J
Mol Cell Cardiol 22, 11991208 ( 1990)
REVIEW
Mechanisms
of Cardiovascular
ARTICLE
Drugs
as Antioxidants”
(Received 30 October 1989, accepted in revised form 29 May 1990)
Introduction The processof oxidation in biological systems incorporates molecular oxygen into biochemical reactions necessaryfor essentialmetabolic processes.Phosphorylation of mitochondria, drug metabolism by mixed function oxidases etc., are such biological mechanismsthat have evolved in aerobic organisms. Mammalian tissuesare composedof a number of molecular “building blocks” that are susceptible to another form of oxidation, i.e. peroxidation. Among the potential sites of peroxidation in tissues are the unsaturated bonds of fatty acids, particularly those in biological membranes that contain many forms of structural lipids. Biological membranes,such as the plasmalemma, also provide hydrophobic binding sitesfor drugs and other substancesthat reside in membranes: e.g. natural antioxidants such as vitamin E and beta-carotene, which interact with lipids and proteins of membranes. In addition, cells contain hydrophilic environments, e.g. cytosol, that provide natural sites for water soluble antioxidants such as ascorbate (vitamin C), and glutathione (GSH). In the normal tissue,the above “natural antioxidants” provide antioxidant protection to the cells and extracellular spaces.In vitamin deficiency states, such as vitamin E deficiency in premature infants [I] or scurvy, a consequence of vitamin C deficiency, characteristic pathological changesoccur. Although the biological requirements for antioxidant vitamins have been studied for a number of years [ZJ, the ability of other synthetic compounds, such asdrugs, to act asantioxidants in tissuesis an area of recent interest. The purpose of this review is to outline someof the recent developments in this area. * Supported by grants from POl-HL-38079, ROl-HL-36418, Company and ONR NOOOl4-K-88+405. Please address all correspondence to: William B. Weglicki, of Medicine, Ross Hall, Room 409, 2300 Eye Street, N.W., 0022-2828/90/101199
+ 10 $03.00/O
Free radical generation and inrrCti~tiOlI
Free radicals were discovered at the turn of the 20th century by Gomberg [A. Subsequently, they were shown to play an important role in oxidation of biochemical components and more recently in cellsand organisms [A. In an oxygen environment, the predominant form of free radicals that are encountered in biology and medicine are oxygen radicals (Table 1) [5J. The term free radical refers to the presence of one or more unpaired electrons; the common convention for designating this electron is R’. Xanthine, hypoxanthine, NADPH, RS- and many other reducing agents may, under certain conditions, generate superoxide radical: 0, + e- --+*02-.
1’) Molecular oxygen itself contains two unpaired electrons and is not as reactive as those compounds referred to as oxygen free radicals, which contain an unpaired electron residing predominantly on an oxygen atom. During normal metabolism, such as oxidative phosphorylation, oxygen is reduced stepwise to produce water; this is accomplished by the mitochondrial electron transport system catalyzing sequential one electron additions to molecular oxygen. Intermediate speciesof one electron reduction of oxygen in solutions are the superoxide radical (‘0, -), hydrogen peroxide (H,O,) and hydroxyl radical (‘OH): O,“-+‘O;=--H,O,Z + Hz0
‘OH’-
(2) All three intermediary speciesare thought to be involved in cytotoxic injury of tissues.The exact mechanismsof free radical generation in AHA
National
Capital
Affiliate,
Director, Division of Experimental Washington, DC 20037, USA. 0
Bristol Medicine,
1990 Academic
Myers
Squibb
Department Press Limited
1200 TABLE
W. B. Weglicki 1. Nomenclature
of related Oxygen species and derived radicals
Species
Name
RH R’ ROO’ ‘OOH ‘0,-
Parent molecule Free radical Peroxy radical Hydroperoxy radical Superoxide radical Hydroxy radical Hydrogen peroxide Alkoxy radical Hydroperoxide Peroxide
‘OH Hz02
RO’ ROOH ROOR ArO’
Origin RH R’ + 02, ROOH 02- + H+
02 + e-, xanthine oxidase,NADPH oxidase Hz023
Phenoxyand aroxy radicals
free radicals in normal tissue have not been determined directly. In the activated white cell, the superoxide radical can be produced by NADPH oxidase during the respiratory burst of phagocytosis. The natural antioxidant enzyme for this process is superoxide dismutasewhich converts superoxide radicals into hydrogen peroxide, which will then be degraded by peroxidases and catalases. Another antioxidant enzyme is glutathione peroxidase which requires GSH in the reaction schemeas follows: + H,O,
nlul.athionc ueroxidnsc , GSSG
+ 2H,O
(3)
Catalase on the other hand transforms H,Os: 2H202
csca’ase bO2 + 2H,O
(4)
When these various antioxidant enzymes and moleculesare deficient, the tissuesmay be rendered more susceptible to peroxidative injury. Even when the tissue levels of the above antioxidant compounds are in the normal range, the protective mechanismscan be overwhelmed by excessive oxidant stress of various types. For these situations, the addition of natural and synthetic antioxidants (such as drugs) to the tissuesmay provide additional protection. Antioxidant
H2O
‘0~~, Superoxidedismutase ROOH, ROOR ROO’
viva are not fully understood, and the levels of
2~s~
et al.
mechanisms
Natural and synthetic antioxidants may intervene at various stages of the peroxidation
Peroxidation Phenolic antioxidants
processsuch as: Initiation:
RH+R’
(5)
R’ + 0s + ROO’
(6)
ROO’ + RH-+ ROOH + R’
(7)
or Propagation:
followed by:
In the absenceof antioxidants peroxy radicals are eliminated by Termination:
ROO’ + ROO’-+ unreactive products (8) The above processesare referred to as autooxidation or peroxidation of an organic substrate [6J. Among the synthetic antioxidants are the various aromatic phenols, ArOH. These compounds inhibit autoxidation as follows: ROO’ + ArOH --+ ROOH + ArO’
(9)
Reaction of antioxidants with peroxy radicals is in fact an electron transfer process[A and not an H atom transfer as indicated by the above overall reaction. Hence, good electron donors such as ascorbate, AH- (E, = 0.28 V) and urate, UHz- (E, = 0.56 V) are excellent antioxidants. Being water soluble, ascorbate and urate are cytosolic or extracellular antioxidants. The reactions of these antioxidants with peroxyl radicals proceed in the following
Drags
FIGURE
1. The chromanoxy
radical
resonant
as Antioxidant8
1201
state of a-tocopherol.
fashion with their rate constants indicated:
followed by:
Ascorbate
E-O-
[ 83 :
ROO’
+ AH - --+ ROO- + ‘AH, (k = 2 x IO6 M-I s-l) followed by: ‘AHcr’AROO-
+ H+, (pKa = 4.2)
+ H+ ++ROOH,
(pKa = 12).
(10) (11) (12)
(pKa = 10-11).
+ UH,- +ROO(k=3 x 106~-‘s-‘)
or by inhibition
+ ‘UH,, (13)
For Vitamin E, E-OH, (ET = 0.48 V) which is a classical membrane associated antioxidant, its mechanism of action differs somewhat from the above reactions [II: ROO’
+ E-OH+ ROO- + E--OH+ (k = lo6 - lo7 M-I s-r) (14) Being at the interface of a membrane, the positive radical E-‘OH+ reacts rapidly with water: E--OH+
+ HsO--,E-0’
+ H,O+
(15)
To give a long lived chromanoxy radical (E-O’), a special class of phenoxy radicals (Ph-0’). Similar to phenoxy radicals, the vitamin E radical exists in multiple resonance states (Fig. 1). Th e resonant state of the radical [IO] gives it specific properties such as an extremely long life due to its low decay rate constant (k = 2 x lo2 M-’ s- ‘) and unreactivity towards oxygen (k < 10e2 M-~ s-l) [ 111. In general, the expected longer life of the vitamin E radical in membranes permits it to be repaired by other reducing agents with lower redox potentials. Such repair reactions of vitamin E radicals in liposomes [ 14 and homogeneous solutions [ 13J have been known for some time. For example, vitamin C is an excellent repairing agent of vitamin E radicals [13]: E-O’ + AH- --+ E-O- + ‘AH, (k = 1.5 X lo6 M-l s-l) (16)
(17)
Another class of antioxidants are the sulfhydryls (thiols) which can repair free radical injury and may act as either hydrogen atom (RSH), or electron (RS-) donors [14]. They promote repair of free radical injury either by restitution: R’ + RSH-+
Urate [9]: ROO’
+ H+*E-OH,
ROO’
RH + RS’
(18)
of peroxidation: + RS-+ROO-
+ RS’
(19)
Thiol-containing compounds are good scavengers of hydroxyl radicals, and thus they may act as protective agents against injury due to hydroxyl radicals, provided that the concentration locally of the thiol is relatively high. *OH + RSH+ H,O + RS’, (k = 10” M-~ S-’
(20)
Other recent data suggest that thiols may participate in enzymatic repair processes to regenerate NADPH in the presence of NADPH and the pentose monophosphate shunt pathway [15J. Antioxidanta4ztivitiesofcardiovascdar agents
In addition to their physiological receptor binding functions, most commonly used cardiovascular agents, being amphiphilic by nature, partition readily into the hydrophobic regions of the cell membrane and thus may affect physical, as well as, chemical properties. We have recently used in vitro approaches to assess whether such drug-membrane interactions affect the sensitivity ofcell membranesto free radical injury [ 161. Beta-blockers and class I antiarrhythmic
drugs
By using highly purified cardiac sarcolemmal membranesasa model system, the antiperoxi-
W. B. Wtglicki
1202 Betao
blockem
=
et al.
Class I agsnts
1
Agents
(200
p~u()
FIGURE 2. Effects of B-blocking and Class I antiarrhythmic agents on sarcolemmal lipid peroxidation. Sarcolemmal membranes (100 pgg/ml) were preincubated with each agent for 10 min at 37°C before the additions of Fe-ADP and DHF. After 20 min of incubation, samples were assayed for MDA formationand expressed as y0 inhibition comparing to controls. Detailed experimental conditions were described in Ref. 16. *P metoprolol > atenolol > sotalol. This antioxidant effect appeared to correlate with hydrophobicity: the correlation coefficient (r) between the antioxidant activities of the betablockers and their lipophilicity expressed as log when octanol/water partition coefficient (Fig. 3) is 0.86 (P < 0.05). In the data presented in Figure 2, high levels (200 PM) of the agents were chosen to amplify the differences between the weak and the strong antioxidant agents. In the caseof propranolol, significant protection against membrane peroxidation could be achieved by
I 0
01 -I
i-J 0 2 3 5 H f
I
Log (octanol/HsO)
2 partition
3
4
coefficient
FIGURE 3. Relationship between partition coefficient (Log Octanol/water) and antioxidant activity of the betablockers. The results were derived from Figure 2 and Ref. 16.
concentrations aslow as 10PM (Fig. 4) [ 171.In another study, the mechanismof propranololinhibited membrane lipid peroxidation was examined by electron spin resonance spintrapping technique [ I7J. We observed that pretreatment of the sarcolemmal membranes with 3-30 PM propranolol prevented the formation of membrane carbon-centered lipid radicals in a log concentration-dependent manner; the EC50 is 6.7 paa[ 1I]. Propranolol also possessespotent membrane stabilizing activity and it was thought that its membrane protective effect against free radicals might be related to this membrane effect. However, both quinidine and lidocaine, which are known to have major membrane stabilizing activity, are relatively ineffective; this suggeststhat a significant antiperoxidative effect due to such membrane
.f E E 1 i! = B E .E u P
18 16 I4 12 IO 8
6
4 2 0
0
IO Propranolol
30 (FM)
FIGURE4.Concentration-dependent protectiveeffect of propranolol on sarcolemmal lipid peroxidation. After 20 min of incubation, samples were assayed for MDA formation. Values are means + S.D. of 4 separate determinations; *P < 0.01 versus controls.
Drugs
as Antioxidants
1203
”
FIGURE 5. Effects of propranolol and structurally related compounds on sarcolemmal lipid peroxidation. Sarcolemmal membranes were preincubated with each agent (200 PM) for 10 min before the additions to Fe-ADP and DHF, and were further incubated for 20 min. MDA determination and other conditions were described under Figure 2. Results were means of 3-8 separated experiments.
activity is unlikely. One major common structural feature among most beta-blockers is the aromatic moiety connected to an ethanolamine chain by an aromatic ether linkage [ l8j. It is believed that much of the hydrophobicity of these agents is contributed by this ring structure, especially if it is unsubstituted like that of propranolol, a few structurally related compounds were studied. Figure 5 demonstrates that the naphthalene moiety alone provided no protection; however when a naphthoxyl linkage was present, as represented by I-methoxy-naphthalene, 65% of antioxidant activity (relative to propranolol) was preserved. However, when the naphthoxyl moiety was removed, asrepresented by 1-naphthaleneacetic acid, no antioxidative activity was observed. These results indicated that both the ring structure and the aromatic oxy-linkage were essentialfor the antioxidant activity of propranolol. Thus, propranolol and the other more lipophilic beta-blockers appear to inhibit membrane lipid peroxidation through a chain breaking mechanism, like the lipophilic natural antioxidant alpha tocopherol. The requirement for an aromatic ring structure is probably due to reduction of redox potential and the resonance-stabilizing capacity similar to that described for alpha tocopherol (Fig. 1). The importance of the aromatic oxy-linkage may be due to its tautomerization into a phenolic group which would inactivate peroxy radicals (reaction 9).
40~
calcium
blockers
or propranolol
FIGURE 6. Comparative inhibitory effects of propranolo1 and calcium channel blockers on sarcolemmal lipid peroxidation. Sarcolemmal membranes were preincubated with each agent (40 PM) for 10 min before ;he final additions of DHF/Fe-ADP: after 20 min of incubation. samples were assayed for MDA formation and expressed as percent inhibition relative to the controls. Values are mean + S.D. of 3-6 separate determinations. * P < 0.05, **P C 0.01 vs controls.
Calcium channel blockers
Studies from Janero et al. [19] described that certain lessspecific calcium blockers (bepridil, prenylamine, flunarizine and cinnarizine) provided inhibitory effects against cardiac liposomal lipid peroxidation that was mediated by xanthine oxidase/hypoxanthine system. We have found that certain specific calcium channel blockers (nifedipine, verapamil, and diltiazem) exhibit various levels of antiperoxidative activities [NJ. Using our sarcolemmal membranes, nifedipine was the most potent of the calcium channel blocking agents (> P-fold more potent than propranolol), followed by verapamil and, to a lesser extent, diltiazem [20] (Fig. 6). Despite their markedly different chemical structures, all three agents do possess aromatic rings, a feature common .to beta-blockers and the conventional chain-breaking antioxidants. Furthermore, verapamil and diltiazem contain electron releasing groups (CHsO), whereas nifedipine has an electron-rich dihydropyridine ring moiety. Also, all three calcium blockers are lipophilic and partition into the phospholipid domains of cardiac membranes; thus, we speculate that the antioxidant effects of theseagents in vitro are mediated by neutralizing the lipid peroxy radicals through an electron transfer processsimilar to “reaction 19”. Additional experiments (unpublished) have been performed in which the incubation medium was removed after the drug pretreat-
1204
W. B. Weglicki et al.
ment but before exposure to the free radical system; propranolol and the three calcium channel blockers were found to be similarly protective against membrane lipid peroxidation Other workers have studied isolated cardiomyocytes exposed to activated oxygen species; Ver Donck et al. [Zl, 22] have shown that both lessspecific (cinnarizine, flunarizine) and specific (nicardipine, verapamil, diltiazem) calcium blockers provided various degrees of cytoprotection. However, nifedipine was reported to be ineffective. This latter result, using cardiomyocytes doesnot agree with our data using sarcolemma from adult cardiomyocytes.
to protect against oxidation of cholesterolcarrying low density lipoprotein appearsto be essentialin this mechanism [24]. It has been postulated that the antioxidant properties of this drug may have relevance in preventing foam cell formation [ 2.51. Angiotensin
converting enzyme (ACE)
inhibitors
ACE inhibitors are a new classof cardiovascular agents for treatment of hypertension and other cardiovascular diseases[26, 271. Captopril, the original member of this class,contains a sulfhydryl moiety which is believed to contribute to the potency of the drug by binding to the zinc moiety of the enzyme [27J. In addition to its ACE inhibition, captopril has been shown to possess anti-inflammatory and Antiatherosclerotic agents: probucol cardioprotective effects which do not appear Probucol is a drug that was developed as an to be shared by other non-sulfhydryl containagent for the treatment of hypercholesterole- ing ACE inhibitors [28J. Recent studies mia and atherosclerosis, but this compound reported that captopril was capable of scavhas no apparent structural similarity to other enging superoxide anions generated from agentsthat lower blood cholesterol. One novel either a purinelxanthine oxidase system or aspect of probucol is its potent antioxidant from activated neutrophils [29]. These free property, which is due to its conjugated phe- radical scavenging effects were shared by two nolic groups. From a structural point of view, other SH-containing (epi-captopril, and probucol consistsof two butylated hydroxytozofenoprilat) but not by non-SH-containing luene moleculesconnected by a -S-C-S- link- (enalaprilat and teprotide) ACE inhibitors. In age (Fig. 7). In a recent study, using our addition, these studiesalso demonstrated that purified sarcolemmal membrane model sys- pretreatment with captopril, but not enalaprilat, significantly attenuated the level of myotem, we found that the antiperoxidative potency of probucol is comparable to that of cardial stunning; it was postulated that this alpha tocopherol [23]. Like alpha tocopherol improvement of post-ischemic contractile and BHT, it appears that probucol inhibits derangement might be related to superoxide lipid peroxidation by a chain-breaking anion scavenging by SH-containing agents mechanism. [291. Recent studies with probucol have conStudies from our laboratory have demonfirmed the antiatherogenic properties of this strated that both captopril and zofenoprilat drug in the WHHL rabbit model; the ability exhibit dose-dependent ( 1O-200 PM) inhibition ( 15560%) of cellular lipid peroxidation in cultured endothelial and smooth muscle cells when exposed to the superoxide and hydroxyl radical generating systems[30, 311. In addition, the induced lossesin cellular viability were also prevented. Since the SH-containing stereoisomerof captopril, which is much less effective as an ACE inhibitor, was found to be equally effective, the protective effects were clearly independent of their ACE inhibition properties. Neither enalaprilat nor lisinopril, the non-SH-ACE inhibitors, produced any FIGURE 7. Structureof probucol. major beneficial effectsat the cellular level. In
Drugs 2 llnr
-
as Antioxidants
-.
Proprenolol
Coptopril
FIGURE 8. Effects of 50 /,u+t propranolol or captopril on DMPO-OH adduct formation in incubation mixture containing F&O4 (50 PM) + Hz02 (2 mM) and DMPO (45 mhi).
separate experiments, the effects of these ACE agents on the levels of hydroxyl radicals produced in our system were assessed by ESR spin-trapping technique using DMPO as the spin-trap. All three SH-containing agents (50 PM) reduced 40-60% of the hydroxyl radical signal (Fig. 8) [31]; whereas both non-SH containing agents (up to 200 P(M) were found ineffective. Therefore in support of the potential anti-radical effect of the -SH group, our data suggest that ACE inhibitors with SH groups are potent scavengers of hydroxyl radical and/or their daughter radicals. In agreement with our findings, a recent study using HPLC methods of measuring hydroxylated products of salicylic acid indicated that captopril was as effective as dimethylthiourea in scavenging hydroxyl radicals [32]. Oxidative
stress and deficiency
antioxidant
Recently, it was demonstrated in rat liver microsomes that the absence of vitamin E potentiates chemiluminescence induced by autoxidation processes [331. The addition of 5Trp-OH, an excellent physiological antioxidant [34], reduced chemiluminescence and protected the microsomal components from autoxidation. This effect, however, was considerably stronger when both vitamin E and 5-Trp-OH were present, indicating an important aspect of these agents: improvement of antioxidant status beyond the normal level of defense. These data suggested that the capacity to protect membranes may be boosted to meet excessive demands imposed by oxidative stress. The possible synergism between
1205
endogenous and added antioxidant agents is not understood at present, but it is clear that the mechanism may not require the repair of the vitamin E radicals. The defense of biological membranes against oxidative stress should have diverse components due to the compositional heterogeneity of the biological material. Hence, both electron donors and H atom donors (e.g. sulfhydryls) should be considered in efforts to design increased antioxidant defenses of various tissues and organs. Conclusions The ability of a number of cardiovascular drugs to act as antioxidants at the subcellular and cellular levels raises additional questions of clinical relevance. One major concern in the in vitro experimental work is that the incubations of membranes and cells with micromolar levels of these agents are required to produce significant protection against oxygen free radical challenge. Although serum levels of propranolol have been reported to approach such levels after relatively high doses, lower levels (< 1 PM) in the serum are more common [351. Since the focus of much of our work has been at the plasma membrane level, particularly the sarcolemma, a key question to be resolved is whether the effective level of each of these antioxidant cardiovascular drugs is reached within the sarcolemma during clinical therapy. We have stressed the greater efficacy of some of the more lipophilic beta-blocking and calcium channel blocking agents as antioxidants. We postulate that the protective effects of these more lipophilic agents, which partition into lipids, may be due in part to their concentration in cardiovascular membranes. Lullman et al. [36, 34 studied the rates and extent of accumulation of different neutral and cationic drugs in isolated atria from the guinea-pig; both the extent of drug accumulation and the time to reach maximal uptake could be related to the lipophilicity of the drugs. Verapamil was accumulated 30fold and the maximum uptake was not achieved until more than 2 h of incubation [36]. In a related study using Purkinje fibers [3fl, a 40-fold accumulation of propranolol was observed; time to equilibrium required 3 h of incubation. Platelets have been shown to accumulate propranolol up to 30-fold over
W. B. Weglicki
1206
plasma concentrations [39]. The above findings suggest that a longer time of incubation may lead to higher intracellular and membrane concentrations of the more lipophilic agents; this process might depend not only on the lipophilicity of the drug, but also on other cellular uptake mechanisms. Recent work by Herbette et al., has approached the above question from another perspective [40, II]. They studied native and model membranes and determined the level of dihydropyridine partitioning into the membranes by X-ray and neutron diffraction. They observed that the calcium blockers concentrated more than 2 orders of magnitude higher into sarcolemma1 membranes than in the aqueous buffer. These data suggest that the predominantly hydrophobic calcium channel blockers and other amphiphilic drugs reside in membranes in relatively high concentrations. Other classes of cardiovascular agents, such as sullhydryl containing ACE inhibitors, seem to have direct antiradical properties in addition to their pharmacological effects. Compared to propranolol, our data have shown that captopril is a much more potent hydroxyl radical scavenger in the aqueous phase. As represented in Figure 7, in a system using DMPO to monitor hydroxyl radical levels generated from the Fenton reagents (Fe?+ + HsOz), 50 FM captopril diminished about 50% of the relative DMPO-OH signal; in contrast, the same level of propranolol provided a minimal scavenging effect (about 5%). However, propranolol has been shown to be quite effective as an anti-peroxidative agent, within the membrane [ ISJ and we have reported that pretreatment of sarcolemmal membranes with propranolol effectively prevented free radical mediated carbon-centered
et al.
radical formation as detected by the lipophilic spin-trap, MNP [ 17J. We suggest that the presence of lipophilic agents such as proprano101 would provide membrane antiperoxidative protection. The presence of hydrophilic agents such as SH-containing ACE inhibitors would intercept oxy-radicals before they reach the cellular sites of injury; these agents concentrate at the endothelial plasmalemma when they bind to the angiotensin converting enzyme and the increased concentration of sulfhydryl moieties may provide protection against free radical injury in the aqueous phase. Increased free radical stress may arise in either the aqueous or membrane compartments. When oxygen derived radicals arise in the aqueous compartment (e.g. blood) they can be detected by the electron spin resource technique using spin trap agents (e.g. DMPO) that “trap” radicals in this compartment; whereas more lipid-soluble agents (e.g. MNP) are able to trap membrane-associated radicals in the hydrophobic or membrane compartment. Since free radicals may be found in both aqueous and membrane compartments, therapy directed at both distributions would seem preferable to prevent free radical-mediated tissue injury.
W. B. Weglicki’, I. T. Mak’ and M. G. Simic2 ‘Division of Experimental Medicine, Department of Medicine, George Washington
National
University Medical School, Washington, D.C. and ‘Center for Radiation Research, Institute of Standards & Technology, Gaithersburg, MD, USA
References 1 2 3 4
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M. G., TAYLOR, K. A. Introduction to peroxidation and antioxidant mechanisms. In: Uxygcn Rndimts in Eiology and lWedicine (M. G. Simic, K. A. Taylor, J. F. Ward, Eds), pp. l-10, New York: Plenum Press, (1988). SIMIC, M. G. Free radical mechanisms in autoxidation processes. J Chem Ed 58, 125-131 (1981). SIMIC,
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7 JOVANOVIC, S. V., SIMIC, M. G. Redox properties of oxy and antioxidant radicals. In: Oxygen Radical in Bzolosy and Medicine (M. G. Simic, K. A. Taylor, J, F. Ward, Eds), pp. 115-122, New York: Plenum Press, (1988). 8 PACKER, J. E., WILLSON, R. L., BAHNEMANN, D., As~us, K. D. Electron transfer reactions of halogenated aliphatic peroxyl radicals: measurement pfabsoiute rate constants by pulse radiolysis. J Chem Sot. Perkin Trans 1 I, 296-299 (1980). 9 SIMIC, M. G., JOVANOVIC, S. V. Antioxidation mechanisms of uric acid. J Am Chem Sot, III, 5778-5782 (1989). IO SIMIC, M. G., JOVANOVIC, S. V., SHIEKHLY, M. A. Heterocyclic resonant radicals. Free Radical Res Commun 6, 113-115 (1989). II HUNTER;. P. L., DESROSIERS, M. F., SIMIC, M. G. The effect of oxygen, antioxidants and superoxide radical on tyrosine phenoxyl radical dimerization. Free Radical BioI Med 6, 581-585 (1989). 12 NIKI, E. Antioxidants in relation to lipid peroxidation. Chemistry and Physics of Lipids 44, 227-253 (1987). R. L. Direct observation of a free radical interaction between vitamin E 13 PACKER, J, E., SLATER, T. F., WILLSON, and vitamin C. Nature [Lond] 278, 737-738 (1979). 14 SIMIC, M. G., HUNTER, E. Reaction mechanism ofperoxyl and C-centered radicals with suifydryls. J Frrp Radicals in Biol and Med 2, 227-230 (1986). I .5 REVESZ, L., EDCREN, M. Glutathione-dependent yield and repair of single-strand DNA breaks in irradiated cells. Br J Cancer [Suppl] 6, 55-60 (1984). I6 MAK, I. T., WEGLICKI, W. B. Protection by B-blocking agents against free radical-mediated sarcolrmmal lipid peroxidation. Circ Res 63, 262-266 (1988). 17 MAK, I. T., ARROYO, C. M., WEGLICKI, W. B. Inhibition ofsarcolemmal carbon-crntered free radical formation by propranolol. Circ Res65, 1151-1156 (1989). 18 CONNOLLY, M. E., KERSTING, F.. DELLERY, C. T. The clinical pharmacology of beta-adrenoceptor-blocking drugs. Prog Cardiovasc Dis 19, 203-234 (1976). 19 JANERO, D. R., BURGHARDT, B., LOPEZ, R. Protection of cardiac membrane phospholipid against oxidativr injury by calcium antagonists. Biochem Pharmacol37, 4197-4203 (1988). 20 MAK, I. T., WECLICKI, W. B. Comparative antioxidant activities of propranolol, nifedipinc, vcrapamil and diltiazem against sarcolemmal membrane lipid peroxidation. Circ Res 66, 1449-1452 (1990). 21 VER DONCK, L., VANREEMPTS, J., VANDEPLASCHE, G., BOGERS, M. A new method to study activated oxygen species induced damage in cardiomyocytes and protection by Ca’+ -antagonists. J MoI Cell Cardiol20, 8 I I --823 ( 1988). 22 BOGERS, M., VER DONCK, L., VANDEPLASCHE, G. Pathophysiology ofcardiomyocytes. Ann NY .4cad Sci 522,633653 j 1988). 23 MAK, I. T., DICKENS, B. F., WEGLICKI, W. B. Potent antiperoxidative effect ofprobucol on isolated mrmhranrs and cultured vascular cells. J Mol Cell Cardiol 21 [SuppI II], 96 (1989) by which 24 NARUSZEWI~Z, M., CAREW, T. E., PITTMAN, R. C., WITZTUM, J. L.. STEINBERG, D. A novel mechanism probucol lowers LDL levels demonstrated in the LDL receptor-deficient rabbit. J Lipid Res 25, 1206- I2 13 ( 1984). 25 STEINBERG, D. Studies on the mechanism of action of probucol. Am J Cardiol57, 16H-21H (1986). 26 LOPEZ-OVEJERO, J. A., SAAL, S. D., D’ANGELO, W. A., CHEICH, J. S., STENZEL, K. H., LARAGH, J. H. Reversal of vascular and renal crises of scleroderma by oral angiotensin converting enzyme blockade. New Engl J Mrd 300, 1417-1419 (1979). 27 CIISHMAN, D. W., CHEUNC, H. S.. SABO, E. F. Development and design of specific inhibitors ofangiotmsinconverting enzyme. Am J Cardiol 4!#, 139(F-I394 (1982). “8 VAN CILST, W. H., DEGRAEFF, P. A., WESSELING, H., DELANGEN, C. D. J. Reduction of rep&&ion arrhythmia in thr ischemic isolated rat heart by angiotensin converting enzyme inhibitors: A comparison of captopril. rnalapril and HOE 498. J Cardiovasc Pharmacol8, 722-728 ( 1986). 29 WESTLIN, W., MULLANE, K. Does captopril attenuate reperfusion-induced myocardial dysfunction hy scavrnging frcr radicals. Circulation 77 [Suppl 61, 130-139 (1988). 30 WECLICKI, W. B., DICKENS, B. F., PFLUG, B. R., MAK, I. T. Protective effects of sulfhydryl-containing ,4CE inhibitors against lipid peroxidation in endothelial and smooth muscle cells. FASEB J 3, A593 (1989). 31 MAK, I. I‘., DICKENS, B. F., WEGLICKI, W. B. Hydroxyl radical scavenging and attenuation of free radical injury endothelial cells by SH-containing ACE inhibitors. Circulation 80, II-242 (1989). 32 BAGCHI, D., PRASAD, R., DAS, D. K. Direct scavenging of free radicals by captopril. an angiotrnsirl c.onvrrting enzyme inhibitor. Biochem Biophys Res Commun 158: 52-57 (1989). 33 CADENAS, E., SIMIC, M. G., SIES, H. Antioxidant activity of 5-hydroxytryptophan, 5-hydroxyindole. and DOPA against microsomal lipid peroxidation and its dependence on vitamin E. Free Radical Res Commun I I-I 7 ( 1989). 34 JOVANOVIC, S. V., SIMIC, M. G. Tryptophan metabolites as antioxidants. Life Chem Rep 3, 124-130 (1985). 35 CRUICKSHANK. J, M. The clinical importance of cardioselectivity and lipophilicity in beta blockrrs. Am Heart J 100, 160-178 (1980). :x5 LULLMANN, H., TIMMERMANS, P. B. M. W. M., WEIKERT, G. M., ZIEGLER, A. Accumulation ofdrugs bv guinea pig isolated atria. Quantitative correlations. J Med Chem 23: 560-565 (1980). 37 LVLLMANN, H., TIMMERMANS, P. B. M. W. M., WEIKERT, G. M., ZIEGLER, A. Accumulation of drugs bv resting or beating cardiac tissue. Eur J Pharmacol60, 277-285 (1979). 38 PRIIETT, J, K., WALLE, T., WALLE, U. K. Propranolol effects on membrane repolarization tissue contcn~ and the influence of exposure time. J Pharmacol Exp Tber 215, 539-543 (1980). 38 WEKSLER, B. B., GILLICK, M., PINK, J. Effect of propranolol on platelet function. Blood 49, 185-196 : 19771
1208 40
41
W. B. Weglicki
et al.
HERBETTE, L. G., VAN ERVE, Y. M. H., RHODES, D. G. Interaction of 1, 4 dihydropyridine calcium channel antagonists with biological membranes: lipid bilayer partitioning could occur before drug binding to receptors. J Mol Cell Cardiol 21, 187-201 (1989). MASON, R. P., GONYE, G. E., CHESTER, D. W., HERBEITE, L. C. Partitioning and location of Bay K8644, 1,4,dihydropyridine in model and biological membranes. Biophys J 55, 769-778 (1989).
Mol Cell Cardiol 22, 11991208 ( 1990)
REVIEW
Mechanisms
of Cardiovascular
ARTICLE
Drugs
as Antioxidants”
(Received 30 October 1989, accepted in revised form 29 May 1990)
Introduction The processof oxidation in biological systems incorporates molecular oxygen into biochemical reactions necessaryfor essentialmetabolic processes.Phosphorylation of mitochondria, drug metabolism by mixed function oxidases etc., are such biological mechanismsthat have evolved in aerobic organisms. Mammalian tissuesare composedof a number of molecular “building blocks” that are susceptible to another form of oxidation, i.e. peroxidation. Among the potential sites of peroxidation in tissues are the unsaturated bonds of fatty acids, particularly those in biological membranes that contain many forms of structural lipids. Biological membranes,such as the plasmalemma, also provide hydrophobic binding sitesfor drugs and other substancesthat reside in membranes: e.g. natural antioxidants such as vitamin E and beta-carotene, which interact with lipids and proteins of membranes. In addition, cells contain hydrophilic environments, e.g. cytosol, that provide natural sites for water soluble antioxidants such as ascorbate (vitamin C), and glutathione (GSH). In the normal tissue,the above “natural antioxidants” provide antioxidant protection to the cells and extracellular spaces.In vitamin deficiency states, such as vitamin E deficiency in premature infants [I] or scurvy, a consequence of vitamin C deficiency, characteristic pathological changesoccur. Although the biological requirements for antioxidant vitamins have been studied for a number of years [ZJ, the ability of other synthetic compounds, such asdrugs, to act asantioxidants in tissuesis an area of recent interest. The purpose of this review is to outline someof the recent developments in this area. * Supported by grants from POl-HL-38079, ROl-HL-36418, Company and ONR NOOOl4-K-88+405. Please address all correspondence to: William B. Weglicki, of Medicine, Ross Hall, Room 409, 2300 Eye Street, N.W., 0022-2828/90/101199
+ 10 $03.00/O
Free radical generation and inrrCti~tiOlI
Free radicals were discovered at the turn of the 20th century by Gomberg [A. Subsequently, they were shown to play an important role in oxidation of biochemical components and more recently in cellsand organisms [A. In an oxygen environment, the predominant form of free radicals that are encountered in biology and medicine are oxygen radicals (Table 1) [5J. The term free radical refers to the presence of one or more unpaired electrons; the common convention for designating this electron is R’. Xanthine, hypoxanthine, NADPH, RS- and many other reducing agents may, under certain conditions, generate superoxide radical: 0, + e- --+*02-.
1’) Molecular oxygen itself contains two unpaired electrons and is not as reactive as those compounds referred to as oxygen free radicals, which contain an unpaired electron residing predominantly on an oxygen atom. During normal metabolism, such as oxidative phosphorylation, oxygen is reduced stepwise to produce water; this is accomplished by the mitochondrial electron transport system catalyzing sequential one electron additions to molecular oxygen. Intermediate speciesof one electron reduction of oxygen in solutions are the superoxide radical (‘0, -), hydrogen peroxide (H,O,) and hydroxyl radical (‘OH): O,“-+‘O;=--H,O,Z + Hz0
‘OH’-
(2) All three intermediary speciesare thought to be involved in cytotoxic injury of tissues.The exact mechanismsof free radical generation in AHA
National
Capital
Affiliate,
Director, Division of Experimental Washington, DC 20037, USA. 0
Bristol Medicine,
1990 Academic
Myers
Squibb
Department Press Limited
1200 TABLE
W. B. Weglicki 1. Nomenclature
of related Oxygen species and derived radicals
Species
Name
RH R’ ROO’ ‘OOH ‘0,-
Parent molecule Free radical Peroxy radical Hydroperoxy radical Superoxide radical Hydroxy radical Hydrogen peroxide Alkoxy radical Hydroperoxide Peroxide
‘OH Hz02
RO’ ROOH ROOR ArO’
Origin RH R’ + 02, ROOH 02- + H+
02 + e-, xanthine oxidase,NADPH oxidase Hz023
Phenoxyand aroxy radicals
free radicals in normal tissue have not been determined directly. In the activated white cell, the superoxide radical can be produced by NADPH oxidase during the respiratory burst of phagocytosis. The natural antioxidant enzyme for this process is superoxide dismutasewhich converts superoxide radicals into hydrogen peroxide, which will then be degraded by peroxidases and catalases. Another antioxidant enzyme is glutathione peroxidase which requires GSH in the reaction schemeas follows: + H,O,
nlul.athionc ueroxidnsc , GSSG
+ 2H,O
(3)
Catalase on the other hand transforms H,Os: 2H202
csca’ase bO2 + 2H,O
(4)
When these various antioxidant enzymes and moleculesare deficient, the tissuesmay be rendered more susceptible to peroxidative injury. Even when the tissue levels of the above antioxidant compounds are in the normal range, the protective mechanismscan be overwhelmed by excessive oxidant stress of various types. For these situations, the addition of natural and synthetic antioxidants (such as drugs) to the tissuesmay provide additional protection. Antioxidant
H2O
‘0~~, Superoxidedismutase ROOH, ROOR ROO’
viva are not fully understood, and the levels of
2~s~
et al.
mechanisms
Natural and synthetic antioxidants may intervene at various stages of the peroxidation
Peroxidation Phenolic antioxidants
processsuch as: Initiation:
RH+R’
(5)
R’ + 0s + ROO’
(6)
ROO’ + RH-+ ROOH + R’
(7)
or Propagation:
followed by:
In the absenceof antioxidants peroxy radicals are eliminated by Termination:
ROO’ + ROO’-+ unreactive products (8) The above processesare referred to as autooxidation or peroxidation of an organic substrate [6J. Among the synthetic antioxidants are the various aromatic phenols, ArOH. These compounds inhibit autoxidation as follows: ROO’ + ArOH --+ ROOH + ArO’
(9)
Reaction of antioxidants with peroxy radicals is in fact an electron transfer process[A and not an H atom transfer as indicated by the above overall reaction. Hence, good electron donors such as ascorbate, AH- (E, = 0.28 V) and urate, UHz- (E, = 0.56 V) are excellent antioxidants. Being water soluble, ascorbate and urate are cytosolic or extracellular antioxidants. The reactions of these antioxidants with peroxyl radicals proceed in the following
Drags
FIGURE
1. The chromanoxy
radical
resonant
as Antioxidant8
1201
state of a-tocopherol.
fashion with their rate constants indicated:
followed by:
Ascorbate
E-O-
[ 83 :
ROO’
+ AH - --+ ROO- + ‘AH, (k = 2 x IO6 M-I s-l) followed by: ‘AHcr’AROO-
+ H+, (pKa = 4.2)
+ H+ ++ROOH,
(pKa = 12).
(10) (11) (12)
(pKa = 10-11).
+ UH,- +ROO(k=3 x 106~-‘s-‘)
or by inhibition
+ ‘UH,, (13)
For Vitamin E, E-OH, (ET = 0.48 V) which is a classical membrane associated antioxidant, its mechanism of action differs somewhat from the above reactions [II: ROO’
+ E-OH+ ROO- + E--OH+ (k = lo6 - lo7 M-I s-r) (14) Being at the interface of a membrane, the positive radical E-‘OH+ reacts rapidly with water: E--OH+
+ HsO--,E-0’
+ H,O+
(15)
To give a long lived chromanoxy radical (E-O’), a special class of phenoxy radicals (Ph-0’). Similar to phenoxy radicals, the vitamin E radical exists in multiple resonance states (Fig. 1). Th e resonant state of the radical [IO] gives it specific properties such as an extremely long life due to its low decay rate constant (k = 2 x lo2 M-’ s- ‘) and unreactivity towards oxygen (k < 10e2 M-~ s-l) [ 111. In general, the expected longer life of the vitamin E radical in membranes permits it to be repaired by other reducing agents with lower redox potentials. Such repair reactions of vitamin E radicals in liposomes [ 14 and homogeneous solutions [ 13J have been known for some time. For example, vitamin C is an excellent repairing agent of vitamin E radicals [13]: E-O’ + AH- --+ E-O- + ‘AH, (k = 1.5 X lo6 M-l s-l) (16)
(17)
Another class of antioxidants are the sulfhydryls (thiols) which can repair free radical injury and may act as either hydrogen atom (RSH), or electron (RS-) donors [14]. They promote repair of free radical injury either by restitution: R’ + RSH-+
Urate [9]: ROO’
+ H+*E-OH,
ROO’
RH + RS’
(18)
of peroxidation: + RS-+ROO-
+ RS’
(19)
Thiol-containing compounds are good scavengers of hydroxyl radicals, and thus they may act as protective agents against injury due to hydroxyl radicals, provided that the concentration locally of the thiol is relatively high. *OH + RSH+ H,O + RS’, (k = 10” M-~ S-’
(20)
Other recent data suggest that thiols may participate in enzymatic repair processes to regenerate NADPH in the presence of NADPH and the pentose monophosphate shunt pathway [15J. Antioxidanta4ztivitiesofcardiovascdar agents
In addition to their physiological receptor binding functions, most commonly used cardiovascular agents, being amphiphilic by nature, partition readily into the hydrophobic regions of the cell membrane and thus may affect physical, as well as, chemical properties. We have recently used in vitro approaches to assess whether such drug-membrane interactions affect the sensitivity ofcell membranesto free radical injury [ 161. Beta-blockers and class I antiarrhythmic
drugs
By using highly purified cardiac sarcolemmal membranesasa model system, the antiperoxi-
W. B. Wtglicki
1202 Betao
blockem
=
et al.
Class I agsnts
1
Agents
(200
p~u()
FIGURE 2. Effects of B-blocking and Class I antiarrhythmic agents on sarcolemmal lipid peroxidation. Sarcolemmal membranes (100 pgg/ml) were preincubated with each agent for 10 min at 37°C before the additions of Fe-ADP and DHF. After 20 min of incubation, samples were assayed for MDA formationand expressed as y0 inhibition comparing to controls. Detailed experimental conditions were described in Ref. 16. *P metoprolol > atenolol > sotalol. This antioxidant effect appeared to correlate with hydrophobicity: the correlation coefficient (r) between the antioxidant activities of the betablockers and their lipophilicity expressed as log when octanol/water partition coefficient (Fig. 3) is 0.86 (P < 0.05). In the data presented in Figure 2, high levels (200 PM) of the agents were chosen to amplify the differences between the weak and the strong antioxidant agents. In the caseof propranolol, significant protection against membrane peroxidation could be achieved by
I 0
01 -I
i-J 0 2 3 5 H f
I
Log (octanol/HsO)
2 partition
3
4
coefficient
FIGURE 3. Relationship between partition coefficient (Log Octanol/water) and antioxidant activity of the betablockers. The results were derived from Figure 2 and Ref. 16.
concentrations aslow as 10PM (Fig. 4) [ 171.In another study, the mechanismof propranololinhibited membrane lipid peroxidation was examined by electron spin resonance spintrapping technique [ I7J. We observed that pretreatment of the sarcolemmal membranes with 3-30 PM propranolol prevented the formation of membrane carbon-centered lipid radicals in a log concentration-dependent manner; the EC50 is 6.7 paa[ 1I]. Propranolol also possessespotent membrane stabilizing activity and it was thought that its membrane protective effect against free radicals might be related to this membrane effect. However, both quinidine and lidocaine, which are known to have major membrane stabilizing activity, are relatively ineffective; this suggeststhat a significant antiperoxidative effect due to such membrane
.f E E 1 i! = B E .E u P
18 16 I4 12 IO 8
6
4 2 0
0
IO Propranolol
30 (FM)
FIGURE4.Concentration-dependent protectiveeffect of propranolol on sarcolemmal lipid peroxidation. After 20 min of incubation, samples were assayed for MDA formation. Values are means + S.D. of 4 separate determinations; *P < 0.01 versus controls.
Drugs
as Antioxidants
1203
”
FIGURE 5. Effects of propranolol and structurally related compounds on sarcolemmal lipid peroxidation. Sarcolemmal membranes were preincubated with each agent (200 PM) for 10 min before the additions to Fe-ADP and DHF, and were further incubated for 20 min. MDA determination and other conditions were described under Figure 2. Results were means of 3-8 separated experiments.
activity is unlikely. One major common structural feature among most beta-blockers is the aromatic moiety connected to an ethanolamine chain by an aromatic ether linkage [ l8j. It is believed that much of the hydrophobicity of these agents is contributed by this ring structure, especially if it is unsubstituted like that of propranolol, a few structurally related compounds were studied. Figure 5 demonstrates that the naphthalene moiety alone provided no protection; however when a naphthoxyl linkage was present, as represented by I-methoxy-naphthalene, 65% of antioxidant activity (relative to propranolol) was preserved. However, when the naphthoxyl moiety was removed, asrepresented by 1-naphthaleneacetic acid, no antioxidative activity was observed. These results indicated that both the ring structure and the aromatic oxy-linkage were essentialfor the antioxidant activity of propranolol. Thus, propranolol and the other more lipophilic beta-blockers appear to inhibit membrane lipid peroxidation through a chain breaking mechanism, like the lipophilic natural antioxidant alpha tocopherol. The requirement for an aromatic ring structure is probably due to reduction of redox potential and the resonance-stabilizing capacity similar to that described for alpha tocopherol (Fig. 1). The importance of the aromatic oxy-linkage may be due to its tautomerization into a phenolic group which would inactivate peroxy radicals (reaction 9).
40~
calcium
blockers
or propranolol
FIGURE 6. Comparative inhibitory effects of propranolo1 and calcium channel blockers on sarcolemmal lipid peroxidation. Sarcolemmal membranes were preincubated with each agent (40 PM) for 10 min before ;he final additions of DHF/Fe-ADP: after 20 min of incubation. samples were assayed for MDA formation and expressed as percent inhibition relative to the controls. Values are mean + S.D. of 3-6 separate determinations. * P < 0.05, **P C 0.01 vs controls.
Calcium channel blockers
Studies from Janero et al. [19] described that certain lessspecific calcium blockers (bepridil, prenylamine, flunarizine and cinnarizine) provided inhibitory effects against cardiac liposomal lipid peroxidation that was mediated by xanthine oxidase/hypoxanthine system. We have found that certain specific calcium channel blockers (nifedipine, verapamil, and diltiazem) exhibit various levels of antiperoxidative activities [NJ. Using our sarcolemmal membranes, nifedipine was the most potent of the calcium channel blocking agents (> P-fold more potent than propranolol), followed by verapamil and, to a lesser extent, diltiazem [20] (Fig. 6). Despite their markedly different chemical structures, all three agents do possess aromatic rings, a feature common .to beta-blockers and the conventional chain-breaking antioxidants. Furthermore, verapamil and diltiazem contain electron releasing groups (CHsO), whereas nifedipine has an electron-rich dihydropyridine ring moiety. Also, all three calcium blockers are lipophilic and partition into the phospholipid domains of cardiac membranes; thus, we speculate that the antioxidant effects of theseagents in vitro are mediated by neutralizing the lipid peroxy radicals through an electron transfer processsimilar to “reaction 19”. Additional experiments (unpublished) have been performed in which the incubation medium was removed after the drug pretreat-
1204
W. B. Weglicki et al.
ment but before exposure to the free radical system; propranolol and the three calcium channel blockers were found to be similarly protective against membrane lipid peroxidation Other workers have studied isolated cardiomyocytes exposed to activated oxygen species; Ver Donck et al. [Zl, 22] have shown that both lessspecific (cinnarizine, flunarizine) and specific (nicardipine, verapamil, diltiazem) calcium blockers provided various degrees of cytoprotection. However, nifedipine was reported to be ineffective. This latter result, using cardiomyocytes doesnot agree with our data using sarcolemma from adult cardiomyocytes.
to protect against oxidation of cholesterolcarrying low density lipoprotein appearsto be essentialin this mechanism [24]. It has been postulated that the antioxidant properties of this drug may have relevance in preventing foam cell formation [ 2.51. Angiotensin
converting enzyme (ACE)
inhibitors
ACE inhibitors are a new classof cardiovascular agents for treatment of hypertension and other cardiovascular diseases[26, 271. Captopril, the original member of this class,contains a sulfhydryl moiety which is believed to contribute to the potency of the drug by binding to the zinc moiety of the enzyme [27J. In addition to its ACE inhibition, captopril has been shown to possess anti-inflammatory and Antiatherosclerotic agents: probucol cardioprotective effects which do not appear Probucol is a drug that was developed as an to be shared by other non-sulfhydryl containagent for the treatment of hypercholesterole- ing ACE inhibitors [28J. Recent studies mia and atherosclerosis, but this compound reported that captopril was capable of scavhas no apparent structural similarity to other enging superoxide anions generated from agentsthat lower blood cholesterol. One novel either a purinelxanthine oxidase system or aspect of probucol is its potent antioxidant from activated neutrophils [29]. These free property, which is due to its conjugated phe- radical scavenging effects were shared by two nolic groups. From a structural point of view, other SH-containing (epi-captopril, and probucol consistsof two butylated hydroxytozofenoprilat) but not by non-SH-containing luene moleculesconnected by a -S-C-S- link- (enalaprilat and teprotide) ACE inhibitors. In age (Fig. 7). In a recent study, using our addition, these studiesalso demonstrated that purified sarcolemmal membrane model sys- pretreatment with captopril, but not enalaprilat, significantly attenuated the level of myotem, we found that the antiperoxidative potency of probucol is comparable to that of cardial stunning; it was postulated that this alpha tocopherol [23]. Like alpha tocopherol improvement of post-ischemic contractile and BHT, it appears that probucol inhibits derangement might be related to superoxide lipid peroxidation by a chain-breaking anion scavenging by SH-containing agents mechanism. [291. Recent studies with probucol have conStudies from our laboratory have demonfirmed the antiatherogenic properties of this strated that both captopril and zofenoprilat drug in the WHHL rabbit model; the ability exhibit dose-dependent ( 1O-200 PM) inhibition ( 15560%) of cellular lipid peroxidation in cultured endothelial and smooth muscle cells when exposed to the superoxide and hydroxyl radical generating systems[30, 311. In addition, the induced lossesin cellular viability were also prevented. Since the SH-containing stereoisomerof captopril, which is much less effective as an ACE inhibitor, was found to be equally effective, the protective effects were clearly independent of their ACE inhibition properties. Neither enalaprilat nor lisinopril, the non-SH-ACE inhibitors, produced any FIGURE 7. Structureof probucol. major beneficial effectsat the cellular level. In
Drugs 2 llnr
-
as Antioxidants
-.
Proprenolol
Coptopril
FIGURE 8. Effects of 50 /,u+t propranolol or captopril on DMPO-OH adduct formation in incubation mixture containing F&O4 (50 PM) + Hz02 (2 mM) and DMPO (45 mhi).
separate experiments, the effects of these ACE agents on the levels of hydroxyl radicals produced in our system were assessed by ESR spin-trapping technique using DMPO as the spin-trap. All three SH-containing agents (50 PM) reduced 40-60% of the hydroxyl radical signal (Fig. 8) [31]; whereas both non-SH containing agents (up to 200 P(M) were found ineffective. Therefore in support of the potential anti-radical effect of the -SH group, our data suggest that ACE inhibitors with SH groups are potent scavengers of hydroxyl radical and/or their daughter radicals. In agreement with our findings, a recent study using HPLC methods of measuring hydroxylated products of salicylic acid indicated that captopril was as effective as dimethylthiourea in scavenging hydroxyl radicals [32]. Oxidative
stress and deficiency
antioxidant
Recently, it was demonstrated in rat liver microsomes that the absence of vitamin E potentiates chemiluminescence induced by autoxidation processes [331. The addition of 5Trp-OH, an excellent physiological antioxidant [34], reduced chemiluminescence and protected the microsomal components from autoxidation. This effect, however, was considerably stronger when both vitamin E and 5-Trp-OH were present, indicating an important aspect of these agents: improvement of antioxidant status beyond the normal level of defense. These data suggested that the capacity to protect membranes may be boosted to meet excessive demands imposed by oxidative stress. The possible synergism between
1205
endogenous and added antioxidant agents is not understood at present, but it is clear that the mechanism may not require the repair of the vitamin E radicals. The defense of biological membranes against oxidative stress should have diverse components due to the compositional heterogeneity of the biological material. Hence, both electron donors and H atom donors (e.g. sulfhydryls) should be considered in efforts to design increased antioxidant defenses of various tissues and organs. Conclusions The ability of a number of cardiovascular drugs to act as antioxidants at the subcellular and cellular levels raises additional questions of clinical relevance. One major concern in the in vitro experimental work is that the incubations of membranes and cells with micromolar levels of these agents are required to produce significant protection against oxygen free radical challenge. Although serum levels of propranolol have been reported to approach such levels after relatively high doses, lower levels (< 1 PM) in the serum are more common [351. Since the focus of much of our work has been at the plasma membrane level, particularly the sarcolemma, a key question to be resolved is whether the effective level of each of these antioxidant cardiovascular drugs is reached within the sarcolemma during clinical therapy. We have stressed the greater efficacy of some of the more lipophilic beta-blocking and calcium channel blocking agents as antioxidants. We postulate that the protective effects of these more lipophilic agents, which partition into lipids, may be due in part to their concentration in cardiovascular membranes. Lullman et al. [36, 34 studied the rates and extent of accumulation of different neutral and cationic drugs in isolated atria from the guinea-pig; both the extent of drug accumulation and the time to reach maximal uptake could be related to the lipophilicity of the drugs. Verapamil was accumulated 30fold and the maximum uptake was not achieved until more than 2 h of incubation [36]. In a related study using Purkinje fibers [3fl, a 40-fold accumulation of propranolol was observed; time to equilibrium required 3 h of incubation. Platelets have been shown to accumulate propranolol up to 30-fold over
W. B. Weglicki
1206
plasma concentrations [39]. The above findings suggest that a longer time of incubation may lead to higher intracellular and membrane concentrations of the more lipophilic agents; this process might depend not only on the lipophilicity of the drug, but also on other cellular uptake mechanisms. Recent work by Herbette et al., has approached the above question from another perspective [40, II]. They studied native and model membranes and determined the level of dihydropyridine partitioning into the membranes by X-ray and neutron diffraction. They observed that the calcium blockers concentrated more than 2 orders of magnitude higher into sarcolemma1 membranes than in the aqueous buffer. These data suggest that the predominantly hydrophobic calcium channel blockers and other amphiphilic drugs reside in membranes in relatively high concentrations. Other classes of cardiovascular agents, such as sullhydryl containing ACE inhibitors, seem to have direct antiradical properties in addition to their pharmacological effects. Compared to propranolol, our data have shown that captopril is a much more potent hydroxyl radical scavenger in the aqueous phase. As represented in Figure 7, in a system using DMPO to monitor hydroxyl radical levels generated from the Fenton reagents (Fe?+ + HsOz), 50 FM captopril diminished about 50% of the relative DMPO-OH signal; in contrast, the same level of propranolol provided a minimal scavenging effect (about 5%). However, propranolol has been shown to be quite effective as an anti-peroxidative agent, within the membrane [ ISJ and we have reported that pretreatment of sarcolemmal membranes with propranolol effectively prevented free radical mediated carbon-centered
et al.
radical formation as detected by the lipophilic spin-trap, MNP [ 17J. We suggest that the presence of lipophilic agents such as proprano101 would provide membrane antiperoxidative protection. The presence of hydrophilic agents such as SH-containing ACE inhibitors would intercept oxy-radicals before they reach the cellular sites of injury; these agents concentrate at the endothelial plasmalemma when they bind to the angiotensin converting enzyme and the increased concentration of sulfhydryl moieties may provide protection against free radical injury in the aqueous phase. Increased free radical stress may arise in either the aqueous or membrane compartments. When oxygen derived radicals arise in the aqueous compartment (e.g. blood) they can be detected by the electron spin resource technique using spin trap agents (e.g. DMPO) that “trap” radicals in this compartment; whereas more lipid-soluble agents (e.g. MNP) are able to trap membrane-associated radicals in the hydrophobic or membrane compartment. Since free radicals may be found in both aqueous and membrane compartments, therapy directed at both distributions would seem preferable to prevent free radical-mediated tissue injury.
W. B. Weglicki’, I. T. Mak’ and M. G. Simic2 ‘Division of Experimental Medicine, Department of Medicine, George Washington
National
University Medical School, Washington, D.C. and ‘Center for Radiation Research, Institute of Standards & Technology, Gaithersburg, MD, USA
References 1 2 3 4
FARRELL, P. M. Vitamin E deficiency in premature infants. J Pediatr 95, 869-878 (1979). &ES, B. N. Dietary carcinogens and anticarcinogens. Science ‘221, 1256-1264 (1983). GOMBERG. M. An instance of trivalent carbon: Triohenvlmethvl. 1 Am Chem Sot 22. 757 (19001 M&OR&J. M., FRIDOWCH, I. The reduction of dytocirome by-milk xanthine oxidke. J ‘Viol bhem
243, 5753
(1968). 5 6
M. G., TAYLOR, K. A. Introduction to peroxidation and antioxidant mechanisms. In: Uxygcn Rndimts in Eiology and lWedicine (M. G. Simic, K. A. Taylor, J. F. Ward, Eds), pp. l-10, New York: Plenum Press, (1988). SIMIC, M. G. Free radical mechanisms in autoxidation processes. J Chem Ed 58, 125-131 (1981). SIMIC,
Drugs
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