Free Radical Biology & Medicine, Vol. 8, pp. 145-152, 1990 Printed in the USA. All rights reserved.

0891-5849/90 $3.00+ .00 © 1990 Pergamon Press plc

Original Contribution MICROSOMAL LIPID PEROXIDATION: THE ROLE OF NADPH--CYTOCHROME P450 REDUCTASE AND CYTOCHROME P450 ALEX SEVANIAN, KERSTIN NORDENBRAND,* EUNJOO KIM, LARS ERNSTER,* and PAUL HOCHSTEIN Institute for Toxicology, University of Southern California, Los Angeles, CA 90033, U.S.A.

(Received 13 April 1989; Revised 29 August 1989; Accepted 13 October 1989) A b s t r a c t - - T h e role of NADPH - - cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP - - Fe 3÷-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPHand A D P - Fe 3÷-dependent lipid peroxidation involves both N A D P H - cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-1inked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for 02 reduction. The first electron is used for reduction of A D P - - F e 3÷ and subsequent addition of O2 to the perferryl radical (ADP--Fe3+-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with 02 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe 2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione. Keywords--Lipid peroxidation, Cytochrome P450, Cytochrome P450 reductase, ADP-Iron, Superoxide, Liposomes, Free radicals

crosomal lipid peroxidation, using purified, reconstitutively active reductase and cytochrome P450 in combination with liposomes prepared from various phospholipids. Another objective was to explain the enhanced rate of NADPH oxidation found in microsomes undergoing active lipid peroxidation, 1-3 a finding that is not accounted for by current schemes for the mechanism of microsomal lipid peroxidation. 1,2,~°-~2An abstract summarizing some parts of this work has been published. ~3

INTRODUCTION

Initiation of microsomal lipid peroxidation by NADPH and A D P - - F e 3+, first described 25 years ago, 1-3 is generally believed to involve NADPH--cytochrome P450 reductase. 4 Evidence supporting this concept is twofold: 1) Antibodies against the reductase inhibit lipid peroxidationS; 2) Purified P450 reductase can initiate NADPH- and A D P - - F e 3÷-dependent lipid peroxidation in liposomes prepared from microsomal phospholipids. 6 However, there is growing evidence that other factors may be involved in initiation reactions, as suggested by the findings that 1) Purified P450 reductase is a poor catalyst of A D P - - F e 3+ reduction by NADPHT'S; 2) In the liposomal system E D T A - Fe 3+, in addition to A D P - - F e 3+, is required for the initiation of lipid peroxidation. Cytochrome P450 can partially replace EDTA--Fe 3+, even with reductase preparation that is not capable of reducing cytochrome P450. 9,j° The purpose of the present study was to attempt to identify any additional component(s) involved in mi-

MATERIALS AND METHODS

Lipid peroxidation was measured by following NADPH oxidation spectrophotometrically at 340 nm, 02 consumption polaro-graphically, and by TBAR formation as described by Ernster and Nordenbrand. 14Liposomes were prepared either from a 4:1 (mol:mol) mixture of bovine phosphotidyl choline (PC) and phosphotidyl ethanolamine (PE) or from total rat-liver phospholipids as described by Sevanian et a1.15 Liposomes were mixed with N A D P H - cytochrome P450 reductase and/or cytochrome P450 at room temperature prior to addition to the reaction medium. Purified rat-liver

*Address correspondence to: Alex Sevanian, Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden. (Permanent address) 145

146

A. SEVAN]ANet al.

/

N A D P H - - c y t o c h r o m e P450 reductase (reconstitutively active) was kindly provided by Dr. M. IngelmanSundberg, Stockholm, and purified rabbit-liver cytochrome P450 (isozyme 2) by Dr. M. J. Coon, Ann Arbor, MI. Rat liver microsomes were prepared as described in reference 14.

ADP-Fe3+(100:I) EDT~-FeZ/ TA_Fe3+(1.1:1)

E

O 10Et-. v "ID ¢1 N .D "ID xO I o.. 5< z

RESULTS

//

ADP-Fe3+(20:1)

! 30

20 iuM Fe 3+

Fig. 1. Reactivity of purified NADPH--cytochrome P450 reductase with A D P - - F e Z." and E D T A - - F e 3+. The incubation medium consisted of 25 mM Tris--C1, pH 7.5, 150 mM KCI, 0.11 mM NADPH, and 5/21 of reductase (30 units/ml). Fe 3÷ chelates were added as indicated, final volume 1 ml and temperature 23°C.

A

ADP-Fe3" ADP(FEe~DTA_Fe3"

EDTff-F~ i

NADPH.....

1

of purified NADPH

reductase

w i t h A D P - - F e 3+ a n d E D T A - - F e 3+

As shown in Figure 1, the enzyme catalyzes the reduction of A D P - - F e 3+ (molar ratio 100: l); halfmaximal reaction rate occurs at an Fe 3+ concentration of approx. 5 /tM, which is similar to the value (6.3 /~M) reported for microsomes. J6 In contrast to microsomes, j6 the purified enzyme is much less active with A D P - - F e 3+ than with E D T A - - F e 3+ (molar ratio 1.1: 1) as substrate (Fig. 1). This difference is even more striking at a molar ratio A D P : F e 3+ of 2 0 : 1 , which gives a very low rate of reaction with the purified enzyme. It should be noted that a molar ratio A D P - - Fe 3+ of 20: 1 has been shown to yield a nearly maximal rate of lipid peroxidation in microsomes at an Fe 3+ concentration of approximately 12/LM Fe 3÷ ,~ and that a molar ratio A D P : Fe 3+ ranging between 20 : 1 and 5 : 1 , at an Fe 3+ concentration of 4 0 - 5 0 0 / ~ M , has been used in experiments with liposomes? 7,10.17Combination of A D P - - Fe 3+ and E D T A - - Fe 3+ gave es-

ADd~P-Fe3+(100:1)

10

-- cytochrome

P450

Reactivity

A D P - F e 3"

""~'~'~

50

18.2~uMMDA

/

pM] 02 1 rain

18.6 pM MDA

Fig. 2. Reconstitution of lipid peroxidation with purified NADPH--cytochrome P450 reductase. The incubation medium consisted of 25 mM Tris--C1, pH 7.5, 150 mM KCI, 0.2 mM NADPH, and 4 0 / 2 M A D P - - F e 3+ (Fe Z+ concentration, ADP:Fe 3+ = 100: 1). 0.75-1.0 mg liposomes prepared from total rat-liver phospholipids were mixed with 0.375 U reductase prior to the addition to the incubation vessel. Final volume 2.5 ml, temperature 30°C. In Figure 2A, 50/2M E D T A - - F e ~* (1.1 : 1); in Figure 2B, 40/2M EDTA--Fe3+; and, in Figure 2C, 50/2M menadione was used. When indicated, 0.3/2M P450, 30/2M BHT or 20/2g SOD were added to the incubation medium.

Microsomal lipid peroxidation

B

ADP-Fe3~"

EDTA_Fe3~

ADP-Fea" ~

~

147

ADP-Fe"* BHT

- - ~ 2.9 jJM MDA

50 p

± ~m,°,

\ 23.6 ~M MDA

ADP_Fe3÷

C

ADP_Fe 3.

19.1 pM MDA ADP-Fe34-

M

Menedlone

~

M

MDA

50 IJMI 0 2 ~ mi

19.0 uM MDA Fig. 2 (Continued)

sentially additive activities with the purified enzyme (Fig. 1). At concentrations above 3 mM, ADP itself inhibited the reductase (determined with cytochrome c as the electron acceptor) in an apparently competitive manner with respect to NADPH (not shown). These data indicate (a) that A D P - - F e 3÷ can serve

as an electron acceptor for the purified N A D P H - cytochrome P450 reductase, with a K,~ value similar to that found with microsomes; (b) that E D T A - - F e 3+ does not serve as an intermediate electron carrier between the reductase and A D P - - F e 3÷ as has been proposed on the basis of experiments with liposomes6,17;

148

A. SEVANIANet al.

and (c) that the A D P : F e 3+ ratio, as well as the net concentration of ADP, (the latter probably relative to the NADPH concentration) are critical parameters in determining the ability of A D P - - F e 3+ to serve as an electron acceptor for the reductase.

In the case of liposomes, menadione gave an effect similar to that of E D T A - - F e 3+, promoting, in combination with A D P - - Fe 3 ~, O2 consumption and TBAR formation (Fig. 2C). This effect was abolished by both BHT and SOD. These results show that both E D T A - - F e -~ and menadione are efficient in supporting lipid peroxidation in liposomes in the presence of NADPH and N A D P H - - c y t o c h r o m e P450 reductase; however, in the former case, the effect probably is due to EDTAFe 2+ (rather than to O j formed through autooxidation of the latter), whereas in the latter case, it is due to 02" itself, possibly by interaction with an endogenous iron component of the microsomes.

Reconstitution of lipid peroxidation with purified NADPH--cytochrome P450 reductase 02 consumption and TBAR formation (indicated as MDA) are observed with liposomes prepared from total liver phospholipids in the presence of P450 reductase, NADPH, A D P - - F e 3+ and E D T A - - F e 3+ (Fig. 2A). ADP - - Fe 3+ in the absence of EDTA - - Fe 3+ gave measurable 02 consumption and MDA formation, whereas E D T A - - F e 3+ in the absence of A D P - - F e 3+ gave none. In the ADP--Fe3+-supplemented system, E D T A - - F e 3+ could be partially replaced by cytochrome P450. These results are in essential agreement with those by Aust et al. 5'6'1°'17 The A D P - - Fe 3÷ and E D T A - - Fe 3÷-dependent 02 consumption and MDA formation were inhibited by the antioxidant butylated hydroxytoluene (BHT) but not by superoxide dismutase (SOD) (Fig. 2B). Menadione (2-methyl-l,4-naphthoquinone) is reduced by NADPH in microsomes, giving rise to an 02 consumption through "redox cycling" via N A D P H - cytochrome P450 reductase with the formation of the autoxidable semiquinone ~8 but no TBAR formation. J2

Effects o f A D P Fe 3+ E D T A - - F e 3+ cvtochrome P450 and liposomes on the rate of oxidation of NADPH by purified NADPH -- cytochrome P450 reductase The oxidation of NADPH by purified reductase incorporated into total rat-liver phospholipid liposomes was slow in the presence A D P - - F e 3+ (100:1) and more rapid in the presence of EDTA:Fe 3+ (1.1: 1) as electron acceptor (Fig. 3.); these results were essentially similar to those obtained in the absence of liposomes (Fig. 1). The two chelates together gave rise to a biphasic rate of NADPH oxidation when incubated

ADP-Fe 3+

pM MDA

EDTA-Fe 3÷

~M MDA

2.0

ADP-Fe 3+ Cyt. P-450

7.8

ADP-Fe3~DTA_Fe 3÷

_