Free Radical Biology and Medicine 97 (2016) 398–407

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Free Radical Biology and Medicine

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

Review article

Redox signaling: An evolution from free radicals to aging$

Henry Jay Forman
Leonard Davis School of Gerontology, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089, United States

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

Article history: Redox biology has evolved from studies of the pathology that involves oxidants to an understanding of
Received 15 June 2016 how oxidants participate in normal as well as aberrant signal transduction. Although the concept that
Received in revised form signal transduction involved changes in the redox state dates from the 1930s, the modern history of
30 June 2016
redox biology began with the discovery of superoxide dismutase by McCord and Fridovich. The initial
Accepted 4 July 2016
Available online 5 July 2016
focus was on free radicals and damage of macromolecules, which remains an important topic. But, over
time it was realized that hydroperoxides, especially H2O2 produced by NADPH oxidases, and electro-
Keywords: philes derived from lipid peroxidation or metabolism, played essential roles in physiologically relevant
Oxidative stress signaling. The mechanisms through which H2O2 and other electrophiles signal became an important area
Redox signaling
of study that provided insight into how these reactive molecules were involved in major signaling
Superoxide
pathways and regulation of transcription factors. Thus, the field of redox signaling that is the overlap of
Hydrogen peroxide
NADPH oxidase signal transduction with redox biology was established. Alterations in redox signaling are observed in
AP-1 aging, but we also now know that redox signaling is essential in physiological homeostasis and that
Nrf2 sustained deviation from redox homeostasis results in disease. This is a review of the history of redox
Aging biology from a personal perspective of nearly fifty years working in this field that hopefully provides
Homeostasis some insights for the reader.
Antioxidant & 2016 Elsevier Inc. All rights reserved.
Air pollution
Thiolate
Glutathione

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

2. Superoxide as a product or intermediate in enzymatic reactions – free radical biology begins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
3. Superoxide dismutase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
4. Mitochondrial superoxide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
5. Pulmonary oxygen and paraquat toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
6. Phagocytic superoxide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
7. Glutathione biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
8. Redox signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
9. Mechanisms of signaling by electrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
10. Air pollution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
11. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
12. Redox signaling chemistry and redox homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
13. Key publications in the field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
14. Summary of lessons learned . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

1. Introduction

From the time of Joseph Priestly and the other co-discovers of

☆
Current major support is from NIH grant ES023864. oxygen in the eighteenth century, the potential toxicity of this

http://dx.doi.org/10.1016/j.freeradbiomed.2016.07.003
0891-5849/& 2016 Elsevier Inc. All rights reserved.
 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407 399

essential gas has been known. But, understanding of the me- in the laboratory next to the Fridovich lab, joined the Feigelson
chanism did not advance significantly until the 1950s when Re- laboratory. Frank introduced us to the concept of using superoxide
becca Gerschman and coworkers noted that oxygen poisoning dismutase as a tool to study biological oxidations [8]. He also in-
resembled the damage from X-rays and proposed that free radicals troduced me to Irwin Fridovich. Frank passed away at a relatively
were involved [1]. Shortly after, Harman proposed that aging could early age, but he had a great positive and lasting influence on
also involve free radicals based upon radiation chemistry [2]. The those who knew him.
modern history of redox biology however, began with the dis- The Spring of 1968, my second semester in graduate school,
covery by Joe McCord and Irwin Fridovich of superoxide dismutase was tumultuous. Early in the semester, a riot broke out and the
[3]. police occupied the Columbia University campus where we at-
I was fortunate to become a postdoctoral fellow in the Frido- tended most of our classes. In April, Martin Luther King was as-
vich laboratory at a time when the field, then known as free ra- sassinated and riots broke out in several cities. During the summer
dical biology, was just beginning to expand. It is a very good thing of 1968, Robert Kennedy was assassinated. Not long after, a riot
to be in the right place at the right time. Is also good to be inspired broke out outside the Democratic convention in Chicago. For a
by brilliant minds. My graduate mentor, Philip Feigelson, my liberal New Yorker, it was a time that becoming radical could have
postdoctoral mentor, Irwin Fridovich, and the person who gave me happened easily. Fortunately, free radical biology was the clear
the opportunity to begin my independent career, Aron Fisher, are choice over radical politics.
the scientists who most influenced my career.
Albert Szent-Gyorgyi, the Nobel Prize winning discover of vi-
tamin C [4], proposed the involvement of free radicals in biological 3. Superoxide dismutase
systems well before the discovery of superoxide dismutase [5].
Szent-Gyorgyi often quoted the statement, attributed to both In 1969, Joe McCord and Irwin Fridovich published their classic
Schopenhauer and Jonathan Swift in various forms, that, “Dis- paper on the discovery that erythrocuprein had SOD activity [3].
covery consists of seeing what everybody has seen and thinking This discovery and the burst of publications from the Fridovich la-
what nobody has thought.” That this statement itself evolved boratory over the next few years (summarized in [9,10], provided a
provides an example of what underlies most of the breakthroughs foundation for the field. A postdoctoral fellowship from the National
in science. We all depend upon what came before, and make ad- Institutes of Health allowed me to work with Irwin and resulted in
vances by seeing things in different ways with the assistance of four publications [11–14]. These papers included determining the
technologies that our predecessors did not have. Likewise, my active site histidines in SOD1, the rate constants for different SODs,
contributions have depended on the work of many others in the and the role of zinc in SOD 1. The findings regarding zinc in struc-
field of redox biology, some of whom I have had the privilege to tural stability of SOD1 were cited fourteen years later in a ground-
work with directly. They are listed at the end of this review. The breaking paper suggesting a role for abnormal zinc binding in SOD1
lesson learned from the words of Szent-Gyorgyi that influenced that causes the enzyme to gain a nitration catalysis function and
my career is to keep your eyes and mind open. thereby participate in the pathology of amyotrophic lateral sclerosis
[15]. The environment in the Fridovich laboratory was wonderful. It
taught me that one should always try to take advantage of being in
2. Superoxide as a product or intermediate in enzymatic re- a place where new development is occurring.
actions – free radical biology begins

4. Mitochondrial superoxide production

The budget consequences of the Vietnam War had a devastat-

ing economic impact on science. Under the circumstances, the best
position offered was a Research Associate position in Kansas City.
Without dwelling on how, at the age of twenty-six I nearly left
academia, thanks to James Kennedy a position at the Veterans
Administration Hospital, which led to a Research Assistant Pro-
fessor appointment at the University of Kansas Medical Center
developed. While at the VA, I began work with James on pyr-
imidine biosynthesis, which unexpectedly led to one of my most
fortunate and important findings.

Irwin Fridovich suggested that xanthine oxidase produced su-

peroxide as an intermediate in the oxidation of sulfite [6], eight
years before he and his student, Joe McCord discovered superoxide
dismutase (SOD) [3]. In 1968, as a graduate student investigating
the catalytic mechanism of tryptophan oxygenase with Philip
Feigelson at Columbia University, we proposed that the enzyme
formed a ferric heme-superoxide intermediate that did not release
superoxide from the enzyme [7]. This began my interest in free
radicals and I fell under the influence the old work of Szent-
Gyorgyi. About that time, Frank Brady, who had done his graduate
work at Duke University with Philip Handler and K.V. Rajagopalan
400 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407

In the early 1970s, Alberto Boveris, Nozumo Oshino, and Britton Rats exposed to 100% oxygen at 1 atmosphere die within
Chance described the production of H2O2 by mitochondria [16]. In 1974, 3 days. Rats exposed for 5 days to 80% oxygen adapt and become
Loschen, Azzi, Richter and Flohe, discovered that succinate oxidation in able to survive indefinitely in 100% oxygen. In the 1970s, Crapo
mitochondria produced O2
 and suggested that it was the source of and Tierney [26], using this model of adaptation to hyperoxia,
the H2O2 [17]. At almost the same time, we discovered that the oxi- demonstrated that increased SOD activity in the lung correlated
dation of dihydroorotate, which is the one step in pyrimidine bio- with adaptation to hyperoxia. About the same time, studies were
synthesis that occurs in mitochondria, also produced O2
 [18]. But, the being made of the toxicity of the herbicide, paraquat, which
more important discovery in that study was that the reaction producing caused lethal lung damage through generation of O2
 con-
O2
 was thermodynamically unfavorable and actually pulled forward tinuously [27]. In 1978, I was recruited to the University of Penn-
by SOD. We did not know what the exact reaction was that produced sylvania by Aron Fisher, who introduced me to pulmonary phy-
O2
 , but soon after a debate began about cytochrome b versus ubi- siology and toxicology. Investigating how different cells in the
quinone as the source. Work by Enrique Cadenas and Alberto Boveris lungs of adapting rats changed in the activities of SOD1 and SOD2
[19] strongly indicated that ubisemiquinone oxidation was the source of along with other enzymes that contributed to antioxidant defense,
O2
 . Christine Winterbourn showed that the reaction of semiquinones we found that the cells that appeared to be most responsive in
with oxygen to produce O2
 was thermodynamically unfavorable and terms of increasing these activities were the granular type II epi-
that SOD could pull the reaction forward [20]. This work explained our
thelial cells [28]. With Tom Aldrich, we also explored how para-
previous finding that SOD promoted the production of O2
 in which
quat accumulates specifically into those cells making type II epi-
Q. − + O2 ⇄ Q + O2.− is displaced to the right by SOD in the reaction
thelial cells the target of the toxicity [29]. It was interesting to
2O2.−+2H + → H2O2 + O2. As later pointed out by Forman and Azzi, this
SOD observe that the cells that adapted best to hyperoxia were the
results in mitochondrial O2
 concentration being negligible [21]. Only specific target of paraquat.
a reaction with a rate constant competitive with the near diffusion
limited rate of SOD, such as the reaction of
NO with O2
 to produce
peroxynitrite [22], would be likely to involve O2
 directly.
Although work on mitochondrial H2O2 and O2
 production con- 6. Phagocytic superoxide production
tinued, and has had a resurgence in the last few years [23], my work in
that area ended when the Biochemistry Study Section of the National
Institutes of Health told me that, “Superoxide production by mi-
tochondria is an uninteresting artefact.” Moving on, I always have
wondered whether my project would have been funded if the “artefact”
was more interesting to those reviewers.

5. Pulmonary oxygen and paraquat toxicity

As mentioned at the beginning of this article, the toxicity of

oxygen has been known since its discovery when Lavoisier noted in
1783 that mice exposed to pure oxygen had damage to their lungs
[24]. Probably the clearest example in terms of human experience is
the toxicity of oxygen therapy for premature infants, which notably
can cause lifelong consequences for lung function and blindness. But,
for a century and a half, the underlying cause of oxygen toxicity The signaling pathway and function we chose to focus on was
remained unknown. While free radicals were proposed by Gersch- the respiratory burst of alveolar macrophages. Bernie Babior dis-
man based on the resemblance to injury from X-rays [1], there was covered in 1973 that phagocytes generated O2
 [30]. We began
little direct evidence. Interest in the field increased during World our studies looking at how paraquat might cause loss of the ability
War II when it was noticed that divers breathing pure oxygen had of macrophages to produce O2
 when stimulated. Through ser-
seizures. In 1945, Haugaard and coworkers however, demonstrated endipity and the careful notebook keeping of June Nelson, we
the loss of activity of enzymes after hyperbaric oxygen exposure [25]. found that in the absence of glucose, paraquat depleted the sub-
strate, NADPH, for the phagocyte oxidase and only later became
toxic to the cells in the presence of glucose [31]. But, paraquat did
not deplete NADH. Babior learned about our results and said that
there was an ongoing debate about whether NADPH or NADH was
used by the phagocyte oxidase and that our work on toxicity
contributed to resolution of this debate in favor of NADPH.
Sometimes one gets very lucky.
In the 1980s free radical biological research was largely focused
on mechanisms of cell injury and death. When I moved into my
first fully independent position, we laboratory began to study the
mechanisms whereby oxidants cause inhibition of signaling, as we
observed changes in macrophage functions in exposure to hyper-
oxia and low exogenous concentrations of hydroperoxides both in
vitro and in vivo without cell death [32,33]. This was important
because we get sick more often than we die! Furthermore, if one is
going to use in vitro models, it is essential needed to avoid using
“the thermonuclear attack model of toxicology” in which cells die
from doses of added hydroperoxides that has no resemblance to
actual physiological or even pathological conditions.
 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407 401

7. Glutathione biosynthesis absent as was the understanding that O2
 and H2O2 were gen-
erated in cells. In the 1970s a few studies showed that H2O2 could
mimic insulin [41,42], but the mechanisms were unknown. Be-
ginning in the late 1980s studies showed that exogenous H2O2
could modulate multiple signaling pathways, but specific targets
were not clear with a few notable exceptions. Gopalakrishna and
Anderson showed that classical protein kinase C activity could be
elevated by H2O2 in the absence of calcium [43], while Lands and
others showed that a lag in the activation of cyclooxygenase [44]
and 5-lipoxygenase [45] was abolished by low concentrations of
H2O2. Lands coined the term “peroxide tone” and suggested that
Glutathione (GSH), γ-L-glutamyl-L-cysteinylglycine, is the re- the production of eicosanoids was regulated by the endogenous
ducing substrate for the glutathione peroxidases and peroxir- level of H2O2 in cells.
edoxin 6 in removal of hydroperoxides, and for the glutathione But, almost all the early work on the activation of cell signaling
S-transferases that help remove toxicants as well as participate in by H2O2 was done through addition of H2O2 to cells. This included
leukotriene C4 synthesis. GSH is synthesized and degraded in a our own work on the respiratory burst of alveolar macrophages
cycle that was described by Alton Meister [34]. Helmut Sies, who [46]. In those studies, we had switched from looking at how hy-
coined the term oxidative stress [35], suggested that GSH oxida- droperoxides inhibited the respiratory burst without killing the
tion to the disulfide (GSSG) and formation of mixed protein-glu- cells to investigating the mechanism underlying an serendipitous
tathione disulfides was the essential determinant of that stress. discovery that low concentrations of hydroperoxides increased
Research in our laboratory on altered cell function led us into subsequent stimulation of the respiratory burst [47], which we
studies of glutathione redox cycling, primarily by Mark Sutherland, eventually found was due to an elevation of intracellular calcium
that were cited recently [36] in an issue of Archives of Biochem- [48] and activation of phosphatidylcholine specific phospholipase
istry and Biophysics dedicated to Helmut Sies upon his retirement C (PC-PLC) [49]. This work was carried out by two graduate stu-
as Editor in Chief of the journal. dents, Judith Murphy and Carolyn Hoyal, and Julio Giron-Calle, a
An increase in total GSH plus GSSG (GSH is almost all in the postdoc.
GSH form in unstressed cells) occurred in many situations where
cells or animals were exposed to sublethal oxidative stress. We
decided to pursue the question of how this occurred. Using redox
cycling quinones, menadione that could be conjugated to GSH and
another, 1,2-dimethoxy-naphthoquinone that could only redox
cycle, Ming Shi, Li Tian, Rui-Ming, and Amir Kugelman demon-
strated that both catalytic and modulatory subunits of the first
enzyme in de novo GSH biosynthesis, glutamate cysteine ligase
(GCL, also known as γ-glutamylcysteine synthetase) could be
transcriptionally induced by mild oxidative stress [37–39]. We also
showed that γ-glutamyltranspeptidase, which is involved in GSH
recycling was also transcriptionally inducible by quinones [40].

One of the most interesting and influential studies of signaling

caused by the addition of exogenous H2O2 was the activation of
The studies of GSH biosynthesis began at the Institute for
Toxicology at the University of Southern California. Paul Hochstein, the NF-κB transcription factor [50]. Realizing that our experi-
Alex Sevanian, Kelvin Davies, Enrique Cadenas, Joseph Landolph mental model cells, macrophages, generated H2O2 upon stimula-
and others in this group that focused on free radical biology pro- tion, my colleague Martine Torres and I wondered if the amount of
vided an outstanding environment for our work. I also was for- H2O2 generated would be sufficient to activate NF-κB in these
tunate to become the Head of the Cell Biology Group at Children’s cells. It was [51]. Working in collaboration with Rayadu Gopa-
Hospital of Los Angeles, where we collaborated with Thomas lakrishna, Nalini Kaul from our laboratory found that the activa-
Coates and Martine Torres among others. The latter became my tion of NF-κB by endogenously produced H2O2 was be due to the
main collaborator for many years until her retirement. Martine, an activation of protein kinase C [52]. We also showed that increased
expert in signal transduction, influenced much of my thinking on tyrosine phosphorylation of many proteins and activation of the
the roles of oxidants in signaling. Retaining relationships over ERK pathway by stimulants of the respiratory burst in macro-
decades has been of great value to my career. With Tom Coates, we phages was H2O2 dependent [53]. At the time we discovered this,
are currently looking at how sickle cell disease alters redox it was widely believed that the only cells that produced H2O2 in
homeostasis in blood, and as described below, we currently col- response to stimulation were phagocytes. Thus, despite our being
laborate with Kelvin Davies on studies of aging. able to publish this work and even get funded by the NIH to study
it, the activation of signaling by endogenously generated H2O2 was
8. Redox signaling considered an oddity of macrophages and other phagocytes. That
changed dramatically in 1999 when David Lambeth and coworkers
In the 1930s Warburg and Szent-Gyorgyi proposed that redox discovered that other cells had a related NADPH oxidase [54] and
reactions regulate cell function, but tools for studying this were then that there were seven mammalian NADPH oxidases labeled
402 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407

as NOXs and DuOXs [55] distributed in every cell type. With that appeared to be responsible for pathology of several diseases [68–71].
discovery, the field of redox signaling now clearly had identified a Poli then demonstrated that low concentrations of HNE could act as
major source in all cell types of H2O2 produced upon stimulation. stimulants of signaling [72–74]. We first learned about HNE when we
While mitochondria, other organelles and some oxidoreductases began to examine the mechanism through which exposure to the
generate H2O2, it remains to be determined whether anything atmospheric pollutant, nitrogen dioxide, caused loss of macrophage
other than the NOX and DuOX proteins are truly involved in sti- function. Working with Michael J. Thomas, Tim Robison and I showed
mulated signaling. The work on redox signaling resulted in one of that NO2 exposure of macrophages resulted in the formation of many
the first reviews [56] and two edited books on the topic [57,58]. aldehydes, and that HNE was the most abundant [75]. Then Rui-Ming
Going back to the suggestion of Lands regarding peroxide tone Liu, Dale Dickinson, Karen Iles, and Hongqiao Zhang showed that HNE
[59], it seems the lipoxygenases and cyclooxygenases have the could induce GSH biosynthesis through transcriptional elevation of
best chance of fitting that description by providing lipid hydro- GCL and GGT gene expression [76,77]. Expression of GCL genes ap-
peroxides in response to stimulation. That hydroperoxides rather peared to be under the control of the AP-1 transcription factor [78].
than other so-called reactive oxygen species are the mediators of It was becoming clear that while generation of H2O2 could in-
signaling has been addressed in detail in recent reviews [60,61]. duce GCL, other electrophiles were actually stronger inducers. Nrf2
In 1999, we moved to the University of Alabama at Birmingham is the transcription factor that appears to be a master rheostat in
where I became the Chair of the Department of Environmental transcriptional regulation of a large number of enzymes involved
Health Sciences and joined the Center for Free Radical Biology in protection of cells from stress. The regulatory element, first
headed by Bruce Freeman and Victor Darley-Usmar. It was for- called the antioxidant response element (ARE) was discovered by
tunate that almost all of the graduate students and postdocs in my Cecil Pickett [79]. Shortly after, a more accurate name for these
laboratory at USC moved to UAB. Both USC and UAB were great group of related sequences, the electrophile response element
environments in which we interacted with some of the best in- (EpRE) was supplied by Violet Daniel [80]. Around the time we
vestigators in the free radical biology field. began to look at HNE-induced GCL expression, Tim Mulcahy de-
At UAB, we continued to investigate how H2O2 generated by monstrated that the two subunits of GCL were regulated by Nrf2 in
cells acts as a signaling molecule, particularly on the activation of response to electrophiles [81,82]. So, we examined whether the
the AP-1 transcription factor [62]. Ichijo and colleagues demon- electrophiles, HNE, curcumin, and 15-deoxy-Δ12,14-prostaglandin
strated that oxidation of thioredoxin (Trx) by addition of H2O2 to J2, the latter in collaboration with Anna-Liisa Levonen and Victor
cells activated ASK1, a kinase that is upstream of both p38MAPK Darley-Usmar, induced GCL gene expression through Nrf2 [83–86].
and JNK [63]. Honglei Liu, a postdoc in our laboratory, showed that What was clear from our studies and those of others was that the
the activation of H2O2 production in macrophages led to activation GCL genes are dually regulated by both AP-1 and Nrf2 [84].
of the JNK pathway through the Trx mediated activation of ASK1 In 2003, we moved to the new University of California campus
[64]. Subsequently, Im and coworkers showed that the oxidation that was being built in Merced, a small city in the agricultural
of Trx in this system is through the activity of peroxiredoxin [65]. heartland of the Central Valley of California. Once again, several
members of the UAB laboratory made the move to UC Merced.
There, surrounded by cows, we continued studies on how both
9. Mechanisms of signaling by electrophiles hydroperoxides and HNE activated the ERK, JNK pathways.
Two new postdocs, Alessandra Rinna and Smadar Levy joined the
lab along with a graduate student, Chris Mahaffey. Smadar discovered
that c-Myc played a role in regulation of Nrf2 by apparently binding to
Nrf2 in the Nrf2-EpRE complex and thereby inhibiting transcription of
several phase II genes. Moreover, c-Myc also appeared to cause de-
gradation of the nuclear pool of Nrf2 [87]. Chris showed that HNE
could induce multidrug resistance protein 3 through activation of
Nrf2 in human bronchial cells and non-small cell lung carcinoma cell
lines, where many of the latter have defective Keap1 that causes
constitutively high MRP3 expression [88,89].

Alessandra demonstrated that activation of H2O2 production

The non-enzymatic oxidation of polyunsaturated lipids leads to caused transient inactivation of protein tyrosine phosphatase 1B
the production of a large variety of products. Hermann Esterbauer (PTP1B) through glutathionylation stimulated by the endogenous
was the discoverer of 4-hydroxy-2-nonenal (HNE), one of the more generation of H2O2 [90]. PTP1B is the enzyme that prohibits acti-
abundant and biologically active products [66,67]. Along with Dia- vation of the ERK pathway by dephosphorylating Raf1. Another of
nzani, Comporti, Poli, Slater, and Esterbauer showed that HNE her studies demonstrated that HNE could activate the JNK
 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407 403

pathway through inhibition of the protein tyrosine phosphatase, in his laboratory in the Leonard Davis School of Gerontology. For
SHP-1 [91]. the following six years, my laboratory was at USC, while all of my
Studies on the pathophysiological role of HNE have continued teaching responsibilities remained at UC Merced. This was an
in a collaboration with Giuseppe Valacchi [92,93]. We first met on undergraduate organic chemistry and a graduate level course in
the tennis courts of the University of California at Davis when signal transduction.
Giuseppe was a postdoc in Carroll Cross’s laboratory. Hongqiao Zhang and Honglei Liu, who moved from UAB to UC
Merced also moved with the laboratory to USC. A very positive
10. Air pollution part of coming back to USC was the opportunity to collaborate
with Kelvin along with Caleb (Tuck) Finch, leaders in aging re-
Collaborations with people at UC Davies led to development of search. USC is also the home of the Southern California Environ-
a joint training program with UC Merced in air pollution. During mental Health Sciences Center, where my lab can interact with
this time, I was appointed by the Governor of California to the many experts in air pollution, including Costas Sioutas and Frank
Governing Board of the San Joaquin Valley Air Pollution Control Gilliland. In 2015, I became a Distinguished Emeritus Professor of
District. Meeting a movie star and the niece of a president, Arnold Biochemistry at UC Merced and the next day went to USC to my
Schwarzenegger and Maria Shriver was nice, but the appointment current position, which involves almost full time research.
was a unique opportunity to see how research could be helpful in At USC, we began a collaboration with Davies and Finch to try
formulating policy that affects the health of the population.
to understand the mechanisms that cause increased susceptibility
Over many decades, there have been a great many studies on
to the deleterious consequences of air pollution in aging. We
the effect of airborne particulates and manufactured nanoparticles
suspected that this involved a decrease in responsiveness of Nrf2
on health [94,95]. In terms of free radical biology, some of the
signaling that had been reported by Tory Hagen and my former
most notable work has been done by Andre Nel and coworkers
[96]. Studies by Andrij Holian on silica induced toxicity interested postdoc and colleague, Rui-Ming Liu [100]. Our studies demon-
us as well [97]. At UC Merced, Honglei Liu and Hongqiao Zhang strated that the age-related loss in Nrf2-regulated transcription
began our studies of the mechanism through which respirable due to chronic exposure to air pollution was observed not only in
silica activates the production of pro-inflammatory cytokines. lungs, but in the cerebellum and liver as well [101]. The results
Their studies implicated activation of PC-PLC in the signaling [98]. also suggested that an increase during aging in c-Myc and Bach 1,
Gayatri Premasekharan, a graduate student working in materials another inhibitor of Nrf2 transcription, may be responsible for the
science with Valerie Leppert, took this work further by demon- loss of inducibility of Nrf2-regulated genes [101]. This work is
strating an essential role for surface iron and the role of lipid raft currently being pursued in our laboratory. We continue also to
disruption in the mechanism [99]. collaborate with colleagues at UC Merced, particularly Valerie
Leppert and Peggy O’Day, who are analyzing the surface chemistry
11. Aging of air pollution nanoparticles and synthesizing model nano-
particles to allow for a more uniform material for the mechanistic
In 1999, Kelvin Davies asked me if I was interested in returning studies we pursue at USC. Our work on signaling in which H2O2
to the University of Southern California and offered to share space production by cells is involved is illustrated in Fig. 1.

Fig. 1. Studies on H2O2 signaling from the Forman laboratory. The studies are for the endogenous production of H2O2. H2O2 produced by Nox2 can activate Src kinases [105]
and cause inactivation of PTP1B through glutathionylation [90]. Together the activation of a Src kinase and inactivation of PTP1B cause ERK activation [53]. H2O2 produced by
Nox2 is also used to oxidize thioredoxin, which allows activation of the ASK1 to JNK pathway. Stimulation of Nox2 also leads to H2O2-dependent NF-κB activation [51]. H2O2
produced by redox cycling of quinones activates Nrf2 and Phase 2 gene expression [37]. Silica activates Nox2 generation of H2O2 that is then used in the Fenton reaction to
initiate localized lipid peroxidation that results in lipid raft disruption, activation of PC-PLC, and NF-κB-dependent cytokine production.
404 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407

12. Redox signaling chemistry and redox homeostasis tion of hydroxyl radical, which no enzyme or small molecule can
do efficiently as
OH reacts with all molecules with rate constants
In 1990, Fulvio Ursini and Matilde Maiorino invited me to come near the limit of diffusion. The likely reason why many dietary
to the University of Padova for a month long visit. The purpose was compounds, including isocyanates and flavonols that have been
to begin a collaboration on the effect of hydroperoxides on sig- called antioxidants work is because they or their metabolites can
naling. Over the past 26 years, the collaboration has evolved, as did alkylate Keap1. By activating Nrf2, transcription of many anti-
the field, toward attempting to understand how hydroperoxides oxidant enzymes as well as the enzymes that provide the sub-
work as signaling molecules, understanding how antioxidants strates for the antioxidant enzymes are elevated. This maintains a
work, and how redox signaling contributes to homeostasis. Too nucleophilic tone in cells. This brings the discussion back to Albert
many people have contributed significantly to these areas to Szent-Gyorgyi. In 1936, he proposed that the dietary phytochem-
mention all of them here. But, the work of many of those con- icals, flavonols, should be called vitamin P foreshadowing again an
tributors are cited in our recent articles. important component of redox biology [103]. Indeed, as with other
In two reviews [60,61], we have argued that among the so- vitamins, it is difficult to demonstrate any positive effect for health
called reactive oxygen species, only H2O2 and other hydroper- with high doses. It remains to be seen whether an insufficiency of
oxides fulfill the requirements for functioning as a second mes- electrophiles in the diet (if that can actually be achieved without
senger. The important points we made in these reviews were general malnutrition) would reveal that they are indeed vitamins.
based on understanding of kinetic constraints that are frequently Our most recent publication [104] concerns the maintenance of
ignored. The targets for hydroperoxide signaling are protein cy- redox homeostasis, which involves signaling by electrophiles in-
steine in their thiolate form (-S-). But, non-enzymatic oxidation of cluding HNE along with hydroperoxide signaling to maintain nu-
thiolates by H2O2 is, in most cases, too slow to be involved in cleophilic tone. The principle arguments we made, besides re-
signaling. Indeed, the rate of non-enzymatic oxidation of GS-, iterating what we described in the prior reviews, are: that redox
which is 105 times as great as the protein thiolate. Thus, the in- signaling acts as a rheostat in a continuous dynamic process of
termediate formation of a sulfenic acid through oxidation of a oxidant production balanced by reduction rather than an on-off
protein thiolate must involve enzymatic catalysis of thiol switch; redox signaling is determined by reaction kinetics and
oxidation. does not rely on reaching thermodynamically defined ratios; redox
In another review with Davies [102], we described how the signaling involves reactions of specific electrophiles with specific
kinetics of antioxidant defenses requires that, with the exception protein thiolates, primarily through enzyme catalyzed reactions;
of vitamin E scavenging of hydroperoxyl radicals, rules out a role and that challenges to redox homeostasis generally stay within the
for scavenging of oxidants including free radicals by non-enzy- bounds of a physiological defined range of electrophiles and nu-
matic mechanisms. Instead, enzymes with fast rate constants re- cleophiles, but that a condition of prolonged and/or enhanced
move superoxide and hydroperoxides. This then prevents forma- exposure to environmental stressors in either the oxidized (pro-

Table 1
Key papers in the history of redox biology – a biased view.

Year Key finding Reference

1954 Gerschman and coworkers notes oxygen poisoning resembles X-ray damage and propose free radical involvement [1]
1956 Harman proposes the free radical theory of aging [2]
1958 Szent-Gyorgyi proposed involvement of free radicals in biological systems [5]
1967 Esterbauer describes the production of 4-hydroxy-2-nonenal (HNE) [66]
1969 McCord and Fridovich discover superoxide dismutase [3]
1970 Meister describes the γ-glutamyl cycle in glutathione biosynthesis [34]
1972 Boveris Oshino, and Chance discover production of H2O2 by mitochondria [16]
1973 Babior and coworkers demonstrate O2
 production by phagocytes [30]
1974 Two labs demonstrate O2
 production by mitochondrial respiratory chain [17,18]
1974 Crapo and Tierney demonstrate adaptation to hyperoxia in rats correlates with increased superoxide dismutase [26]
1976 Cadenas and Boveris show that ubisemiquinone oxidation is the source of mitochondrial O2
 [19]
1977 Murad demonstrates nitric oxide induction of cyclic GMP [106]
1979 Ignarro demonstrates muscle relaxation by nitro compounds including NO [107]
1981 Furchgott describes endothelial derived relaxing factor (EDRF) [108]
1983 Lands demonstrates peroxide tone regulates cyclooxygenase [44]
1984–1985 Comporti, Dianzani, Poli, Slater, and Esterbauer demonstrate pathology due to HNE [66,67,70]
1985 Sies defines oxidative stress [35]
1987 Poli demonstrates that HNE acts as a stimulant of signaling [72]
1989 Gopalakrishna and Anderson demonstrate H2O2 activation of classical protein kinase C [43]
1991 Schreck and coworkers demonstrate NF-κB activation by addition of exogenous H2O2 to lymphocytes [50]
1991 Rushmore and Pickett discover the antioxidant response element (aka EpRE) [79]
1994 Forman and coworkers demonstrate that quinones induce glutamate cysteine ligase (GCL) [38]
1995 Mulcahy and coworkers show that EpRE regulates [81]
1996 Kaul and Forman demonstrate activation of NFκB by endogenously generated H2O2 in macrophages [51]
1996 Venugopal and Jaiswal identify Nrf2 as regulator of EpRE [109]
1998 Denu and Tanner demonstrate reversible inactivation of PTP1B by H2O2 [110]
1999 Lambeth and coworkers discover the NOX DuOX family of enzymes is found in almost all cells [55]
1999 Chock demonstrates glutathionylation of PTP1B [111]
2001 Kensler, Yamamoto and coworkers demonstrate link of Nrf2 deficiency to carcinogenesis [112]
2002 Talalay, Yamamoto, and coworkers demonstrate Keap1 modification by electrophiles [113]
2003 Nel and coworkers demonstrate a role for oxidants in signaling by air pollution particle exacerbation of asthma [114]
2003 Yamamoto, Hayes and coworkers demonstrate Keap1 facilitates Nrf2 degradation [115]
2004 Levonen, Darley-Usmar and coworkers show HNE conjugates to Keap1 [116]
2004 Hagen and coworkers demonstrate loss of Nrf2 signaling in aging [100]
2006 Forman and coworkers demonstrate selective and reversible PTP1B glutathionylation during signaling [90]
 H.J. Forman / Free Radical Biology and Medicine 97 (2016) 398–407 405

inflammatory) or reductive direction results in a new steady state Finch, Todd Morgan, and Corinne Spickett.
that is abnormal. Thus, while adaptation may preserve function, it Important contributions were also made by several under-
results a shift away from physiological redox homeostasis. graduate students, notably Mark Posner, Eric Rotman, Kim Folde-
nauer, Natalie Court, Albert Shih, and Sam Chung.
A number of scientists with whom I never published a research
13. Key publications in the field paper, but whose work inspired mine included Paul Hochstein,
Helmut Sies, Dean P. Jones, Alton Meister, Leopold Flohé, Regina
Table 1 shows a biased list of key publications in the field. Brigelius-Flohé, Christine Winterbourn, Sue Goo Rhee, Leslie Poole,
These are some of the very many key citations that influenced me. Ron Mason, Bruce Freeman, James Crapo, Barry Fanburg, Joe
Citations on nitric oxide have been included in this table although McCord, Hara Misra, Giuseppe Poli, Lars Ernster, Garry Buettner,
not described in the text. This is because the author worked in this Herman Esterbauer, Timothy Mulcahy, Britton Chance, Giovanni
field only as a collaborator, and had the pleasure of Lou Ignarro as Mann, Balaraman Kalyanaraman, Giuseppi Poli, Meg Tarpey,
a frequent visitor during a period of several years when a group of Shannon Bailey, Rakesh Patel, Joe Beckman, Doug Ruden, and Al-
LA radicals met in his home for monthly chalk talks. Studies on bert Szent-Gyorgyi. Of course, there are thousands of other sci-
H2S and other important areas are not cited. While that work entists whose work has influenced the field of redox chemistry
certainly influenced my thinking, I did not want to have thousands and biology and I apologize to all whose work should have been
of references here. My Mendeley database has over 10,000 entries, acknowledged here, but where my memory failed.
most of which could have been included. But, I warned you that Finally, I thank the National Institutes of Health, the California
this was biased! The table ends a decade ago because history has Tobacco Smoke Research Program, and the Berger Foundation for
taught me that one needs the perspective of time to see what is providing the resources for our work. Current major support is
the most significant work. from NIH grant ES023864.

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