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REVIEW
An assessment of techniques to manipulate oxidative
stress in animals
Rebecca E. Koch* and Geoffrey E. Hill
Department of Biological Sciences, Auburn University, 101 Life Sciences Hall, Auburn, AL 36830, USA
Summary
1. Physiological ecologists require techniques for controlled oxidative challenges in live ani-
mals to facilitate the study of oxidative stress.
2. Techniques for manipulating oxidative stress include agents that increase generation of
pro-oxidants, such as paraquat, diquat, radiation, heavy metals, dietary oxidized lipids, and
tert-butyl-hydroperoxide, as well as genetic (RNAi) and chemical (buthionine sulfoximine)
knock-downs that target specific antioxidants.
3. We critically assess both currently used and potentially useful methods for inducing sys-
temic oxidative challenge in animals. We provide a resource for biologists to select the most
robust methods for oxidative challenge in their study system, to improve interpretation of
results within the context of cellular mechanisms and to maximize effectiveness of experiments
while minimizing unintended side effects.
Key-words: antioxidants, experimental challenge, oxidative damage, paraquat, reactive oxy-
gen species
Introduction
Free radical biology was once the exclusive domain of bio-
chemists and biomedical researchers, but beginning with
the publication of an influential paper by von Schantz
et al. (1999), physiological ecologists began to consider the
role of free radicals in a host of biological processes,
including life-history evolution, condition-dependent sig-
nalling and senescence (Costantini 2008, 2014; Monaghan,
Metcalfe & Torres 2009; McGraw et al. 2010; Hill & John-
son 2012). High concentrations of pro-oxidants, most
prominently including reactive oxygen species (ROS), have
negative effects on fundamental cellular processes because
they compromise genomic stability and disrupt protein or
lipid structures. The capacity to resist and recover from
states of increased oxidative stress can be a central compo-
nent of an animal’s fitness.
Pro-oxidant molecules, such as ROS produced during
oxidative phosphorylation (OXPHOS) and the activity of
redox enzymes, are more than damaging by-products: they
serve as essential signalling molecules that regulate mito-
chondrial activity and other processes (Seifried et al. 2007;
Valko et al. 2007; Sena & Chandel 2012). In a healthy
organism, oxidative damage from pro-oxidants is largely
held in check by the activity of antioxidants. Under states
of increased oxidative stress, however, pro-oxidant mole-
cules are generated at a rate that exceeds the capacity for
antioxidant molecules to mitigate their effects (Scherz-
Shouval & Elazar 2007). Importantly, there is individual
variation in susceptibility to oxidative stress such that
some individuals appear better able to avoid oxidative
damage and its accompanying harmful effects, even when
faced with stress-inducing environmental challenges (Mon-
aghan, Metcalfe & Torres 2009). Discovering the causes
and consequences of this variability is a main focus of
oxidative stress ecology.
To disentangle the effects of oxidative stress from the
confounding effects of other physiological processes,
researchers have looked for methods to experimentally
manipulate levels of oxidative stress by performing oxida-
tive challenges. The ideal oxidative challenge is a relatively
simple and cost-effective treatment that is accessible to and
safe for use by organismal biologists outside of the
biomedical sciences. Perhaps most critically, effective chal-
lenges must induce a measurable change in oxidative stress
without causing significant side effects. As the field of
oxidative stress ecology continues to expand, there is a
need for a critical assessment of techniques for manipula-
tion of oxidative stress.
In this review, we assess the pros and cons of different
methods that have been shown or that show promise to be
*Correspondence author. E-mail: rek0005@auburn.edu
©2016 The Authors. Functional Ecology ©2016 British Ecological Society
Functional Ecology 2016 doi: 10.1111/1365-2435.12664
used to manipulate oxidative stress in live animals, particu-
larly non-model vertebrate species. As we assess tech-
niques, we emphasize the mechanism of action of each
potential oxidative challenge with the purpose of describ-
ing the effects that may make a treatment more or less
suitable for particular experimental designs, and we
describe potential confounding side effects of the challenge
that may occur independently of oxidative stress pathways
and interfere with results interpretation. The primary goal
of this study was to provide a resource for organismal
biologists and ecologists that highlights the complex effects
of oxidative challenges used to experimentally study oxida-
tive stress in animals.
Pro-oxidant generators
Because oxidative stress depends on both the rate of pro-
oxidant generation and the counteracting effects of antioxi-
dants, experimental oxidative challenges can target either
production of ROS or suppression of antioxidants. Treat-
ments that increase ROS production are particularly useful
in studies that aim to quantify individual ability to defend
against pro-oxidants while possessing their full suite of
antioxidant defences. However, by their nature, substances
that increase ROS levels tend also to increase the risk of
non-targeted tissue damage or death of experimental sub-
jects and may pose additional risks to researchers perform-
ing the administration. It is therefore critical to consider
the specific biochemical means of effect, the clinical symp-
toms of exposure and the potential side effects of methods
that increase pro-oxidant generation.
PARAQUAT
One of the most common means to induce oxidative stress
via increased ROS production in vertebrate species is
ingestion or injection of paraquat (1,10-dimethyl-4,40-bipyr-
idinium dichloride), an herbicide that is well studied
because of its toxic effects on humans following accidental
exposure (Suntres 2002; Dinis-Oliveira et al. 2008). Once
inside the body, paraquat spreads rapidly and causes
effects largely through ROS generation via redox cycling
and subsequent disruption of redox pathways (Dinis-Oli-
veira et al. 2008; Table 1; see Appendix S1 and Fig. S1 in
Supporting Information). Because it rapidly increases ROS
generation and oxidative damage in the same redox sys-
tems involved in naturally induced states of increased
oxidative stress, paraquat is a promising option for an
oxidative challenge; however, researchers should be mind-
ful of the potential for selective impacts on organs, partic-
ularly the lungs and brain (Appendix S1). As a result of
harmful clinical effects to exposed wildlife and agricultural
workers, the use of paraquat in agriculture has been
banned or discouraged in a number of European countries
(InfoCuria 2007; Kerv
egant et al. 2013). However, para-
quat may yet be an effective tool within oxidative stress
ecology when paired with careful use of protective equip-
ment for personal safety and thorough use of pilot studies
to determine doses that induce increased ROS production
without causing organ failure or fatality.
Surprisingly little is known about the effects of low
doses of paraquat administered in an experimental setting
because most biomedical studies have examined model sys-
tems receiving high doses to mimic acute paraquat poison-
ing (e.g. Fukushima et al. 1994; Tawara et al. 1996) or to
induce the symptoms of diseases associated with exception-
ally high oxidative stress (e.g. Andreyev, Kushnareva &
Starkov 2005; Cicchetti et al. 2005; McCormack et al.
2005; Gomez, Bandez & Navarro 2006). Because of inter-
est in determining the ecological effects of agricultural
paraquat use, the effects of low doses of paraquat on non-
mammalian and non-model systems are perhaps best
understood for wild game birds (reviewed in Hoffman
et al. 1987; Table 2) and fish (Gabryelak & Klekot 1985;
Parvez & Raisuddin 2006). Importantly, these studies
reveal wide variation in susceptibility to paraquat exposure
depending on dose, species and duration of exposure. It is
therefore important that researchers perform pilot studies
on their own species to identify experimental designs that
are effective –and ethical –in inducing a controlled
increase in ROS generation.
As of February 2016, paraquat had been used in five
ecologically oriented experimental studies, all on bird spe-
cies (Galvani et al. 2000; Isaksson & Andersson 2008;
Meitern et al. 2013; Lucas, Morales & Velando 2014; Gir-
audeau et al. 2015; Table 2), which varied widely in both
their experimental approaches and their findings. Two of
these studies used dosage trials to find a concentration of
paraquat that caused no apparent clinical symptoms in
their focal species (Isaksson & Andersson 2008; Lucas,
Morales & Velando 2014); one study chose an arbitrary
low dosage well below the species’ LD-50 (Galvani et al.
2000); and, two based their doses on that of Isaksson &
Andersson (2008; Meitern et al. 2013; Giraudeau et al.
2015). Giraudeau et al. (2015) also tested the dose on three
individuals of their study species prior to the start of the
main experiment. Unfortunately, it is difficult to draw con-
clusions from these studies about the effectiveness of para-
quat as an oxidative challenge because the findings are
generally mixed (Table 2). It is often unclear whether a
lack of significant treatment effect is due to an ineffective
oxidative challenge (too low or too high a dose) or due to
a true lack of relationship between oxidative stress and the
focal variables.
For instance, Isaksson & Andersson (2008) were the first
to introduce the use of paraquat as an oxidative challenge
to the field of behavioural ecology. They exposed juvenile
great tits (Parus major) to paraquat in the drinking water
and found no relationship between paraquat exposure and
concentration of carotenoid pigments in plasma or feath-
ers. They concluded that their experiment provided evi-
dence against the widespread hypothesis that oxidative
stress depletes circulating carotenoids and hence reduces
the coloration of carotenoid-based ornaments (Møller
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
2R. E. Koch & G. E. Hill
et al. 2000; Alonso-Alvarez et al. 2008; Perez-Rodriguez
2009; Perez-Rodriguez, Mougeot & Alonso-Alvarez 2010;
Alonso-Alvarez & Galvan 2011; but see Costantini & Møl-
ler 2008; Hartley & Kennedy 2004). While this study was a
pivotal first step in the use of chemical challenges within
oxidative stress ecology, it did not incorporate measures of
oxidative stress into the experiment, so it is difficult to
parse how antioxidant capacity or oxidative damage levels
may have been affected in the tissues important to carote-
noid metabolism and coloration.
Table 1. Summary of the potential oxidative challenges discussed within this review, including their desired effects and some potential
problems to consider before their use in ecological experiments.
Challenge Target effect Method(s) of dosing
Main mechanism for
increased oxidative
stress Other effects Potential problems
Paraquat ROS
generation
Ingestion, injection,
dermal application,
environmental
water exposure
(aquatic organisms)
Redox cycling: Accepts
electron from strong
reducing enzyme,
passes electron to
oxygen to form
superoxide anion and
regenerate oxidized
form (Fig. S1)
Disrupts redox
signalling through
oxidative damage and
by accepting electrons
meant for other
functions
Causes localized damage to
lungs; interacts with
microglia to induce
increased neural damage,
particularly to
dopaminergic neurons;
researchers must take
precautions to prevent
exposure
Diquat ROS
generation
Same as paraquat Same as paraquat Same as paraquat More reactive –and
potentially more
damaging –than
paraquat; researchers
must take precautions to
prevent exposure
Heavy metals ROS
generation
Ingestion, injection,
environmental
water exposure
(aquatic organisms)
Can generate ROS
through redox
reactions, or interact
with antioxidant
structures to inhibit
function
Reacts with and
compromises cellular
pathways
independently of
oxidative stress
Organ-specific toxicity;
oxidant-independent
effects
Ionizing
radiation
ROS
generation
Exposure in
biomedical setting
Forms ROS from
ionizing water found
in the body
Also damages molecular
structures
Oxidant-independent
effects; bodywide
induction of ROS may
not represent an oxidative
stress response
experienced in natural
conditions
tBHP ROS
generation
Environmental
water exposure
(aquatic
organisms),
injection
Acts as a pro-oxidant
similarly to H
2
O
2
Interacts with
mitochondrial
permeability transition
pore
The specific locations of
tBHP activity remain
largely unexplored
Oxidized
dietary
lipids
ROS
generation
Ingestion Ingested oxidized lipids
cause oxidative
damage through
further reactions
May compromise lipid
assimilation, interfere
with structures
independently of ox.
stress
Response varies greatly
among species with
changing consumption
and absorption; non-
target effects on lipid
absorption and cellular
structures may confound
results
BSO Reduced
glutathione
levels
Injection Binds to glutathione
synthetase to inhibit
glutathione production
Affects activity of other
antioxidants that rely
on glutathione
pathways
Not appropriate for studies
requiring activity of all
naturally occurring
antioxidants
RNAi Knocked-
down
antioxidant
gene
expression
Injection Degrades mRNA
related to expression
of a specific
antioxidant
None Requires development of
specific RNAi, not
currently available for
most vertebrate species;
not appropriate for
studies requiring activity
of all naturally occurring
antioxidants
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
Assessment of oxidative stress challenges 3
Table 2. Summary of the experimental parameters and results of studies involving controlled paraquat exposure in bird species, the taxa most commonly involved in paraquat experiments within
ecology. Doses are expressed in g paraquat per L drinking water (g L
1
), mg paraquat per kg animal body weight (mg kg
1
) or ppm (paraquat parts per million parts substrate).
Species Dose(s) Administration Duration Symptoms Source(s)
Mallard (Anas
platyrhynchos)
1048 ppm Diet 5 days 50% mortality Heath et al. (1972), Hill
& Camardese (1986)
American kestrel (Falco
sparverius)
10, 25 or 60 mg kg
1
Oral dose 10 days Reduced skeletal growth in all doses; 44%
dead within 4 days on highest dose
Hoffman et al. (1987)
Northern bobwhite
(Colinus virginianus)
981 ppm Diet 5 days 50% mortality Heath et al. (1972), Hill
& Camardese (1986)
25 or 100 ppm Drinking water 60 days No clinical symptoms in either dose Bunck, Bunck & Sileo
(1986)
Japanese quail
(Coturnix japonica)
970 ppm Diet 5 days 50% mortality Heath et al. (1972), Hill
& Camardese (1986)
100 ppm Drinking water 7 days 100% mortality Paulov (1977)
10 mg kg
1
Intraperitoneal injection 7 days Increased glutathione peroxidase activity and
decreased glutathione levels in blood;
increased lipid peroxidation (MDA) in lung
but not blood
Galvani et al. (2000)
Ring-necked pheasant
(Phasianus colchicus)
1468 ppm Diet 5 days 50% mortality Heath et al. (1972), Hill
& Camardese (1986)
Domestic turkey
(Meleagris gallopavo)
100 mg kg
1
Intraperitoneal injection Single dose 50% mortality; anorexia, diarrhoea,
inactivity
Smalley (1973)
20 mg kg
1
Intravenous injection Single dose
290 mg kg
1
Oral dose Single dose
500 mg kg
1
Dermal application Single dose
Domestic chicken
(Gallus gallus)
262 mg kg
1
Diet Single dose 50% mortality Clark, McElligott &
Hurst (1966)
40 ppm Drinking water 14 days Increased numbers of eggs did not hatch or
produced abnormal chicks
Fletcher (1967)
Yellow-legged gull
(Larus michahellis)
23mgkg
1
Oral dose Single dose Higher total antioxidant capacity, but no
change in lipid peroxidation (MDA),
protein carbonyl levels, total reactive
oxygen metabolites or mass; all oxidative
stress assays were blood-based
Lucas, Morales &
Velando (2014)
Great tit (Parus major)15, 075, 038, 019
or 009 g L
1
Drinking water 6 weeks All but lowest dose were lethal or caused
clinical symptoms of distress (mass loss,
decreased activity); lowest dose had no
effect on plasma carotenoid concentrations
or ornamental plumage coloration
Isaksson & Andersson
(2008)
(continued)
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
4R. E. Koch & G. E. Hill
Several years later, Meitern et al. (2013) exposed Euro-
pean greenfinches (Carduelis chloris) to either the same or
twice the dose of paraquat previously used by Isaksson &
Andersson (2008), and they measured multiple aspects of
oxidative damage and antioxidant capacity. However,
50% of the finches on the higher of the two doses died
during the experiment. Those birds surviving the high dose
had increased levels of DNA damage and the antioxidant
glutathione in erythrocytes, but no significant change in
any of six other measurements related to oxidative stress;
none of the birds on the low dose had any measurable
change in oxidative damage or antioxidant measurements
(Table 2). While it appears that the lower of the two doses
(identical to that used on great tits in Isaksson & Ander-
sson 2008) was insufficient to cause a measurable response
in greenfinches, the higher dose was evidently too high for
this species, causing the death of birds that were unable to
withstand the paraquat challenge. It is uncertain whether
the surviving birds on the higher dose had generally non-
significant measurements of oxidative stress because they
recovered from the challenge before blood was collected,
because they underwent a hormetic response to the
increased ROS exposure, or because these measures never
were elevated due to treatment. While the study of Meitern
et al. (2013) is a valuable addition to the literature on
oxidative stress ecology, performing a pilot study to isolate
a suitable dose for this species prior to experimentation
may have both prevented unintended bird mortality and
increased the quality and quantity of data.
The primary challenges with using paraquat to induce
oxidative stress in birds and other non-model species are
1) finding an appropriate dose and 2) accounting for non-
target effects, such as accumulation in lungs or toxicity at
low doses (Table 2). Paraquat may be a useful tool for
inducing increased oxidative stress, but researchers should
carefully consider the effects of dose, means of dosage and
exposure duration of treatment on their study species –as
well as proper use of protective equipment to minimize
risk of exposure to the researchers themselves –before
using the technique.
DIQUAT
Diquat (1,10-ethylene-2,20-bipyridylium; Table 1) is similar
in both structure and function to paraquat and has also
been used to induce increased ROS generation in animals
(Sewalk, Brewer & Hoffman 2000; Xu et al. 2007; Alonso-
Alvarez & Galvan 2011). Because diquat is not commonly
used in agriculture, exposure to humans, wildlife and live-
stock is infrequent and its toxic effects are less well studied,
though they generally have been found to be comparable
to the effects of paraquat (Gage 1968; Rawlings, Wyatt &
Heylings 1994; Jones & Vale 2000; Drechsel & Patel 2009).
Clinically, the main difference between acute paraquat and
diquat poisoning is that diquat does not accumulate in the
lungs, and instead primarily causes damage to the kidneys
and liver (Rose, Smith & Wyatt 1974; Jones & Vale 2000).
Table 2 (continued)
Species Dose(s) Administration Duration Symptoms Source(s)
European greenfinch
(Carduelis chloris)
01or02gL
1
Drinking water 7 days Significant mass loss in both doses; 50%
mortality in higher dose; in higher dose:
higher levels of DNA damage (comet assay)
and glutathione, but no change in levels of
protein carbonyls, lipid peroxidation
(MDA), overall antioxidant protection
(TAC and OXY), uric acid or carotenoids;
no oxidative stress effects on low dose; all
oxidative stress assays were blood-based.
Meitern et al. (2013)
House finch
(Haemorhous mexicanus)
01gL
1
Drinking water 28 days Significant mass loss in all treated birds;
increase in oxidative damage (TBARS) and
trend towards decrease in activity levels in
birds supplemented with carotenoids, but
not unsupplemented birds; no effect on
plumage hue or plasma carotenoid levels.
Giraudeau et al. (2015)
MDA, malondialdehyde; TAC, total antioxidant capacity; OXY, oxygen radical absorbance; TBARS, thiobarbituric acid-reactive substances.
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
Assessment of oxidative stress challenges 5
For this reason, diquat may be a useful alternative if
experimental subjects are found to be particularly prone to
paraquat-caused respiratory damage.
At the cellular level, diquat induces ROS generation by
the same redox cycling reactions that characterize para-
quat (Wong & Stevens 1986; Rawlings, Wyatt & Heylings
1994; Drechsel & Patel 2009). However, diquat has a lower
activation energy than paraquat, so redox cycling is more
readily triggered in diquat than in paraquat. As a conse-
quence, diquat can cause stronger effects more quickly and
at lower doses (Baldwin et al. 1975; Witschi et al. 1977;
Suleiman & Stevens 1986; Drechsel & Patel 2009). Thus, if
diquat is used as an agent to induce oxidative stress in live
animals, it is especially important to determine an appro-
priate dosing treatment. Accordingly, Galv
an & Alonso-
Alvarez (2009) and Alonso-Alvarez & Galvan (2011) per-
formed thorough dosage trials prior to experiments with
diquat, and they were able to minimize negative side effects
while still inducing an increase in oxidative stress in their
animal subjects. In their pilot studies of red-legged par-
tridges (Alectoris rufa), they divided 36 birds into groups
receiving one of four diquat doses in their drinking water,
and measured consumption as well as mass loss and a
blood-based measure of oxidative damage (lipid peroxida-
tion). Based on this dose assessment, they were able to
increase oxidative damage in their main experiment with-
out causing the birds to lose weight or show other signs of
distress (Galv
an & Alonso-Alvarez 2009; Alonso-Alvarez
& Galvan 2011). These experiments demonstrate how a
potentially toxic chemical such as diquat can be used with
minimal harm to the animals while manipulating the target
variable. To better characterize the mechanisms by which
diquat induced an increase in the measure of blood lipid
peroxidation, however, it would be valuable to assess how
other measures of oxidative damage, ROS production and/
or antioxidant capacity varied after diquat exposure.
Though the studies of Galv
an & Alonso-Alvarez (2009)
and Alonso-Alvarez & Galvan (2011) successfully used
diquat in their experiments after careful pilot study, para-
quat may generally be a better choice than diquat as an
oxidative challenge because diquat is both less well studied
and more reactive –and hence potentially more toxic –
than paraquat.
HEAVY METALS
Another means by which ROS production can be
increased in an organism is through ingestion or injection
of heavy metals (Table 1). The effects of transition metals
on biological systems are well studied because of the harm-
ful effects of overdose on organisms from plants to fish to
humans (Sorensen 1991; Stohs & Bagchi 1995; Jezierska &
Witeska 2001; Clemens 2006; Jomova & Valko 2011).
Although metal ions are essential for basic cellular pro-
cesses and form the catalytic centres of many critical
enzymes, high concentrations of metals can induce ROS
production and can also interfere with antioxidants (Ercal,
Gurer-Orhan & Aykin-Burns 2001). However, metal ions
can also interact directly with cellular components to
impair other physiological pathways independently of
oxidative stress, which may confound the results of an
experimental challenge (Bal & Kasprzak 2002; Kasprzak
2002; Leonard, Harris & Shi 2004). Exposing experimental
subjects to heavy metals through ingestion or injection is
therefore not an effective means to manipulate systemwide
ROS levels in a controlled fashion because of the direct
interactions of metal ions with non-redox cellular path-
ways.
RADIATION
Another potential means for inducing systemic increase in
free radical levels in animals is controlled exposure to ion-
izing radiation using medical machinery (Table 1). The
connections between ionizing radiation and ROS genera-
tion are intimate and direct (Lane 2002), and because
total-body radiation potentially affects all parts of an
organism uniformly, it avoids the issue of unintended
localization of pro-oxidants and oxidative damage that
may occur in other treatments.
Radiation is well studied for its effects on human health
(Weiss & Landauer 2003; Hendry et al. 2009; Morgan &
Sowa 2009; Goodhead 2010). As a result, many of the
molecular processes triggered by radiation, the pathologi-
cal effects of high doses and the cellular pathways respon-
sible for mediating radiation exposure are well
characterized (Riley 1994; Weiss & Landauer 2003; Spitz
et al. 2004; Kim, Chandrasekaran & Morgan 2006; Dauer
et al. 2010; Szumiel 2012).Low-energy radiation, such as
from UV and visible light, is not adequate as an experi-
mental inducer of ROS because such radiation does not
pass beyond the skin of animals and does not directly gen-
erate ROS from water contained in the body (Riley 1994;
Ichihashi et al. 2003). High-energy ionizing radiation (such
as gamma rays; Riley 1994), on the other hand, is better
suited as a generalized oxidative challenge due to its ability
to stimulate systemwide increased ROS.
Ionizing radiation induces damage at the cellular level
both directly (through primary effects of ionization itself)
and indirectly (through ROS generation). Primary radia-
tion effects occur when high-energy radiation ionizes mole-
cules by causing the ejection of an outer-shell electron.
This ionization can cause cellular damage directly, such as
by breaking DNA strands. However, because animal cells
predominately contain water, radiation is most likely to
ionize water rather than critical cellular molecules (Riley
1994; Lane 2002). Once ionized, water rapidly reacts with
other water molecules to form the superoxide radical and
subsequently other ROS, including hydrogen peroxide and
hydroxyl radicals (Riley 1994; Lane 2002).Thus, radiation
generates pro-oxidants through the ionization of water
molecules present everywhere in the body.
Within the ecological literature, most studies investigat-
ing the effects of ionizing radiation have focused on
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
6R. E. Koch & G. E. Hill
changes in the molecular and morphological phenotypes,
as well as survival, of species at sites of the nuclear power
plant accidents at Chernobyl in 1986 (Møller & Mousseau
2006) and Fukushima in 2011 (Beresford & Copplestone
2011; Mousseau & Møller 2012; Strand et al. 2014). A
recent meta-analysis assessing the effects of chronic low-
dose radiation on wildlife found that exposure to radioac-
tive contaminants tended to cause small-to-moderate
increases in oxidative damage and decreases in antioxidant
capacity, though responses varied significantly among spe-
cies (Einor et al. 2016).
The caveat to using ionizing radiation as a pro-oxidant
stimulant is the potential for ROS generation in locations
and rates not typically found in a wild animal’s body,
which may induce physiological responses that are not
standard oxidative stress responses (Szumiel 2012). The
recovery from and prevention of radiation damage is gen-
erally mediated by signalling pathways that cause upregu-
lation of antioxidants and DNA repair mechanisms, or
activation of autophagy or apoptosis of damaged struc-
tures (Spitz et al. 2004; Dauer et al. 2010; Pazhanisamy
et al. 2011; Szumiel 2012). These responses are characteris-
tic of a defensive response during most states of high
oxidative stress (Sies 1997; Valko et al. 2007). However,
high-dose radiation can also trigger the release of inflam-
matory cytokines, which in turn activate ROS production
and innate immune activity, which is not a typical response
to increased oxidative stress (Iyer & Lehnert 2000; Kim,
Chandrasekaran & Morgan 2006; Jang et al. 2013). The
effects of low doses of ionizing radiation, as might be uti-
lized in oxidative challenges within ecological experiments,
are less well understood than the pathological effects of
high doses, but may be more comparable to naturally stim-
ulated states of increased oxidative stress than are the
effects of high doses.
Low doses of ionizing radiation may have a hormetic
(or ‘radioadaptive’) effect, causing the downregulation of
cellular processes that produce ROS and upregulation of
antioxidant production to boost resistance to future oxida-
tive challenge (reviewed in Szumiel 2012; Spitz et al. 2004),
which has been observed in bird species occupying the
Chernobyl area (Galv
an et al. 2014). Much as in other
instances of increased pro-oxidant burden, increased
antioxidant levels correlate with a decrease in the detri-
mental effects of radiation (Spitz et al. 2004; Pazhanisamy
et al. 2011; Jang et al. 2013). In fact, many of the reactions
to low-dose radiation exposure are representative of
response to oxidative damage and increased ROS abun-
dance, rather than radiation-specific effects, though empiri-
cal data in non-model species are lacking. One of the few
studies to examine the response of wild animals to a con-
trolled exposure of low-dose, whole-body ionizing radia-
tion found that gamma radiation treatment consistently
increased DNA oxidative damage, though there were sig-
nificant differences in how two different songbird species
responded to varying levels of radiation over varying time
periods (Luloff et al. 2011). These results provide impor-
tant baseline data for how response may vary in a dose-
and duration-dependent manner, at least in songbirds, and
again emphasize the importance of assessing how any par-
ticular study species may respond to radiation. Further
experimentation is needed to determine how other aspects
of oxidative stress may vary during low-dose treatment,
and whether any non-target damage incurred from the pri-
mary effects of radiation is observed.
With careful study of potential side effects, low doses of
ionizing radiation may offer a useful method for increasing
systemwide ROS levels. Furthermore, and important to
the logistical feasibility of performing such trials, the
machinery required to expose animals to ionizing radiation
is often available in major research institutions for medical
use. However, additional research is needed to gauge
whether even low doses of ionizing radiation cause too
much direct damage to biological molecules to isolate the
effects of oxidative stress alone, or whether the body’s
response to systemwide pro-oxidant stimulation differs sig-
nificantly from the typical pathways of response to
increased oxidative stress. Until these questions can be
more definitively answered with respect to the non-model
systems used within ecology, radiation may be best suited
for captive experiments aimed at inducing especially strong
oxidative challenges without the immediate toxic side
effects associated with many chemical treatments.
TBHP
A fourth potential means to elevate systemwide ROS in
animals is exposure to tert-butyl-hydroperoxide (tBHP), a
membrane-permeable, stable analog of hydrogen peroxide
that is metabolized within the cell into unstable forms that
can induce oxidative damage (Table 1; Piret et al. 2004;
Oh et al. 2012; Slamenova et al. 2013). The cellular effects
of tBHP have been found to be comparable to those of
H
2
O
2
exposure, though tBHP has the logistical advantage
of remaining chemically stable until administered to cells
or tissue (Spector et al. 2002; Alia et al. 2005; Slamenova
et al. 2013). Cellular exposure to tBHP not only induces
oxidative damage, but also stimulates mitochondrial mem-
brane depolarization, cytochrome crelease and apoptotic
signalling through direct interaction with the mitochon-
drial permeability transition pore in the outer mitochon-
drial membrane (Haidara et al. 2002; Piret et al. 2004).
These latter effects of tBHP occur independently of ROS
generation, but the apoptotic signals generated are typical
of a mitochondrial response to high rates of oxidative
damage (Hamanaka & Chandel 2010). High concentra-
tions of tBHP, however, can induce cell necrosis rather
than apoptosis (Haidara et al. 2002; Oh et al. 2012),
underscoring the importance of careful dose determination
to avoid harmful effects beyond increased oxidative stress.
tBHP is primarily known from biomedical research on
isolated cell lines in which cell cultures are bathed in a
solution containing tBHP to test the efficacy of various
antioxidants and to determine the specifics of how cells
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
Assessment of oxidative stress challenges 7
respond to oxidative damage (e.g. Liu et al. 2002; Lazze
et al. 2003). However, only a limited number of studies in
vertebrates –specifically, in rats –have administered tBHP
in vivo, finding increased oxidative damage and apoptosis
in liver tissue after intraperitoneal injection of tBHP (Liu
et al. 2002; Oh et al. 2012). While these studies indicate
the potential for tBHP as an inducer of oxidative damage,
further research is needed to investigate other potential tis-
sue-specific effects of the chemical in vertebrates and how
effects vary among species and methods of administration.
Interestingly, tBHP has recently gained popularity as an
oxidative stress inducer outside of the biomedical literature
in studies of the remarkable longevity of bivalve molluscs,
such as ocean quahog clams (Arctica islandica; Ungvari
et al. 2011) and freshwater pearl mussels (Margaritifera
margaritifera; Ridgway et al. 2014). These studies indicate
that tBHP may be particularly useful in aquatic systems
where subjects can be exposed to the chemical by adding
tBHP to the water surrounding the animal. In the pub-
lished studies on bivalves, oxidative damage and apoptosis
were increased in the gill tissue exposed directly to the dis-
solved tBHP (Ungvari et al. 2011, 2013; Ridgway et al.
2014). Though only tested in invertebrates to date, these
methods may prove to be broadly applicable to a variety
of aquatic species.
In summary, tBHP represents a promising avenue of
future research and may prove a valuable and safe inducer
of widespread oxidative damage, particularly given easy
administration to aquatic organisms, though the specific
effects of the chemical in most systems and the potential
for the chemical to induce widespread oxidative stress after
injection remain to be examined.
OXIDIZED DIETARY LIPIDS
Another means to induce increased oxidative stress has
emerged through the study of animals raised for human
consumption, particularly poultry and fish. Because of the
effects of increased oxidative damage on a variety of
aspects of both animal health and meat quality, research-
ers have manipulated the oxidation state of lipids in cap-
tive animal diets both to examine the effect of diet quality
on the animal’s oxidative stress levels and to gauge the effi-
cacy of various dietary antioxidants in preventing
increased oxidative stress (Table 1). Dietary lipids with
high concentrations of polyunsaturated fatty acids (PUFA)
are prone to oxidation (Frankel 1980; Kubow 1992; Sar-
gent et al. 1999). While PUFA are essential nutrients, the
by-products of their peroxidation via heat or chemical
catalysis can be damaging both through the formation of
reactive radicals and through the direct covalent interac-
tions between some peroxidation products, such as 4-
hydroxynonenal, and biological molecules (Kubow 1992;
Bacot et al. 2007; Awada et al. 2012). Adding oxidized
(often, heat-treated) lipids to the diet has been shown to
slow growth rate and increase oxidative damage in many
fish species (Nakano et al. 1999; Peng et al. 2009; Yuan
et al. 2014) and poultry (Zhang et al. 2010; Ac
ßıkg€
oz et al.
2011; Delles et al. 2014). Similar experiments have also
been performed in mammal species, including the domestic
pig (Shi-bin et al. 2007) and rat (Izaki, Yoshikawa &
Uchiyama 1984; Keller, Brandsch & Eder 2004), and con-
sumption of oxidized lipids and their by-products is con-
sidered a human health risk (Kanner 2007).
Providing experimental subjects with dietary oxidized
lipids to induce oxidative challenge has the advantage of
being a simple experimental design, requiring no injections
and posing no safety risks to researchers, that has been
widely performed in several vertebrate species. However,
this technique may not be suitable for broad application
within studies of oxidative stress ecology because of the
large variation in assimilation and responses observed
among subjects (e.g. Tocher et al. 2003). This variation
may be due at least in part to differences in the consump-
tion and absorption of dietary oxidized lipids (Tocher
et al. 2003; Dong et al. 2011), but it is also important to
consider that the conditions in which the lipids oxidize
may cause variation in the products consumed by the ani-
mals, some of which may have toxic properties that can
potentially damage the digestive tract membrane (Kaneda
& Miyazawa 1987; Engberg et al. 1996). Moreover, the
antioxidant and other properties of the diet beyond its oxi-
dized lipid content can influence the effects of treatment on
oxidative stress (Kubow 1992; Kanner 2007; Awada et al.
2012).
Administration of dietary oxidized lipids can be an effec-
tive means of inducing increased oxidative stress and is an
important method for determining the effects of diet prop-
erties on the health and quality of animals; it may also be
particularly useful if a study is interested in examining the
antioxidant properties of the gut, or interactions between
dietary oxidants and antioxidants. If used as an oxidative
challenge, it is important to account for the fact that some
oxidized lipids and their products may be assimilated
directly into the body, inflating quantitative measurements
intending to estimate lipid oxidative damage to the body
(Awada et al. 2012). However, the effects of dietary oxi-
dized lipids are subject to variation from a variety of
sources that may be difficult to control, and use of this
technique outside of agricultural and model species is lar-
gely unstudied. Thus, we do not recommend this technique
for use as a general oxidative stress challenge.
Antioxidant knock-downs
As an alternative to increasing ROS production directly,
decreasing the levels of antioxidants may induce a con-
trolled oxidative challenge by shifting the oxidative bal-
ance towards increased levels of pro-oxidants. Such a
manipulation has rarely been explored within ecological
studies because, until recently, accessible methods did not
exist or were not commonly known. However, a chemical
manipulation –buthionine sulfoximine (BSO) –and the
rapidly advancing field of RNAi research both offer useful
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
8R. E. Koch & G. E. Hill
methods to eliminate antioxidants within the animal body
without additional side effects. Importantly, neither of
these techniques poses any risk of harm to the researcher
administering the oxidative challenge.
BSO
Injection of BSO is an effective means to systemically
lower levels of glutathione (c-glutamylcysteinylglycine),
one of the most important antioxidants in the bodies of
animals (Table 1; Anderson 1998). BSO is unusual among
chemical agents because of its specificity: once injected, it
suppresses glutathione production throughout the body by
irreversibly binding to c-glutamylcysteine synthetase, an
enzyme involved in the initial catalytic steps of glutathione
synthesis (Griffith & Meister 1979; Griffith 1982).
Because BSO is rapidly cleared from an animal’s system,
chronic effects of BSO on glutathione synthesis are not
expected, but repeated injections may continuously deplete
glutathione levels. A study investigating the effects of
chronic BSO injection in rats found that intraperitoneal
injection twice daily for 30 days caused 85% reduction in
c-glutamylcysteine synthetase activity in the lung tissue,
but only a 36% decrease in glutathione levels (Thanislass,
Raveendran & Devaraj 1995). However, BSO treatment in
this study increased lipid peroxidation and decreased the
levels of a multitude of other cellular antioxidants. These
effects were likely due to both a decrease in the antioxidant
activity of glutathione and a handicap to other antioxi-
dants, particularly ascorbic acid, which rely on glutathione
reactions for proper function (Meister 1992; Thanislass,
Raveendran & Devaraj 1995). This study illustrates the
cascading effects of BSO-driven glutathione depletion, and
how even a modest decrease in glutathione appears to shift
the balance of pro- and antioxidants to induce a measur-
able increase in oxidative stress.
Within the ecological literature, Galv
an & Alonso-
Alvarez (2008) and Romero-Haro & Alonso-Alvarez
(2015) have performed controlled experiments in which
they injected nestling great tits or zebra finches (Taeniopy-
gia guttata), respectively, with four doses of BSO over six
days and measured the effects of treatment on adult phe-
notype. Importantly, BSO exposure did not affect adult
body condition or survival in either species, though both
studies found complex interactions between glutathione
depletion, ornamentation and oxidative state. In particular
–and in contrast to the decreased antioxidant levels found
in the study of rats (Thanislass, Raveendran & Devaraj
1995) –treated nestlings but not always adults showed in-
creased total antioxidant capacity, despite lower glu-
tathione levels. Two additional studies on adult
greenfinches (H~
orak et al. 2010) and domestic canaries
(Serinus canaria; Costantini et al. 2015) followed similar
designs of repeated BSO injections and found that BSO
successfully increased a measure of plasma oxidative stress
and decreased erythrocyte glutathione levels, though the
former study found no effect of treatment on plasma total
antioxidant capacity. These studies exemplify the variation
in how different species (and different sexes, ages) may
respond to depletion of a key antioxidant, but all four
show clear evidence that BSO exposure did indeed
decrease glutathione levels within the animal body and
cause measurable changes in oxidative damage and/or the
activity of other antioxidants.
The advantage of BSO is that it targets one antioxidant,
causing limited effects on other cellular components. As
with all techniques, however, the usefulness of BSO as an
oxidative challenge in a particular experimental design will
depend on the nature of the research questions being
addressed. While BSO may be an effective means to increase
pro-oxidant burden on the cellular antioxidants that remain
after glutathione depletion, the challenge may not induce
overall increased oxidative stress if those other antioxidants
are able to successfully compensate for the inhibition of glu-
tathione (Galv
an & Alonso-Alvarez 2008; H~
orak et al.
2010). In addition, BSO may not be appropriate for studies
interested in the response of an animal’s total antioxidant
defences to oxidative challenge because suppressing glu-
tathione handicaps one major pathway of pro-oxidant resis-
tance. On the other hand, a BSO challenge may be
particularly well suited to answering questions about the
specific function of glutathione in antioxidant response, or
to examining the ability of other cellular antioxidants to
upregulate in response to increased pro-oxidant burden.
RNAI
A promising but largely untested option for a targeted
manipulation of antioxidants is RNA interference (RNAi;
Table 1). This technique causes post-transcriptional degra-
dation of specific mRNAs, reducing expression of the cor-
responding proteins. RNAi techniques are rapidly being
developed for vertebrate models and even non-model sys-
tems (reviewed in Sifuentes-Romero, Milton & Garc
ıa-
Gasca 2011; Perrimon, Ni & Perkins 2010), though the
technique remains most commonly employed in inverte-
brate species. mRNA knock-downs via RNAi are invalu-
able tools for isolating the function of particular gene
sequences and have been fundamental to advancing the
fields of functional genomics and developmental biology.
A recent study on copepods (Tigriopus californicus) exem-
plifies how RNAi methodology may have application to
evolutionary ecology: Barreto, Schoville & Burton (2014)
developed a method of using double-stranded RNA to
suppress the expression of five target genes by at least
50%; they then used this technique to determine that
inhibiting a heat shock protein gene caused a significant
increase in mortality of copepods during thermal stress,
which is relevant to the survival of this species to high tem-
peratures in tidal pools. RNAi techniques have also been
used successfully to knock-down the genes for antioxi-
dants, including mitochondrial superoxide dismutase in
Drosophila (Kirby et al. 2002; Martin, Jones & Grotewiel
2009; Martin et al. 2009).
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
Assessment of oxidative stress challenges 9
While the methodology for using RNAi in the non-
model vertebrate systems often important to ecological
studies remains largely in development, RNAi techniques
represent an important future avenue for reducing the
function of antioxidants and other cellular proteins in
order to induce increase oxidative stress. As with BSO, the
specificity of RNAi represents a valuable opportunity to
manipulate the activity of target enzymes without risking
the broad spectrum of clinical effects that can be caused by
harmful compounds like paraquat.
Conclusions
Researchers currently have at least six methods for increas-
ing ROS production –paraquat, diquat, heavy metals,
radiation, oxidized dietary lipids and tBHP –and at least
two methods for decreasing production of antioxidants –
BSO and RNAi –as means to manipulate oxidative stress.
Selecting the most suitable oxidative challenge method for
a particular experiment depends largely on balancing two
primary concerns: (i) the biochemical and physiological
effects of the challenge suit the research goals of the study
with minimal non-target effects and (ii) the risk of acciden-
tal overdose to animal subjects and the safety concerns to
researchers administering the challenge are minimized.
Given these parameters, paraquat is the best option for
inducing increased ROS production within animals, but its
toxic chemical properties necessitate particular care during
dosage trials and diligent use of personal protective equip-
ment. Conversely, BSO and RNAi offer non-toxic alterna-
tives to paraquat, but knocking-down the activity of
specific antioxidants may not be appropriate for particular
experimental questions. We therefore recommend that
researchers carefully evaluate the type of oxidative stress
challenge that will best address their research goals and
select the corresponding method that minimizes the risk of
accidental harm.
Regardless of the oxidative challenge used, we encour-
age researchers performing oxidative challenges to measure
at least some aspects of oxidative damage, antioxidant
capacity and/or ROS production to ascertain that the
treatment caused the desired response. Such measurements
not only reveal whether or not the oxidative challenge had
an effect, but also the nature of the effect (Monaghan,
Metcalfe & Torres 2009). Many established methods exist
for measuring different components of oxidative stress
(Mateos & Bravo 2007), and in vivo methods for measur-
ing the specific rate of ROS production from subcellular
sites, such as the mitochondria, may become increasingly
useful to pinpoint the effect of oxidative challenge (Stier
et al. 2013). With conscientious application of pilot study,
choice of appropriate treatment method, and validation of
effects, oxidative challenges can yield invaluable informa-
tion about the interactions among oxidative stress and
other physiological pathways –a core component of
oxidative stress ecology.
Acknowledgements
The authors would like to thank an anonymous reviewer, David Costantini,
Caroline Isaksson, Antoine Stier and the Hill, Hood, and Wada labs at
Auburn University for constructive feedback on this manuscript. R.E.K.
was supported by NSF GRFP during manuscript preparation.
Data accessibility
This manuscript does not use data.
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Received 23 August 2015; accepted 15 March 2016
Handling Editor: David Costantini
Supporting Information
Additional Supporting Information may be found online in the
supporting information tab for this article:
Appendix S1. Biochemical mechanisms of paraquat activity.
Fig. S1. Redox cycling of paraquat.
©2016 The Authors. Functional Ecology ©2016 British Ecological Society, Functional Ecology
Assessment of oxidative stress challenges 13
