Brazilian Journal of Medical

Animal adaptations and Biological

for oxidative stress Research (1996) 29: 1715-1733 1715
ISSN 0100-879X

Oxidative stress: animal

adaptations in nature
K.B. Storey Institute of Biochemistry and Department of Biology,
Carleton University, Ottawa, Ontario, Canada K1S 5B6

Abstract

Correspondence
K.B. Storey
Institute of Biochemistry and
Department of Biology
Carleton University
Ottawa, Ontario
Canada K1S 5B6
Fax: (613) 520-4389
E-mail: kbstorey@ccs.carleton.ca
Presented at the II Workshop in
Comparative Animal Physiology,
Serra Negra, SP, Brasil,
August 21-23, 1995.
Research supported by the National
Institute of General Medical
Sciences, USA (No. GM43796).
Received July 8, 1996
Accepted August 1, 1996
As a consequence of aerobic life, an organism must deal with the Key words
-
continuous generation of reactive oxygen species (O2 , H2O2, •OH) as • Reactive oxygen species
byproducts of metabolism and defend itself against the harm that these • Free radical damage
• Lipid peroxidation
can do to cellular macromolecules. Organisms protect themselves
• Anoxia tolerance
from such damage with both enzymatic and nonenzymatic antioxidant
• Freeze tolerance
defenses. However, the reperfusion injuries noted after ischemic
• Estivation
insult in mammalian organs and ascribed to a burst of reactive oxygen
• Ischemia
species produced when oxygenated blood is reintroduced demonstrate
that the antioxidant defenses of many organisms can be overwhelmed.
Although unusual among most mammals, many organisms routinely
experience wide variation in oxygen availability to their tissues due to
factors such as environmental oxygen lack, breath-hold diving, extra-
cellular freezing, or apnoeic breathing patterns in arrested metabolic
states. In recent studies using various animal models (anoxia-tolerant
turtles, freeze-tolerant snakes and frogs, estivating snails) our labora-
tory has explored the adaptations of antioxidant defenses that allow
such organisms to deal with rapid changes in tissue oxygenation with
little or no accumulation of damage products. The key to successful
transitions in several systems is the induction, during the oxygen-
limited state, of elevated activities of antioxidant and associated
enzymes, such as catalase, superoxide dismutase, glutathione-S-trans-
ferase, and glutathione peroxidase, so that damage during the reintro-
duction of oxygen (such as lipid peroxidation) is minimized. How-
ever, animals that are excellent facultative anaerobes, such as freshwa-
ter turtles, appear to deal with the potential of oxidative stress during
the anoxic-aerobic transition by maintaining constitutively high anti-
oxidant defenses (e.g. enzyme activities similar to those of mammals
and much higher than those of anoxia-intolerant lower vertebrates)
that can readily accommodate the burst of reactive oxygen species
generation when breathing is renewed.

Introduction one that few organisms can live without in-

Oxygen is indispensable to the lives of
most organisms on earth, with the mitochon-
drial ATP production that is linked to the
reduction of O2 to H2O being the primary
energy-producing pathway of the cell and
definitely. Although more than 90% of the
O2 taken up by the human body is used by
mitochondrial cytochrome oxidase which
adds four electrons onto each O2 molecule to
form water (O2 + 4H+ + 4e- → 2H2O) (1),
oxygen is also used as a substrate by numer-

Braz J Med Biol Res 29(12) 1996

1716 K.B. Storey

ous other enzymes. For example, Jones (2) In vivo much of the hydroxyl radical
listed 30 enzymes other than cytochrome production comes from the reduction of H2O2
oxidase that use oxygen in mammalian kid- by superoxide (the Haber-Weiss reaction)
ney, each with differing substrate affinities which is, in fact, a two-step process cata-
for oxygen and involved in a wide variety of lyzed by transition metals (Fe3+, sometimes
metabolic processes including the metabo- Cu3+) and involving the Fenton reaction.
lism of biological amines, prostaglandins,
Fe3+ + O2 → Fe 2+ + O 2
-
purines, steroids, amino acids, and carnitine.
Fe2+ + H 2O2 → Fe3+ + OH + •OH
-
Fenton reaction
Many of these reactions generate reactive
- -
oxygen species (ROS) as their products in- O2 + H2O2 → O2 + OH + •OH Haber-Weiss reaction
-
cluding superoxide (O2 ) and hydrogen per-
Numerous studies have shown that toxic-
oxide (H2O2). In addition, ROS are derived
ity of superoxide and hydrogen peroxide is
from other cellular activities including the
highly dependent on the presence of iron or
autooxidation of various small molecules
copper and that the nature and extent of
(e.g. flavins, catecholamines, hydroqui-
damage initiated by these species is related
nones), the microsomal cytochromes P450
to the subcellular location of these metals
and b5, microsomal flavoprotein reductases,
(1). All cellular components are susceptible
and superoxide leakage from the electron
to attack by ROS, particularly by •OH. At-
transport chain (3,4). The rate of ROS gen-
tack on proteins can lead to the modification
eration is closely related to oxygen con-
of amino acids, oxidation of sulfhydryl groups
sumption and proportional to the amount of
leading to conformational changes, altered
mitochondria in the tissue. In mammalian
enzymatic activity, crosslinking, peptide bond
systems, such as rat liver or pigeon heart, at
cleavage as well as carbohydrate modifica-
physiological O2 concentration, it has been
tion in glycoproteins, loss of metal in
estimated that about 1-4% of consumed oxy-
- metalloproteins, altered antigenicity, and in-
gen is converted to O2 and H2O2 at the mito-
creased proteolytic susceptibility (7,8). ROS
chondrial level due to electron leaks (5,6).
attack also causes DNA strand breaks and
Superoxide and hydrogen peroxide are rela-
base modifications (leading to point muta-
tively unreactive and long-lived in biologi-
tions) (8). Polyunsaturated fatty acids, such
cal systems but their danger lies in the fact
as arachidonic acid, appear to be particularly
that they readily give rise to highly reactive
susceptible to radical attack. Abstraction of
hydroxyl radicals (•OH) which are involved
a hydrogen atom by a radical attack leaves
in numerous forms of damage to cellular
behind a carbon-centered lipid radical (con-
macromolecules (4). In some instances, the
jugated diene) which further reacts with oxy-
destructive actions of ROS are actually ben-
gen to form a peroxyl radical (lipid hydro-
eficial to an organism; for example, phago-
peroxide). This can then abstract a hydrogen
cytic cells generate bursts of ROS to kill
atom from an adjacent fatty acid side chain
engulfed microorganisms. More generally,
and set off an autocatalytic chain reaction
however, ROS must be rapidly eliminated to
that converts many membrane lipids into
minimize their destructive nature.
lipid hydroperoxides (lipid-OOH). The pres-
Reactive oxygen species are intermedi-
ence of lipid hydroperoxides in a membrane
ates of the univalent reduction of oxygen:
disrupts its function by altering fluidity and
-
O2 + e- → O2 superoxide allowing ions such as Ca2+ to leak across the
-
O2 + e + 2 H → H2O2
- +
hydrogen peroxide membrane; the consequences of this include
H 2O 2 + e- + H+ → H2O + •OH hydroxyl radical activation of phospholipases, “membrane bleb-
• OH + e + H → H2O
- +
bing” and eventual membrane rupture (1). In

Braz J Med Biol Res 29(12) 1996

Animal adaptations for oxidative stress 1717

addition, peroxyl radicals can also attack and
damage membrane proteins. These hydroper-
oxides are eventually broken down to a series
of low molecular weight products (such as
alkanes, alkenes, hydroxy or epoxy deriva-
tives, ketones, or polyhydroperoxides) which
may themselves prove toxic to the cell (3,9).
Because of the damaging effects of ROS,
all cells maintain antioxidant defenses. Three
levels of protection have been considered: 1)
prevention of ROS formation, 2) termina-
tion of the ROS using free radical scavengers
or antioxidant enzymes, and 3) repair of
damaged cellular components. An important
aspect of prevention is the segregation or
chelation of metals that can catalyze •OH
formation, such as by iron binding to ferritin
(10). Nonenzymatic antioxidants include glu-
tathione, alpha-tocopherol (vitamin E), ascor-
bic acid, beta-carotene, and uric acid; these
are mostly considered to be chain-breaking
antioxidants in that they interrupt the auto-
catalytic spread of radical reactions (11).
The tripeptide glutathione is extremely im-
portant in the antioxidant defenses of the cell
and has multiple roles: as a substrate for
antioxidant enzymes, as an independent scav-
enger of hydroxyl and singlet oxygen, a func-
tion in the reactivation of some enzymes
inhibited under oxidizing conditions, and a
role in vitamin E regeneration (3,12). In-
deed, the ratio of reduced to oxidized glu-
tathione (GSH/GSSG) in the cell is a good
indicator of the level of oxidative stress.
Enzymes involved in antioxidant defenses
exist as a coordinated system and include
superoxide dismutase (SOD) which catabo-
lizes superoxide radicals and catalase (CAT)
and glutathione peroxidase (GPOX) which
with catalase appears to be the primary en-
zyme involved in organic hydroperoxide and
H2O2 removal, respectively (15). Also involved
in the removal of peroxides is alkyl hydroper-
oxide reductase (AHR) which converts lipid
hydroperoxides and other alkyl hydroperox-
ides to their corresponding alcohols, using
either NADH or NADPH as the reducing
agent (16,17). Secondary enzymes in antioxi-
dant defense include those of glutathione me-
tabolism. Glutathione S-transferase (GST) cata-
lyzes the conjugation of reduced glutathione
(GSH) to nucleophilic xenobiotics or cellular
components damaged by ROS attack which
leads to their detoxification. NADPH-depend-
ent glutathione reductase (GR) replenishes the
GSH substrate for GPOX and GST from oxi-
dized glutathione (GSSG).
The overproduction of ROS has been
linked to a number of clinical disease states
(3,18). Of particular interest to my labora-
tory is the well-documented role of ROS in
ischemia-reperfusion injury in mammals
(1,19). Studies on the effects of the interrup-
tion of circulation to organs (particularly
brain) not only revealed damage due to en-
ergy stress when oxidative phosphorylation
free radical generation
GS-electrophile
electrophile glutathione
superoxide S-transferase
dismutase
O2- + H
+
H2 O2
catalase GSH +
NADP
glutathione
reductase
H2 O + O 2
selenium-
dependent GSSG NADPH
Fenton glutathione
reaction peroxidase
Fe 2+

degrade hydrogen peroxide and hydroperox- H2 O + O2

ides, respectively (Figure 1). GPOX has two

-
forms. The selenium-dependent GPOX is Fe 3+ + OH + •OH peroxidation

widely believed to be the key enzyme in

Haber-Weiss reaction
vertebrate peroxide detoxification (13,14).
Fe salt catalyst
O2- + H2 O2
-
Se-independent peroxidase activity is a func- O2 + OH + •OH

tion of one class of glutathione S-transferase Figure 1 - Summary of the pathways for the generation of reactive oxygen species and of
isozymes; in insects this activity together the actions of some of the enzymes involved in antioxidant defenses in the cell.

Braz J Med Biol Res 29(12) 1996

1718
was disrupted (20), but also showed that
other forms of damage were initiated during
the recovery period when oxygenated blood
was reintroduced. Ischemia appears to
“prime” tissues to respond to reoxygenation
-
with a burst of O2 and H2O2 generation (21)
and reperfusion damage has been clearly
linked to ROS formation. Several sources of
the reperfusion-stimulated ROS generation
have been suggested. One is the electron
transport chain which produces few oxyradi-
cals when the cytochrome chain is kept in a
relatively oxidized state by the action of the
cytochrome oxidase catalyzing the 4-elec-
tron transfer to reduce oxygen to water, but
which generates more oxyradicals (appar-
ently from the ubiquinone-cytochrome b re-
gion) when the electron transport chain be-
comes highly reduced (22,23). During is-
chemia, accumulated electrons are available
for oxyradical formation using whatever oxy-
gen remains and can also readily react with
oxygen when perfusion begins. The action
of xanthine oxidase may be another con-
tributor to ROS generation since adenylate
degradation during ischemia can elevate the
levels of xanthine and hypoxanthine which,
when O2 is reintroduced, are oxidized with
-
the production of O2 and H2O 2 (21,24). An-
other consequence of ischemia is the col-
lapse of plasma membrane potential differ-
ences which leads to uncontrolled Ca2+ in-
flux that activates phospholipases to liberate
free fatty acids. Polyunsaturated fatty acids,
such as arachidonic acid, are highly suscep-
tible to radical attack and, with the reintro-
duction of oxygen during perfusion, can set
off a chain reaction of membrane lipid
peroxidation (19). Elevated cell Ca2+ could
also be responsible for setting up the poten-
tial for xanthine oxidase-mediated damage
since the Ca2+-activated protease, calpain,
cleaves the peptide bond in xanthine dehy-
drogenase that converts the enzyme to the
oxidase form (21). Other reported forms of
ischemia-reperfusion injury include protein
oxidation (carbonyl formation) (25) and glu-
Braz J Med Biol Res 29(12) 1996
K.B. Storey
tathione release from tissues (26). Another
related situation where ischemic damage is
well known is the hypothermic and cryo-
preservation of tissue and organ explants
removed from the body for subsequent use
in transplant therapy. Fuller et al. (9) sum-
marize the evidence for the involvement of
ROS damage as one of the injuries caused by
cold ischemia and reperfusion. For example,
excised hearts that were perfused with free
radical scavengers (e.g. mannitol, iron chela-
tors) showed fewer pathological changes af-
ter cold storage and improved function after
transplantation. Studies with kidney showed
altered glutathione status and the accumula-
tion of lipid peroxidation products with ex-
tended cold storage. Furthermore, the post-
storage function of the kidney was improved
by the addition of allopurinol, iron chelators,
or SOD and CAT when they were added
both to the organ perfusate and to the recipi-
ent at the time when blood flow to the organ
was restarted. Addition of SOD to liver
homogenates in phosphate buffered saline
reduced the extent of lipid peroxidation dam-
age (measured as thiobarbituric acid reac-
tive substances, TBARS) found after freez-
ing storage of tissues at -20oC; TBARS were
also reduced after -20oC storage if the ho-
mogenizing buffer was changed to sucrose/
EDTA indicating that metal-catalyzed reac-
tions are involved in ROS generation in fro-
zen homogenates (27).
Animal adaptation to variation in
oxygen availability in nature
Mammals in general, as well as most of
their organs, are quite intolerant of anoxia
and/or ischemia partly because their me-
tabolism is ill-equipped to endure the energy
shortfall that occurs when mitochondrial ATP
production is blocked and partly because of
injuries that arise due to a burst of ROS
formation when oxygen is reintroduced.
Clearly, in mammalian systems, the antioxi-
dant defenses of the organism can be over-
Animal adaptations for oxidative stress
whelmed by rapid and large changes in tis-
sue ROS levels. However, although unusual
for most mammals, many organisms rou-
tinely experience wide variation in oxygen
availability to their tissues due to factors
such as environmental oxygen lack, breath-
hold diving, extracellular freezing, or ap-
noeic breathing patterns in arrested meta-
bolic states. To cope with these situations,
many lower vertebrates and invertebrates
have well-developed tolerances for anoxia
and ischemia that allow them to endure these
stresses as part of their normal life (28). For
example, various gill-breathing intertidal
marine invertebrates routinely experience
cyclic bouts of oxygen deprivation with the
tides and have evolved excellent capacities
for facultative anaerobiosis that, in fact, al-
low them to survive for days or weeks at a
time without oxygen (29). Among verte-
brates, anoxia tolerance is highly developed
in various species of freshwater turtles that
dive routinely and also hibernate underwa-
ter; species of the Chrysemys and Trache-
mys genera, for example, can survive for 3-4
months submerged in deoxygenated water at
3oC (30,31). Freeze-tolerant animals have to
deal not only with anoxia but also with is-
chemia for when extracellular body fluids
freeze, all circulation is cut off and indi-
vidual cells must rely on internal fermenta-
tive fuel reserves to survive for perhaps days
or weeks until they thaw again. Freeze toler-
ance is quite common among cold-hardy
insects in northern latitudes and is also a
strategy used by several species of woodland
frogs and some hatching turtles for winter
survival (32). In addition, many other types
of animals, while not facing such extremes
of anoxia or ischemia, experience wide varia-
tion in oxygen availability in their normal
life and endure wide cycles of normoxic and
hypoxic conditions. For example, estivating
animals (such as various land snails and
burrowing toads) have this experience since
they use apnoeic breathing patterns to mini-
mize body water loss during long-term dor-
1719
mancy (33-35) and diving animals (such as
seals and whales) experience profound hy-
poxia in many organs due to circulatory
readjustments that preferentially direct oxy-
genated blood to the skeletal muscles and
brain (36). Hibernating mammals also expe-
rience hypoxia due to apnoeic breathing while
dormant and experience a rapid 10-20-fold
increase in oxygen consumption during
arousal when they rewarm their bodies from
ambient back to 37oC over just a few minutes
(37).
Studies in my laboratory focus on the
biochemical adaptations that allow animals
to withstand environmental extremes. Two
of our interests are the adaptations that sup-
port anoxia tolerance and freeze tolerance in
nature. More recently, we have also begun to
explore the metabolic regulation and molec-
ular adaptations that underlie estivation and
mammalian hibernation, two states of aero-
bic dormancy where hypoxic conditions gen-
erally prevail due to apnoeic breathing pat-
terns (32,38). The common thread linking
survival in all of these situations is metabolic
rate depression, the ability to lower meta-
bolic rate in the stressed state to at least 20-
30%, and sometimes as little as 1-5%, of the
corresponding normal resting state (28,38).
This profound metabolic arrest allows ani-
mals to greatly extend the time that limited
internal reserves of fuel can sustain life. In
addition, other biochemical adaptations deal
with the particular circumstances of differ-
ent situations; for example, among anoxia-
tolerant animals, marine molluscs use alter-
native pathways of substrate fermentation to
enhance the yield of ATP under anaerobic
conditions whereas freshwater turtles have
greatly improved the buffering capacity of
their tissues to deal with extreme lactic aci-
dosis. Freeze-tolerant animals produce high
concentrations of sugars or polyhydric
alcohols that act as cryoprotectants to pro-
tect their cells during freezing. Studies have
included analysis of the protective strategies
that deal with the stress itself, the regulation
Braz J Med Biol Res 29(12) 1996
1720
of the intermediary metabolism and fuel use
to adapt to the energy needs of the stress
condition, and the mechanisms that orches-
trate the coordinated suppression of the rates
of all cellular processes so that homeostasis
is preserved in the stress state.
Apart from the common use of metabolic
rate depression to maximize survival time
based on a fixed internal metabolic fuel sup-
ply, all of these stress states also share a
reduced oxygen availability and/or consump-
tion that is either imposed by the environ-
ment (e.g. by freezing of body fluids, sub-
mergence of lung breathing species) or is
“voluntary” (e.g. due to apnoeic breathing in
the dormant state). For animals in all of these
situations, therefore, the transition back to
normal, active metabolism is accompanied
by a rapid and large increase in oxygen
uptake, concentration and consumption by
the organism. These transitions are function-
ally analogous to the reperfusion situation
that occurs after ischemic insult in mam-
mals. We reasoned, then, that animals that
naturally experience wide variation in oxy-
gen availability should express biochemical
adaptations that deal not only with the period
of oxygen deprivation but also with the con-
sequences of reintroducing high concentra-
tions of oxygen into their systems. If ROS
overgeneration is a problem in situations of
mammalian ischemia-reperfusion, then it
could also be a potential problem during
metabolic recovery from anoxia, freezing
and estivation and one to be dealt with by
biochemical adaptations of the organism.
Our studies over the last 2-3 years, therefore,
have been analyzing the antioxidant defenses
in anoxia-tolerant, freeze-tolerant, and esti-
vating animals to determine how they cope
with wide variation and rapid changes in
oxygen availability and rates of ROS forma-
tion. We will begin the present discussion
with an analysis of the evidence for lipid
peroxidation damage arising from these stress
states and then discuss the adaptive changes
in antioxidant defenses, both enzymatic and
Braz J Med Biol Res 29(12) 1996
K.B. Storey
nonenzymatic, that are expressed by animals
to cope with oxidative stress.
Measurements of lipid peroxidation
damage
Lipid peroxidation has been reported as a
major contributor to the loss of cell function
under oxidative stress situations. For ex-
ample, peroxidation attack on microsomal
membranes can lead to calcium release and
uncontrolled activation of calcium-depend-
ent proteases and lipases (39,40), whereas
attack on mitochondrial membranes can al-
ter permeabilities and induce a disruption of
cellular energetics (41). In addition, an accu-
mulation of lipoperoxidation products under
some pathological conditions indicates the
probable involvement of oxygen radicals in
these disorders (3,42,43).
The reactions below summarize the auto-
oxidation of lipids (LH) leading to hydroper-
oxide (LOOH) formation:
LH + R• → L• [1]
L• + O2 → LOO • [2]
LOO• + LH → L• + LOOH [3]
where R represents an initiator radical (such
as •OH) and LOO • is a lipid peroxide radical.
The general sequence of events in the per-
oxidation of polyunsaturated fatty acids is as
follows. The first stage in the peroxidation
sequence is the removal (usually by •OH but
also by other radicals) of a hydrogen atom
from a methylene (-CH 2-) group, typically
from one adjacent to an existing double bond
in the fatty acid. This results in an unpaired
electron on the carbon which is then stabi-
lized by a molecular rearrangement to pro-
duce a conjugated diene (alternating double-
single-double bonds). This reacts with oxy-
gen to form a peroxy radical which then
abstracts a hydrogen atom from another lipid
molecule (initiating a chain reaction) and
becomes a lipid hydroperoxide (R-OOH).
Transition metal complexes (particularly
iron) can then catalyze the fission of the O-O
Animal adaptations for oxidative stress
bond of the hydroperoxide producing an
alkoxy radical which readily causes beta-
scission of the chain to cleave off hydrocar-
bons and aldehydes of varying sizes, one of
these degradation products being malondial-
dehyde.
A variety of different methods have been
used to assess the extent of peroxidative
damage to lipids (44). Some of them, such as
the iodometric assay and peroxidase-cata-
lyzed reactions (45,46), directly quantify
LOOH. Other methods quantify the damage
done by peroxidation including the forma-
tion of conjugated dienes and of various
decomposition products (such as malondial-
dehyde, lipofuchsin, alkenes and light emis-
sion by Russel reactions) (45,47). The meth-
ods developed to date all have limitations of
reproducibility, sensitivity, or accuracy. For
example, the spectrophotometric determina-
tion of conjugated dienes is widely used but
it has been shown that much of the conju-
gated-diene material in tissues does not con-
tain the hydroperoxide functional group, sug-
gesting that the method does not exclusively
measure the products of lipid peroxidation
(3,48). Malondialdehyde and others are quan-
tified as TBARS (49) but this popular assay
has been criticized for a lack of specificity
and accuracy (50), although many of the
problems are caused by inappropriate modi-
fications of the assay (51).
In an attempt to improve the methodol-
ogy for quantifying lipid peroxidation dam-
age, we recently adapted a method for use in
the determination of LOOH in animal tissue
extracts that appears to directly measure li-
pid peroxides (52). The FOX assay (ferrous
oxidation/xylenol orange method) (53,54) is
based on the oxidation of Fe(II) by LOOH at
acid pH in the presence of the Fe(III)-
complexing dye, xylenol orange; complex
formation is then quantified by spectropho-
tometric absorbance at 580 nm. Previous use
of the assay was limited to the measurement
of peroxides in irradiated solutions (55) or in
low density lipoprotein (LDL) or membrane
1721
preparations (53,54) but we demonstrated
that the assay effectively assessed LOOH
content in heterogeneous tissue extracts (52).
Fe(III)xylenol orange complex formation
increased linearly with increasing amounts
of methanolic tissue extract added and a
significant correlation (r = 0.88; P<0.005)
was demonstrated between the levels of lipid
peroxides indicated by the FOX assay and by
the TBARS assay. Figure 2 shows that this
correlation holds up well for extracts from a
variety of tissue types and multiple animal
species. For example, the three tissues with
the lowest TBARS content (turtle skeletal
muscle, rat lung, and ground squirrel white
adipose tissue) also showed the lowest lev-
els of FOX equivalents.
In a new study, we have used this assay
and compared it with two other methods in
order to assess the potential extent of
peroxidative damage to lipids occurring in
the anoxia-tolerant turtle Trachemys scripta
over the course of anoxia exposure and aero-
bic recovery (Willmore WG and Storey KB,
unpublished data). Each method quantifies
the damage at a different stage in the
peroxidation process. As described above,
the FOX assay appears to directly quantify
lipid hydroperoxides. The conjugated-diene
assay directly assesses the content of these
early products of the peroxidation process
Figure 2 - Correlation between
the xylenol orange method,
100 measured as cumene hydroper-
oxide (CHP) equivalents, and the
TBARS (nmol/g wet weight)
80 MH
thiobarbituric acid reactive sub-
stance (TBARS) assay for quanti-
fying the levels of lipid peroxida-
60 TL
tion in different animal tissues.
SL
ML The solid and dashed lines are
40 MK computer-generated linear corre-
RB lations (r = 0.88; P<0.005) and
SWAT
20 95% confidence intervals, re-
TM spectively, calculated from the
RLn means of values (N = 9); where
0
0 3000 6000 9000 error bars are not indicated, they
CHP equivalents (nmol/g wet weight) are smaller than the symbol. Tis-
sues are: RLn, rat lung; SWAT,
squirrel white adipose tissue; TM, turtle red muscle; RB, rat brain; SL, squirrel liver; TL,
turtle liver; MK, mouse kidney; ML, mouse liver, and MH, mouse heart (From Ref. 52, with
permission).
Braz J Med Biol Res 29(12) 1996
1722
using second-order derivative spectroscopy
over the region (centered on 233-234 nm)
where these structures absorb UV light (56).
The TBARS assay measures the complex
formed (absorbance at 532 nm) between
thiobarbituric acid and various aldehydes
that are terminal products of the peroxidation
process (malondialdehyde is a prominent
product and the standard used for the assay)
(47). Table 1 shows the results of these
determinations in liver samples from control
and 20-h anoxic (submerged in N2-bubbled
water) turtles and from turtles submitted to
24-h aerobic recovery after anoxia (Willmore
WG and Storey KB, unpublished data). The
levels of peroxidation products indicated by
the different methods cannot be quantita-
tively compared but they all indicated the
same qualitative result. Except for a signifi-
cantly lower level of conjugated dienes in
liver from anoxic turtles, the results were
consistent in indicating a lack of accumu-
lated lipid peroxidation damage during ei-
ther anoxia exposure or the aerobic recovery
period after anoxia. Furthermore, when FOX
and TBARS analyses were performed on
three turtle organs (kidney, red muscle and
white muscle) the same result was found,
although the levels of damage products were
overall much lower in these organs than in
the liver (Willmore WG and Storey KB,
Table 1 - Assessment of lipid peroxidation products in turtle T. scripta liver by three
methods, and comparison of samples from aerobic controls, 20-h anoxia exposure,
and 24-h aerobic recovery after anoxia.
The FOX assay measures lipid hydroperoxides directly whereas conjugated dienes are
an early product of peroxidative damage. Thiobarbituric acid (TBA) reactive substances
quantify malondialdehyde and other aldehydes that are terminal products of
peroxidative damage. Lipid hydroperoxides are reported as cumene hydroperoxide
equivalents and conjugated dienes are reported as relative second-order derivative
absorbance units (d 2A/mg lipid). *P<0.05 compared to aerobic control liver (Dunnett
test) (Willmore WG and Storey KB, unpublished data).
Tissue Condition Lipid hydroperoxides Conjugated dienes TBA
(nmol/g wet weight) (d2A/mg lipid x 10-5) (nmol)
Liver Control 5230 ± 131 (3) 13.1 ± 1.78 (11) 71.0 ± 1
Anoxic 4216 ± 86 (3) 8.15 ± 1.11 (11)* 85.9 ± 1
Recovered 5624 ± 783 (3) 9.39 ± 1.21 (12) 89.0 ± 2
Braz J Med Biol Res 29(12) 1996
K.B. Storey
unpublished data). Indeed, turtle liver was
the only organ tested where significant lev-
els of conjugated dienes could be detected.
Overall, these results indicate that the rein-
troduction of oxygen after anoxia exposure
does not stimulate an accumulation of lipid
peroxidation products in organs of anoxia-
tolerant turtles. Although we did not attempt
to assess damage to other macromolecules
(e.g. proteins or DNA), the data indicate that
animals that deal with aerobic/anoxic transi-
tions naturally have developed mechanisms
to minimize or prevent oxidative damage as
the result of ROS formation when oxygen is
abruptly reintroduced. As will be discussed
later for the turtle, these mechanisms appear
to be the maintenance of constitutively high
activities of antioxidant enzymes and of glu-
tathione.
The same conclusion could be drawn
when the FOX and TBARS assays were
applied to assess potential peroxidation dam-
age in organs of the wood frog Rana sylvatica
over a course of freezing and thawing expo-
sure in vivo (57). No evidence of accumu-
lated damage products could be found in
liver, skeletal muscle, brain or kidney after
24 h of freezing or up to 4 h of thawing.
Therefore, it appears that neither the natural
ischemic excursion imposed by the freezing
of extracellular body fluids nor the reperfu-
sion event associated with thawing and the
reestablishment of circulation leads to an
accumulation of lipid peroxidation products
in the organs of freeze-tolerant frogs. As for
the anoxia-tolerant turtles, this again sug-
gests that animals that are well adapted to
survive anoxic or ischemic insults have in-
cluded, as part of this adaptation, mecha-
nisms that prevent or minimize the produc-
tion of ROS during tissue reoxygenation.
We also assessed the production of lipid
peroxidation damage products during the
arousal of land snails from dormancy. Esti-
vation is a response to desiccating condi-
tions and is often associated with limited
food availability and high environmental tem-
Animal adaptations for oxidative stress
peratures. Snails can easily sustain dormancy
for a year or more but high humidity or rain
breaks dormancy and snails become active
again within as little as 10-20 min. Arousal
includes a rapid increase in ventilation rate
and oxygen consumption and a reactivation
of metabolic pathways (34,38,58). Figure 3
shows the changes in the levels of TBARS in
the hepatopancreas of the land snail Otala
lactea during the first few minutes of arousal
(after 30 days of estivation) (59). Lipid
peroxidation damage clearly increased when
snails were aroused from dormancy, the level
of TBARS rising by 25% within 20 min in
the hepatopancreas. This suggests that this
tissue is sensitive to ROS generation when
the rate of oxygen consumption is abruptly
increased during arousal. Foot muscle, by
contrast, showed no change in TBARS dur-
ing arousal. Comparable situations where
the rate of oxygen consumption increases
rapidly have also been shown to result in an
increase in TBARS in mammalian tissues
under posthypoxic conditions (60), during
thermogenesis in brown adipose tissue (61),
and during acute aerobic exercise in skeletal
muscles (61,62). These situations seem to be
associated with high flux generation of ROS
from mitochondria. In the snail hepatopan-
creas other sources of ROS might also be
considered such as cytochrome P450 (which
functions in detoxification of xenobiotics)
and xanthine oxidase (XO). Indeed, XO ac-
tivity rose by 3-fold in O. lactea hepatopan-
creas over 35 days of estivation (63) and,
furthermore, xanthine is known to accumu-
late during estivation in snails (64). Thus,
XO activity might be a source of ROS gen-
eration during arousal after estivation, al-
though maximal XO activity is actually quite
low in the snail hepatopancreas (0.03 nmol
isoxanthopterin min-1 mg protein-1 or 1.2
mU/g wet weight after 35 days of estivation)
and H2O 2 generation by this enzyme should
be easily dealt with by hepatopancreas cata-
lase activity which is several orders of mag-
nitude higher (63).
1723
Glutathione status
Glutathione is considered to be one of the
most important components of the antioxi-
dant defense of living cells. The reduced
tripeptide GSH is a hydroxyl radical and
singlet oxygen scavenger, and participates in
a wide range of cellular functions such as
protein and DNA synthesis, intermediary
metabolism, and transport (3,65,66). Since
glutathione is present in high intracellular
concentrations, there is a high probability
that reactive oxygen species (such as super-
oxide, singlet oxygen and hydrogen perox-
ide) will be quenched by reaction with glu-
tathione before they can initiate their chain
reaction-damaging effects (67). Reduced glu-
tathione acts as a hydrogen donor and as
such is a substrate of key antioxidant en-
zymes including the Se-dependent glu-
tathione peroxidase, GPOX, which removes
hydrogen peroxide and glutathione S-trans-
ferases, GSTs, which in turn catalyze conju-
gation reactions between glutathione and
ROS-damaged cellular components. The Se-
independent peroxidase activity of GSTs is
also used in the removal of organic hydro-
peroxides (68,69). When oxyradicals are
present in large amounts, GSSG formation
Figure 3 - Changes in the con-
TBARS (nmol/g wet weight)
35 centration of damage products
a
(thiobarbituric acid reactive sub-
30 stances, TBARS) (upper panel)
and in the activities of superox-
25 ide dismutase (SOD) (lower
panel, open circles) and glu-
20
0 20 40 60 80 100 24 h
tathione peroxidase (GPOX)
(lower panel, filled circles) in O.
lactea hepatopancreas with time
120
a
U/mg SOD or mU/mg GPOX
after arousal from 3 months of
100 estivation. Data are reported as
80
means ± SEM; N = 4-7; where
error bars are not indicated, they
60 a are smaller than the symbol.
aP<0.05 compared to the value
40
a determined at 0 min (Dunnett
a a test) (From Ref. 59, with per-
20
a mission).
0
0 20 40 60 80 100 24 h
Time (min)
Braz J Med Biol Res 29(12) 1996
1724
Table 2 - Levels of reduced (GSH) and oxidized (GSSG) glutathione in liver and skeletal muscle
of different species.
Data are reported as means ± SEM. For turtles, muscle is white skeletal muscle; for snails the
“liver” is the hepatopancreas. Data are taken from Refs. 57,59,79 and Willmore WG and
Storey KB, unpublished data.
Species
Turtle, Trachemys scripta
Frog, Rana sylvatica
Frog, Rana pipiens
Snake, Thamnophis sirtalis
Snail, Otala lactea
Braz J Med Biol Res 29(12) 1996
K.B. Storey
exceeds its clearance and the ratio of re- weight) reported for liver glutathione in a
duced to oxidized glutathione (GSH/GSSG) variety of other ectothermic vertebrates (fish
decreases. Hence, this ratio is frequently and amphibians) (70). Indeed, turtle liver
used as an indicator of the level of oxidative values were closer to the levels in avian liver
stress in cells. In addition, GSSG itself can (2.5-3.7 µmol/g wet weight) but still lower
exert deleterious effects through nonspecific than values reported for mammalian liver (6-
reactions with the free sulfhydryl groups of 8 µmol/g wet weight) (70). High redox buffer
proteins to form mixed disulfides, reactions capacity, attributed to high numbers of re-
that can lead to inactivation of enzymes pos- duced sulfhydryl groups present in blood
sessing sulfhydryl groups at their active sites proteins, has been postulated as a mech-
(3). The maintenance of GSH levels, and anism preventing reperfusion injury in the
thereby the reducing environment of the cell, hypoxia-resistant turtlePhrynops hilarii (71)
is crucial, therefore, to organisms that peri- and the results obtained for T. scripta further
odically undergo oxidative stress. Thus, from support this idea. Glutathione pools in frog
the analysis of the cellular levels of glu- and snake organs were lower but the freeze-
tathione and the effects of stress on the pools tolerant frog species, R. sylvatica, had a
of GSH and GSSG and their ratio we can higher pool size than did the freeze-intoler-
assess both the extent of oxidative stress ant R. pipiens and this is particularly striking
imposed under different metabolic systems in the comparison of skeletal muscle values.
and the level of constitutive antioxidant de- Substantially higher glutathione pools were
fenses maintained by different organisms. also found in most other organs of R. sylvatica
The levels of GSH and GSSG in liver and compared with R. pipiens (57) which sug-
skeletal muscle from control (unstressed) gests that the ability to endure and to recover
animals of several species are shown in Table from freezing and from the ischemia that it
2. What is immediately obvious is that these imposes is supported by elevated levels of
levels are highest in the species with well- glutathione in all organs of the freeze-toler-
developed anoxia tolerance, the turtle T. ant anuran. The data in Table 2 show that the
scripta. Furthermore, the liver glutathione estivating snail species O. lactea also main-
content of T. scripta is substantially higher tains a substantial pool of glutathione in its
than the range of values (0.9-2.2 µmol/g wet tissues.
The effect of stress on tissue glutathione
pools has also been evaluated in these sys-
tems. In turtles, total glutathione content
decreased significantly during anoxia expo-
sure in four organs, dropping to 49, 67, 54
and 59% of control values in liver, heart,
white muscle, and kidney, respectively; how-
Liver Skeletal muscle ever, red muscle and brain pools were unaf-
fected (Willmore WG and Storey KB, un-
GSH GSSG GSH GSSG
(nmol/g wet weight) (nmol/g wet weight)
published data). The reduced total glutathione
content of these organs also persisted over
3350 ± 262 10.4 ± 3.0 1535 ± 195 6.0 ± 0.9 the 24-h aerobic recovery period. Differen-
1293 ± 152 12.2 ± 3.1 216 ± 17 <1.5 tial changes in GSH and GSSG contents,
963 ± 84 <1.5 19.6 ± 1.8 <1.5 however, meant that the GSH/GSSG ratio
1015 ± 92 74 ± 14 454 ± 43 40 ± 4 increased in most tissues during anoxia ex-
2178 ± 280 461 ± 36 634 ± 100 177 ± 2
posure, a finding that is consistent with the
more reduced state of many other cellular
Animal adaptations for oxidative stress
components (e.g. cytochromes, NADH)
when oxygen is depleted (72). The reason
for the large net decrease in glutathione lev-
els in most organs during anoxia is not yet
known. In their detoxification role, GSTs
conjugate reduced glutathione to electrophilic
substrates including xenobiotics, lipid hy-
droperoxides (including prostaglandins), and
nucleotide hydroperoxides. Hence, a gradual
accumulation of damage products during
anoxia may possibly be controlled in this
manner, with the further disposal of these
products and regeneration of glutathione
postponed until the return to aerobic condi-
tions when ATP availability is no longer
limiting. However, there was no evidence of
the accumulation of lipid peroxidation prod-
ucts in turtle organs during anoxia (Willmore
WG and Storey KB, unpublished data), so
the identity of any damage products remains
to be established. Analysis of the glutathione
status of wood frog organs showed that nei-
ther freezing nor thawing had major effects
on the system and indicated, therefore, that
little or no oxidative stress occurred during
this ischemia-reperfusion event (57). Over-
all, the GSH/GSSG ratio was unchanged in
most organs during freezing and, although
the tissue concentrations of both compounds
increased during freezing, this effect was
explained by the organ dehydration that oc-
curs as water moves into extra-organ spaces
to freeze. Hence, the levels of all metabolites
(when quantified per gram wet weight) tend
to increase in organs from frozen frogs. Lev-
els of GSH and GSSG in Otala lactea tissues
showed little evidence of stress during the
arousal process. The total pool size of these
compounds remained constant in 30-day es-
tivating snails and over a 24-h time course of
arousal, although changes in TBARS and
enzyme activities indicated some oxidative
stress during arousal (59). However, over 24
h of arousal the GSSG content actually fell
about 50% in both hepatopancreas and
muscle. A higher GSSG content (and a lower
GSH/GSSG ratio) during estivation could
1725
suggest oxidative stress under the hypoxic
conditions of dormancy itself but more likely
reflect a limitation in GSSG reconversion to
GSH due to limiting NADPH availability in
the metabolically suppressed state. Taken
together, the data indicate that animals that
deal naturally with wide variations in oxy-
gen availability and consumption have ad-
justed their tissue glutathione pools to higher
levels than nontolerant organisms so that
during stress and recovery they can buffer
any oxidative stress that occurs with mini-
mal change (or even no change) in the glu-
tathione pools or the GSH/GSSG ratio.
Antioxidant enzymes
How do organisms that experience wide
variation in oxygen availability and/or inter-
vals of natural ischemia (e.g. during freez-
ing) deal with the accompanying wide varia-
tion in the production of reactive oxygen
species? Three possible strategies can be
suggested. The first would be to maintain
constitutively high levels of antioxidant de-
fenses (enzymes, low molecular weight anti-
oxidants) so that any stress can be dealt with
effectively. The second would be to elevate
antioxidant defenses as a rapid response to
anoxia/ischemia stress so that these are in
place in anticipation of the overgeneration
of ROS during the aerobic recovery period.
The third possible strategy would be to en-
dure an accumulation of damage products
during the recovery period and emphasize
mechanisms that rapidly dispose of ROS-
damaged products. As discussed above, we
have found little evidence for the accumula-
tion of damage products in animals with
well-developed tolerances for anoxia or is-
chemia; neither anoxia-tolerant turtles nor
freeze-tolerant frogs showed accumulation
of lipid peroxidation damage products dur-
ing the recovery from these stresses
(Willmore WG and Storey KB, unpublished
data; 57). Land snails showed a small and
short-lived increase in TBARS during arousal
Braz J Med Biol Res 29(12) 1996
1726 K.B. Storey

from estivation (59). These data suggest
that the third option (letting damage prod-
ucts accumulate and then initiating repair
mechanisms) is probably not a strategy that
is used and indeed, intuitively, this is a poor
idea since the repair of damaged systems
would require specific metabolic machin-
ery and a considerable investment of meta-
bolic energy. Indeed, as a principle in com-
parative biochemistry in general, adapta-
tion is generally focused on preventive meas-
ures that minimize the disruption of the
“normal” metabolism rather than on repair
mechanisms that alleviate accumulated dam-
age. Of the first two strategies, then, which
is used? Our studies to date suggest that
both are used in different systems with the
“choice” between them apparently deter-
mined by the extent to which a species
experiences oxidative stress naturally. Thus,
our data suggest that a good facultative
anaerobe deals with the inevitable burst of
ROS generation during the anoxic to aero-
bic transition by maintaining high activities
of antioxidant enzymes and a large glu-
tathione pool constitutively. Animals that
only infrequently experience oxidative
stress, however, appear to initiate adaptive
changes in their antioxidant defenses dur-
ing the stress period that minimize oxida-
tive damage during recovery. These differ-
ent strategies are illustrated in the four cases
presented below.
It is instructive to begin with an examina-
tion of the constitutive activities of antioxi-
dant enzymes in liver and muscle of the five
species. Table 3 shows the maximal activi-
ties of SOD, CAT and GPOX (Se-depend-
ent) in liver and muscle of control animals.
Tissues from the anoxia-tolerant turtle T.
scripta clearly have very high activities of
these enzymes compared with those of gar-
ter snakes and leopard frogs, species that
only rarely encounter wide and rapid varia-
tion in oxygen availability in nature. Thus,
for example, in liver the ratio of SOD activi-
ties in the turtle, leopard frog and snakes was
100:29:17, CAT was 100:39:31, and GPOX
was 100:35:52. Values for liver enzyme ac-
tivities in the freeze-tolerant wood frog, how-
ever, were quite similar to those in the turtle
indicating that the antioxidant defenses of
frogs are well prepared to deal with potential
ROS insult during reperfusion after thawing.
Se-dependent GPOX has also been meas-
ured in Rana perezi liver (73) and the activ-
ity (54-80 U/mg protein) was about the same
as in R. pipiens and considerably lower than
that in R. sylvatica (Table 3). Again this
indicates that the high GPOX activities found
in R. sylvatica are specific for this species,
probably related to freeze tolerance. Muscle

Table 3 - Activities of superoxide dismutase (SOD), catalase (CAT), and Se-dependent glutathione peroxidase (GPOX)
in vertebrate liver and muscle from different species (control animals).

Data are reported as means ± SEM. For turtles, muscle is white skeletal muscle; for snails the “liver” is the
hepatopancreas. Data are taken from Refs. 57,59,79 and Willmore WG and Storey KB, unpublished data.

Species Liver Skeletal muscle

SOD CAT GPOX SOD CAT GPOX

(U/mg) (U/mg) (mU/mg) (U/mg) (U/mg) (mU/mg)

Turtle, T. scripta 48.6 ± 5.8 229 ± 8.3 298 ± 2 34.1 ± 2.2 55 ± 5.6 32 ± 1
Wood frog, R. sylvatica 37 ± 3.4 214 ± 17 136 ± 15 20.1 ± 2.4 1.3 ± 0.1 19 ± 2.4

Leopard frog, R. pipiens 14 ± 2 86 ± 8.5 70 ± 7 6.4 ± 0.5 1.2 ± 0.2 9.9 ± 0.6

Garter snake, T. sirtalis 8.3 ± 0.7 71.8 ± 5.5 155 ± 7 3.38 ± 0.22 22.9 ± 2.8 65.6 ± 10

Land snail, O. lactea 78.4 ± 14.0 177 ± 13 25.8 ± 6.9 42.1 ± 6.2 5.9 ± 1.0 4.2 ± 0.4

Braz J Med Biol Res 29(12) 1996

Animal adaptations for oxidative stress
antioxidant enzymes showed a similar pat-
tern of high activities in turtle, slightly lower
values in wood frog and still lower in leop-
ard frog and snake. Exceptions to the pat-
tern, however, were the very low activities of
muscle CAT in both frog species and high
GPOX activity in garter snake muscle. Val-
ues for white skeletal muscle of turtles are
shown in Table 3 as this muscle is the closest
comparison for frog and snake skeletal
muscle but activities of antioxidant enzymes
were even higher in red skeletal muscle from
turtles, as would be expected of a tissue with
a higher myoglobin content and greater de-
pendence on aerobic respiration (Willmore
WG and Storey KB, unpublished data). Com-
parisons can also be made between turtle
liver and mammalian liver. GPOX activities
were similar in turtle and rat liver, both being
about 6-fold higher than in ground squirrels
(74). Activities of SOD in turtle liver were
also very similar to those of mammalian
liver but CAT activity in turtle liver was
lower than in mammals (74). It appears,
then, that despite the much lower aerobic
metabolic rate of ectothermic turtles com-
pared with endothermic mammals, anoxia-
tolerant turtles have generally elevated anti-
oxidant enzyme activities within the ranges
characteristic of mammalian tissues and con-
siderably higher than the ranges seen in other
ectothermic lower vertebrates. This would
provide turtle organs with a high constitutive
level of antioxidant defenses that would serve
to prevent or buffer damage due to bursts of
reactive oxygen species generation during
reoxygenation after submergence. Antioxi-
dant enzyme activities were also generally
high in tissues of the pulmonate land snail,
O. lactea, indicating a good constitutive ca-
pacity for dealing with ROS generation. Ac-
tivities of Se-dependent GPOX in snail tis-
sues were low compared with those in verte-
brates, but, as noted previously, this is also
true of insects and GPOX is replaced in
insects by the Se-independent activity asso-
ciated with GST (15). The same may also be
1727
true of molluscs and other invertebrates.
Anoxia tolerance in freshwater turtles
Freshwater turtles are excellent faculta-
tive anaerobes and can survive under a nitro-
gen gas atmosphere for many hours at warm
temperature and for as long as 3-4 months at
3oC. Their anoxia tolerance is used naturally
during diving and for winter hibernation at
the bottom of ponds (30). Biochemical
mechanisms supporting metabolic rate de-
pression and anaerobic metabolism have been
extensively studied in Trachemys and Chry-
semys species and the turtle brain has been
widely used as a model system for investi-
gating the events of brain response to anoxic
or ischemic insult, to further the understand-
ing of the damage done by stroke in mamma-
lian brain. Turtles are clearly adapted to
withstand anoxic insults that are unpredict-
able both in onset and duration and, as such,
their metabolism appears to be optimally
prepared at all times to deal with both the
metabolic consequences of anoxia and with
the potential for oxidative stress upon recov-
ery. The high constitutive activities of anti-
oxidant enzymes in turtle organs appear to
be key in this regard. However, we won-
dered whether turtles would also respond to
individual anoxic excursions with modifica-
tions of their antioxidant systems to further
improve their ability to efficiently deal with
ROS stress during aerobic recovery. There-
fore, we compared the maximal activities of
antioxidant enzymes in organs of the turtle
Trachemys scripta from control (aerobic at
7oC), anoxic (20 h at 5oC) and aerobic recov-
ered (24 h returned to 7oC) animals (Willmore
WG and Storey KB, unpublished data). The
results showed that anoxia exposure did,
indeed, lead to a few selected changes in
antioxidant defenses: SOD decreased in liver
by 30%, CAT decreased in heart by 31%,
CAT and total GPOX decreased in kidney
(by 68 and 41%), and CAT and SOD de-
creased in brain (by 80 and 15%) (Willmore
Braz J Med Biol Res 29(12) 1996
1728
WG and Storey KB, unpublished data). These
data were consistent with a reduced poten-
tial for oxidative damage during anoxia. Most
anoxia-induced changes were reversed dur-
ing aerobic recovery, although brain enzyme
activities remained suppressed. Some spe-
cific changes occurred also during the recov-
ery period, with SOD rising to 45% above
initial control activities in heart whereas
GPOX was decreased by 50% in heart. The
results show that enzymes that deal directly
with free radicals are modulated in some
tissues to track the predictable changes in
ROS production during anoxia and recov-
ery. One of the targets of anoxia-induced
metabolic rate depression, therefore, may be
the biosynthesis of antioxidant enzymes.
Enzymes related to glutathione metabolism
also showed selected changes during anoxia
and recovery in turtles (Willmore WG and
Storey KB, unpublished data). During an-
oxia exposure, GR activity increased by 52
and 80% in liver and red muscle, respec-
tively. Strikingly, the enzyme gamma-
glutamyl transpeptidase, which is involved
in glutathione degradation, was reduced to
only 2% of control values in anoxic kidney,
a change that would help to suppress glu-
tathione turnover during anoxia, a time when
the oppositely-directed action of glutathione
synthetase would also be limited by the avail-
ability of ATP as a substrate. The kidney is
the major organ responsible for glutathione
removal from blood circulation and at least
50 to 65% of the net plasma glutathione
turnover occurs there (75,76). Thus, by re-
stricting the synthesis and degradation of
glutathione in anoxia, the net use of the
tripeptide could be directed towards conju-
gation reactions in detoxification systems.
Freeze tolerance in wood frogs
The wood frog Rana sylvatica has been
extensively studied by us as a model of
vertebrate freeze tolerance and as a guide to
the types of metabolic adjustments that must
Braz J Med Biol Res 29(12) 1996
K.B. Storey
be made in order to permit the freezing pres-
ervation of mammalian organs for transplant
(32,77). As noted above, wood frogs display
considerably higher constitutive activities of
antioxidant enzymes in their tissues com-
pared with the same organs of the freeze-
intolerant leopard frog and these appear to
gear them to deal well with potential ROS
overgeneration during thawing. As with an-
oxia stress in turtles, we also wondered
whether whole animal freeze/thaw exposures
would stimulate any acute changes in organ
antioxidant enzyme activities that would fur-
ther prepare the animal to deal with ROS
generation during thawing and reperfusion.
The results showed that SOD activity de-
creased significantly during freezing in
muscle, kidney, and heart of wood frogs,
falling to 77, 43, and 43% of control values,
respectively (57). However, SOD activity in
kidney and heart recovered to control levels
after 24 h of thawing but this activity re-
mained suppressed in skeletal muscle. This
SOD response parallels the effects of an-
oxia/recovery in turtles and again suggests
that SOD may be very sensitive to acute
changes in superoxide levels. Organ catalase
activities in frogs were unaltered throughout
freezing and thawing but total glutathione
peroxidase activity (Se-dependent and -in-
dependent) increased significantly by 1.2- to
2.5-fold during freezing in all R. sylvatica
tissues. Upon thawing GPOX remained high
in liver and brain but showed a decreasing
trend in the other three tissues. Changes in
Se-dependent GPOX activity paralleled those
of total GPOX in four tissues but in liver the
activity remained constant throughout the
experimental course. These responses by
GPOX appear to anticipate an overgeneration
of H2O 2 and organ hydroperoxides during
thawing. Changes in GST activity in wood
frogs during freezing and thawing were highly
tissue dependent. In liver and brain, the ac-
tivity increased throughout to peak after thaw-
ing but in kidney and heart the maximal
activity decreased during freezing and in-
Animal adaptations for oxidative stress
creased again after thawing. GST activity
did not change significantly in skeletal muscle
during the freeze-thaw cycle.
Anoxia and freezing stress in garter snakes
The garter snake Thamnophis sirtalis is the
most northernly distributed reptile in North
America and throughout its Canadian range
must have well-developed strategies for deal-
ing with winter cold. It appears that the pri-
mary mechanism of winter survival for this
species is to hibernate in well-protected under-
ground dens where the temperature is buffered
and does not often fall below 0oC; indeed,
snakes are known to migrate many miles to
congregate by the hundreds in a few selected
den sites. However, as part of our studies of
animal freeze tolerance, we determined that
garter snakes have a significant ability to with-
stand freezing. Animals recovered after sev-
eral hours of freezing at -2.5oC with about
50% of total body water frozen as extracellular
ice (78). Such a capacity would not support
long-term hibernation within the dens but
would be effective in allowing animals to
endure short, overnight frosts when they are
active above ground in both spring and au-
tumn. Garter snakes also have a substantial
ability to endure anoxia exposure and can
survive for 2 days under a nitrogen gas atmos-
phere at 5oC. To determine how the antioxi-
dant defenses of garter snakes responded to
stress we measured the activities of five en-
zymes (SOD, CAT, GPOX, GR and GST) in
liver, skeletal muscle and lung of control (aero-
bic at 5oC), frozen (5 h at -2.5oC) and anoxic
(10 h at 5oC) snakes (79). Enzyme activities in
control liver and skeletal muscle of snakes are
shown in Table 3 and stress had the most
substantial effects on enzyme activities in these
organs. Freezing exposure stimulated defenses
against the overgeneration of H2O2 in muscle
and lung; muscle CAT activity increased by
183% and GPOX by 52% during freezing
whereas lung CAT increased by 63% during
freezing. Anoxia, on the other hand, stimulat-
1729
-
ed the defense against overgeneration of O2
with increases in SOD of 188% in liver and
56% in muscle. Neither of the stress states are
ones that should themselves be associated with
elevated levels of ROS since in both states
oxygen in the tissues is depleted. This is sup-
ported by a lack of change in the GSH/GSSG
ratio in either frozen or anoxic tissues, which
shows that there is no increase in the consump-
tion of GSH by antioxidant reactions during
either freezing or anoxia exposure (79). It
appears, therefore, that the elevation of anti-
oxidant defenses under stress is an adaptive
response that anticipates the overgeneration of
oxyradicals at the termination of the freezing
or anoxia exposures. Analogous anticipatory
responses have been documented in other sys-
tems as follows: 1) the activities of antioxidant
enzymes in mammalian lung are enhanced
during late gestation in preparation for the
elevated oxygen pressure that will be experi-
enced after birth (80,81), and 2) antioxidant
defenses increase in brown adipose tissue of
hibernating ground squirrels for protection
against ROS generation during the periods of
intense uncoupled respiration that generate
heat in the organ to rewarm the animal during
arousal (82,83). Thus, for a species that only
intermittently experiences anoxia or freezing-
induced ischemia in nature, it appears that the
appropriate strategy to deal with oxidative
stress during the recovery period is to elevate
the activities of key antioxidant enzymes while
in the anoxic or frozen state. This is an energy-
expensive option in a situation where only
glycolytic ATP generation is available, but for
dealing with infrequent exposures to anoxia or
freezing, this is more efficient overall than
maintaining constitutively high antioxidant ac-
tivities as occurs in turtles.
Estivation in land snails
Another example of an anticipatory re-
sponse by antioxidant defenses is seen in the
case of estivation in the land snail Otala
lactea. In response to a shortage of food and
Braz J Med Biol Res 29(12) 1996
1730
water, snails enter into a dormant state char-
acterized by a sharply reduced rate of oxy-
gen consumption and an apnoeic breathing
pattern that reduces water loss. Again, ROS
damage during estivation itself would not be
expected since the constitutive antioxidant
defenses of the snail should be sufficient to
deal with the much lower rate of ROS forma-
tion that would accompany the 3-10-fold
decrease in oxygen consumption in the dor-
mant state (34). Snails were submitted to two
cycles of 30-day estivation followed by a 24-
h arousal and the activities of five enzymes
(SOD, CAT, GPOX, GST and GR) were
monitored (59). With the exception of GR,
estivation/arousal had major effects on the
antioxidant potential of both foot muscle
and hepatopancreas (Table 4). In foot muscle,
CAT, SOD and GST activities were all high
during dormancy but decreased substantially
(by 43, 40, and 47%, respectively) after snails
were aroused for 24 h. However, when snails
were allowed to re-enter estivation and were
then sampled after a second 30-day period of
estivation, enzyme activities were again el-
evated (but fell again when snails were
aroused for a second time). In the hepato-
pancreas, the activity of SOD was also re-
Table 4 - Effect of estivation and arousal on the activities of selected antioxidant enzymes in
foot muscle and hepatopancreas of the land snail Otala lactea.
Dormant-1 snails were sampled after 30 days of continuous estivation at 20-22o C. Snails
were then aroused for 24 h and active-1 animals were sampled. The cycle was then repeated
with snails allowed to estivate again for 30 days (dormant-2) and then aroused for 24 h (active-
2). Data for active-1 and active-2 cycles were extremely similar and were combined as the
“active” group. Data are reported as means ± SEM (N = 8 for active and N = 4 for dormant
groups), all data reported per mg protein. *All values for active snails are significantly lower
than the corresponding values for either dormant group (P<0.05). Data are taken from Ref. 60,
with permission.
Dormant-1 Active Dormant-2
Foot muscle
Catalase (U/mg) 5.9 ± 1.0 3.4 ± 0.1* 5.2 ± 0.1
Superoxide dismutase (U/mg) 42.1 ± 6.2 25.2 ± 1.5* 39.3 ± 6.7
Glutathione S-transferase (mU/mg) 239 ± 54 126 ± 20* 206 ± 25
Hepatopancreas
Superoxide dismutase (U/mg) 78.4 ± 14.0 50.0 ± 5.9* 89.2 ± 9.7
Glutathione peroxidase (mU/mg) 25.8 ± 6.9 9.3 ± 1.6* 20.5 ± 2.4
Braz J Med Biol Res 29(12) 1996
K.B. Storey
duced (by 37%) in aroused snails compared
with dormant snails. GPOX activity was also
affected in the hepatopancreas, activities
being about twice as high in estivating as in
aroused snails (Table 4). We proposed that
the increase in antioxidant enzyme activities
in O. lactea tissues during estivation was a
protective mechanism against oxidative stress
during arousal (59). Overall oxygen con-
sumption rates are low during dormancy,
only 15-30% of the corresponding values for
active snails (34,58) and hence high rates of
ROS generation during dormancy would not
be expected. During the first few minutes of
arousal, however, oxygen consumption
quickly rises to peak at about twice the value
that is typical of steady-state active snails.
This peak occurs at about 10 min after the
foot emerges from the shell (58) but we have
found that estivation-induced inactivation of
pyruvate kinase (a regulatory enzyme of gly-
colysis) is reversed within as little as 10 min
in foot muscle and pyruvate dehydrogenase
(which gates carbohydrate entry into the mi-
tochondrial metabolism) was fully reacti-
vated within 60 min (84,85). Thus, a major
metabolic reorganization to activate the aero-
bic metabolism clearly occurs very quickly
during arousal and the elevation of antioxi-
dant enzyme activities during dormancy
would clearly facilitate the rapid disposal of
accompanying ROS produced as the result
of high rates of oxygen consumption early in
arousal.
For a better understanding of the pattern
of enzymatic changes during arousal, espe-
cially during the early minutes when oxida-
tive stress would predictably be greatest,
activities of hepatopancreas SOD and GPOX
were monitored over time after arousal was
initiated by spraying snails with water (59).
As Figure 3 shows, SOD activity rose quickly
over the first few minutes of arousal, in-
creasing by about 70% after 40 min. Subse-
quently, enzyme activity fell over time to the
lower values seen after 24-h arousal. How-
ever, GPOX responded differently, with its
Animal adaptations for oxidative stress 1731

activity steadily decreasing over the first 40
min of arousal and remaining suppressed
over the remainder of the active period. These
results show a particular need to increase the
superoxide scavenging abilities of the hepato-
pancreas quickly during arousal, again indi-
cating that ROS generation probably in-
creases rapidly during the early minutes of
arousal and suggesting also that SOD activ-
ity is the limiting enzymatic activity in the
disposal of ROS in the tissue.
Concluding remarks
Biochemical adaptations have permitted
many animal species to endure a variety of
situations where oxygen availability to tissues
is highly variable including numerous stresses
that impose anoxia or ischemia. Hence, many
species deal naturally with rapid transitions
from situations of little or no oxygen to high
rates of oxygen uptake and consumption, con-
ditions that are associated with a rapid in-
crease in the production of reactive oxygen
species. Although oxidative damage is clearly
a problem in situations such as ischemia-re-
perfusion in mammals, animals that deal with
similar stresses naturally have developed meta-
bolic adjustments to deal with oxidative stress
and incorporated these biochemical adapta-
tions as part of their strategy for survival.
Animals that repeatedly experience prolonged
periods of anoxia or ischemia appear to deal
with the problem of oxidative stress during
recovery by maintaining a high antioxidant
defense capacity at all times including both
elevated activities of antioxidant enzymes and
large cellular pools of glutathione. Where oxi-
dative stress is a more intermittent phenome-
non, the adaptive strategy appears to be el-
evated antioxidant defenses during the low
oxygen stress in anticipation of a rapid in-
crease in reactive oxygen species formation
during the recovery from stress. The data from
animal models make it clear that adaptations
of antioxidant defenses are a fundamental and
widespread requirement for all animals that
deal with natural variation in oxygen availabil-
ity, and they show that these adaptations are
just as important and integral to overall animal
survival as are the well-studied biochemical
adaptations of fermentative metabolism and
metabolic rate depression that ensure survival
under oxygen restriction.
Acknowledgments
Thanks are due to recent members of my
laboratory, including M. Hermes-Lima, W.
Willmore, and D. Joanisse for their contribu-
tions to the research reviewed here, and to
J.M. Storey for assistance in the preparation
of the manuscript.

References

1. Halliwell B (1992). Reactive oxygen spe-
cies and the central nervous system. Jour-
nal of Neurochemistry, 59: 1609-1623.
2. Jones DP (1986). Renal metabolism dur-
ing normoxia, hypoxia, and ischemic in-
jury. Annual Review of Physiology, 48:
33-50.
3. Halliwell B & Gutteridge JMC (1989). Free
Radicals in Biology and Medicine.
Clarendon Press, UK.
4. Kleinveld HA, Swaak AJG, Hack CE &
Koster JF (1989). Interactions between
oxygen free radicals and proteins. Scandi-
navian Journal of Rheumatology, 18: 341-
352.
5. Turrens JF, Freeman BA, Levitt JG &
Crapo JD (1982). The effect of hyperoxia
on superoxide production by lung submi-
tochondrial particles. Archives of Bio-
chemistry and Biophysics, 217: 401-410.
6. Konstantinov AA, Peskin AV, Popova EY,
Khomutov GB & Ruuge EK (1987). Super-
oxide generation by the respiratory chain
of tumor mitochondria. Biochimica et Bio-
physica Acta, 894: 1-10.
7. Stadtman ER (1992). Protein oxidation and
aging. Science, 257: 1220-1224.
8. Sies H (1993). Strategies of antioxidant
defense. European Journal of Biochemis-
try, 215: 213-219.
9. Fuller BJ, Gower JD & Gree CJ (1988).
Free radical damage and organ preserva-
tion: fact or fiction. A review of the inter-
relationship between oxidative stress and
physiological ion disbalance. Cryobiology,
25: 377-393.
10. Balla G, Jacob HS, Balla J, Rosenberg M,
Nath K, Apple F, Eaton JW & Vercellotti
GM (1992). Ferritin: a cytoprotective anti-
oxidant strategem of endothelium. Jour-
nal of Biological Chemistry, 267: 18148-
18153.
11. Cadenas E (1989). Biochemistry of oxy-
gen toxicity. Annual Review of Biochem-
istry, 58: 79-110.

Braz J Med Biol Res 29(12) 1996

1732
12. Reddy RC, Scholz RW, Thomas CE &
Massaro EJ (1982). Vitamin E dependent
reduced glutathione inhibition of rat liver
microsomal lipid peroxidation. Life Sci-
ences, 31: 571-579.
13. Simmons TW & Jamall IS (1988). Signifi-
cance of alterations in hepatic antioxidant
enzymes, primacy of glutathione peroxi-
dase. Biochemistry Journal, 251: 913-917.
14. Raes M, Michels C & Remacle J (1987).
Comparative study of the enzymatic de-
fense systems against oxygen-derived
free radicals: the key role of glutathione
peroxidase. Free Radical Biology and
Medicine, 3: 3-7.
15. Ahmad S, Beilstein MA & Pardini RS
(1989). Glutathione peroxidase activity in
insects: a reassessment. Archives of Insect
Biochemistry and Physiology, 12: 31-49.
16. Storz G, Jacobson FS, Tartaglia LA, Mor-
gan RW, Silveira LA & Ames BN (1980).
An alkyl hydroperoxide reductase induced
by oxidative stress in Salmonella typhi-
murium and Escherichia coli : genetic char-
acterization and cloning of ahp. Journal of
Bacteriology, 171: 2049-2055.
17. Jacobson FS, Morgan RW, Christman MF
& Ames BN (1989). An alkyl hydroperox-
ide reductase from Salmonella typhimurium
involved in the defense of DNA against
oxidative damage. Journal of Biological
Chemistry, 264: 1488-1496.
18. Halliwell B, Gutteridge JMC & Cross CE
(1992). Free radicals, antioxidants, and hu-
man disease: Where are we now? Jour-
nal of Laboratory and Clinical Medicine,
119: 598-620.
19. Traystman RJ, Kirsch JR & Koehler RC
(1991). Oxygen radical mechanism of
brain injury following ischemia and reper-
fusion. Journal of Applied Physiology, 71:
1185-1195.
20. Hochachka PW (1986). Balancing conflict-
ing metabolic demands of exercise and
diving. Federation Proceedings, 45: 2948-
2952.
21. McCord JM (1985). Oxygen-derived free
radicals in post-ischemic tissue injury.
New England Journal of Medicine, 312:
159-163.
22. Dawson TL, Gores GJ, Nieminen A-L,
Herman B & Lemasters JJ (1993). Mito-
chondria as a source of reactive oxygen
species during reductive stress in rat
hepatocytes. American Journal of Physi-
ology, 264: C961-C967.
23. Rifkind JM, Abugo O, Levy A, Monticone
R & Heim J (1993). Formation of free
radicals under hypoxia. In: Hochachka PW,
Lutz PL, Sick TJ, Rosenthal M & van den
Thillart G (Editors), Surviving Hypoxia:
Mechanisms of Control and Adaptation.
CRC Press, Boca Raton.
Braz J Med Biol Res 29(12) 1996
24. Greene EL & Paller MS (1992). Xanthine
oxidase produces O-2 in posthypoxic injury
of renal epithelial cells. American Journal
of Physiology, 263: F251-F255.
25. Ayene IS, Al-Mehdi AB & Fisher AB
(1993). Inhibition of lung tissue oxidation
during ischemia/reperfusion by 2-mercap-
to-propionylglycine. Archives of Biochem-
istry and Biophysics, 303: 307-312.
26. Rigobello MP & Bindoli A (1993). Effect of
pyruvate on rat heart thiol status during
ischemia and hypoxia followed by reper-
fusion. Molecular and Cellular Biochemis-
try, 122: 93-100.
27. Whiteley GS, Fuller BJ & Hobbs KEF
(1992). Deterioration of cold-stored tissue
specimens due to lipid peroxidation:
modulation by antioxidants at high sub-
zero temperatures. Cryobiology, 29: 668-
673.
28. Lutz PL & Storey KB (1996). Adaptations
to variations in oxygen tension by verte-
brates and invertebrates. In: Lutz PL &
Storey KB (Editors), Handbook of Com-
parative Physiology. CRC Press, Boca
Raton.
29. Storey KB (1993). Molecular mechanisms
of metabolic arrest in mollusks. In:
Hochachka PW, Lutz PL, Sick TJ,
Rosenthal M & van den Thillart G (Edi-
tors), Surviving Hypoxia: Mechanisms of
Control and Adaptation. CRC Press, Boca
Raton.
30. Ultsch GR (1989). Ecology and physiology
of hibernation and overwintering among
freshwater fishes, turtles and snakes. Bio-
logical Reviews, 64: 435-516.
31. Storey KB (1996). Metabolic adaptations
supporting anoxia tolerance in reptiles:
recent advances. Comparative Biochem-
istry and Physiology B, 113: 23-35.
32. Storey KB, Mosser DD, Douglas DN,
Grundy JE & Storey JM (1996). Biochem-
istry below OoC: nature’s frozen verte-
brates. Brazilian Journal of Medical and
Biological Research, 29: 293-307.
33. Loveridge JP & Withers PC (1981). Me-
tabolism and water balance of active and
cocooned African bullfrogs Pyxicephalus
adspersus. Physiological Zoology, 54:
203-214.
34. Barnhart MC (1987). Discontinuous car-
bon dioxide release and metabolic depres-
sion in dormant land snails. Journal of
Experimental Biology, 128: 123-138.
35. Pinder AW, Storey KB & Ultsch GR (1992).
Estivation and hibernation. In: Feder ME
& Burggren WW (Editors), Environmental
Biology of the Amphibia. University of
Chicago Press, Chicago.
36. Hochachka PW & Somero GN (1984). Bio-
chemical Adaptation. Princeton University
Press, Princeton.
K.B. Storey
37. Wang LCH (1989). Ecological, physiologi-
cal, and biochemical aspects of torpor in
mammals and birds. In: Wang LCH (Edi-
tor), Advances in Comparative and Envi-
ronmental Physiology. Vol. 4. Springer-
Verlag, Heidelberg.
38. Storey KB & Storey JM (1990). Faculta-
tive metabolic rate depression: molecular
regulation and biochemical adaptation in
anaerobiosis, hibernation, and estivation.
Quarterly Review of Biology, 65: 145-174.
39. Orrenius SD, McConkey DJ, Bellomo G &
Nicotera P (1990). Role of Ca2+ in toxic
cell killing. Trends in Pharmacological Sci-
ences, 10: 281-285.
40. Geeraerts MD, Ronveaux-Dupal M-F,
Lemasters JJ & Herman B (1991). Cyto-
solic free Ca2+ and proteolysis in lethal
oxidative injury in endothelial cells. Ameri-
can Journal of Physiology, 261: C889-
C896.
41. Bindoli A (1988). Lipid peroxidation in mi-
tochondria. Free Radical Biology and
Medicine, 5: 247-261.
42. Kunio Y, Komura S, Ihara N, Abe H,
Konishi H & Arichi S (1985). Serum lipid
peroxide levels in rats with inherited cata-
racts. Journal of Applied Physiology, 7:
202-206.
43. Wade CR, Jackson PG, Highton J & Van
Rij AM (1987). Lipid peroxidation and
malonaldehyde in the synovial fluid and
plasma of patients with rheumatoid arthri-
tis. Clinica Chimica Acta, 164: 245-250.
44. Halliwell B & Chirico S (1993). Lipid
peroxidation: its mechanism, measure-
ment, and significance. American Journal
of Clinical Nutrition, 57 (Suppl): 715S-
725S.
45. Slater TF (1984). Overview of methods
used for detecting lipid peroxidation.
Methods in Enzymology, 105: 283-293.
46. Cramer GL, Miller JF, Pendleton RB &
Lands WEM (1991). Iodometric measure-
ment of lipid hydroperoxides in human
plasma. Analytical Biochemistry, 193:
204-211.
47. Uchiyama M & Mihara M (1978). Determi-
nation of malonaldehyde in tissues by
thiobarbituric acid test. Analytical Bio-
chemistry, 86: 271-278.
48. Smith CV & Anderson RE (1987). Meth-
ods for determination of lipid peroxidation
in biological samples. Free Radical Biol-
ogy and Medicine, 3: 341-344.
49. Bird RP & Draper HH (1984). Comparative
studies on different methods of malonal-
dehyde determination. Methods in Enzy-
mology, 105: 299-305.
50. Bonnes-Taourel D, Guerrin M-C &
Torreilles J (1992). Is malonaldehyde a
valuable indicator of lipid peroxidation?
Biochemical Pharmacology, 44: 985-988.
Animal adaptations for oxidative stress
51. Lapanna D & Cuccurullo F (1993). TBA
test and “free” MDA assay in evaluation
of lipid peroxidation and oxidative stress
in tissue systems. American Journal of
Physiology, 265: H1030-H1031.
52. Hermes-Lima M, Willmore W & Storey
KB (1995). Quantification of peroxides in
animal tissues based on Fe(II)-xylenol or-
ange formation. Free Radical Biology and
Medicine, 19: 271-280.
53. Jiang Z-Y, Woollard ACS & Wolf SP
(1991). Lipid hydroperoxide measurement
by oxidation of Fe2+ in the presence of
xylenol orange. Comparison with the TBA
assay and an iodometric method. Lipids,
26: 853-856.
54. Jiang Z-Y, Hunt JV & Wolf SP (1992). Fer-
rous ion oxidation in the presence of
xylenol orange for detection of lipid hy-
droperoxide in low density lipoprotein. An-
alytical Biochemistry, 202: 384-389.
55. Michaels HB & Hunt JW (1978). Determi-
nation of peroxides and hydroperoxides in
irradiated solutions of nucleic acid con-
stituents and DNA. Analytical Biochemis-
try, 87: 135-140.
56. Corongiu FP & Milia A (1983). An im-
proved method for determining diene
conjugation in autoxidized polyunsatu-
rated fatty acids. Chemistry and Biology
Interactions, 44: 289-297.
57. Joanisse DR & Storey KB (1996). Oxida-
tive damage and antioxidants in Rana
sylvatica, the freeze-tolerant wood frog.
American Journal of Physiology (in press).
58. Herreid CF (1977). Metabolism of land
snails (Otala lactea) during dormancy,
arousal, and activity. Comparative Bio-
chemistry and Physiology A, 56: 211-215.
59. Hermes-Lima M & Storey KB (1995). Anti-
oxidant defenses and metabolic depres-
sion in a pulmonate land snail. American
Journal of Physiology, 268: R1386-R1393.
60. Dwaliwal H, Kirshenbaum LA, Randhawa
AK & Singal PK (1991). Correlation be-
tween antioxidant changes during hypoxia
and recovery on reoxygenation. American
Journal of Physiology, 261: H632-H638.
61. Barja de Quiroga G (1992). Brown fat ther-
mogenesis and exercise: two examples
of physiological oxidative stress? Free
Radical Biology and Medicine, 13: 325-
340.
62. Ji LL & Fu R (1992). Responses of glu-
tathione system and antioxidant enzymes
to exhaustive exercise and hydroperox-
ide. Journal of Applied Physiology, 72:
549-554.
63. Hermes-Lima M & Storey KB (1995). Xan-
thine oxidase and xanthine dehydrogen-
ase from an estivating snail. A possible
role in the induction of oxidative stress.
Zeitschrift für Naturforschung. C: Bio-
sciences, 50: 685-694.
64. Speeg KV & Campbell JW (1968). Purine
biosynthesis and excretion in Otala (= He-
lix) lactea: an evaluation of the nitrogen
excretory potential. Comparative Bio-
chemistry and Physiology, 26: 579-595.
65. Meister A & Anderson ME (1983). Glu-
tathione. Annual Review of Biochemistry,
52: 711-760.
66. Deneke SM & Fanburg BL (1989). Regula-
tion of cellular glutathione. American Jour-
nal of Physiology, 257: L163-L173.
67. Jones DJ, Eklow L, Thor H & Orrenius S
(1981). Metabolism of hydrogen peroxide
in isolated hepatocytes: relative contribu-
tion of catalase and glutathione peroxi-
dase in decomposition of endogenous
generated H2O2. Archives of Biochemis-
try and Biophysics, 210: 505-516.
68. Tan KH, Meyer DJ, Coles B & Ketterer B
(1986). Thymine hydroperoxide, a sub-
strate for rat Se-dependent glutathione
peroxidase and glutathione transferase
isoenzymes. FEBS Letters, 207: 231-233.
69. Tan KH, Meyer DJ, Coles B, Gillies N &
Ketterer B (1987). Detoxication of
peroxidized DNA by glutathione trans-
ferases. Biochemical Society Transac-
tions, 15: 628-629.
70. Lopez-Torres M, Perez-Campo R,
Cadenas S, Rojas C & Barja G (1993). A
comparative study of free radicals in ver-
tebrates. II. Non-enzymatic antioxidants
and oxidative stress. Comparative Bio-
chemistry and Physiology B, 105: 757-
763.
71. Reischl E (1986). High sulfhydryl content
in turtle erythrocytes: is there a relation
with resistance to hypoxia? Comparative
Biochemistry and Physiology B, 85: 723-
726.
72. Lutz PL, McMahon P, Rosenthal M & Sick
TJ (1984). Relationships between aerobic
and anaerobic energy production in turtle
brain in situ. American Journal of Physiol-
ogy, 247: R240-R247.
73. Perez-Campo R, Lopez-Torres M & Barja
de Quiroga G (1990). Thermal acclima-
tion, hydroperoxide detoxifying enzymes
and oxidative stress in the lung and liver
of Rana perezi. Journal of Thermal Biol-
ogy, 15: 193-199.
1733
74. Buzadzic B, Spasic MB, Saicic ZS,
Radojicic R, Halliwell B & Petrovic VM
(1990). Antioxidant defenses in the
ground squirrel Citellus citellus. 1. A com-
parison with the rat. Free Radical Biology
and Medicine, 9: 401-406.
75. Griffith OW & Meister A (1979). Glu-
tathione: interorgan translocation, turn-
over and metabolism. Proceedings of the
National Academy of Sciences, USA, 76:
5606-5610.
76. Haberle D, Wahlander A & Sies H (1979).
Assessment of the kidney function in
maintenance of plasma glutathione con-
centration and redox state in anaesthe-
tized rats. FEBS Letters, 108: 335-340.
77. Storey KB & Storey JM (1992). Natural
freeze tolerance in ectothermic verte-
brates. Annual Review of Physiology, 54:
619-637.
78. Churchill TA & Storey KB (1992). Freezing
survival of the garter snake Thamnophis
sirtalis parietalis. Canadian Journal of Zo-
ology, 70: 99-105.
79. Hermes-Lima M & Storey KB (1993). Anti-
oxidant defenses in the tolerance of freez-
ing and anoxia by garter snakes. Ameri-
can Journal of Physiology, 265: R646-
R652.
80. Frank L & Sosenko IRS (1987). Prenatal
development of lung antioxidant enzymes
in four species. Journal of Pediatrics, 110:
106-110.
81. McElroy MC, Postle AD & Kelly FJ (1992).
Catalase, superoxide dismutase and glu-
tathione peroxidase activities of lung and
liver during human development. Bio-
chimica et Biophysica Acta, 1117: 153-
158.
82. Buzadzic B, Spasic MB, Saicic ZS,
Radojicic R, Petrovic VM & Halliwell B
(1990). Antioxidant defenses in the
ground squirrel Citellus citellus. 2. The
effect of hibernation. Free Radical Biol-
ogy and Medicine, 9: 407-413.
83. Barja de Quiroga G, Lopez-Torres M,
Perez-Campo R, Abelenda M, Paz Nava M
& Puerta ML (1991). Effect of cold accli-
mation on GSH, antioxidant enzymes and
lipid peroxidation in brown adipose tis-
sue. Biochemical Journal, 277: 289-292.
84. Whitwam RE & Storey KB (1990). Pyru-
vate kinase from the land snail Otala
lactea: regulation by reversible phospho-
rylation during estivation and anoxia. Jour-
nal of Experimental Biology, 154: 321-337.
85. Brooks SPJ & Storey KB (1992). Proper-
ties of pyruvate dehydrogenase from the
land snail, Otala lactea: control of enzyme
activity during estivation. Physiological Zo-
ology, 65: 620-633.
Braz J Med Biol Res 29(12) 1996