Capitolul IV: Stresul oxidativ

IV.1. Noiuni generale

Stresul oxidativ reprezint un fenomen complex, chimic i fiziologic, care nsoete

practic stresul biotic i abiotic n plantele superioare i se dezvolt ca un rezultat al
supraproduciei i acumulrii de specii reactive la oxigen (SRO).

Cercetrile recente au clarificat originile stresului oxidativ din plante i arat c, pe

lng cloroplastul clasic, sursele mitocondriale i peroxisomale, SRO sunt sintetizate de
oxidazele NADPH i peroxidaze.

Oxidative stress is a complex chemical and physiological phenomenon that

accompanies virtually all biotic and abiotic stresses in higher plants and develops as a result
of overproduction and accumulation of reactive oxygen species (ROS). This review revises
primary mechanisms underlying plant oxidative stress at the cellular level. Recent data have
clarified the origins of oxidative stress in plants, and show that apart from classical
chloroplast, mitochondrial and peroxisome sources, ROS are synthesized by NADPH
oxidases and peroxidases. ROS damage all major plant cell bio-polymers, resulting in their
dysfunction. They activate plasma membrane Ca2+-permeable and K+-permeable cation
channels as well as annexins, catalyzing Ca2+ signaling events, K+ leakage and triggering
programed cell death. Downstream ROS-Ca2+-regulated signaling cascades probably include
regulatory systems with one (ion channels and transcription factors), two (Ca2+-activated
NADPH oxidases and calmodulin) or multiple components (Ca2+-dependent protein kinases
and mitogen-activated protein kinases). Intracellular and extracellular antioxidants form
sophisticated networks, protecting against oxidation and shaping stress signaling. Research
into plant oxidative stress has shown great potential for developing stress-tolerant crops. This
can be achieved through the use of directed evolution techniques to prevent protein oxidation,
bioengineering of antioxidant activities as well as modification of ROS sensing mechanisms.
(Vadim Demidchik, 2015)

The mechanisms of action of many environmental agents commonly involve oxidative

stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic
contaminant that has been implicated in a variety of oxidant-dependent toxicological
responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn2+
does not generate reactive oxygen species (ROS) through redox cycling. Objective: To
characterize the role of oxidative stress in zinc-induced toxicity. Methods: We used an
integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific
fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5,6,6-tetrachloro-
1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted
form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells.
Results: Zinc treatment in the presence of the Zn2+ ionophore pyrithione of A431 skin
carcinoma cells preloaded with the H2O2-specific indicator PG1 resulted in a significant
increase in H2O2 production that could be significantly inhibited with the mitochondrial
inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as
the source of zinc-induced H2O2 formation by the observation that exposure to zinc caused a
loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure
of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also
demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from
mouse hearts. Conclusion: Taken together, these findings show a disruption of mitochondrial
integrity, H2O2 formation, and a shift toward positive redox potential in cells exposed to zinc.
These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative
stress in toxicological responses.( Wan-Yun Cheng, Haiyan Tong, Evan W. Miller,
Christopher J. Chang, James Remington, Robert M. Zucker, Philip A. Bromberg, James M.
Samet and Thomas P.J. Hofer , Environmental Health Perspectives, Vol. 118, No. 7 (JULY
2010), pp. 902-908).

Specii reactive de oxigen

2.2. REACTIVE OXYGEN SPECIES: COMPLEX ROLES IN NORMAL

PHYSIOLOGY
ROS molecules are simply oxygen-containing molecules that are reactive. They can be
divided into free-radical ROS and nonradical ROS. Free-radical ROS have unpaired electrons
in their outer orbits; examples of such molecules are superoxide and hydroxyl radical.
Nonradical ROS do not have unpaired electrons; however, these are chemically reactive and
can be converted into free-radical ROS. One example of a nonradical ROS is hydrogen
peroxide.
2.2.1. Role of Reactive Oxygen Species in Cell Signaling
To survive, cells must sense their immediate surroundings and change their activity according
to their microenvironment. This is accomplished through cell signaling. A basic signaling
pathway relays a signal through the cell by modulating the activities of proteins along the
pathway. A mediator or second messenger is a molecule that promotes (or inhibits) a step
in a signaling pathway. Functions of ROS have been described at different locations of
signaling pathways. The ROS molecules have been described as the very first stimulus that
starts the cascade of a signaling pathway, the initiator, and also as the last step of a signaling
pathway, the so-called effector. Furthermore, ROS can also be involved somewhere between
the start and the end of the signaling pathway, either as the molecule that relays the signal
itself or by promoting a step in the signaling pathway. In both cases, ROS can be seen as the
mediator in the particular pathway (for review, see the work by Hancock [2009]). However,
for ROS to function as signaling mediators, they should be produced where and when they are
needed, sensed by some mechanism, and should be rapidly removed to stop the signal from
being sustained.

2.2.2. Production of Reactive Oxygen Species

ROS molecules are created during the reduction of oxygen to water. The addition of one
electron to oxygen creates superoxide, whereas further reduction gives hydrogen peroxide.
Production of ROS can also be a consequence of endogenous or exogenous stimuli, including
ultraviolet (UV) radiation, chemotherapy, environmental toxins, and exercise (Blomhoff
2005). Deliberate production of ROS occurs in different cellular compartments from enzymes
such as nicotinamide adenine dinucleotide phosphate (NADPH), oxidases (NOX and dual
oxidase [DUOX]), nitric oxide (NO) synthase (NOS), xanthine oxidase, and from the electron
transport chain of the mitochondria.

There are seven NADPH oxidases (i.e., NOX1 to NOX5 and DUOX1 and DUOX2). These
are transmembrane proteins that produce superoxide or hydrogen peroxide. The oxidases
NOX1 through 5 produce superoxide by the transfer of an electron through the membrane
from NADPH to oxygen. The enzymes DUOX12 are calcium-dependent enzymes and
produce hydrogen peroxide directly by virtue of a peroxide-like subunit located on the outer
side of the membrane in addition to the transfer of an electron from NADPH. The enzymes
further differ in their cellular compartmentalization, their upstream activators, and the
associated subunits. Known inducers of NOX are growth factors, cytokines, and vitamin D
(Brown and Griendling 2009; Chen et al. 2009; Leto et al. 2009).

Mitochondria have traditionally been thought to produce ROS only as an unwanted by-
product of energy production in the electron transfer chain. However, deliberate ROS
production also occurs from the mitochondria. This occurs at least partially by the inhibition
of cytochrome c oxidase by NO leading to increased superoxide production without affecting
energy production. Mitochondrial superoxide dismutase converts superoxide to hydrogen
peroxide, which can cross the membrane and take part in cytosolic signaling (Brookes et al.
2002).
2.2.3. How Are Reactive Oxygen Species Perceived?
ROS can alter the production, stability, or function of proteins. The redox status may alter the
activity of transcription factors in the nucleus. In general, the reduced transcription factor
binds to deoxyribonucleic acid (DNA) and promotes transcription, whereas an oxidized
transcription factor will not be able to bind to DNA and thus will not promote transcription.
Furthermore, the stability of proteins can be affected by the oxidation of proteasomes.
Oxidation of proteasomes may render them inactive and unable to degrade proteins, thus
maintaining or increasing the level of proteins. Finally, the function of proteins and molecules
can be modified through oxidation by the following three different strategies: (1) Proteins,
such as thioredoxin, can be oxidized, resulting in alteration of the activity of the protein
directly. (2) The oxidation targets a chaperone protein that usually inhibits protein activity; on
oxidation, the protein can dissociate from its inhibitor and thus become active. (3)
Phosphatases and kinases can be targets for oxidation, and subsequently alter the activity of
proteins through posttranslational modifications. Protein tyrosine phosphatases are often
inactivated by oxidation, whereas the different kinases are generally activated. The most
common targets of oxidation are cysteine residues, but other amino acids like tyrosine and
methionine can also be targets. Further oxidation of target molecules may lead to irreversible
oxidative damage. Oxidized cysteine residues can be protected from further oxidation by the
formation of thiol bridges.

In phagocytosis, ROS is an effector that is produced by NOX2 inside the phagosome to kill
phagocytozed microbes. Targets of ROS in signaling pathways include transcription factors,
redox sensors, and phosphatases/kinases. Transcription factors include Nrf2, NF-B, p53, AP-
1, cyclic adenosine monophosphate response element binding (CREB), HomeoboxB5, and
nuclear receptors such as the estrogen receptor. Redox sensors include thioredoxin,
glutharedoxins, peroxiredoxins, glutathione, and redox effector factor-1 (Ref-1), whereas
phosphatases/kinases include PTP, Akt, JNK, ERK, Src, and CDK (Brown and Gutteridge
2007; Halliwell and Gutteridge 2007; Kamata et al. 2005; Kiley and Storz 2004; Trachootham
et al. 2008). To counteract the possible toxic effects of ROS and enable ROS to act in
signaling pathways, intricate systems of antioxidants have evolved. This system is highly
specialized in terms of both removal of specific ROS and compartmentalization of the
different antioxidants. For a discussion of various antioxidant systems, please see the
excellent book by Halliwell and Gutteridge (2007).

Hancock J.T. The role of redox mechanisms in cell signaling. Mol Biotechnol. 2009;43:162
6. [PubMed] [Reference list]
Blomhoff R. Dietary antioxidants and cardiovascular disease. Curr Opin Lipidol. 2005;16:47
54. [PubMed] [Reference list]
Brown D.I, Griendling K.K. Nox proteins in signal transduction. Free Radic Biol Med.
2009;47:123953. [PMC free article] [PubMed] [Reference list]
Chen K, Craige S.E.E, Keaney J.F Jr. Downstream targets and intracellular
compartmentalization in Nox signaling. Antioxid Redox Signal. 2009;10(11):246780. [PMC
free article] [PubMed] [Reference list]
Leto T.L, Morand S, Hurt D, Ueyama T. Targeting and regulation of reactive oxygen species
generation by Nox family NADPH oxidases. Antioxid Redox Signal. 2009;10(11):260719.
[PMC free article] [PubMed] [Reference list]
Brookes P.S, Levonen A.L, Shiva S, Sarti P, Darley-Usmar V.M. Mitochondria: Regulators of
signal transduction by reactive oxygen and nitrogen species. Free Radic Biol Med.
2002;33:75564. [PubMed] [Reference list]
Halliwell B, Gutteridge J.M. Free Radicals in Biology and Medicine. 4th. New York: Oxford
University Press Inc; 2007. [Reference list]
Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M. Cell. Vol. 120. 2005. pp. 64961.
[PubMed] [Reference list]
Kiley P.J, Storz G. Exploiting thiol modifications. PLoS Biol. 2004;(2) e400. [PMC free
article] [PubMed] [Reference list]
Trachootham D, Lu W, Ogasawara M.A, Nilsa R.D, Huang P. Redox regulation of cell
survival. Antioxid Redox Signal. 2008;8(10):134374. [PMC free article] [PubMed]
[Reference list]