Oxidants and Antioxidants: State of the Art AALT BAST, Ph.D., GUIDO
R. M. M. HAENEN, Ph.D., CEES J. A. DOELMAN, Ph.D., Amsterdam,The Netherlands
Reactive oxygen species are regarded as merely pernicious. This is incorrect for they play a pivotal role in many physiologic reactions, such as cytochrome P450-mediated oxidations, regulation of the tone of smooth muscle, and killing of microorganisms. An imbalance in oxidant-antioxidant activity is involved in many free radical-mediated pathologies, e.g., ischemia-reperfusion and asthma. In an attempt to alleviate these pathologies with antioxidants, it should be noted that these compounds are neither specific nor mere antioxidants. Associated with antioxidant activity is a pro-oxidant action. In the development of new antioxidant therapies, the important question of how these drugs are incorporated in or commensurate with existing integrated physiologic radical-defense systems should be addressed.
From the Department of Pharmacochemistry,Vrije Universiteit, Amsterdam, The Netherlands. Requestsfor reprints should be addressedto Prof. Dr. A. Bast, Departmentof Pharrnacochemistry,Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
36--2S
scussing free radicals in physiology, we fre-
Di~uently consider oxygen free radicals. In 1924 it was established that molecular oxygen (02) contains unpaired electrons• It should not be described as 0 = 0 , in which all electrons are paired, but as • 0 - - 0 .. Although 02 has a radical nature and can even be called a diradical (two unpaired electrons), it does not have extreme reactivity, due to quantum-mechanical restrictions. The electron structure causes 02 to form water by a stepwise (univalent) reduction with four electrons: 02 + 4H ÷ 4e-
> 2H20
(1)
Figure 1 shows the protonated and unprotonated intermediate forms occurring during reduction of oxygen, but singlet oxygen, 102, formed by the input of energy has not been shown. Several forms of singlet oxygen exist and most important biologically is the one in which all electrons are paired. Frequently, the term "oxygen radicals" is wrongly used, being assigned to all reactive intermediate oxygen species including those molecular forms that are not radicals. It is appropriate to speak of "reactive oxygen species" instead of "oxygen free radicals." Articles, books, and congress proceedings devoted to reactive oxygen species promulgate the view that these reactive oxygen species should be regarded as extremely reactive and therefore deleterious to the organism. Note that radical chemistry has played an essential role in the origin of aerobic life forms. Also obvious is that radical reactions are an integral part of the homeostasis in cellular processes. It appears that enzymes and factors that were able to restrain radical reactions were already present early in evolution. The organism was equipped with an ancient balance of oxidants and antioxidants in order to cope with free radical biochemistry. Oxidants and antioxidants have well-defined functions and reside in specific cellular compartments and a disturbance in the delicate oxidantantioxidant balance results in many pathophysiologic conditions.
REACTIVE OXYGEN SPECIES AND IRON The organism has many strategies to prevent the propagation of radical reactions. It is possible to
September 30, 1991 The American Journal of Medicine Volume91 (suppl 3C)
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/ BAST ET AL
.5-5.
0 2 ground state
+e
0 2 superoxide anion radical
I0-0. +e
0%" peroxide ion
I ~- ~ I
+ 2 H+~
H202
hydrogen peroxide
+ H+ ~ r
• OH
hydroxyl radical
+2H
H20
+e
m
O;
and
I0"
0 2-
m
and
I01
+
te 2 I01
2 02.
+4H+ --
2
H20
Figure 1, The univalentreductionof oxygen.
scavenge reactive oxygen directly; for example, the enzyme superoxide dismutase (SOD) catalyzes the conversion of 02 "" 202 • +2H + SOD ~ H20z + 02
(2)
The heme-containing enzyme catalase transforms HzO~ into water and oxygen: 2H202
catalase
) 2H20 + 02
(3)
Glutathione peroxidase (GSH-Px) also reduces peroxides. The selenium-dependent GSH-Px reduces H202 as well as organic hydroperoxides. The selenium independent GSH-Px accepts hydroperoxides as substrate: ROOH + 2GSH
GSH-Px )
ROH + H20 + GSSG
(4) In a physiologic environment, both initiation as well as propagation of radical reactions appear to be restrained to a large extent. An important reaction that should not disseminate is the transformation of
Hz02 into a hydroxyl radical (. OH) by traces of transition ions such as iron or copper• This Fenton reaction proceeds as follows: Fe 2+ + H20e
> Fe 3÷ + .OH + OH-
(5)
• OH is extremely reactive. Although it is still uncertain whether . OH or an iron-oxygen complex is the ultimate reactive initiating species, it is evident that safe storage of iron ions is crucial [1]. Two thirds of the 4 g of iron present in an adult is stored in hemoglobin. Ten percent of the iron is found in myoglobin and a small portion in ironcontaining enzymes and in the transport protein transferrin. The remainder is present in intracellular storage proteins such as ferritin and hemosiderin. The glycoprotein transferrin binds 2 tool of Fe 3+ per mole. Under normal conditions, transferrin is only 30% bound with iron. The free plasma concentration of iron is therefore almost nil. The cellular uptake of iron, which is needed, for example, for synthesis of iron-containing enzymes, probably occurs through pinocytosis of the transferrin-iron complex. In the cell, iron is immediately stored in ferritin. The storage capacity of this protein is
September30, 1991 The AmericanJournal of Medicine Volume91 {sup# 3C)
3C-3S
SYMPOSIUM ON OXIDANTS AND ANTIOXIDANTS/ BAST ET AL
Fe3+ ' (ROH)Fe 3+
RH (RH)Fe 3+
XOOH
XOH
(RH)Fe 3+ (XOOH)
(R)(FeOH)3+,~------~ (RH)(FeOH)3+(XO')
(R•H)(FeO)3+
~
~~
e
02
(RH)Fe3"~O2-)
(RH)Fe3+(O22")~ H20
2 H+
large, viz. 4,500 mol of iron per mole of protein. Binding of iron to ferritin prevents its participation in the Fenton reaction but its storage is not completely assured. At pH 6, iron dissociates from ferritin, and in the microenvironment of the phagocytes, a pH value uric acid + NADH + H ÷
(7)
Conversion of XD into XO occurs rapidly upon mild proteolysis. During ischemia, degradation of adenosine triphosphate (ATP) forms hypoxanthine and in this way XO is supplied with a substrate during the reperfusion process. Note that the XO hypothesis has been questioned [14]. Activated neutrophils may adhere to the slightly damaged vascular endothelial layer longer than in the control situation. The neutrophil-induced damage is in the first instance directed to the vascular endothelium. Excessive levels of catecholamines, released during ischemia/hypoxia, also serve as a source for
September30, 1991 The AmericanJournalof Medicine Volume91 (suppl3C)
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/BASTET AL
oxygen radicals, arising from oxidation of the catecholamines. It is suggested that the changes in the lipid bilayer underlie the conversion from reversible myocardial damage into irreversible lesions. The activation of phospholipases, the detergent-like actions of accumulated fatty acids and lysophospholipids, together with the activation of lipid peroxidation have been called the "lipid triad" [15]. During lipid peroxidation of ~oG and ~o3 fatty acids, biologically active fatty-acid breakdown products are formed. Among these products, the hydroxyalkenals (e.g., 4-hydroxy-2,3-transnonenal) are produced in large amounts. There is evidence that these aldehydes have a number of properties that explain the injurious effects of free radical pathology [16]. The aldehydes have been suggested to act as "toxic second messengers" for the primary free radicals [16]. During detoxication of lipid peroxidation products, such as lipid hydroperoxides, aldehydes, and H2Oe, glutathione (GSH) is consumed, and extensive GSH-consumption imperils the function of proteins that depend on a critical sulfhydryl moiety. Oxidation of these proteins may occur, resulting for instance in the dysfunction of Ca2+-ATPases [17]. Oxidative stress induces nicotinamide adenine dinucleotide (NAD) depletion. The mechanism responsible for the fall in intracellular NAD levels is probably poly(ADP-ribose) polymerase, which uses NAD as substrate. This enzyme is activated under conditions of DNA strand breakage, which may occur in cells exposed to oxidative stress [18]. In addition to dysfunction of Ca2+-ATPase, clustering of lipid hydroperoxides and formation of lysophospholipids may result in calcium overload (Figure 5). This has recently been underscored by reports [19] on heart mitochondrial pores activated by oxidative stress. An increase in intracellular Ca 2+ levels leads to activation of Ca2+-dependent enzymes such as proteases and phospholipase that lead to worsening of oxidative damage. It has also been found that ischemia in the heart causes a decline in the enzymatic defense against free radicals in heart tissue [20]. Strong evidence for the supposed invoivement of free radicals in postischemic reperfusion injury comes from spin-trapping experiments [21]. The production of 0~- - could be related to the severity of ischemia in this way. The metabolic effect of oxygen radicals in heart tissue has been recently reviewed [22]. Drug-induced cardiotoxicity can sometimes also be related to the formation of reactive oxygen species. Clinical use of the effective anticancer drug, doxorubicin, is limited by cardiomyopathy that develops after cumulative dosing [23]. Oxygen radicals play a role in doxorubicin-induced cardiotoxic-
ity [24] via redox cycling of the compound. Moreover, it has recently been shown that the semiquinone free radicals, formed upon one-electron reduction of doxorubicin, are effective in releasing iron ions from the iron storage protein ferritin [25]. Iron chelators prevent the doxorubicin-induced cardiomyopathy.
Lungs In lung pathology, conditions such as emphysema, bronchopulmonary dysplasia, pneumoconiosis, bleomycin toxicity, paraquat toxicity, butylhydroxytoluene toxicity, mineral dust toxicity, cigarette smoke toxicity, respiratory distress syndrome, and asthma have been related to oxygen radicals. Asthma can be regarded as the manifestation of an inflammatory reaction. Lungs from patients with severe asthma show a shedding or loss of epithelium, thickening of the basal membrane, subepithelial fibrosis, accumulation of inflammatory cells, widespread mucus plugging, submucosal edema, and smooth muscle hyperplasia. Moreover, the observed asthmatic lung hyperreactivity [26] to a specific stimuli, such as methacholine, histamine, cold air, or exercise, has been proposed to be a consequence of the inflammatory reaction [27]. Since the effect of reactive oxygen species closely mimics the pathology of asthma, their involvement in the etiology of the disease has been suggested. It is reported that inhalation of xanthine/XO in cats causes a hyperreactivity of the airways to acetylcholine in vivo [28]. Hyperreactivity of the rat tracheal smooth muscle has been observed after cumene hydroperoxide incubation or of guinea pig tracheal smooth muscle after hypochlorite incubation (unpublished data). Also, leukotriene D 4 (LTD4) exposure of guinea pig tracheal smooth muscle in vitro causes histamine hyperreactivity that can be blocked by SOD, showing the involvement of O~ • in the induction of hyperreactivity by LTD4 [29]. A fi-adrenoceptor dysfunction has been observed after exposure of airways tissue to reactive oxygen species in vitro. H202, for example, induced an autonomic imbalance between muscarinic (contracting) and fi-adrenoceptor (relaxing) responses in favor of the muscarinic receptor response in rat tracheal tissue in vitro [30] (Table I). Epithelial damage and mucus hypersecretion, important features of asthma, are caused by reacrive oxygen species. The eosinophil peroxideH2Oe-halide system destroys human nasal epithelial cells in vitro [31]. We observed that hypochlorite induces a methacholine hyperreactivity of guinea pig tracheal tissue in vitro, which is proba-
September30, 1991 The AmericanJournal of Medicine Volume9i (suppl3C)
3C-7S
SYMPOSIUMON OXIDANTSAND ANTIOXIDANTS/BASTET AL TABLE I Effect of H202 (10 -3 M) on the Methacholine and the (-)lsoprenaline Concentration-Response Curve in Rat Tracheal and Lung Parenchymal Tissue Control (-log ECso/Era~x%) Methachoiine Trachea Lungstrips Isoprenaline Trachea Lung strips
Pretreated (-tog ECso/Em,,%)
5.54 +- 0.06/100 5.03 -+ 0.04/100
5.40 + 0.09/39 _+6* 4.71 -+ 0.04"/86 _+9
7.85 +- 0.05/100 7.35 +- 0.08/100
Not detectable/O* 6.91 -+ 0.12"/39 + 14"
Note: The -log EC5ovalues (-+SEM) and maximal effects (-+SEM) of either methacholine or (-)isoprenaline Cumulative concentration-response curves before and after treatment af the rat tracheal and lung parenchymal strips with H~02 are shown (*p Enz-Cu e+ + Ira. +H20
(16)
Enz-Ou + + H202
(17)
Enz represents the Cu-containing SOD and ImH the imidazole moiety of histidine at the active site of the enzyme. The use of spin-trap experiments itrefutably proved the formation of • OH by SOD in combination with H202 [46]. The CuZn-SOD comprises a positively charged channel that ends near the active site at the Cu ion. This channel conducts the substrate O F ' , which also explains the high rate for the dismutation reaction (2). Small molecules, such as cyanide and azide anions, may also move through this channel and subsequently bind to the metal ion and protect the CuZn-SOD against inactivation by H~02. In the presence of H20~, SOD acts as a prooxidant. H202 has a low affinity for SOD [46]. This enables a beneficial integration between catalase and peroxidases and between catalase and SOD, offering protection.
TABLE II GSH-Dependent En7ymes That Offer Protection Against Lipid Peroxidation GSH-DependentEnzymes
Substrate
Se-GSH peroxidase Se-phospholipidhydroperoxideGSH peroxidase CytosolicGSHtransferase MicrosomalGSHtransferase Freeradical reductase
H202/LOOH H2OjLOOH/PLOOH LOOH LOOH/PLOOH vit E,
GSH: glutathione; LOOH: lipid hydroperoxide; PLOOH: phospholipid hydroperoxide; vit E ." vitamin E radical (~-chromanoxyl radical).
Termination reactions, in which the peroxylsulpenyl radical (GSO0 • ) reacts with a second radical, form oxygen that can be in the singlet state [50]: GSO0. +GSO0.
> GSSG + 202
(20)
Hence, a generally accepted antioxidant, such as GSH, may possess pro-oxidant activity under certain conditions.
PHARMACOTHERAPEUTICIMPLICATIONS Thiols Thiol (SH) groups are essential in the protection against the deleterious effects of reactive oxygen species. The tripeptide GSH (~-Glu-Cys-Gly) is the pivot in various protective systems (Table II). In addition, the SH group is important for the function of many proteins. The Ca2+-ATPases, for example, contain an essential thiol group. Impairment of this thiol moiety leads to increased intracellular levels of Ca 2+ (Figure 5), which frequently precedes cellular necrosis. Hormone receptors involved in maintaining Ca 2+ homeostasis often contain a critical SH moiety [47]. To protect SH groups of proteins, high concentrations of the reducing GSH are necessary. It is difficult to estimate the level of GSH needed, since thiols may exhibit pro-oxidant and antioxidant actions [47]. In model systems, the pro-oxidant activity of, for example, the thiol GSH can be simply explained [48,49] as involving the reduction of Fe 2+. Radical-mediated oxidation of GSH in the presence of oxygen may form singlet oxygen [50]: GSH + R.
> GS- + R H
(18)
The thiyl radical (GS') may react with oxygen: GS' +02
> GSO0'
(19)
Note that the pro-oxidant activity of vitamin C, vitamin E, SOD, and thiols has often been established in simple in vitro systems. The relevant question of whether the pro-oxidant capacity is also expressed in a delicately integrated physiologic system remains unanswered. The biochemical interplay of the radical-protecting systems may obviate the pro-oxidant action of the separate parts. Antioxidant pharmacotherapy ideally encorepasses specific acting drugs. Targeting of antioxidant drugs to locations with undesirable excessive radical formation could impart specificity, thus preventing interaction with physiologically important radical-mediated processes. In the development of new therapeutic antioxidants, the question of how these compounds are incorporated in or commensurate with existing integrated physiologic radicaldefense systems should be addressed. Although novel, exciting therapeutic antioxidants have been designed (Figure 10), the above aspects have not been fully appreciated. In our opinion, close collabOration between the triad of molecular pathophysiologist (for an understanding of the molecular mechanism of the free radical diseases), food physiologist (explaining the normal delicate interplay of antioxidants in physiology), and the medicinal chemist (designer of new drugs) will eventually lead to the development of new, specific, and effective antioxidants.
September 30, 1991 The American Journal of Medicine Volume 91 (suppl 3C)
3C-119
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/ BASTET AL
(C113)3(" °
IIO
CH-"-Cil~ICI ~O~C211s
©
(cH3hc
/--N
ethyl 3,5-di-tert-butyl-4-hydroxycinnamate
COOII
/
CI13--~
" "
R. M. M. HAENEN, Ph.D., CEES J. A. DOELMAN, Ph.D., Amsterdam,The Netherlands
Reactive oxygen species are regarded as merely pernicious. This is incorrect for they play a pivotal role in many physiologic reactions, such as cytochrome P450-mediated oxidations, regulation of the tone of smooth muscle, and killing of microorganisms. An imbalance in oxidant-antioxidant activity is involved in many free radical-mediated pathologies, e.g., ischemia-reperfusion and asthma. In an attempt to alleviate these pathologies with antioxidants, it should be noted that these compounds are neither specific nor mere antioxidants. Associated with antioxidant activity is a pro-oxidant action. In the development of new antioxidant therapies, the important question of how these drugs are incorporated in or commensurate with existing integrated physiologic radical-defense systems should be addressed.
From the Department of Pharmacochemistry,Vrije Universiteit, Amsterdam, The Netherlands. Requestsfor reprints should be addressedto Prof. Dr. A. Bast, Departmentof Pharrnacochemistry,Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
36--2S
scussing free radicals in physiology, we fre-
Di~uently consider oxygen free radicals. In 1924 it was established that molecular oxygen (02) contains unpaired electrons• It should not be described as 0 = 0 , in which all electrons are paired, but as • 0 - - 0 .. Although 02 has a radical nature and can even be called a diradical (two unpaired electrons), it does not have extreme reactivity, due to quantum-mechanical restrictions. The electron structure causes 02 to form water by a stepwise (univalent) reduction with four electrons: 02 + 4H ÷ 4e-
> 2H20
(1)
Figure 1 shows the protonated and unprotonated intermediate forms occurring during reduction of oxygen, but singlet oxygen, 102, formed by the input of energy has not been shown. Several forms of singlet oxygen exist and most important biologically is the one in which all electrons are paired. Frequently, the term "oxygen radicals" is wrongly used, being assigned to all reactive intermediate oxygen species including those molecular forms that are not radicals. It is appropriate to speak of "reactive oxygen species" instead of "oxygen free radicals." Articles, books, and congress proceedings devoted to reactive oxygen species promulgate the view that these reactive oxygen species should be regarded as extremely reactive and therefore deleterious to the organism. Note that radical chemistry has played an essential role in the origin of aerobic life forms. Also obvious is that radical reactions are an integral part of the homeostasis in cellular processes. It appears that enzymes and factors that were able to restrain radical reactions were already present early in evolution. The organism was equipped with an ancient balance of oxidants and antioxidants in order to cope with free radical biochemistry. Oxidants and antioxidants have well-defined functions and reside in specific cellular compartments and a disturbance in the delicate oxidantantioxidant balance results in many pathophysiologic conditions.
REACTIVE OXYGEN SPECIES AND IRON The organism has many strategies to prevent the propagation of radical reactions. It is possible to
September 30, 1991 The American Journal of Medicine Volume91 (suppl 3C)
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/ BAST ET AL
.5-5.
0 2 ground state
+e
0 2 superoxide anion radical
I0-0. +e
0%" peroxide ion
I ~- ~ I
+ 2 H+~
H202
hydrogen peroxide
+ H+ ~ r
• OH
hydroxyl radical
+2H
H20
+e
m
O;
and
I0"
0 2-
m
and
I01
+
te 2 I01
2 02.
+4H+ --
2
H20
Figure 1, The univalentreductionof oxygen.
scavenge reactive oxygen directly; for example, the enzyme superoxide dismutase (SOD) catalyzes the conversion of 02 "" 202 • +2H + SOD ~ H20z + 02
(2)
The heme-containing enzyme catalase transforms HzO~ into water and oxygen: 2H202
catalase
) 2H20 + 02
(3)
Glutathione peroxidase (GSH-Px) also reduces peroxides. The selenium-dependent GSH-Px reduces H202 as well as organic hydroperoxides. The selenium independent GSH-Px accepts hydroperoxides as substrate: ROOH + 2GSH
GSH-Px )
ROH + H20 + GSSG
(4) In a physiologic environment, both initiation as well as propagation of radical reactions appear to be restrained to a large extent. An important reaction that should not disseminate is the transformation of
Hz02 into a hydroxyl radical (. OH) by traces of transition ions such as iron or copper• This Fenton reaction proceeds as follows: Fe 2+ + H20e
> Fe 3÷ + .OH + OH-
(5)
• OH is extremely reactive. Although it is still uncertain whether . OH or an iron-oxygen complex is the ultimate reactive initiating species, it is evident that safe storage of iron ions is crucial [1]. Two thirds of the 4 g of iron present in an adult is stored in hemoglobin. Ten percent of the iron is found in myoglobin and a small portion in ironcontaining enzymes and in the transport protein transferrin. The remainder is present in intracellular storage proteins such as ferritin and hemosiderin. The glycoprotein transferrin binds 2 tool of Fe 3+ per mole. Under normal conditions, transferrin is only 30% bound with iron. The free plasma concentration of iron is therefore almost nil. The cellular uptake of iron, which is needed, for example, for synthesis of iron-containing enzymes, probably occurs through pinocytosis of the transferrin-iron complex. In the cell, iron is immediately stored in ferritin. The storage capacity of this protein is
September30, 1991 The AmericanJournal of Medicine Volume91 {sup# 3C)
3C-3S
SYMPOSIUM ON OXIDANTS AND ANTIOXIDANTS/ BAST ET AL
Fe3+ ' (ROH)Fe 3+
RH (RH)Fe 3+
XOOH
XOH
(RH)Fe 3+ (XOOH)
(R)(FeOH)3+,~------~ (RH)(FeOH)3+(XO')
(R•H)(FeO)3+
~
~~
e
02
(RH)Fe3"~O2-)
(RH)Fe3+(O22")~ H20
2 H+
large, viz. 4,500 mol of iron per mole of protein. Binding of iron to ferritin prevents its participation in the Fenton reaction but its storage is not completely assured. At pH 6, iron dissociates from ferritin, and in the microenvironment of the phagocytes, a pH value uric acid + NADH + H ÷
(7)
Conversion of XD into XO occurs rapidly upon mild proteolysis. During ischemia, degradation of adenosine triphosphate (ATP) forms hypoxanthine and in this way XO is supplied with a substrate during the reperfusion process. Note that the XO hypothesis has been questioned [14]. Activated neutrophils may adhere to the slightly damaged vascular endothelial layer longer than in the control situation. The neutrophil-induced damage is in the first instance directed to the vascular endothelium. Excessive levels of catecholamines, released during ischemia/hypoxia, also serve as a source for
September30, 1991 The AmericanJournalof Medicine Volume91 (suppl3C)
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/BASTET AL
oxygen radicals, arising from oxidation of the catecholamines. It is suggested that the changes in the lipid bilayer underlie the conversion from reversible myocardial damage into irreversible lesions. The activation of phospholipases, the detergent-like actions of accumulated fatty acids and lysophospholipids, together with the activation of lipid peroxidation have been called the "lipid triad" [15]. During lipid peroxidation of ~oG and ~o3 fatty acids, biologically active fatty-acid breakdown products are formed. Among these products, the hydroxyalkenals (e.g., 4-hydroxy-2,3-transnonenal) are produced in large amounts. There is evidence that these aldehydes have a number of properties that explain the injurious effects of free radical pathology [16]. The aldehydes have been suggested to act as "toxic second messengers" for the primary free radicals [16]. During detoxication of lipid peroxidation products, such as lipid hydroperoxides, aldehydes, and H2Oe, glutathione (GSH) is consumed, and extensive GSH-consumption imperils the function of proteins that depend on a critical sulfhydryl moiety. Oxidation of these proteins may occur, resulting for instance in the dysfunction of Ca2+-ATPases [17]. Oxidative stress induces nicotinamide adenine dinucleotide (NAD) depletion. The mechanism responsible for the fall in intracellular NAD levels is probably poly(ADP-ribose) polymerase, which uses NAD as substrate. This enzyme is activated under conditions of DNA strand breakage, which may occur in cells exposed to oxidative stress [18]. In addition to dysfunction of Ca2+-ATPase, clustering of lipid hydroperoxides and formation of lysophospholipids may result in calcium overload (Figure 5). This has recently been underscored by reports [19] on heart mitochondrial pores activated by oxidative stress. An increase in intracellular Ca 2+ levels leads to activation of Ca2+-dependent enzymes such as proteases and phospholipase that lead to worsening of oxidative damage. It has also been found that ischemia in the heart causes a decline in the enzymatic defense against free radicals in heart tissue [20]. Strong evidence for the supposed invoivement of free radicals in postischemic reperfusion injury comes from spin-trapping experiments [21]. The production of 0~- - could be related to the severity of ischemia in this way. The metabolic effect of oxygen radicals in heart tissue has been recently reviewed [22]. Drug-induced cardiotoxicity can sometimes also be related to the formation of reactive oxygen species. Clinical use of the effective anticancer drug, doxorubicin, is limited by cardiomyopathy that develops after cumulative dosing [23]. Oxygen radicals play a role in doxorubicin-induced cardiotoxic-
ity [24] via redox cycling of the compound. Moreover, it has recently been shown that the semiquinone free radicals, formed upon one-electron reduction of doxorubicin, are effective in releasing iron ions from the iron storage protein ferritin [25]. Iron chelators prevent the doxorubicin-induced cardiomyopathy.
Lungs In lung pathology, conditions such as emphysema, bronchopulmonary dysplasia, pneumoconiosis, bleomycin toxicity, paraquat toxicity, butylhydroxytoluene toxicity, mineral dust toxicity, cigarette smoke toxicity, respiratory distress syndrome, and asthma have been related to oxygen radicals. Asthma can be regarded as the manifestation of an inflammatory reaction. Lungs from patients with severe asthma show a shedding or loss of epithelium, thickening of the basal membrane, subepithelial fibrosis, accumulation of inflammatory cells, widespread mucus plugging, submucosal edema, and smooth muscle hyperplasia. Moreover, the observed asthmatic lung hyperreactivity [26] to a specific stimuli, such as methacholine, histamine, cold air, or exercise, has been proposed to be a consequence of the inflammatory reaction [27]. Since the effect of reactive oxygen species closely mimics the pathology of asthma, their involvement in the etiology of the disease has been suggested. It is reported that inhalation of xanthine/XO in cats causes a hyperreactivity of the airways to acetylcholine in vivo [28]. Hyperreactivity of the rat tracheal smooth muscle has been observed after cumene hydroperoxide incubation or of guinea pig tracheal smooth muscle after hypochlorite incubation (unpublished data). Also, leukotriene D 4 (LTD4) exposure of guinea pig tracheal smooth muscle in vitro causes histamine hyperreactivity that can be blocked by SOD, showing the involvement of O~ • in the induction of hyperreactivity by LTD4 [29]. A fi-adrenoceptor dysfunction has been observed after exposure of airways tissue to reactive oxygen species in vitro. H202, for example, induced an autonomic imbalance between muscarinic (contracting) and fi-adrenoceptor (relaxing) responses in favor of the muscarinic receptor response in rat tracheal tissue in vitro [30] (Table I). Epithelial damage and mucus hypersecretion, important features of asthma, are caused by reacrive oxygen species. The eosinophil peroxideH2Oe-halide system destroys human nasal epithelial cells in vitro [31]. We observed that hypochlorite induces a methacholine hyperreactivity of guinea pig tracheal tissue in vitro, which is proba-
September30, 1991 The AmericanJournal of Medicine Volume9i (suppl3C)
3C-7S
SYMPOSIUMON OXIDANTSAND ANTIOXIDANTS/BASTET AL TABLE I Effect of H202 (10 -3 M) on the Methacholine and the (-)lsoprenaline Concentration-Response Curve in Rat Tracheal and Lung Parenchymal Tissue Control (-log ECso/Era~x%) Methachoiine Trachea Lungstrips Isoprenaline Trachea Lung strips
Pretreated (-tog ECso/Em,,%)
5.54 +- 0.06/100 5.03 -+ 0.04/100
5.40 + 0.09/39 _+6* 4.71 -+ 0.04"/86 _+9
7.85 +- 0.05/100 7.35 +- 0.08/100
Not detectable/O* 6.91 -+ 0.12"/39 + 14"
Note: The -log EC5ovalues (-+SEM) and maximal effects (-+SEM) of either methacholine or (-)isoprenaline Cumulative concentration-response curves before and after treatment af the rat tracheal and lung parenchymal strips with H~02 are shown (*p Enz-Cu e+ + Ira. +H20
(16)
Enz-Ou + + H202
(17)
Enz represents the Cu-containing SOD and ImH the imidazole moiety of histidine at the active site of the enzyme. The use of spin-trap experiments itrefutably proved the formation of • OH by SOD in combination with H202 [46]. The CuZn-SOD comprises a positively charged channel that ends near the active site at the Cu ion. This channel conducts the substrate O F ' , which also explains the high rate for the dismutation reaction (2). Small molecules, such as cyanide and azide anions, may also move through this channel and subsequently bind to the metal ion and protect the CuZn-SOD against inactivation by H~02. In the presence of H20~, SOD acts as a prooxidant. H202 has a low affinity for SOD [46]. This enables a beneficial integration between catalase and peroxidases and between catalase and SOD, offering protection.
TABLE II GSH-Dependent En7ymes That Offer Protection Against Lipid Peroxidation GSH-DependentEnzymes
Substrate
Se-GSH peroxidase Se-phospholipidhydroperoxideGSH peroxidase CytosolicGSHtransferase MicrosomalGSHtransferase Freeradical reductase
H202/LOOH H2OjLOOH/PLOOH LOOH LOOH/PLOOH vit E,
GSH: glutathione; LOOH: lipid hydroperoxide; PLOOH: phospholipid hydroperoxide; vit E ." vitamin E radical (~-chromanoxyl radical).
Termination reactions, in which the peroxylsulpenyl radical (GSO0 • ) reacts with a second radical, form oxygen that can be in the singlet state [50]: GSO0. +GSO0.
> GSSG + 202
(20)
Hence, a generally accepted antioxidant, such as GSH, may possess pro-oxidant activity under certain conditions.
PHARMACOTHERAPEUTICIMPLICATIONS Thiols Thiol (SH) groups are essential in the protection against the deleterious effects of reactive oxygen species. The tripeptide GSH (~-Glu-Cys-Gly) is the pivot in various protective systems (Table II). In addition, the SH group is important for the function of many proteins. The Ca2+-ATPases, for example, contain an essential thiol group. Impairment of this thiol moiety leads to increased intracellular levels of Ca 2+ (Figure 5), which frequently precedes cellular necrosis. Hormone receptors involved in maintaining Ca 2+ homeostasis often contain a critical SH moiety [47]. To protect SH groups of proteins, high concentrations of the reducing GSH are necessary. It is difficult to estimate the level of GSH needed, since thiols may exhibit pro-oxidant and antioxidant actions [47]. In model systems, the pro-oxidant activity of, for example, the thiol GSH can be simply explained [48,49] as involving the reduction of Fe 2+. Radical-mediated oxidation of GSH in the presence of oxygen may form singlet oxygen [50]: GSH + R.
> GS- + R H
(18)
The thiyl radical (GS') may react with oxygen: GS' +02
> GSO0'
(19)
Note that the pro-oxidant activity of vitamin C, vitamin E, SOD, and thiols has often been established in simple in vitro systems. The relevant question of whether the pro-oxidant capacity is also expressed in a delicately integrated physiologic system remains unanswered. The biochemical interplay of the radical-protecting systems may obviate the pro-oxidant action of the separate parts. Antioxidant pharmacotherapy ideally encorepasses specific acting drugs. Targeting of antioxidant drugs to locations with undesirable excessive radical formation could impart specificity, thus preventing interaction with physiologically important radical-mediated processes. In the development of new therapeutic antioxidants, the question of how these compounds are incorporated in or commensurate with existing integrated physiologic radicaldefense systems should be addressed. Although novel, exciting therapeutic antioxidants have been designed (Figure 10), the above aspects have not been fully appreciated. In our opinion, close collabOration between the triad of molecular pathophysiologist (for an understanding of the molecular mechanism of the free radical diseases), food physiologist (explaining the normal delicate interplay of antioxidants in physiology), and the medicinal chemist (designer of new drugs) will eventually lead to the development of new, specific, and effective antioxidants.
September 30, 1991 The American Journal of Medicine Volume 91 (suppl 3C)
3C-119
SYMPOSIUM ON OXIDANTSAND ANTIOXIDANTS/ BASTET AL
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