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Toxicity of oxygen from naturally occurring redox-active pro-oxidants.

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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. Although much of the biochemistry of O2activation
to ROS has been studied in vertebrates, especially mammals, it is clear that
invertebrates such as insects resemble all other aerobic organisms in their
response to ROS.
114
Pardini
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