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(The FASEB Journal. 1999;13:667-675.)
© 1999 FASEB

Research Communications

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice

PAUL K. WITTING, KNUT PETTERSSONa, ANNE-MARGRET ÖSTLUND-LINDQVISTa, CHRISTER WESTERLUNDa, ANNIKA WESTIN ERIKSSONa and ROLAND STOCKER*

Biochemistry Group, Heart Research Institute, Sydney, Australia; and

a Astra Hässle, Mölndal, S-43183, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of ~200 µM, increased plasma total cholesterol slightly and decreased plasma and aortic {alpha}-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.—Witting, P. K., Pettersson, K., Östlund-Lindqvist, A.-M., Westerlund, C., Westin Eriksson, A., Stocker, R. Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.


Key Words: antioxidants • coantioxidation • LDL • {alpha}-tocopherol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE OXIDATIVE MODIFICATION of low density lipoproteins (LDL)1 within the arterial wall is implicated as a crucial early step in atherogenesis. According to the 'oxidation theory' 1-3) , oxidized LDL affords foam cell formation, is cytotoxic, and instigates various proatherogenic processes including transcriptional (dys)regulation, up-regulation of adhesion molecule expression, inactivation of endothelium-derived relaxing factor, monocyte activation, recruitment of smooth muscle cells, and platelet activation 3-8) . Lipid peroxidation is one of the earliest processes occurring during in vitro LDL oxidation induced by most, though not all, oxidants (9) . Increasing the antioxidant defense against lipid peroxidation may therefore attenuate the initial stages of atherogenesis. Indeed, some 10-13) though not all antioxidants 14-18) inhibit atherogenesis in various animal models. However, whether this antiatherogenic effect is related to an inhibition of lipoprotein lipid peroxidation within the vessel wall has not been addressed.

The presence of oxidized lipids in atherosclerotic lesions is well documented (see, for example, ref 19 and references therein). Surprisingly, oxidized lipids coexist with relatively normal concentrations of {alpha}-tocopherol ({alpha}-TOH) (20) , the major endogenous antioxidant associated with LDL. This can be explained by recent advances in the understanding of the molecular mechanism underlying the early, {alpha}-TOH-containing stage of in vitro LDL lipid peroxidation and its inhibition by antioxidants. During this early stage, LDL lipid peroxidation follows established chemistry reminiscent of emulsion polymerization and termed tocopherol-mediated peroxidation (TMP): thus, the fate of {alpha}-tocopheroxyl radical, rather than {alpha}-TOH alone, governs whether significant lipid peroxidation occurs (9 , 21-23 ). LDL lipids are most effectively protected from oxidation in the presence of both {alpha}-TOH and suitable reducing substances (hereafter referred to as coantioxidants), which 'export' the radicals from oxidizing LDL particles into the aqueous phase and convert them into nonradical products (9 , 21-23 ). It has been proposed 21-23) that if arterial LDL lipid oxidation indeed causes atherosclerosis, coantioxidants may be antiatherosclerotic.

We have developed tests to screen compounds for coantioxidant activity; this provided a library of natural and synthetic compounds that effectively inhibit LDL lipid peroxidation in vitro under conditions where TMP prevails (23 , 24 ). Active agents are characterized by a low anti-TMP index and a high {alpha}-tocopheroxyl radical-reducing ability (24) . The bisphenolic probucol metabolite 3,3',5,5'-tetra-tert-butyl-4,pr-bisphenol (H 212/43) is an effective coantioxidant (Table 1 ), somewhat more active than butylated hydroxytoluene (BHT), a coantioxidant that inhibits atherosclerosis in rabbits (25 , 26 ). Compared with {alpha}-TOH, H 212/43 is a kinetically inferior radical scavenger that only moderately increases the 'lag-time' of LDL undergoing oxidation induced by high concentrations of Cu2+ (27) . These properties make H 212/43 an ideal compound to conceptually test whether coantioxidants can inhibit lipoprotein lipid peroxidation in vivo and, if so, how this relates to atherosclerosis. We therefore supplemented apolipoprotein E- and LDL receptor gene double knockout (apoE-/-;LDLr-/-) mice with standard or H 212/43-fortified chow and assessed circulating and aortic lipid (per)oxidation as well as the size of atherosclerotic lesions assessed in the descending thoracic aorta. The results obtained show that H 212/43 effectively prevents both processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All reagents were of the highest purity available. Phosphate buffer (50 mM, pH 7.4, 50 mM) and Dulbecco's phosphate buffer saline were prepared from nanopure water and stored over Chelex-100 (BioRad, Richmond, Calif.) at 4°C for at least 24 h to remove contaminating transition metals (28) . Cholesteryl linoleate (Ch18:2) and cholesteryl arachidonate (Ch20:4), together referred to as cholesteryl esters (CE), unesterified cholesterol, formal saline (containing 4% formaldehyde), BHT, EDTA, ascorbate, and D-isoascorbate were obtained from Sigma (St. Louis, Mo.). {alpha}-TOH (purity 96%) was a gift from Henkel Corporation (Sydney, Australia). {alpha}-Tocopherylquinone ({alpha}-TQ, purity 99%) was from Kodak (Sydney, Australia). Probucol was obtained from Jucker Pharma (Stockholm, Sweden). The peroxyl radical generator 2,2'-azobis(2-amidino-propane)-hydrochloride and H 212/43 were from Polysciences (Warrington, Pa.). [1{alpha}, 2{alpha}(n)-3H Ch18:2 ([3H]-Ch18:2, 48 Ci/mmol) was from Dupont (Boston, Mass.). [3H]-Ch18:2 hydroxide ([3H]-Ch18:2-OH) was prepared by oxidation of [3H]-Ch18:2 with rabbit reticulocyte 15-lipoxygenase (a gift from Dr. Dagmar Heydeck, Berlin, Germany) and subsequent reduction with NaBH4. H 330/68 (3,3',5,5'-tetra-tert-butyl-4,pr-diphenoquinone) was prepared by oxidation of H 212/43 using (diacetoxyiodo)benzene in methanol (29) . {alpha}-Tocotrienol was purified by high-performance liquid chromatography (HPLC) (30) from palmvitee (Palm Oil Research Institute of Malaysia). Authentic Ch18:2 hydroperoxide (Ch18:2-OOH) was prepared (31) and stored in ethanol at -20°C. Glass homogenizers with matching Teflon pistons were from Wheaton (Edwards Instruments, Sydney, Australia).

Diets
Standard R3-mouse chow was obtained from Lactamin (Stockholm, Sweden) and used as purchased or after fortification with H 212/43 at 0.03% (w/w), a level of supplementation shown in pilot experiments to afford circulating concentrations of the drug of ~200 µM and considered suitable to test the effect of the coantioxidant on atherogenesis in this animal model.

ApoE-/-;LDLr-/- mice
Male apoE-/-;LDLr-/- mice (59 total), obtained from Bommice (Ejby, Denmark), were derived from apoE-/-;LDLr-/- mice (Jackson Laboratories, Bar Harbor, Maine). ApoE-/- mice, originally from a strain developed by Maeda and co-workers (32) , were back-crossed six times to C57BL/6J mice. LDLr-/- mice were from a strain developed by Goldstein and co-workers (33) . Mice were confirmed deficient in apoE and LDLr by Southern and Western blotting (not shown). Animals were maintained on standard R3 chow from weaning to 8 wk of age (young controls, n=9). Thereafter, mice received R3 chow with (n=25) or without supplemented H 212/43 (n=25) for an additional 14 wk. At 22 wk of age, 10 and 15 mice of each group were used for biochemical and histological analyses, respectively. Due to the small amount of material, it was necessary to pool aortas to yield sufficient tissue for biochemical analyses.

Blood sampling and preparation of plasma and serum
Blood samples from control and drug-treated apoE-/-;LDLr-/- mice (~1 ml) were taken by direct cardiac puncture. Briefly, animals were anesthetized (5% isoflurane in water, v/v); the thoracic and abdominal cavities were opened and blood was drawn from the left ventricle into heparinized tubes. Plasma, obtained by centrifuging blood samples at 1000 x g at 4°C for 10 min, was divided into two aliquots and frozen to below -70°C in liquid N2 until analysis for lipids, antioxidants, and ex vivo plasma lipid oxidizability. Preliminary studies showed that such storage did not significantly affect the parameters analyzed.

Perfusion and fixation of aortic vessels
Mouse aortas were excised as follows. After bleeding, the heart was perfused with Dulbecco's phosphate-buffered saline containing 100 µM BHT and 1 mM EDTA (maximum pressure 80 mm Hg) through the left ventricle, the right side chamber being opened to allow flow. For histological samples only (see text below), the vasculature was subsequently fixed with formal saline. The hearts and entire aortas from all treatment groups were removed and immediately cleaned of fat and connective tissue. Aortas for biochemical analyses were frozen immediately (-70°C) without formalin fixation, as preliminary experiments showed that such treatment caused artefactual oxidative modification as judged by complete depletion of ascorbate and the presence of large quantities of CE-O(O)H in control aortic homogenates (not shown).

Evaluation of atherosclerosis
Aortic lesions were assessed in segments centered around the third pair of intercostal artery branches in the descending thoracic aorta. Briefly, the fixed aortas were dehydrated in ethanol, cleared with xylene, and embedded in paraffin. Serial sections (10 in total; each 2–3 µm thick and 100 µm apart) were cut and stained using Weigert's hematoxylin-van Gieson. Aortic thickening was assessed as the total volume of intima in the segment investigated in H 212/43-treated vs. control samples.

Briefly, aortic volumes were determined by planimetry, using a Lucivid device (MicroBrightField, Colchester, Vermont, Canada) attached to a Leitz DRM microscope that allowed the superposition of a computer monitor onto the cross sectional image. Planimetry was performed using Microvid Software (MikroMakro AB, Gothenburg, Sweden) in a blinded fashion using coded samples. Briefly, the external and internal elastic laminae and the endothelial lining were highlighted using the mouse-operated cursor. The enclosed area for each section was then calculated, assuming the intima defines the lesion boundary and the internal elastic lamina area defines the media. Cross-sectional areas of intima (i.e., area after subtracting the lumen area from that defined by the internal elastic lamina) were estimated serially along the length of the aortas and finally expressed as a volume for the aortic section measured. Mean volumes obtained from the same aortas on different occasions varied by <5%.

Preparation of aortic homogenates
Cleaned aortic segments were thawed, blotted, pooled (n=7 or 8), weighed, and added to 2 ml of argon-flushed phosphate-buffered saline (to give ~40 mg wet tissue/ml) containing BHT (100 µM) and EDTA (1 mM). The tissue was minced with scissors, and isoascorbate (5 µM) and {alpha}-tocotrienol (1 µM) were added as internal standards for ascorbate and vitamin E (including {alpha}-TQ), respectively; the samples were transferred to a polytetrafluoroethylene-lined glass tube and homogenized at 4°C for 5 min using a Teflon piston rotating at 500 r.p.m. This homogenization procedure has been optimized previously for extraction of aortic lipids without substantial oxidation of lipid- and water-soluble antioxidants (20) and verified here for ascorbate, {alpha}-TOH, and Ch18:2 (not shown). For recovery of oxidized lipids, [3H]-Ch18:2-OH was incorporated into human LDL (34) and added to the vessel prior to homogenization. Analysis of spiked homogenate showed 94 ± 1.3% recovery of the label (mean±range for two separate experiments), indicating an efficient extraction of oxidized lipids. For ascorbate measurement, raw homogenate (50 µl) was added to metaphosphoric acid (5% v/v, 50 µl) and frozen on dry ice. Immediately before HPLC analysis, the aliquots were thawed and diluted with phosphate buffer (50 µl, 250 mM, pH 7.4) to adjust the pH. For analyses of lipids, the remaining homogenate (~1.8 ml) was divided into 4 x 450 µl aliquots and each was extracted with chilled methanol (2 ml) and hexane (10 ml). Hexane phases were combined and evaporated to dryness and the residue was resuspended in isopropanol (200 µl), as described 35, 36) .

Oxidation of mouse plasma
Oxidation of plasma, pooled from >=3 mice was carried out by addition of the peroxyl radical generator (final concentrations 5 mM) and incubating the reaction mixture at 37°C under air. Aliquots (50 µl) of the reaction mixture were removed, extracted in methanol/hexane (1:5, v/v) 35, 36) , and the consumption of antioxidants and accumulation of lipid oxidation products were determined.

Analysis of lipid and water-soluble compounds
Analyses of oxidized and nonoxidized lipids were carried out by reverse phase (RP)-HPLC as described 20, 31, 35, 36) , except that in some instances UV234 nm rather than postcolumn chemiluminescence detection was used to measure Ch18:2-OOH and the corresponding hydroxides (together referred to as CE-O(O)H), which show similar retention times under these chromatographic conditions. {alpha}-TQ, {alpha}-TOH, {alpha}-tocotrienol, D-isosascorbate, and ascorbate were determined by HPLC with electrochemical detection (20) . For oxidation of plasma, unesterified cholesterol (which remained unoxidized in these experiments) was used as the internal standard for all polyunsaturated, lipid-soluble components analyzed. H 212/43 and H 330/68 were analyzed by RP-HPLC: flow 1.5 ml/min, 100% solvent A (MeCN/MeOH/H2O 10:10:3, v/v/v) for 0–15 min monitored at 270 nm, followed by 50% solvent A and B (MeCN/MeOH 1:1, v/v) for 15–22 min at 242 nm, then 100% B for 22–28 min at 420 nm. H 212/43 and H 330/68 eluted at 9 and 27 min, respectively. All compounds were quantified by peak area comparison with authentic standards. Where indicated, total cholesterol and triglyceride were assayed enzymatically (Boehringer, Mannheim, Germany).

Statistics
As data sets contained a significant number of values at or near zero, the Wilcoxon two-sample test was used to determine differences between groups of data. T tests were not suitable, as data sets did not follow a log-normal distribution and log transformations could not be performed. Statistical difference was accepted at the {alpha} <0.05 level. Ranking of data was performed using Mystat Statistical Software (Course Technology Inc., Cambridge, Mass.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ApoE-/-;LDLr-/- mice readily develop detectable lesions after only 15 wk of standard chow diet (A. Westin Eriksson, L. Amrot-Fors, and K. Pettersson, unpublished results), indicating that these animals are suitable to study both early events in atherogenesis and its inhibition. Table 2 summarizes the plasma levels of lipids, {alpha}-TOH, and H 212/43 in mice receiving a control or H 212/43-fortified diet. There was an age-dependent increase in plasma total cholesterol, triglycerides and vitamin E as judged by comparing data from 8- and 22-wk-old control animals. After 14 wk intervention, plasma levels of H 212/43 plus H 330/68 reached 216 µM. Supplementation of the diet with H 212/43 significantly increased total plasma cholesterol, whereas triglycerides were unchanged and vitamin E levels decreased significantly compared to age-matched controls (Table 2) .


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  		Table 2. Plasma lipid, vitamin E, and drug levels in apoE-/-;LDLr-/- micea

Samples of pooled plasma from H 212/43-treated mice were markedly resistant to peroxyl radical-induced ex vivo lipid peroxidation compared with age-matched controls (Fig. 1 ). Thus, even after 12 h of oxidation at 37°C, {alpha}-TOH remained unaltered (Fig. 1A ) with <1 µM CE-OOH detected. By contrast, ~70% of {alpha}-TOH was consumed and >30 µM CE-OOH accumulated in the corresponding control plasma (Fig. 1B ), fully consistent with plasma lipid peroxidation proceeding via TMP (9) . Separate studies showed that this resistance to peroxyl radial-induced ex vivo lipid peroxidation was directly attributable to H 212/43, as the bisphenol rather than {alpha}-TOH was consumed during the period of oxidation (not shown). The corresponding oxidation product, H 330/68, was formed stoichiometrically from H212/43. The former is incapable of acting as a (co)antioxidant, as judged by its high anti-TMP index and inability to cause the decay of {alpha}-tocopheroxyl radical (Table 1) .



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  		Figure 1. Resistance of plasma from apoE-/-;LDLr-/- mice receiving H 212/43 to lipid peroxidation initiated by aqueous peroxyl radicals. Pooled plasma obtained from at least three mice receiving control diet ({bullet}) or diet supplemented with H 212/43 ({blacksquare}) was treated with 5 mM AAPH and incubated at 37°C. At the times indicated, aliquots were removed and analyzed for {alpha}-TOH (A) and CE-OOH (B;). 100% {alpha}-TOH levels correspond to 28 ± 3 and 20 ± 2 µM for control and H 212/43 plasma, respectively. Data represent the mean ± SD (n=4 independent experiments).

For biochemical analyses it was necessary to pool aortas to yield sufficient material to detect the various lipids and antioxidants, despite the use of HPLC with sensitive detection. As a result of this limitation, tissue parameters were determined as the means of duplicate analyses on two separate pools of aortas of each the control and H 212/43-treated mice. The results (Table 3 ) show that concentrations of ascorbate, unesterified cholesterol and CE in aortic homogenates were similar in the two treatment groups, although a marginal decrease in CE was seen in a subgroup of drug-treated mice with high plasma levels of H 212/43 (see legend to Table 3 ). By contrast, the levels of aortic {alpha}-TOH were lower in drug-treated than in age-matched controls.


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  		Table 3. Aqueous and lipophilic parameters of apoE-/-;LDLr-/- mouse aortaea

Aortic tissue of control mice contained significant amounts of oxidized lipids, with approximately 0.15% of the CE present as CE-O(O)H (Table 3) . Strikingly, the level of these oxidized lipids were 10- and 1000-fold lower in aortas from drug-treated animals with low and high plasma levels of H 212/43, respectively, particularly when expressed as percent lipid (Table 3) . Figure 2 shows representative traces of HPLC with postcolumn chemiluminescence detection (see Materials and Methods). CE-OOH, detected in the organic extracts of aortas of control but not H 212/43-treated mice, eluted between 8 and 10 min. Treatment of the control samples with sodium borohydride eliminated these chemiluminescence-positive peaks (not shown), indicating their nature as hydroperoxides. {alpha}-TQ, an additional marker of biological lipid oxidation (20) , was also decreased in aortas of H 212/43 vs. control mice (Table 3) , although the extent of this inhibition was much less than that observed for CE. Linear regression analyses indicated that there were no significant correlations (R<±0.5) between any of the plasma and tissue parameters measured (not shown).



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  		Figure 2. Inhibition of aortic lipoprotein lipid peroxidation in apoE-/-;LDLr-/- mice receiving H 212/43. Pooled aortas from seven or eight mice obtained from control or H 212/43-treated mice were homogenized; lipid was extracted and subjected to HPLC with postcolumn chemiluminescence detection for analysis of CE-OOH, as described in Materials and Methods. Chromatograms correspond to aortas of age-matched control (a or b) or drug-treated mice with low or high plasma levels of H 212/43 (c and d, respectively). Under the conditions used, CE-OOH eluted between 8 and 9.5 min. The chemiluminescence negative peaks between 4 and 5.5 min correspond to the elution of tocopherols.

The intimal volume in the descending thoracic aortas of control apoE-/-;LDLr-/- mice fed the standard chow increased by more than 10-fold from 8 to 22 wk of age (Fig. 3 ). Administration of H 212/43 for 14 wk substantially decreased the lesion size, as judged by a significant decrease in aortic volume compared with age-matched controls, although the intimal volume in the drug-treated (older) animals remained higher than that determined for young control mice (Fig. 3) .



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  		Figure 3. Inhibition of atherosclerosis in apoE-/-;LDLr-/- mice receiving H 212/43. Lesion formation was assessed by changes to intimal volume when compared with young and age-matched controls receiving standard chow for 8 and 22 wk, respectively. *Statistically significant difference with {alpha} < 0.025 (Wilcoxon test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates, for the first time, that administration of a coantioxidant (H 212/43) increases the overall (co)antioxidant capacity of circulating plasma lipoproteins and strongly inhibits both aortic lipid (per)oxidation and atherogenesis in apoE-/-;LDLr-/- mice. These results directly support the notion that coantioxidants effectively inhibit lipoprotein lipid peroxidation in vivo. As coantioxidant act by eliminating {alpha}-tocopheroxyl radical from oxidizing lipoprotein particles (23) , the results imply that TMP is a relevant mode of in vivo lipoprotein lipid peroxidation.

For the present study, we chose apoE-/-;LDLr-/- mice to test the efficacy of H 212/43 as a coantioxidant and antiatherosclerotic agent. Even without feeding a fat-fortified diet, apoE-/-;LDLr-/- mice develop lesions in the dorsal aorta at a greater rate than do the more commonly used apoE-/- mice (A. Westin Eriksson, L. Amrot-Fors, and K. Pettersson, unpublished results), although the lipoprotein profiles in both strains of mice are comparable. The present study demonstrates that even when maintained on a standard chow, aortas of apoE-/-;LDLr-/- mice contain significant CE-O(O)H (Table 3 , Fig. 2 , the primary and major lipid peroxidation products formed during in vitro oxidative modification of lipoproteins induced by radical oxidants. Nevertheless, the proportion of CE present as CE-O(O)H was at least 20-fold lower than that seen in advanced human lesions (20) . This, together with the comparatively lower extent of {alpha}-TOH oxidation (see below), indicates that from a redox point of view as well, lesions in apoE-/-;LDLr-/- mice after 2 wk represent early stages of the disease. It has been shown previously that the early stages of peroxidation of mouse lipoprotein lipids can proceed via TMP (37) . Together, these findings indicate that apoE-/-;LDLr-/- mice are suitable to test the effect of inhibition of aortic lipoprotein lipid peroxidation in the early stages of atherogenesis.

Coantioxidants can effectively inhibit peroxyl radical-induced LDL lipid peroxidation in vitro even if they are kinetically inferior radical scavengers compared to {alpha}-TOH (23 , 24 ). The observed inhibition of in vivo lipid peroxidation by H 212/43 (Table 3) was particularly pronounced when peroxidation was expressed as primary oxidation product per parent CE. At least during the early stages of atherosclerosis and relevant to the present study, aortic CE are present predominantly within intimal lipoproteins (38) , so that our measure of lipid peroxidation most likely reflects that of extracellular lipoprotein particles. Aortas from drug-treated mice were divided into two pools based on the plasma concentration of H 212/43, and the extent of oxidation of aortic CE was lower in the vessels derived from the high vs. low plasma H 212/43 mice. This suggests that inhibition of aortic lipoprotein lipid peroxidation increased with increasing tissue concentrations of the coantioxidant. Unfortunately, the limited amount of material available did not allow determination of tissue levels of H 212/43.

Treatment of mice with H 212/43 also decreased aortic accumulation of {alpha}-TQ, although the relative extent of this antioxidant effect of the bisphenol was markedly lower than that seen for CE-OOH (Table 3) . This is not surprising, as {alpha}-TQ (but not CE-OOH) is also formed by nucleophilic oxidants, not expected to be scavenged effectively by the bisphenol (39) . {alpha}-TQ is the major oxidation product of {alpha}-TOH present in advanced human plaques, where it accounts for up to 10% of the vitamin (A. Terentis, D. Liebler, R. Stocker, unpublished results). By comparison, in apoE-/-;LDLr-/- mice {alpha}-TQ accounted for around 1% of the {alpha}-TOH. This suggests that in the aortas of these animals, most of the endogenous vitamin E remains intact, yet significant lipoprotein lipid peroxidation occurs. This is consistent with aortic lipid (per)oxidation occurring, at least in part, via TMP and perhaps as a result of a imbalance of too few available coantioxidants relative to tissue {alpha}-TOH. If so, supplementation of apoE-/-;LDLr-/- mice and possibly other animals with {alpha}-TOH alone may not necessarily prevent in vivo lipid peroxidation, analogous to the in vitro situation with human LDL (9 , 21 , 22 ). Unfortunately, none of the previous animal vitamin E supplementation studies has directly assessed the effect of the treatment on lipoprotein lipid peroxidation in the vessel wall. What is clear is that supplementation of animals with {alpha}-TOH alone generally fails to decrease atherosclerosis unless the vitamin is used at such high concentration that it causes a hypolipidemic effect (11 , 15 , 17 , 40 ).

A limitation of the present study is that the biochemical analyses could not be performed on the aortic sections used for histology, so that our interpretations rely on interanimal comparisons. To obtain sufficient material for the biochemical analyses in apoE-/-;LDLr-/- mouse aortas, it was necessary to pool samples, thereby limiting the results to those derived from two separate pools. However, the results and apparent trends obtained are consistent with H 212/43 inhibiting aortic lipid peroxidation in a dose-dependent manner.

The close association of inhibition of aortic lipoprotein lipid peroxidation and reduced atherosclerosis strongly supports, though does not prove, a causative role for aortic lipoprotein lipid peroxidation in atherogenesis in apoE-/-;LDLr-/- mice. We observed recently that there was no accumulation of oxidized lipids in aortas of Watanabe heritable hyperlipidemic rabbits treated with either probucol or H 212/43 (P. K. Witting, K. Pettersson, A. M. Östlund-Lindqvist, C. Westerlund, M. Wågberg, R. Stocker, unpublished results). In the probucol-treated animals, the plasma concentration of H 212/43 was high and it is likely that the metabolite H 212/43 was responsible for the inhibition of aortic lipid peroxidation in rabbits, as in the present mouse study. However, in contrast to apoE-/-;LDLr-/- mice, the extent of atheromatous lesions found in H 212/43-treated rabbits was not reduced compared to that of controls (P. K. Witting, K. Pettersson, A. M. Östlund-Lindqvist, C. Westerlund, M. Wågberg, R. Stocker, unpublished results). Together, these findings suggest that inhibition of aortic lipid peroxidation may not generally result in a reduction in atherosclerosis.

The effects of synthetic antioxidants on atherogenesis in mouse models of atherosclerosis are somewhat contradictory. N,N'-diphenyl-phenylenediamine, an effective coantioxidant (24) and radical scavenger (41) , was reported to reduce lesion formation (10) . By contrast, probucol, a moderately active radical scavenger (41 , 42 ) that lacks coantioxidant activity (24) , increases lesion volumes in mice (16 , 42 , 43 ). This could be explained if H 212/43 were the active agent and apoE-/-;LDLr-/- mice (in contrast to rabbits) were unable to metabolize probucol into H 212/43. Thus, it would be interesting to test whether probucol prevents aortic lipid peroxidation in mice, and how this relates to probucol metabolism and the extent of atherosclerosis. In humans, probucol is poorly metabolized into H 212/43 and has failed to reduce femoral atherosclerosis (44) .

In addition to differences in metabolism of probucol, the response to H 212/43 of Watanabe rabbits and apoE-/-;LDLr-/- mice also differed in the plasma and aortic concentration of {alpha}-TOH. Thus, the bisphenol significantly decreased circulating and tissue vitamin E in the mice (Table 3) , whereas it increased plasma and aortic {alpha}-TOH in rabbits (P. K. Witting, K. Pettersson, A. M. Östlund-Lindqvist, C. Westerlund, M. Wågberg, R. Stocker, unpublished results). The reason for and meaning of this, if any, are not presently known. Although {alpha}-TOH can be a prooxidant for lipoprotein lipids under some conditions (9) , such activity is unlikely to be responsible for the difference in the atherosclerotic outcome of H 212/43-treated mice vs. rabbits, as in both animals the bisphenol prevented aortic lipid peroxidation. A potential redox-independent anti-atherosclerotic activity of vitamin E (45 , 46 ) also appears unlikely to be important, because in our mouse and rabbit studies there was a positive correlation between the concentrations of vitamin E and the extent of atherosclerosis.

In summary, our results show that a kinetically inferior antioxidant, capable of interacting with lipoprotein associated {alpha}-TOH, effectively inhibits both in vivo lipoprotein lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice. While these results strongly support a causative role for aortic lipoprotein lipid peroxidation in atherogenesis in this animal model, they do not prove such a relationship. Additional intervention studies, involving other animal models and/or (co)antioxidants, are needed to verify or disprove the generality of a causative relationship between aortic lipoprotein lipid peroxidation and atherogenesis.



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  		Table 1.


   ACKNOWLEDGMENTS
 
This work was supported in part by Astra Hässle and National Health and Medical Research grant 970998 to R.S.


   FOOTNOTES
 
* Correspondence: Biochemistry Group, The Heart Research Institute, 145 Missenden Road, Camperdown NSW 2050, Australia. E-mail r.stocker{at}hri.org.au

1 Abbreviations: ApoE-/-, apolipoprotein E gene knockout; apoE-/-;LDLr-/-, apoE and low density lipoprotein receptor gene double knockout; BHT, butylated hydroxytoluene; CE, cholesteryl esters; CE-O(O)H, cholesteryl ester hydroxides plus hydroperoxides; Ch18:2, cholesteryl linoleate; H 212/43, (3,3',5,5'-tetra-tert-butyl-4,pr-bisphenol); H 330/68 (3,3',5,5'-tetra-tert-butyl-4,pr-diphenoquinone); HPLC, high-performance liquid chromatography; LDL, low densitylipoproteins; TMP, tocopherol-mediated peroxidation; {alpha}-TOH,{alpha}-tocopherol; {alpha}-TQ, {alpha}-tocopherylquinone.

Received for publication September 9, 1998. Revision received November 23, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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