carbonyl tripletmediated hydrogen abstraction followed by peroxidic oxygen insertion in a concerted reaction between the nucleophilic CN-, the substrate, and the 1-hydroxy hydroperoxide. In the absence of substrate the excited carbonyl triplet could relax thermally or, in the presence of endogenously generated or exogenously added fluorescers, transfer its energy to these species by resonance and emit light. Our previous studies [2,3,6] have all been carried out using single, pulse additions of NADPH and/or ascorbic acid to buffered oxygenated microsomal suspensions. Recognizing that microsomal drug oxidation studies are usually made with NADPH-regenerating systems, and that NADPH-mediated processes such as drug oxidation, lipid peroxidation, and chemiluminescence (NADPH can serve as a fluorescer to which carbonyl triplets may transfer their energy) could be sustained by maintaining the concentration of NADPH, we began studies to evaluate the influence of an NADPH-regenerating system on these several processes. The results of these investigations, which we have reported preliminarily [7], represent the subject of this paper. Methods and materials

Isolation o f microsomes. Male Sprague-Dawley rats, 150--250 g, were killed by decapitation and exsanguinated. Each liver was rapidly removed and placed in ice-cold (0--4°C) 0.25 M sucrose. All subsequent manipulations were made at 0--4°C. The liver was weighed, minced and homogenized in 0.25 M sucrose

114 (1 : 2, w/v) utilizing a Tri-R Stir-R variable speed m o t o r set at maximum rev./ min and using a size C Thomas glass homogenizer tube with a smooth teflon pestle. The homogenate was centrifuged at 8700 X g for 20 min in a Beckman Model L Ultracentrifuge using a Type 30 rotor. The supernatant was recentrifuged (Beckman Model L Ultracentrifuge, Type 50 rotor) at 74 000 × g for 90 min. The resulting microsomal pellet was resuspended in the original volume of 0.25 M sucrose, divided into 2-ml aliquots and stored at --20°C for not longer than 15 days prior to use. Analytical methods. Protein was determined by the m e t h o d of Lowry et al. [8]. Lipid peroxidation was measured using a 0.5 ml aliquot of the appropriate microsomal system by the thiobarbituric acid assay of Ottolenghi [9], with absorbances read at 535 nm. All concentrations given are before the addition of KCN. The microsomal chemiluminescence studies were made in a darkened room illuminated with red lights and at ambient temperature (21--22°C). Microsomal preparations were placed in dark adapted 20 ml Packard glass scintillation vials. Incubation conditions. In most experiments each vial contained 0.2 ml microsomes (2.5--4.2 mg protein), 20 mM sodium pyrophosphate, 27 mM D-glucose 6-phosphate (Sigma, m o n o s o d i u m salt), 1 mM NADPH (Sigma, tetrasodium salt), and 0.1 M sodium phosphate buffer, pH 7.4; final volume 2.8 ml. Alterations in this 'standard assay system' are indicated in the legends. Each preparation was gassed with 100% oxygen for one minute prior to the light emission studies which were initiated by the addition of either NADPH or NADP ÷ and glucose-6-phosphate dehydrogenase. The KCN release of the pyrophosphate suppressed NADPH-elicitable microsomal light was effected by the injection of 0.5 ml of 0.1 M KCN (freshly prepared), which when added to 2.8 ml microsomal suspensions gave a final KCN concentration o f 15 mM. Chemiluminescence measurements. Chemiluminescence was measured with a Packard Scintillation Spectrometer, Model 3320, operated in the out-of-coincidence summation m o d e as described b y Stanley and Williams [10]. The following additional reagents were also used (from Sigma): D-galactose 6-phosphate (disodium salt), a-D-glucose 1-phosphate (dipotassium salt), D-fructose 6-phosphate (disodium salt), D-gluconic acid (potassium salt), glucuronic acid lactone (grade IX), and thiobarbituric acid. All other chemicals were o f reagent grade or higher. Results

As reported previously [2] and repeated here a suspension of oxygenated rat liver microsomes in phosphate buffer induced by NADPH addition yields a chemiluminescence accompanied by an accumulation o f lipid peroxides as measured b y the thiobarbituric acid assay. The addition of KCN to such peroxidized microsomes results in a 'flash' of chemiluminescence (Fig. 1, curve a). In contrast, when the oxygenated microsomal suspensions were examined with the NADPH-regenerating system, neither chemiluminescence nor lipid peroxidation resulted until after the addition o f KCN. Moreover, chemiluminescence which displayed after the addition of KCN, did not appear as a flash,

115

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but as a relatively slow and progressively increasing emission. More remarkable however, was the complete absence of lipid peroxidation as measured by the thiobarbituric acid assay either before or after KCN addition (Fig. 1, curve b). It should be emphasized that pyrophosphate and glucose 6-phosphate have no influence on the thiobarbituric acid assay for lipid peroxidation. A peroxidized microsomal preparation gave the same thiobarbituric acid assay values when pyrophosphate and glucose 6-phosphate were added subsequent to peroxidation as that obtained in the absence of pyrophosphate and glucose 6-phosphate (see Table I). In order to establish which component(s) of the NADPH-regenerating system was responsible for the prevention of chemiluminescence prior to KCN addition and the lipid peroxidation, a series of c o m p o n e n t deletion experiments was made in which various components were singly omitted. Prevention of the p r e ~ y a n i d e emission and lipid peroxidation was attributable to the presence of pyrophosphate (Fig. 2, curve a). Additionally the KCN release of the pyrophosphate-suppressed chemiluminescence appeared to require the presence of glucose 6-phosphate (Fig. 2, curve d). The omission of either MgC12 or nicotinamide failed to appreciably alter the chemiluminescence or lipid peroxidation from the results found using complete incubation system (Fig. 2, curves b and c). In subsequent experiments microsomes examined in the m e d i u m containing

116

TABLE I LIPID PEROXIDATION DATA FOR VARIOUS MICROSOMAL SYSTEMS Lipid p e r o x i d a t i o n was m e a s u r e d o n a 0 . 5 m l a l i q u o t t a k e n i m m e d i a t e l y b e f o r e t h e a d d i t i o n o f K C N , a n d again a t the e n d o f the i n c u b a t i o n . E a c h e n t r y r e p r e s e n t s t h e a v e r a g e o f five e x p e r i m e n t s , ± the s t a n d a r d deviation. lcm A b s o r b a n c e (A 53 $ )

Simple buffered system * S t a n d a r d assay s y s t e m S i m p l e b u f f e r e d s y s t e m * with glucose 6 - p h o s p h a t e ( 2 7 m M ) a n d p y r o s p h o s p h a t e ( 2 0 m M ) a d d e d a f t e r t h e i n c u b a t i o n was c o m p l e t e

Before KCN

After KCN

0.99 ± 0 . 1 6 0 . 0 2 ± 0.01 1 . 2 0 ± 0.07

0.80 ± 0.10 0.02 ± 0.00 0.87 ± 0.02

* C o n t a i n s 0.8 m g m i c r o s o m a l p r o t e i n / m l , 1 m M N A D P H , a n d 0.1 M p h o s p h a t e b u f f e r , p H 7.4; final v o l u m e 3.0 ml.

only pyrophosphate, glucose 6-phosphate, and bulk addition of NADPH, gave pre- and post-KCN light and lipid peroxidation results similar to those found using the complete NADPH-regenerating system and served to define the minimal requirements for the KCN release of the pyrophosphate suppressed,

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117 NADPH-elicitable-chemiluminescence w i t h o u t accompanying lipid peroxidation. We will use the term 'KCN-released chemiluminescence' to refer to this t y p e of chemiluminescence. The requirements for pyrophosphate, NADPH, and KCN appear to be specific. The replacement of p y r o p h o s p h a t e b y other chelators such as EDTA, EGTA, or oxalic acid in equimolar amounts inhibited not only NADPHinduced lipid peroxidation b u t also blocked chemiluminescence including the KCN-released chemiluminescence. KOH, KC1, 1,4
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NADPH. As shown in Fig. 5, several hexose-phosphates and other sugars can be substituted for glucose 6-phosphate with varying degrees of effectiveness with galactose 6-phosphate being most effective on a molar basis.

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Further characterization of this system demonstrated that the integral light emitted in the KCN-released chemiluminescence was proportional to the microsomal protein concentration (Fig. 6) and the concentration of glucose 6-phosphate dehydrogenase {Fig. 7). In Fig. 7 the integral light in relative units are indicated on the temporal traces for the chemiluminescence. The b o t t o m curve 12

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in this figure represents the light response with no enzyme added. As these microsomes were not washed we interpret this response as due to contaminating glucose 6-phosphate dehydrogenase. Fig. 8 depicts the temporal traces, with superimposed relative light integrals, for the KCN-released chemiluminescence as a function of the concentration of NADPH. The data indicate that a certain minimum concentration of NADPH appears necessary to permit the KCN release of the light and that the integral light appears to increase as the NADPH concentration increases. Boiled microsomes (10 min) do not support NADPH-induced chemiluminescence or lipid peroxidation in the absence of pyrophosphate [11] nor is KCN able to release chemiluminescence in systems containing boiled microsomes in the presence of pyrophosphate and glucose 6-phosphate. Discussion While we have refrained from using the term 'bioluminescence' to describe the light emission we have observed, we nevertheless believe these emissions to result from an enzymatic process(es), this based on the following observations: (1) There is a direct correlation between microsomal protein and integral light production. (2) Boiled microsomes will not emit KCN-elicitable light in these pyrophosphate systems. It is known that boiled microsomes are capable of supporting a non-enzymatic ascorbic acid-induced lipid peroxidation, but do not support NADPH-induced lipid peroxidation [ 11 ]. (3) This reaction has an absolute specificity for NADPH. On purely chemical considerations it would be expected that NADH could substitute for NADPH in a non-enzymatic system. Hochstein and Ernster [12] examined the influence of varying pyrophosphate concentrations on microsomal oxygen uptake and lipid peroxidation. Low concentrations of pyrophosphate (0.001--0.1 mM) were found to stimulate oxygen uptake and malondialdehyde formation. However both these pro-

121

cesses were inhibited by higher pyrophosphate concentrations, inhibition approaching 100% at 4.0 mM. Since our system contained a high concentration of pyrophosphate (20 mM) the inhibition of lipid peroxidation was predictable. However, the ability of KCN to induce a chemiluminescence in pyrophosphatecontaining systems was totally unexpected, especially in light of McCay's suggestion [13] that microsomal chemiluminescence may result from 102 and other products of lipid peroxide breakdown. Poyer and McCay [14] stressed that Fe (III) is probably a requirement for oxygen uptake by microsomes incubated with NADPH in the absence of substrate. However, the involvement of contaminating Fe (III) in the KCN-released microsomal chemiluminescence appears to be precluded on at least three counts: (1) the absence of lipid peroxidation as'measured by the thiobarbituric acid assay, the presence of which is interpreted as reflecting contaminating metal catalyzed processes; ( 2 ) t h e high concentration of pyrophosphate used (20 mM) which, in spite of containing contaminating iron [15], suppresses lipid peroxidation; and (3) the presence of KCN (15 mM), itself an effective complexation Lewis base, which may form inactive mixed chelates particularly with the transition metals. The most puzzling requirements for the KCN-released light concern the role of cyanide and the hexose phosphates. The variety of biological reactions in which cyanide is known to participate, including interaction with cytochromes, activation or inhibition of various enzymes, metal complexation, etc., makes it difficult to pinpoint the molecular role of this compound in the chemiluminescent phenomenon. The failure of all attempts to substitute compounds of similar structure or function for cyanide suggests a specific action for cyanide in our system. The observation that NADH can not substitute for NADPH tends to rule out any significant involvement of the cyanide-sensitive factor of the microsomal NADH-dependent fatty acid desaturase system. Despite several superficial similarities between the effects of CN- on ascorbic acid biosynthesis [16] and our microsomal systems, preliminary data [17] tend to rule out these reactions as the site of action of cyanide in our system. The requirement for the hexose or derivative is also difficult to explain. The action of glucose 1-phosphate in our system (Fig. 5) tends to preclude the involvement of the carbonyl function. The inability of mannitol to substitute for glucose 6-phosphate in these systems appears to eliminate free radical scavenger activity as the role of the hexoses. The simplest and most plausible explanation for the sugar requirement is that the sugars help maintain high levels of NADPH via appropriate oxidative reactions. Glucose-6-phosphate dehydrogenase is relatively specific for glucose 6-phosphate [18] and would not be expected to have significant activity with the other hexoses used. However, the microsomal NAD(P)-dependent hexose-6-phosphate dehydrogenase exhibits significant activity with galactose 6-phosphate, glucose 6-phosphate and glucose [19], a pattern in agreement with the effect of these sugars on the KCN-induced chemiluminescence (Fig. 5). Metzger et al. [20] as well as Hori and Takahashi [21] have reported that this enzyme is active only in the presence of detergent, acetone, certain enzymes or following sonication. However, other workers demonstrated activity in microsomes which had been only frozen and thawed twice [ 22,23].

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Previous work from this laboratory [3,11] has demonstrated that microsomes incubated in the presence of NADPH or ascorbic acid accumulated a substance(s) which reacted with KCN to yield chemiluminescence, gas evolution, methylene blue or nitroblue tetrazolium reduction, aniline hydroxylation and p-nitroanisole O-demethylation. These observations suggest the involvement of a lohydroxyperoxide. This substance(s} is extractable into chloroform/methanol, but the extract is negative in the thiobarbituric acid assay. However the absence of thiobarbituric acid reactive material does not necessarily preclude the presence of other organic peroxides. We are keenly interested in determining the emission spectrum of the KCNinduced chemiluminescence as an aid to identifying the emitting species in our system. However technical difficulties have prevented us from doing so at this time. Of several potential emitting species present in our microsomal preparations (e.g. NADPH, the NADP-CN complex, Schiff bases, flavins) our attention has focused on flavin derivatives, particularly in light of the recent observation of Hastings and Nealson [24] that a flavin peroxide or derivative {e.g., a 1-hydroxyhydroperoxide) is involved in the light emitting reaction of certain luminous bacteria. Interestingly, Hastings has pointed out this reaction is also a mixed function oxidase reaction [25]. Acknowledgements This work was supported in part by Pharmacological Sciences Training Grant 5T32GM07177, NIH Bio. Med. Res. Support Grant 5507 RR05377-16 and a grant from the Ethyl Corporation. We also wish to thank Dr. James E. Muldrey, Jr. for his help in the preparation of this manuscript. References 1 2 3 4

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