Archives of Insect Biochemistry and Physiology 29:lOl-118 (1 995) Toxicity of Oxygen From Naturally Occurring Redox-Active Pro-Oxidants Ronald S. Pardini Department of Biochemisty, Univevsity of Nevada, Reno, Nevada The survival of all aerobic life forms requires the ground-state of molecular oxygen, 02.However, the activation of O2 to reactive oxygen species (ROS) is responsible for universal toxicity. ROS are responsible in deleterious intracellular reactions associated with oxidative stress including membrane lipid peroxidation, and the oxidation of proteins and DNA. Redox-active allelochemicals such as quinones and phenolic compounds are involved in activating O2 to its deleterious forms including superoxide anion free radical, 0 2 * - , hydrogen peroxide, H 2 0 2 , and hydroxyl radical, 'OH. Molecular oxygen is also activated in biologically relevant photosensitizing reactions to the singlet form, ' 0 2 . The insect lifestyle exposes them to a broad diversity of pro-oxidant allelochemicals and, like mammalian species, they have developed an elaborate antioxidant system comprised of chemical antioxidants and a bank of antioxidant enzymes. We have found that an insect's antioxidant adaptation to a particular food correlates well with its risk of exposure to potential pro-oxidants. o 1995 WiIey-Liss, Inc. Key words: oxidative stress, reactive oxygen species, lipid peroxidation, protein oxidation, DNA oxidation, antioxidants, oxidant repair enzymes, redox-active flavonoids INTRODUCTION Ground-state molecular O2is an interesting molecule from both a chemical and a biochemical perspective. This paper is an overview of the basic chemistry and biochemistry of oxygen relative to its role in oxidative stress and its repair in biological systems. More detailed reviews on this topic have been recently published (Cadenas, 1995; Demple and Harrison, 1994; Dean, 1991; Cheeseman, 1993). Some of the by-products of the activated forms of O2 are deleterious and exert debilitating oxidative stress (Fridovich, 1983). The toxicity of O2which occurs in all aerobic organisms is called endogenous oxidafive stress. Furthermore, this stress is exacerbated by exogenous oxidative Acknowledgments: This is a contribution of the Natural Products Laboratory, and was supported in part by competitive USDA, NSF, and NlEHS grants. Received September 19, 1994; accepted November 30, 1994. Address reprint requests to R.S. Pardini, Department of Biochemistry/MS 330, University of Nevada, Reno, NV 89557-001 4. 0 1995 Wiley-Liss, Inc. 102 Pardini challenge exerted by dietary or other environmental pro-oxidant substances. The potential of endogenous and exogenous oxidative damage requires that all aerobic organisms possess antioxidant defense mechanisms. Also, I have presented some data on insects which demonstrate that the basic processes of 0, activation, and the ensuing toxicity, and protective mechanisms are very similar to the well-studied group of higher animals, the vertebrates. CHEMISTRY OF OXYGEN Ground-state molecular oxygen 0, has two unpaired e- in parallel spin which The paramagnetic makes it paramagnetic and imparts to it a triplet state (302)*. state is revealed in electron spin resonance spectroscopy (ESR; Green and Hill, 1984; Cadenas, 1989). The strong 0-0 bond coupled with the parallel spins of the unpaired e- make '0, chemically less active as an oxidant than expected. Thus, in aerobic reactions it is necessary to weaken the 0 to 0 bond and/or to remove the forbidden spin restriction. This is achieved either by 1e- reduction of Ob or via an energetic process such as photochemical or thermal activation. The first process results in the formation of superoxide anion radical (O,*-),and the second transformation produces the singlet form of O2(0 = 0, 'Ago2, or 'OJ, which not only weakens the primary 0-0 bond (more stable than the double bond), but also removes the spin restriction. In these activated forms, O2 becomes very reactive, and can readily participate in chemical/biological reactions. IMPLICATIONS OF OXYGEN ACTIVATION The 4 electron reduction of O2 to H20 gives aerobic organisms an enormous energetic advantage over anaerobic organisms, that is, 18 times greater energy is derived from the oxidative metabolism of glucose compared to anaerobic glycolysis. However, incomplete reduction of O2gives rise to oxidation states of ROS which possess deleterious reactivity with aerobic cell constituents. Therefore, the energetic advantage of the aerobic lifestyle is maintained at the expense of the evolution and elaboration of a highly sophisticated antioxidant defense system. The equations below exemplify this dilemma. 0, + e- + O,*02*+ H' -+ HO,' (hydroperoxyl radical) H02' + e- + H' + H202 H202+ e- + -OH + 'OH 'OH + e- + H20 (eq. 1) (eq. 2) (eq. 3) (ey. 4) (eq. 4 4 *Abbreviations used: A, dehydroascorbate; A', ascorbyl radical; AFRR, ascorbyl free radical reductase; AH2, ascorbate; a-T-O', a-tocopheroxyl radical; a-T-OH, a-tocopherol; CAT, catalase; ESR, electron spin resonance spectroscopy; GPOX, selenium-dependent glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; CSSC, oxidized glutathione; GST, glutathione S-transferase; GSTPX, selenium-independent glutathione peroxidase; HNE, 4-hydroxynonenal; H202, hydrogen peroxide; HQ2*,hydroperoxyl radical; L', lipid radical; LOO', lipid pcroxyl radical; LOOH, lipid hydroperoxide; NO, nitric oxide; " 0 2 , nitrogen dioxide; 0 2 * - ( superoxide anion radical; 'OH, hydroxyl radical; PH-GPOX, phospholipid glutathione peroxidase; PUFA, polyunsaturated fatty acid; R', organic radical; ROS, reactive oxygen species; SOD, superoxide dismutase; SQ', semiquinone radical; U-, urate; '02,singlet oxygen; 302, triplet state molecular oxygen. Oxidative Stress 103 Superoxide anion radical exists in equilibrium with HOz' with a pK, of 4.8. The above equations represent the sequential 4 e- reduction of oxygen which terminates with the formation of water. Both hydroxyl radical ('OH) and lo2 are the most toxic forms of ROS, however, numerous other ROS are also known. The relevant ones are summarized in Table 1. BIOLOGICAL SOURCES FOR THE PRODUCTION OF ROS Some known sources of 02*production are listed in Table 2. Redoxactive chemicals such as catechols can be converted from their fully reduced form to the semiquinone radical (SQ') intermediate form via 1 eoxidation or by enzymatic processes (e.g., flavoproteins), and then to the fully oxidized quinone form. This process is reversible and is known as the redox cycling path (Powis, 1987). However, the reactive SQ' intermediate may react with 02,transferring its unpaired e- which in turn generIn addition there are various mechanisms by which the ates the 02*-. A classical example of this is the enzymes listed in Table 2 generate 02*-. reaction catalyzed by xanthine oxidase (eq. 5 ) . Xanthine + H20 + O2+ Urate + 02*- (eq. 5 ) The generation of hydrogen peroxide (H202)is from either the HO,' radical as shown in eq. 3, or from direct dismutation of 02*(eq. 6). In addition, H202may also be generated by a direct 2 e- reduction of ground state oxygen catalyzed by many peroxisomal flavin enzymes such as glucose oxidase, urate oxidase, and D-amino oxidase as shown (eq. 7 ) . a-D-amino oxidase + H20+ O2+ a-0x0 acid + NH3 + H202 (eq. 7 ) The *OHradical may be generated by transition metals such as Fe or Cu in the metal catalyzed Haber-Weiss reaction, better known as the Fenton reaction (Halliwell and Gutteridge, 1990b; eqs. 8-10). TABLE 1. Radical and Non-Radical Reactive Oxygen Species (ROS) Radical species Alkoxyl Hydroperoxyl Hydroxyl Nitrogen dioxide Peroxyl Superoxide Nitric oxide Non-radical species RO'/LO'" HOz' 'OH NOz' ROO'/LOO.a Oz.'NO Hydrogen peroxide Hypochlorous acid Ozone Peroxynitrite Singlet oxygen "R = any organic molecule, whereas L denotes an unsaturated lipid molecule. HZOZ HOCl 0 3 ONOO'02 104 Pardini TABLE 2. Biological Sources for the Ox*-Production Autoxidation of redox-active chemicals Enzymatic sources Catecholamines Hemoglobin (slowly liberates O;-) Leukoflavins Myoglobin (slowly liberates O,'-) Reduced ferredoxin Tetrahydropterins Thiols Aldehyde oxidase Cytochrome P-450 reductase Dihydroorotic dehydrogenase Galactose oxidase Indoleamine dioxygenase Mitochrondrial respiratory chain NADH dehydrogenase NADPH-oxidase (phagocytes) Ubiquinone-cytchrome C reductase Xanthine dehydrogenase/oxidase 9'-+ Fe3+-+Fe2++ O2 H202+ Fez++ Fe3++ 'OH + -OH Overall Fenton Reaction: 02'-+ H,O, + 'OH + -OH (eq. 8) (eq. 9) (eq. 10) It is noteworthy that the reaction depicted in eq. 8 is slow (k = 1 x 106M-ls-l). Ascorbate and glutathione (GSH) occur at higher concentration than 02*-, and these reductants, as well as exogenous flavonoids, can speed up the production of *OHradical by reducing Fe3+to Fez+,which can directly split H202 to the products shown in eq. 9. Another mechanism postulated for the simultaneous production of both 'OH and nitrogen dioxide radical (NO,') is from a concentration and pH dependent interaction between 0,'- and nitric oxide ('NO) radicals (Radi et al., 1991): 02*+ *NO+ ONOO(eq. 11) ONOO- + H++ ONOOH (eq. 12) ONOOH + NO2' + 'OH (eq. 13) The first reaction proceeds fast at a rate of 3.4 x 107M-'s-', resulting in the strong oxidant peroxynitrite, which undergoes protonation to form peroxynitrous acid (eq. 12). The acid is unstable with a tlh > 1 s, and its monolytic scission generates the radicals shown in eq. 13. It should be noted that this mode of *OHradical production is a relatively recent discovery in that for nearly four decades, the Fenton reaction was considered the primary source of 'OH. Further research is needed here to clarify to what extent the two mechanisms are involved in the production of 'OH. DELETERIOUS REACTIONS OF ROS Lipid Peroxidation Lipid peroxidation is considered the most studied deleterious process that accounts for the majority of the oxidative stress mediated pathologies (Mannervik, 1985). Polyunsaturated lipids such as polyunsaturated fatty acids (PUFAs) are among the most oxidant-labile components in biological systems. The process has also been extensively studied because of the food Oxidative Stress 105 industries' interest in oxidative rancidity of foods, especially oils and fats (Gutteridge and Halliwell, 1990). Lipid peroxidation is a chain reaction and the *OH radical is very potent in abstracting an H atom from an organic molecule. The methylene-carbon is particularly susceptible for H abstraction (Horton and Fairhurst, 1987). The reaction produces a lipid or organic radical (L' or Re), thus lipid peroxidation is initiated via H abstraction followed by radical reaction with O2as shown below (eqs. 14-16). LH + 'OH + L' + H20 L' + 0' + LOO' LOO' + LH + LOOH + L' Once the lipid peroxyl radical (LOO') is formed, it reacts with another lipid molecule to form a lipid hydroperoxide (LOOH) and another L' radical which can continue to propagate this chain reaction. This chain reaction "may be thousands of events long" (McCord, 1985), causing destruction of many PUFAs unless chain-breaking antioxidants intercept this process. Lipid peroxidation may also be initiated by the attack of other radicals, and by '0' which forms hydroperoxides, endoperoxides, and dioxetans essentially by direct insertion of lo2into an unsaturated carbon skeleton (Singh, 1989).Generally, all peroxides are unstable, for example an endoperoxide may rearrange to an LOOH, or break down to chain propagating LOO' radical. Figure 1 illustrates how PUFAs such as linoleic acid are peroxidized and then decomposed into highly reactive products. An early event in the peroxidation injury is the formation of fatty acid acyl hydroperoxides during the peroxidation of membrane phospholipids. This leads to the stimulation of phospholipases Al and A' and phospholipase C activities. The removal of fatty acyl hydroperoxide by phospholipase leads to the formation of fatty acyl hydroperoxide and lysophospholipid. Lysophospholipid is a detergent and its formation in the membrane can be destructive unless it is reacylated from a pool of fatty acyl CoA's via acyl transferase activity or degradated by lysophospholipid lipase to glycerol phosphate and free fatty acid. The overall activity of phospholipase is beneficial as it releases fatty acid hydroperoxides from the membrane phospholipids which are subsequently detoxified by antioxidant enzymes and converted to innocuous products. If not removed, the phospholipid hydroperoxides break down into cytotoxic carbonyl products which are listed in Table 3. Of these, malondialdehyde is well known for its deleterious cross-linking properties, while 4-hydroxynonenal (HNE) is toxic via its reactivity with thi01s (GSH and protein-SH). This reactivity is responsible for the inhibition of many enzymes involved in DNA and RNA synthesis, protein synthesis, mitochondrial respiration, glycolysis, and the ER ca'' pump (Comporti, 1985; Witz, 1989; Esterbauer et al., 1982; Benedetti and Comporti, 1987). Consequently, it inhibits cell division, causes mutagenesis, and possesses chemotactic properties. The damages to peroxidized membranes are numerous and are as follows: (1) loss in membrane fluidity; (2) loss in selective permeability, and under extreme damage, the membrane integrity is lost; (3) water soluble oxidant-in- 106 Pardini Linoleate Peroxidation H I # 0 i" o k " nK/Lipid Radical Fig. 1 . Linoleate peroxidation. duced degradative products, such as aldehydes, diffuse from membranes into other subcellular compartments; (4) dialdehyde degradation products of LOOHs may cross-link proteins, causing protein aggregation that forms lipofuscin, i.e., aging pigments; (5) oxidized lipid products inhibit many enzyme functions and may react with DNA to form DNA adducts. The cumulative effects of lipid peroxidation are associated with pathological conditions that include atherosclerosis, hemolytic anemia, ischemia/reperfusion injuries, cancer, and aging (Cerutti, 1985; Harmon, 1956; Gebicki, 1991; Freeman, 1991; McCord and Omar, 1991). Oxidative Modification of Proteins Proteins are vulnerable to oxidative damage (Levine et al., 1981; Davies, 1986, 1987; Dean, 1991). Oxidative damage to proteins may occur by two TABLE 3. Aldehyde Products of Membrane Lipid Peroxidation 4-Hydroxy alkenals 2-Alkanals N-Alkanals 4-OH Hexenal 4-OH Nonenal 4-OH 25-nona-dienal Acrolein Pentanal Hexenal Octenal Nonenal Propanal Butanal Pentanol Hexanal Nonanal Other Malondialdehyde Butanone 2,4-decadienal Oxidative Stress 107 separate mechanisms as follows; (1) interactions between lipid peroxidation products and proteins as described above; and (2) the site-specific reactions of HzOzand Fez+at a metal binding site on the enzyme that leads to the production of ROS ('OH, ferryl, perferryl; Stadtman, 1990, 1991). According to Stadtman (1990,1991), 'OH, and iron as ferryl and perferryl species, present at the catalytic center of enzymes may also oxidatively damage proteins as follows. The e N H 2 of a lysyl residue serves as one of the several ligands for Fez+binding. The protein-Fez+complex then reacts with H202to form protein-Fe2*complex, -OH and the *OHradical. The 'OH then rapidly abstracts an H atom from the methylene carbon adjacent to the amino group of the lysyl residue forming a carbon based radical on the methylene carbon. The transfer of an e- of this carbon-centered radical to Fe3+produces an Fez+imino group complex, which upon spontaneous hydrolysis produces a protein aldehyde, NH3 and Fez'. Thus, many proteins are oxidized in this manner (Table 41, which renders them catalytically inactive and susceptible to neutral proteinase degradation. The oxidative modification of numerous enzymes leads to loss of their catalytic activity. Some known ones are listed in Table 5. Oxidative Damage to DNA, RNA, and Repair of Oxidatively Damaged DNA Initially the damage to DNA is analogous to lipid peroxidation in that DNA bases such as thymine are readily peroxidized to thymine-OOH. DNA and RNA, including mRNA and tRNA, are all susceptible to oxidative damage, and the damage has been amply demonstrated in vivo (Fridovich, 1978; Adelman et al., 1988; Kasai et al., 1986; Simic et al., 1989; Richter et al., 1988; Richter, 1988). They include: single strand breaks, double strand breaks, sister chromatid exchange, DNA-DNA cross-links, DNA-protein cross-links, base modifications (saturation, ring opening, ring contraction, and ring hydroxylation), and damage to sugar and phosphate backbone which may also lead to strand breaks (Simic et al., 1989; Teebor et al., 1988). Oxidative damage to DNA may disrupt DNA replication, transcription and translation causing mutations, senescence, and cellular death (Harmon, 1956; Schraufstatter et al., 1988; Ames, 1989; Ames et al., 1991; Harmon, 1981; Simic et al., 1989; Spector et al., 1989). Under normal physiological conditions, it has been estimated that in nuclear DNA one base out of 224,000 bases is oxidatively modified, while in the mitochondria1 DNA one out of 8,000 bases is modified (Ames et al., 1991). TABLE 4. Oxidative Modification of Amino Acid Residues of Proteins Amino acid residues Lys, Arg, Pro Histidyl Arginyl, Prolyl Prolyl LYSYl Methionyl Cysteinyl Modified residues Carbonyl Asparaginyl Glutamylsemialdehyde Glutamyl, pyroglutamyl 2-amino adipylsemialdehyde Methionine sulfoxamide Disulfide derivative Mixed disulfides Protein to protein crosslinks 108 Pardini TABLE 5. Some Enzymes That Are Oxidatively Inactivated* Acetyl CoA hydrolase Acetylcholine esterase Albumin Alkaline phosphatase Alcohol dehydrogenase Carbamoly-P synthetase Catalase Creatine kinase Enolase Fructose diphosphatase Glucose-6-I’ dehydrogenase Glutamine dehydrogenase Glutamine synthetase Glyceraldehyde dehydrogenase Hexokinase Lactic hydrogenase Lactoperoxidase Leucyl-t-RNA synthetase Lysozyme Ornithine decarboxylase 6-phosphogluconate dehydrogenase Phosphoglucomutase a-1-Proteinase inhibitor Phosphogluclomutase Pyruvate dehydrogenase Ribnuclease A and B Superoxide dismutase Tyrosyl-t-RNA-synthetase *Data from Floyd and Carney (1993). During oxidative stress, enzymes involved in the repair of oxidatively damaged DNA are increased in activity (Demple and Harrison, 1994). In general, these repair activities are categorized as either excision repair (Spector et al., 1989; Teebor et al., 1988; Wallace, 1988; Doetsch et al., 1986) or direct repair processes (Ketterer and Meyer, 1989; Teebor et al., 1988). DNA glycosylases, endonucleases, and exonucleases have been reported to preferentially cleave oxidatively modified bases from damaged DNA. Following this, DNA polymerase replaces the excised bases and DNA ligases seal new 3’ and 5’ ends (Teebor et al., 1988; Lindahl, 1987). Modified bases such as thymine glycol and 8-hydroxy guanosine can be measured in urine and have been used to monitor oxidative damage to DNA in vivo. The direct repair of oxidatively damaged bases by glutathione peroxidase, glutathione S-transferase, and DNA methylase have been reported. Oxidative damage to DNA, especially the mitochondria1 DNA, is implicated in cancer and aging (Ames, 1989; Ames et al., 1991). ANTIOXIDANT AND REPAIR MECHANISMS Antioxidant Molecules Non-protein antioxidant molecules are important in inhibiting oxy radical chain initiation and for breaking chain propagation. The lipophilic antioxidants are tocopherols like a-tocopherol (a-T-OH), tocotrienols, p-carotene, bilirubin, and lycopene. The hydrophilic antioxidants include ascorbic acid (AH,), GSH, plasma proteins, thiols, and urate. The literature is vast, therefore, a few examples are given in detail to demonstrate how these molecules act as antioxidants. Alpha-tocopherol is a good scavenger of many free radicals, especially the peroxyl radicals (eq. 17) and this property is crucial in preventing chain initiation and breaking the chain propagation steps of lipid peroxidation. Should the radical scavenging antioxidants fail, then the hydroperoxides formed may be reduced by peroxidase activity (Cadenas, 1989). Oxidative Stress ROO' /LOO' + a-T-OH + ROOH/LOOH + a-T-0' 109 (eq. 17) However, this antioxidant function could deplete the supply of a-T-OH, which turns into a free radical species, the a-tocopheroxyl radical (a-T-0'). Alphatocopherol is regenerated by AH2(eq. 18).This reaction occurs at the expense of AH2which is converted into the ascorbyl radical (A') as depicted in eq. 18. a-T-0' + AH2 + a-T-OH + A' (eq. 18) In plants and some animals such as insects, the enzyme ascorbyl free radical reductase (AFRR) regenerates AH2from A' (Felton and Duffy, 1992).An AFRRlike activity has been reported from some mammalian species such as the rat (colon and renal tissues), but indications are that the enzyme is not homologous to the plant enzyme and may be GSH rather than NADPH dependent (Choi and Rose, 1989; Rose, 1989). Carotenoids are excellent quenchers of excited state *02 and other singlet and triplet excited state pro-oxidants such as the naturally occurring psoralens, e.g., 8-methoxypsoralen (Ahmad and Pardini, 1990b).Although 0-carotene is the most ubiquitous and cited quencher (quenching constant 13-30 k, (109M-'s-'), plasma lycopene is an even more efficient quencher of excited-state oxidants (31 k,; Di Mascio et al., 1989). Moreover, unpublished data from our laboratory has provided evidence that lutein (or hydroxy p-carotene which is the sole carotenoid of vertebrate retina), may have this role in many insect species despite its lower quenching constant of 8-21 k,. This conclusion is based on tissue analysis of larvae of three insect species, the black swallowtail butterfly, Papilio polyxenes (an adept feeder of plants containing high levels of 8-methoxypsoralen), southern armyworm moth, Spodopfera eridania (a generalist feeder with potential of encountering photoactive dietary pro-oxidants), and cabbage looper moth, Trichoplusia ni (behaviorally avoids ingestion of photoactive pro-oxidants) (Ahmad, 1992; Ahmad and Pardini, 1990a; Ahmad et al., unpublished data, 1993). Ascorbate is an outstanding antioxidant of oxy radicals as well as H202as shown (eq. 191, where A represents dehydroascorbate. H202 + AH2 + 2 H2O + A (eq. 19) A GSH-dependent enzyme dehydroascorbate reductase is well known from plants and insects which reduces A back to AH2 (Dalton et al., 1986; Summers and Felton, 1993). Another example of a good soluble antioxidant is the urate (U, Hochstein et al., 1984). Urate can not only scavenge oxy radicals, it is an important stabilizer of AH2by chelating free or protein-bound iron. In mammalian species nitrogenous waste is mainly excreted as urea, with low levels of U-, while in most terrestrial insect species the predominant form of excretion (80+%) is U- and other purines and only trace amounts of urea. Clearly then, urate may be a more significant antioxidant in insects and uricolytic birds, although this aspect has not received much attention. 110 Pardini Antioxidant Enzymes In addition to antioxidant molecules, antioxidant enzymes are important preventive antioxidants which act to reduce the formation of free radicals and ROS and increase cell survival (Michiels et al., 1994). The enzymes involved are superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidases (GPOXs), selenium-independent peroxidase activity of glutathione S-transferase isoenzymes (GSTPX),and glutathione (GSSG) reductase (GR). The reactions catalyzed by these enzymes are as follows: SOD: 0 2 . - + 02'- + 2Hz -+ HZ02 + 0 2 CAT: H202 + HZ02 + H20 + 0 2 GPOX: (1)H202 + GSH -+ 2 HzO + GSSG (2) LOOH + 2 GSH + HzO + LOH + GSSG GSTPX: LOOH + 2 GSH -+ H,O + LOH + GSSG GR: GSSG + NAD(P)H + H' + 2 GSH + NAD'(P) (eq. 20) (eq. 21) (eq. 22) (eq. 23) (eq. 24) (eq. 25) SOD occurs in all aerobic organisms, and in eukaryotes it is present in cytosol and mitochondira. In mammalian species, the cytosolic form is a CuZn enzyme, while the mitochondrial form is an Mn enzyme. In prokaryotes and plants Fe-SOD is present. SOD catalyzed dismutation of 02*(eq. 20) occurs at rates near diffusion-control limit, which is lo4 times faster than spontaneous dismutation (Fridovich, 1983). CAT is a tetrameric hemoprotein and each subunit contains a protoheme IX group. This enzyme primarily occurs in peroxisomes, where H202production is high. Since H202can easily cross from cell membranes and cytosol into peroxisomes, its peroxisomal location is therefore strategic (Chance et al., 1979).CAT reduces 2 HZ02as per eq. 21 formed from the dismutation of 02*radicals by SOD, and H202formed in peroxisomes by a direct 2 e- reduction of 0, as exemplified in eq. 7. GPOX is a primarily mitochondrial and cytosolic selenoprotein. The cytosolic dimeric protein possesses selenocysteine at its active site and decomposes H202and hydroperoxides of PUFAs at nearly the same rate (eqs. 22-23). It is a physiologically important detoxification system due to the low K, for H202and other peroxides. In order for the GPOX to attack ROOH/LOOH, these products must be cleaved off from the membranes. Phospholipase C is activated during oxidative stress and cleaves hydroperoxides from membranes to facilitate the GPOX activity which reduces them to innocuous alcohols, and thus acts to decompose hydroperoxides and prevent their degradation into toxic products such as a propagators of the lipid peroxidation chain reaction. Recently, a monomeric specific GPOX has been characterized and named phospholipid GPOX (PH-GPOX). There is much interest in this enzyme since it attacks membrane LOOHs directly without the need for cleavage from membranes. PH-GPOX is specific for phospholipid hydroperoxides (Weitzel et al., 1990). GSTPX activity of dimeric multifunctional glutathione S-transferases is also important in the destruction of ROOHs/LOOHs but not Hz02(eq. 24) in mammalian microsomes, nuclei, and other subcellular compartments. The enzyme Oxidative Stress 11 1 is considered crucial in invertebrates, since selenium-GPOXs that are characteristic of vertebrates are not present (Ahmad and Pardini, 1988, 1989; Simmons et al., 1989; Weinold et al., 1990). The flavoprotein enzyme, GR, recycles GSSG, formed by GPOX, GSTPX, and other oxidizing mechanisms, back to GSH (eq. 251, using NAD(P)H as reductant (Racker, 1955; Zeigler, 1985). Finally, NAD(P)H levels are restored in cells by several systems which reduce NAD+(P)to NAD(P)H, e.g., the glucose-&phosphate and glucosed-phosphate dehydrogenase system of the hexose-phosphate pathway. Antioxidant Proteases Intracellular neutral proteases are multicatalytic enzymes that are not well characterized. The ongoing research suggests that these enzymes recognize oxidized proteins, and preferentially degrade the oxidized proteins (Davies, 1986; Stadtman, 1992). There is considerable debate regarding this intracellular proteolytic antioxidant system. The terminologies such as simultaneous usage of ”neutral and alkaline proteases,” “macroperoxyproteinase,” specific vs. non-specific enzymes, and specific vesicles for storage of these proteases are areas that warrant clarification. Antioxidant Proteins Several proteins act as antioxidants based on their property as transition metal storage, transport, or scavenging. Among these are albumin, ceruplasmin, metallothioneins, and ferritins or ferrins, including apoferritin, phytoferritin, transferrin, and lactoferrin. Albumin is a Cu scavenger (Halliwell and Gutteridge, 1990a), while ceruplasmin with its ferrioxidase activity converts Fez+to Fe3+for storage in ferritins (Samokyszyn et al., 1989). Metallothioneins scavenge a wide variety of metals and metalloids (Halliwell and Gutteridge, 1990b) and possess antioxidant activity (Sato and Bremner, 1993).The ferritins are iron storage proteins, whereas various ferrins are iron transport proteins (Law et al., 1992; Guttcridge and Quinlan, 1993). By sequestering metals that have the potential to derive the Fenton reaction, these proteins prevent the formation of ‘OH radical, and thereby attenuate lipid peroxidation. INSECTS AND ANTIOXIDANT SYSTEMS We have examined the antioxidant enzymatic defense systems of three insect species including larvae of T. ni (cabbage looper), s. eridania (southern army worm), and P. pdyxenes (black swallowtail butterfly; for review see Ahmad and Pardini, 1990b). In general, the endogenous activities of SOD, CAT, GST, and its peroxidase activity, GSTPX, correlate well with the natural feeding habits of the insect larvae relative to their normal dietary exposure to pro-oxidant allelochemicals. Unique observations in an insect model include the high endogenous and broad subcellular activity of CAT, and the lack of GPOX activity. The activity of GSTPX is unusually high in our insect model, suggesting that it replaces the function of mammalian GPOX activity in insects. In spite of the differences noted above, insects possess an antioxi- 112 Pardini dant enzyme armament that is strategically similar to the antioxidant enzyme systems described in vertebrates. Properties of Redox-Active Flavonoids and Impact on Insect Antioxidant Systems Figure 2 shows the structure of a ubiquitous flavonoid, quercetin (3,5,7,3',4'pentahydroxyflavone). Our studies have demonstrated that this redox-active flavonol induces oxidative stress (Hodnick et al., 1986,1988,1989,1994;Elliot et al., 1992), and the bases for this conclusion are as follows: (1) the presence of a catechol moiety; (2) its redox potential which is -30 to +60 (vs. SCE; Hodnick et al., 1988); (3) it autoxidizes to produce flavonoid free radicals H2O2and *OH and reacts with mitochondria to produce the ROS, 02*-, (Hodnick et al., 1986,1989,1994; Canada et al., 1990); (4) it inhibits the mitochondrial respiration in complex I (NADH-CoQ reductase; Hodnick et al., 1987) and complex I1 (succinate-CoQ reductase; Hodnick et al., 1986); and (5) it inhibits glutathione reductase in vitro (Elliot et al., 1992)and in vivo in T. ni, S. eridania, and I? polyxenes (Pritsoset al., 1988,1990;Ahmad and Pardini, 1990a). Our studies involving a three-species insect model whose relative sensitivity to quercetin is in the order T. ni > S. eridania > P. polyxenes, have demonstrated that quercetin's toxicity is via oxidative stress. The basal levels of the antioxidant enzymes of these species, and the degree of enzyme (especially SOD) induction are congruent with their relative sensitivity (Ahmad and Pardini, 1990a,b). Feeding quercetin increased the activity of SOD in T. ni and S. eridania larvae (Ahmad and Pardini, 1990a; Pritsos et al., 1990). In addition, feeding diethyl dithiocarbamate (DETC), a known inhibitor of CuZnSOD, renders P. polyxenes susceptible to quercetin while inhibiting its SOD activity (Pritsos et al., 1991). These findings demonstrate that the primary response of mid fifth-instar larvae of these two insect species to sub-lethal doses of quercetin is increased activity of SOD and the essential role of SOD is as a protectant from pro-oxidant allelochemicals. Despite this, our unpublished data show that lipid peroxidation is barely and non-significantly higher in quercetin-fed vs. control insects (Ahmad and Pardini, unpublished data). P" OH Fig. 2 . 2-; Structure of redox active flavonoid, quercetin Oxidative Stress 113 Protection by tocopherols, tocotrienols, ascorbate, carotrnoids urate, bilirubin. albumin. thiols SOD, Catdldbe. G m X GST. GSTPX, DT-Didphorase Lipids Proteins DNA Carbohydrates 4 \ '\ Biosynthesis Toxic Aldehydes Protein Carbonyl Protein Crosslinks DNA-DNA Crosslinks Protein-DNA Crosslinks Oxidation of Bases Strmdbreaks L : Cytotoxicity I ' Degradation & Damage Lipid Acylation Protein Svnlhesis Synthesis \ DNP DNA Pn1 Polymerase1 LIgdbt: Ligase \--/' Removal phospholipase A2 & C macroxyproteinase(MOP) endolexo-nucleases DNA glycosylases Excretion of Irreversibly Damaged Products Fig. 3. Oxidative challenge, toxicity, and repair. Quercetin is also known for its antioxidant properties and it can complex with metals (Pratt, 1993; Hanasaki et al., 1994). According to Borg and Schaich (19881, lipid peroxidation is in part propagated by metal-catalyzed breakdown of lipid peroxides to chain propagating LOO', LO' radicals. It is conceivable that the failure to demonstrate the accumulation of lipid peroxidation product such as malondialdehyde during quercetin exposure is because quercetin may also attenuate lipid peroxidation. Nonetheless, these studies have demonstrated the potential of quercetin to act as a pro-oxidant as well as an antioxidant. This scenario requires further investigation. CONCLUSION In conclusion, it is evident that the aerobic lifestyle represents a delicate biochemical balance between intracellular oxidative challenge and the production of ROS and a complex antioxidant protection and repair system, as summarized in Figure 3. 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