GREGORY B. BULKLEY
FREE RADICALS AND REACTIVE OXYGEN SPECIES
The evolution of a scientific concept
Over the past few
decades, free radicals, highly reactive and thereby destructive molecules, have
come to be appreciated increasingly for their importance to human health and
disease. Many common and lifethreatening human diseases, including
atherosclerosis, cancer, and aging, have free radical reactions as an
underlying mechanism of injury. Over this period of time, our conceptual
understanding of the interaction of such reactive oxygen species (ROS) with
living organisms has undergone a remarkable evolution that is perhaps
instructive of how human knowledge of a scientific concept develops and
matures. As this understanding has evolved, it has provided unprecedented
opportunities for improving the quality and even length of human life.
FREE RADICALS
Molecules are
made up of one or more atomic nuclei surrounded by orbiting electrons. The
electrons are arranged in orbitals, depending upon their respective distances
from these nuclei. In most molecules, the electrons within each orbital are paired
with another electron that’s spinning in the
opposite direction. (The concept of “spin,” distinct from the concept of electrical charge, is less
of a
literal description of the electron’s actual
behavior than an analogy that helps us visualize this unseen world in familiar
terms.) The paired electrons keep the molecule relatively stable (at a lower
energy state) and thereby less reactive.When one or more electrons, especially
within the outer orbital, is/are unpaired with respect to spin, the molecule
becomes relatively unstable (at a higher energy state) and consequently more
reactive with other molecules.
A free radical
is a molecule with one or more unpaired electrons in its outer orbital. Many of
these molecular species are oxygen (and sometimes nitrogen) centered. Indeed,
the molecular oxygen we breathe is a free radical. These highly unstable
molecules tend to react rapidly with adjacent molecules, donating, abstracting,
or even sharing their outer orbital electron( s). This
reaction not only changes the adjacent, target molecule, sometimes in profound
ways, but often passes the unpaired electron along to the target, generating a
second free radical or other ROS, which can then go on to react
with a new target. In fact, much of the high reactivity of ROS is due to their
generation of such molecular chain reactions, effectively amplifying their
effects many fold.
The changes
wrought on adjacent molecular targets can vary in magnitude, but because many
of the components of the living cell are particularly susceptible to free
radical injury, the molecular chain reactions can have substantial effects on
the structure and function of living tissue.As a consequence, natural selection
has driven the evolution of a number of intracellular defense mechanisms to
neutralize or control the potentially destructive reactivity of ROS. These
include molecules that react preferentially with ROS without passing that
reactivity along. Some of these are simple molecules like vitamins E and C,
while some are enzymes like superoxide dismutase (SOD) and catalase, which
catalyze such electron- quenching reactions. Often these compounds are referred
to collectively as free radical scavengers.
ROS AND HUMAN
HEALTH
Because our
bodies are continuously exposed to free radicals and other ROS, from both
external sources (sunlight, other forms of radiation, pollution) and generated
endogenously, ROS-mediated tissue injury is a final common pathway for a number
of disease processes.
Radiation injury represents an important
cause of ROS-mediated disease. Extreme examples include the physical-chemical
reactions within the center of the sun and at the center of a thermonuclear
blast. With respect to more commonly encountered levels of radiation, depending
upon the situation, about two-thirds of the sustained injury is mediated not by
the radiation itself, but by the ROS generated secondarily. This applies not
only to the acutely toxic forms of radiation injury, but the long-term,
mutagenic (and hence carcinogenic) effects as well. Thus, the victims of the
thermonuclear explosion in Hiroshima, both those who died instantly from the
blast at ground zero and those in the suburbs who developed leukemia and lymphomas
years later, were victims of free radical injury.
An important
clinical application of this principle is encountered regularly in the
treatment of cancer by radiation therapy. Large tumors often outgrow their
blood supplies and tumor cells die within the center, despite being
well-oxygenated at the periphery. Between these two regions is an area of tumor
that is poorly oxygenated, yet remains viable. Radiation therapy of such tumors
is particularly effective at the periphery, where an abundant concentration of
oxygen is available to form tumorcidal ROS. The poorly oxygenated center is
injured to a significantly smaller degree.While the dead cells in the center
don’t survive anyway, the poorly oxygenated, yet
viable, cells between these two areas can survive a safe dose of radiation
therapy, and thereby seed a later local recurrence of the tumor. This is a
major reason why many large tumors are treated by a combination of radiation
therapy (to kill the tumor at its advancing edges) and surgical removal of the
bulk of the tumor, including these particularly dangerous remaining cells.
Cancer and other malignancies all entail
unconstrained cell growth and proliferation based upon changes in the cell’s genetic information. In most cases, for example, one or more genes that
normally constrain cell growth and replication is/are mutated,
or otherwise inactivated. These genetic deficiencies correspond directly with
deletions and sequence changes in the genetic code, resident in the cell’s DNA. A frequently seen final common cause of such DNA damage is free
radical injury. Of the myriad injuries sustained by our DNA on a daily basis,
most are repaired by normal DNA repair mechanisms within the cell, while some
result in cell death. Since such injuries are sporadic and distributed somewhat
randomly across the genome, most lethal DNA injuries are clinically
inconsequential, resulting in the loss of a few cells among millions. However,
when a single cell sustains an injury that impairs growth regulation, it can
proliferate disproportionately and grow rapidly to dominate the cell population
by positive natural selection. The result is a tumor, frequently a malignant
one, where the constraint of growth and proliferation is particularly
deficient. Therefore, free radical injury to the genetic material is a major
final common pathway for carcinogenesis.
ROS can be
generated within the cell not only by external sources of radiation, but also
within the body as a byproduct of normal metabolic processes. An important
source of endogenous free radicals is the metabolism of some drugs, pollutants,
and other chemicals and toxins, collectively termed xenobiotics. While some of
these are directly toxic, many others generate massive free radical fluxes via
the very metabolic processes that the body uses to detoxify them. One example
is the metabolism of the herbicide paraquat.At one time,
drug enforcement authorities used this herbicide to kill marijuana plants. Growers
realized they could harvest the sprayed crop before it wilted, and still sell
the paraquatlaced product. Many who smoked this product subsequently died of a
fulminant lung injury. Fortunately, this approach has been abandoned as a
particularly inhumane way to solve the drug problem.
While the
paraquat story is a particularly striking example of a metabolic mechanism of
free radical toxicity, many commonly encountered xenobiotics, including
cigarette smoke, air pollutants, and even alcohol are toxic, and often
carcinogenic to a large degree by virtue of the free radicals generated by
their catabolism within our bodies.Moreover, there is accumulating evidence
that a diet rich in fruits and vegetables, which are high in natural
antioxidants, and low in saturated fat (a particularly vulnerable target for
damage by ROS), reduces the risk of atherosclerosis and cancer.
Atherosclerosis is a complex process that
leads to heart attack, stroke, and limb loss by the plugging of the arteries
with atherosclerotic plaque. This plaque is a form of oxidized fat. When free
radicals react with lipids, the consequence is lipid peroxidation, the same
process by which butter turns rancid when exposed to the oxygen in the
air.While a number of factors influence the development and severity of
atherosclerosis, a major factor is the ROS-mediated peroxidation of our low
density lipoproteins (LDLs, or “bad cholesterol”). The dietary
approach to the prevention of heart disease and stroke is based partially on
adding dietary antioxidants to limit LDL oxidation, as well as decreasing the
intake of fat itself. These approaches already have made significant inroads
into the mortality from heart disease, but offer the potential for safe
pharmacological prevention in the future that is not as dependent upon
willpower as are diet and exercise.
Degenerative neurological diseases affect millions
of Americans. A number of these diseases, including amyotrophic lateral
sclerosis (ALS, or Lou Gehrig’s disease),
Parkinson’s disease, and Alzheimer’s disease, appear to have ROS
toxicity as a central component of their underlying mechanism of nerve cell
destruction. Unfortunately, there is little evidence that simply eating more
dietary or even pharmocologic antioxidants will prevent or arrest the neural
degeneration; not surprisingly the mechanism is too complex to lend itself to
such a simplistic remedy. Nevertheless, improving our understanding of these
complex injury mechanisms offers a real potential for improved clinical
outcomes in the near future.
Ischemia/reperfusion injury is a
particularly fascinating example of ROS-mediated disease. When an organ is
deprived of its blood supply (ischemia) it is injured, not just by the
temporary loss of oxygen, but also by the ROS that are generated by reaction
with the oxygen that is reintroduced at reperfusion, when the blood supply is
restored. In some clinical situations, we can prevent this injury by giving
antioxidants, sometimes even after the period of ischemia, but just
prior to reperfusion. For example, the preservation of kidneys, livers, and
other organs in solutions that contain antioxidants, as well as other agents,
is now routine prior to their transplantation. Another
example is the use of drugs that block the function of free radical generating
enzymes prior to stopping the heart for cardiac surgery. These drugs help
prevent reperfusion injury when the heart is restarted and flow is restored. This
reperfusion injury mechanism also has been found to play an important role in
patients suffering from multiple organ failure after trauma, massive surgery,
or shock. Multiple organ failure is now the leading cause of death in intensive
care units, and extensive efforts are under way to understand better how ROS
contribute to this syndrome.
Aging is a remarkably complex process that has
managed to remain relatively opaque to scientific understanding. However,we now understand it to be a process per se, i.e., a
series of controlled mechanisms, and not just the passive accumulation of wear
and tear over the years. Put simply, our bodies age in
the ways that are far more complex and more regulated than the processes by which
our automobiles wear out. But if aging is a series of processes, it’s logical to conclude that it is potentially controllable, or at least
modifiable. One of the most important of these processes is comprised of an
accumulation of the molecular injuries that are mediated by free radicals and
other ROS. For example, since structural lipids are the primary component of
our cell membranes, the integrity of which defines cell viability, aging is
partially a matter of our going rancid as our lipids are progressively
oxidized. While this is an oversimplification of this complex process, it
reflects the optimism of some investigators of the aging process.
Recent studies
indicate that the therapeutic manipulation of ROS metabolism can actually
extend the total life span of mice to a significant degree. This was the first
time that life span has been successfully altered experimentally by
treatment.When one considers that the demographic, and consequent social, economic,
and ecological impacts of even a 10 percent increase in human life span, a
likely eventuality within the next decade or two, would far exceed that of a
100 percent cure for cancer (which is far less likely), the importance of this
potential becomes evident.
NORMAL
BIOLOGICAL FUNCTION
Given the large
impact that ROS can have on biological systems, it is not surprising that natural
selection has found ways to use this reactivity to the organism’s advantage. Accordingly, a number of biochemical mechanisms have
evolved to do just this. For example, every high school science student learns
that we use oxygen to burn our food to produce the energy we need to function. Effectively,
the energy stored within the complex carbon molecules within our food is used
to reduce oxygen to water and, in the process, to produce ATP, a molecular form
of high energy storage which is used by the cell to run a whole host of
functions. This process takes place within the mitochondria, intracellular
organelles whose function it is to make this energy by a process termed oxidative
phosphorylation. In the absence of oxygen, our cells are forced to make
energy by anaerobic fermentation, a mechanism only one-third as
efficient as aerobic oxidative phosphorylation.
Interestingly,while students learn (correctly) that aerobic metabolism is
normal, and the anaerobic pathway is a special situation, evolutionarily it is
the other way around. For most of the period that life has existed on Earth
(about two billion years) there were only negligible levels of oxygen in the
atmoshphere, and virtually every organism made energy by anaerobic
fermentation. When oxygen first began to appear in the environment, a waste
product of plant photosynthesis, it proved to be highly toxic and was literally
of no earthly use. Indeed, evolutionary biologists tell us that the antioxidant
defense mechanisms that we see in cells today evolved very early, in parallel
with the appearance of oxygen in the environment. Many believe that the
mitochondrial mechanism of oxidative phosphorylation evolved later, first as a
highly efficient mechanism to scavenge these highly toxic oxygen molecules. (If
oxygen were a new drug, it would be far too toxic to be approved by the US Food
and Drug Administration.) Having evolved primarily as a defense mechanism
against ROS-mediated tissue injury, the process of oxidative phosphorylation
appears to have been exploited later by natural selection because it was more
efficient for making ATP. We are now so dependent on this high-efficiency
system that humans and most mammals can survive only for brief periods without
a continuous supply of oxygen.
Free radicals are
also an important component of the body’s
defense systems. One of the most important determinants of survival in nature
is the ability to avoid being eaten, not only by large carnivores, but by
microorganisms in the course of an infection. In fact, more people die each
year the world over from infectious diseases than from any other cause. Complex
organisms, including humans, have highly developed immune systems that counter
this threat. Major components of that defense system are macrophages,
cells that circulate throughout the body and engulf and kill bacteria and other
microorganisms by phagocytosis. The leukocytes (white blood cells) in our blood
are an important example, although about 80 percent of the killing of
circulating bacteria is effected by Kupffer cells,
specialized macrophages within the liver. An essential component of this
process is the killing of the bacteria that have been engulfed within a
vacuole, termed the phagosome, without damaging the surrounding cell. The major
mechanism of this killing is the generation of ROS within the phagosome by a
highly specialized enzyme on its inner membrane surface. The active,
microbicidal product of this system is hypochlorous acid (HOCL), the active
ingredient of Clorox® bleach. It is truly remarkable that we
disinfect our bloodstreams with the same chemical that we use to disinfect our
bathrooms. Here, natural selection has driven our bodies to employ
opportunistically the toxic, destructive properties of ROS to our advantage as
a defense against microbial invasion.
We’ve also learned that ROS play a critical role in cell signaling. We now
know that organisms use very low fluxes of ROS to send control messages within
cells (intracellular signaling) and between cells (intercellular signaling) in
the course of normal body function.While such signaling has long been
appreciated, it traditionally has been thought to be effected exclusively by
cytokines and other large, complex signaling molecules. Only recently have we
come to realize that small ROS can function in the same way. A prominent
example of this is the molecule nitric oxide, actually a free radical, the
function of which was the basis for the Nobel Prize in Physiology or Medicine
in 1998. This tiny molecule is a major controller of blood flow and blood
pressure throughout the body and also carries signals between nerve cells. It
is the basis for the action of Viagra®, which works through the
control of blood flow through the penis.
Signaling by
ROS, however, operates somewhat differently than more conventionally understood
signaling. Cytokines are large molecules with relatively large, convoluted
surfaces, with variations in electrical charge across the surface. These effect
signaling by docking with receptors, complimentarily shaped and charged
molecular surfaces on the target cells. The analogy is a key fitting into a
lock and opening a door. This paradigm provides the essential element for cell
signaling: specificity. If every cytokine activated every receptor the
result would be chaos, not control.
This need for
specificity presents a real problem for those who believe that ROS act as
signaling molecules. ROS are small, relatively simple molecules that are so
reactive that they often react with the first adjacent molecule they encounter,
often over distances measured in angstroms and within nanoseconds. Indeed,
their reactivity is promiscuous, not specific. Consequently, signaling
specificity cannot be based upon molecular specificity. We have
hypothesized that the necessary specificity of ROS-mediated signaling is effected by subcellular compartmentalization, providing
spatial proximity between the source of ROS generation and its target. This is
in fact how the organism protects itself against oxidant injury by endogenously
generated ROS: The compartmentalization of phagocytic microbicide within the
phagosome is one example. Another is provided by the mechanism of energy (ATP)
generation by oxidative phosphorylation described earlier. Not only is this
mechanism compartmentalized within the mitochondria, it is effected by each of
the component molecules (cytochromes) being arrayed on the mitochondrial membrane,
directly adjacent to one another to form the respiratory chain. This allows the
unpaired electrons to be passed directly from molecule to adjacent molecule as
oxygen is reduced to water. Because these electrons are so con- fined, they do
not escape into the cytoplasm to react nonspecifically with other molecules.
If the
specificity of molecular signaling by ROS is based upon subcellular
compartmentalization, it presents real challenges to the investigator. Traditional
biochemical techniques require the homogenization of cells and the separation
of pools of particular components by differential density centrifugation of
these components. Obviously, such an approach would destroy the very
architectural structure that we wish to study. It would be analogous to try to
understand how a
Our current research
centering on this problem of ROS-mediated signaling involves imaging these
oxidant fluxes at the subcellular level in intact, living cells in (near) real
time using advanced imaging techniques, including confocal fluorescence
microscopy. We are optimistic that this approach will facilitate future studies
of this kind.
HISTORICAL
DEVELOPMENTS IN THE ROLE OF ROS
The evolution of
our understanding of the role of free radicals and other ROS in biology and
medicine is a fascinating story in its own right. Physicists had long
appreciated that violent free radical reactions were taking place within the
sun and within a thermonuclear blast. Obviously, such extreme conditions are
incompatible with any form of life as we understand it. As we began to control
and use radiation for less violent purposes, we soon came to understand that
some of the more subtle effects of radiation upon the body, including radiation
sickness and carcinogenesis, were also caused by free radicals. Nevertheless,
this conceptual framework was focused upon exogenous, physicalchemical sources
of ROS generation, and emphasized the destructive nature of their interaction
with living things. Indeed, this mindset was so strong that the idea that such
violently reactive molecules could be generated by living organisms was
considered ridiculous.
In 1969, when
Joe McCord and Irwin Fridovich published a paper indicating a common, nearly
ubiquitous intracellular protein (SOD) functioned primarily as a free radical
scavenger, thereby suggesting that the routine defense against endogenously
generated ROS was a major function of the cell, they were ridiculed by many of
their colleagues.Today, their discovery is recognized as the basis of a whole
new field of biology and medicine.
It took about a
decade for the scientific community as a whole to appreciate the importance of
McCord and Fridovich’s contribution, and this
was greatly facilitated in 1980, when Neil Granger and his colleagues first
described free radical-mediated reperfusion injury in the cat small intestine. From
here, the pendulum swung rapidly toward the other extreme: Ischemic disease,
especially heart attack and stroke, were (and continue to be) major threats to
our lives, especially within industrialized countries where the drug markets
are lucrative. Unfortunately, in many clinical situations the first opportunity
to treat comes at the end of a period of ischemia but prior to reperfusion
(e.g., cardiopulmonary resuscitation, CPR). If tissue has already died during
the ischemic period, nothing can bring it back to life. But if much of the
injury that had been heretofore ascribed to the period of ischemia was actually
sustained at reperfusion, after treatment could be initiated, some of
the damage might be prevented. The therapeutic (and economic)
implications were enormous: Injuries thought to be irreversible at the point of
treatment might not yet have occurred! Not surprisingly, a feeding frenzy
ensued, catapulting a small cadre of previously obscure (and often ridiculed)
free radical biologists into sudden prominence.
Some of these
reactions were truly excessive, some humorous, and some unfortunate. On the
humorous side, I recall sitting at a scientific meeting in Point Clear,
Far less
humorous consequences included predictably premature clinical trials of SOD for
myocardial infarction. Fortunately, no one was harmed in these trials, but a
great deal of money and effort was wasted. Over time, it has become clear that
while the reperfusion component of post-ischemic injury is quantitatively quite
important in some clinical situations, it is less so in many others, including
heart attack and stroke. The real value of the work in this field has been, in
my view, a far better understanding of basic physiology, upon which future
advances in the treatment of disease will be based. Viagra® is
indeed a prototype for this developmental process.
Even in the
1980s and early 1990s, our perspective of free radical biology emphasized their
destructive capacity. I sometimes think we envisioned them as tiny blowtorches
running around the body burning things, while our bodies chased after them with
antioxidant fire extinguishers. The analogy clearly is appropriate for
microbicide and reperfusion injury, but it is also too narrow to encompass the
full range of the impact that ROS have on biology and medicine. In the past few
years the appreciation of the role of small fluxes of highly compartmentalized
ROS in controlling normal cell function has greatly expanded this horizon.
What have we
learned from this experience? Beyond the obvious examples of human weakness,
some general observations appear appropriate. One is an appreciation of the
strikingly different impact that an agent can have on biological systems,
depending upon the dose. In massive quantities, ROS can vaporize living things;
in moderate quantities they can kill pathogens to our advantage; and in tiny
quantities at precise locations they can act very precisely as controlling
molecular switches. Similarly, it is perhaps human nature to classify agents in
our environment as “good” or “bad” for us,
but nature doesn’t always make such distinctions. This mindset clearly has
slowed our progress of understanding free radical biology in the past.
Perhaps the most
striking lesson we can take from this is the awesome, opportunistic power of
natural selection to drive the evolution of complicated mechanisms such as the
mitochondrial respiratory chain, phagocytic microbicide, and even cell
signaling to exploit the violent reactivity of ROS to the organism’s survival advantage. This is truly mind-boggling. Finally, this narrative
illustrates how biased and narrow- minded we scientists can be, just like other
people. I frequently am amused by the lengths we must go to show the absence of
a conflict of financial interest, or even the appearance of one, when
peer-reviewing a paper for publication or a grant for funding. Most scientists
are far more subject to bias driven by their preconceived ideas and their own
pet ideas and hypotheses than by financial considerations.
The study of
free radicals and other ROS in biology and medicine is endlessly fascinating
and quite intellectually rewarding. It can carry one rapidly from the
ridiculous to the sublime.At the ridiculous level are the imaginative
advertisements for antioxidant supplements, for the diet, to rub on the skin,
or even as an enema (!) to counteract the ravages of aging. They don’t and can’t work, at least not yet.
At the other end
of the spectrum is human sexuality, which owes a lot more to ROS than Viagra®.
There is a species of yeast (Saccromyces pombe) that reproduces by asexual
budding in its normal, anaerobic environment. This is efficient, but does not
allow for the recombination of genes characteristic of sexual
reproduction, which produces the genetic variations that are acted upon by
natural selection to drive evolution.When these yeast cells are exposed to the
normal levels of oxygen of an aerobic environment, they are severely
stressed by the ROS that are generated, and this triggers sexual
reproduction. Helen Bernstein at the
![[photo of Gregory B. Bulkley]](bulkley.jpg)
Gregory B.
Bulkley (CC ’90) is Mark M. Ravitch
Professor of Surgery at
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