Free Radical Biology and Medicine 65 (2013) 232–233

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Hypothesis Papers

Free radical paradoxes

Stefan I. Liochev
8 Foxlair Court, Durham, NC 27712, USA

art ic l e i nf o a b s t r a c t

Article history: Unlike bigger and more advanced animals, Caenorhabditis elegans does not generate NO, yet it was
Received 5 May 2013 recently found that NO produced by chemical or biological sources exerts profound effects in that animal,
Received in revised form leading to increased life span and thermotolerance. The biological source was Bacillus subtilis, a natural
10 June 2013
food for C. elegans. Yet once in the cell, NO might react with superoxide, leading to the production of the
Accepted 13 June 2013
Available online 19 June 2013
potentially toxic peroxynitrite. In this paper, a number of paradoxes that are involved in that situation are
discussed. It is also argued that their solution might lead to a sizeable advancement of our knowledge of
Keywords: what constitutes oxidative stress and what role oxidative stress plays in the development of pathological
Superoxide processes and aging.
Superoxide dismutase & 2013 Elsevier Inc. All rights reserved.
Nitric oxide
Peroxynitrite
C. elegans
Aging
Hydrogen peroxide
E. coli
B. subtilis

The traditional view in free radical biology posits that free
radicals, reactive oxygen species (ROS), and other related agents,
such as peroxynitrite, are potentially toxic and play a role as causative
agents in a variety of pathological processes and in aging. There are,
however, some puzzling observations that seem to contradict this
view. In particular, a mutant of C. elegans completely devoid of
superoxide dismutases (SODs) had the same life span as the wild
type [1], although the general health of these mutants was severely
impacted. Even more interestingly, some of the mutants that lacked
some but not all SODs actually lived longer than the wild type [1].
Recently, I discussed some possible explanations for these
findings [2]. For example, a point was made that the increase of
superoxide radical level alone as a result of lack of SODs will
mostly result in metabolic slowdown. The more permanent and/or
difficult to repair damage caused by superoxide should be due
to a formation of more reactive species. Thus, superoxide (by
increasing the intracellular [free iron]) and hydrogen peroxide
cooperate in producing hydroxyl radical or similar equivalently
reactive species [2–4]. In the SOD-null C. elegans several hydrogen
peroxide-consuming enzymes were upregulated [1] which mini-
mized such toxic pathways [2]. In addition, superoxide participates
in the creation of the potentially very toxic peroxynitrite through
an extremely fast reaction with nitric oxide (NO) [5]. Other aspects
Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutases
E-mail address: liochev@yahoo.com
critically evaluated were that the experiments described [1] were
performed with nonadapted animals being fed unbalanced and
suboptimal diet (E. coli), and the role of adaptation in the
phenomena observed [2].
Recently, I became aware of an excellent paper by Gusarov et al.
[6] that inspired me to write this hypothesis paper, which in turn
reinforces the original review [2]. Thus C. elegans which is a very
tiny animal is in addition a very unusual animal that does not
produce nitric oxide and E. coli does not produce it either (at least
not under the experimental conditions in Ref. [6]). In contrast B.
subtilis produces nitric oxide and feeding C. elegans with B. subtilis,
a more natural food source for that animal, extended its life span.
That nitric oxide was the factor for that effect was unequivocally
established and it appears likely that nitric oxide exerts this life-
extending effect by triggering some adaptations [6]. It appears that
for a very tiny animal it is preferable to obtain a signaling species
from the natural food rather than to synthesize it! It also appears
that B. subtlis produces enough NO, which exerts profound effects
within the cells of C. elegans.
In this regard, the absence of SOD either in that animal and/or
in its food (B. subtilis) should lead to decreased bioavailability of
nitric oxide and/or increased production of peroxynitrite [7]. It
should also be recalled that the increase of the level of superoxide
in one compartment can damage targets in another compartments
by generating peroxynitrite, which like NO is able to transverse
membranes and in this way SOD might exert cross-compartment
protection [8].

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2013.06.027
 S.I. Liochev / Free Radical Biology and Medicine 65 (2013) 232–233
Thus, there are a number of reasonable expectations of what
might occur in different situations. For example, it is likely that
SOD-deficient mutants of C. elegans will have a shorter life span
than the wild type when fed B. subtilis, and may be even shorter
when fed SOD-deficient B. subtilis. However, the latter prediction
assumes that the increased peroxynitrite production in B. subtilis
will be the toxic factor. If the peroxynitrite formation in the cells of
C. elegans is more toxic, then feeding SOD-deficient mutants of C.
elegans with SOD-null mutants of the bacilli may be less toxic than
feeding them with wild-type B. subtilis. It is also likely that wild-
type animals fed SOD-deficient B. subtilis may have a shorter life
span than, or be otherwise inferior to, such animals that are fed
with wild-type microorganisms. This should be due to decreased
bioavailability of NO, since in the SOD-deficient B. subtilis a
significant part of NO will disappear in reaction with superoxide.
In this situation the peroxynitrite formed by the SOD-deficient
Bacillus may be toxic both for itself and for the wild-type worm.
It is likely, that the evolution of SODs both in that animal and its
natural food led to the best from two worlds. Thus, wild-type
C. elegans benefits from nitric oxide supplied from the food source,
while being protected from the bad behavior of the ugly child of
the superoxide and nitric oxide, all of this while enjoying a
potentially poisonous meal. One of the reasons for the production
of NO by B. subtilis might be an attempt to poison relatively SOD-
deficient tiny competitors and predators by using the fact that all
of them unavoidably produce superoxide aerobically.
It seems obvious that apparent conclusions about the role of
free radicals and reactive species in aging and in pathological
conditions that are based on observations involving these unusual
animals should not be automatically accepted as valid for other
animals.
Obvious or not, one might rightly ask how the correct solution
of the discussed puzzles might contribute to forming a better
informed view of what is oxidative stress and what role the latter
plays in the development of pathological processes and in aging.
Well, despite their premature, in my opinion, denial of the
validity of the free radical theory of aging, the authors of Ref. [1]
and other groups have produced many excellent papers that
describe exciting data. Thus there are five (!) SODs in C. elegans.
Their role in protecting the worm against the toxicity of a variety
of oxidative stress-causing agents has been established to a
significant extent and in some cases even the contribution of the
individual SODs has been studied.
Still the role of all these SODs of C. elegans remains puzzling to
a significant extent. Some reasonable explanations include that
they protect separate compartments; the concentration of oxygen
and therefore the potential for superoxide production in the tiny
worm is likely much higher than in most of the cells of bigger and
more advanced animals, etc.
Here is one more speculation: Some agents like paraquat, for
example, cause increased superoxide production and decrease of
233
the concentrations of the reduced form of essential redox couples.
As a consequence the free iron concentration and the rate of
hydrogen peroxide production are also increased, all of this
potentially extremely damaging. In another situation, increased
NO production does not increase superoxide production but
should lead to a decrease of superoxide level. We are not out of
the woods, however, since in that situation both the rate of
peroxynitrite production and its level will increase. Therefore, it
could be that some of the SODs of C. elegans evolved to protect that
worm from the first mechanism of toxicity of superoxide, while
others from the other one. In the case of the C. elegans (plus
chemical and especially biological source) system NO is not
produced randomly. In fact, NO must first cross some distance
and several membranes before reaching the mitochondrial matrix
of the worm. Thus it is likely that the SOD(s) more proximal to the
NO source evolved to prevent the formation of peroxynitrite, while
those some distance and several membranes away evolved to
protect against the iron-dependent superoxide toxicity. In this
regard information about possible regulation of the SODs of C.
elegans by agents that might cause oxidative stress, such as NO and
peroxynitrite, is essentially lacking, as well. However, a reasonably
informed view concerning the role of NO, superoxide, reactive
oxygen species, etc., in the aging of this worm, for example, or in
aging in general can be formed only after sufficient accumulation
of such data.
Therefore, it might be worthy for readers in possession of the
needed skills as well as of the needed species and strains to
explore these matters and perform the indicated here experi-
ments, as well as other experiments that arise logically from that
interesting paper [6] and the discussion made there and above.
Major improvement of our understanding about many puzzles
including about what causes aging may be the reward.
References
[1] Raamsdonk, J. M.; Hekimi, S. Superoxide dismutase is dispensable for normal
animal lifespan. Proc. Natl. Acad. Sci. USA 109:5785–5790; 2012.
[2] Liochev, S. I. Reactive oxygen species and the free radical theory of aging. Free
Radic. Biol. Med. 60:1–4; 2013.
[3] Liochev, S. I.; Fridovich, I. The role of O2− in the production of HO: in vitro and
in vivo. Free Radic. Biol. Med. 16:29–33; 1994.
[4] Liochev, S. I. The mechanism of Fenton-like reactions and their importance for
biological systems: a biologist's view. Metal Ions Biol. Syst. 36:1–39; 1999.
[5] Beckman, J. S.; Koppenol, W. H. Nitric oxide, superoxide, and peroxynitrite: the
good, the bad, and ugly. Am. J. Physiol. 271:C1424–C1437; 1996.
[6] Gusarov, I.; Gautier, L.; Smolentseva, O.; Shamovsky, I.; Eremina, S.; Mironov, A.;
Nudler, E. Bacterial nitric oxide extends the lifespan of C. elegans. Cell 152:818–-
830; 2013.
[7] Liochev, S. I.; Fridovich, I. Superoxide and nitric oxide: consequences of varying
rates of production and consumption: a theoretical treatment. Free Radic. Biol.
Med. 33:137–141; 2002.
[8] Liochev, S. I.; Fridovich, I. Cross-compartment protection by SOD1. Free Radic.
Biol. Med. 38:146–147; 2005.