Findings in redox biology: From H 2 O 2 to oxidative stress

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Starting to reflect, one's mind goes back in time. What were the personal pivotal influences and decisions that, in retrospect, led to a path of curiosity, enthusiasm, and dedication in science? Reflection, however, also leads toward the future, asking what current developments may help shape the coming age in our science. This is a challenging task. My answer basically is that each new generation of scientists "stands on the shoulders of giants" (humbly adding incrementally to knowledge), and progress is facilitated by novel questions. Advances in methodology help provide new answers, which, in turn, generate new questions. Curiosity and a prepared mind are the two main ingredients. Leaving room for serendipity, groups of scientists join in their new interest, crossfertilize the area, and, with élan and a reasonable dose of competition, move the field forward.
On the sunny side Born in 1942 in Goslar, the town of Kaiser Barbarossa, I grew up together with my younger brother Eckhart at the Western rim of the Harz mountains in the nearby small city of Seesen in Northern Germany. Ernst Sies, our father, was then working at "Sonnen-Werke" (Sun Company), a food company, of which he was to become CEO after the Second World War. He was fortunate not to be in military action, because maintenance of food production and distribution was essential. When in 1945 the line between the British and the Soviet occupation zones (Iron Curtain) was drawn, it went through the middle of the Harz mountains: we had the good fortune to be on the "sunny side," just about 25 miles to the west of it. . .
Living near meadows and hillside forests, I was exposed early to the delights of nature, which made lasting impressions. My parents, Ernst and Ilse Sies, fostered my sense of appreciation of the wonderful treasures provided by nature, as did my first teacher in Elementary School, which I entered in 1948. This teacher, Georg Henkel, contributed a great deal to my positive outlook and confidence: he had a group of us first-graders sit across from each other at a table, gave us problems to solve, such as venturing out to the forest and collecting certain plants and then drawing them in color, doing flower pressings, and presenting the group's work to the class. It was fun! The class model fostered curiosity and recognized individual contributions. Bottom line: you can find out things for yourself, digest what you find, and get recognition for your achievements even though you will make mistakes. I still have contact with Georg, who is approaching his 99 th birthday these days.
The Elementary School in Seesen happened to be where the convoys of US troops made their stop on the way from Frankfurt to Berlin. GIs came over to the school fence and handed us Wrigley's chewing gum, playing great jazz tunes from the American Forces Network (AFN) radio. The experience shaped my interest in the Anglo-American outlook. A self-assembly ship made of balsa wood from a US care package for Christmas 1948 also made a lasting impression.
Another "sunny side" was the Jacobson-Gymnasium in Seesen, founded in 1801. We had good teachers in Latin, physics, math, geology, literature, and history. The school was equipped with a Steinway grand piano. (

My path in biochemistry and molecular biology
The sequential lines of my research activity-how did they come about, and how did I get into biochemistry (1)? What happened between then and now ( Fig. 1), and what were the influences? In the early 1960s, when I entered university as a medical student, redox biochemistry, which would become the research focus throughout my career, was a flourishing field. Reduction-oxidation reactions are at the core of fundamental life processes. The discovery of oxygen was in the 18th century (e.g. Scheele, Priestley, Lavoisier). Energy conversion of sunlight employs redox processes in photosynthesis, and oxidation reactions drive aerobic metabolism. Hydrogen peroxide (Thénard) and selenium (Berzelius) were discovered in the early 19th century, and the elucidation of respiration and redox metabolism occurred in the early 20th century (Warburg, Wieland, Krebs, Szent-György). Based on major discoveries from the previous decades, many new questions had become answerable. As young ones, we were fascinated by the recent breakthroughs.
After graduating from Jacobson-Gymnasium in 1961, I started in academia by enrolling at the University of Tübingen as a medical student and at the studium generale at the Leibniz-Kolleg, which provided a broader base in science and humanities. (The first curriculum of biochemistry worldwide was introduced at Tübingen in 1962; before that, one would study either chemistry or medicine to embark in biochemistry). The trimester theme was "symmetry," with excellent young docents from disciplines as far apart as logic, crystallography, and sociology. For example, one of the docents was Ralf Dahrendorf, later director of the London School of Economics. Experimentally, I was asked to determine the optical rotation of a thenuncharacterized rare sugar by polarimetry, a seemingly pedestrian first step into biochemistry. Tübingen University was a bustling place. One of my fellow students was Bert Sakmann, from Stuttgart. We met on the tennis court and went sailing together at Lake Constance. In our discussions on how to continue our studies, we noted the attractiveness, scientific and otherwise, of Munich, and we enrolled at the Ludwig-Maximilians-Universität. Bert leaned to physiology, joining the Creutzfeld neurophysiology laboratory at the Max Planck Institute of Psychiatry. His studies led him to develop an exciting pathway for fundamental research, the patch-clamp technique; he was awarded a Nobel Prize in Physiology or Medicine in 1991. I chose to go into biochemistry. Theodor Bücher (Fig. 2), a student of Otto Warburg's, was about to take over the chair that had been held by Adolf Butenandt. Bücher was still at the University of Marburg, where he had laid fundamental ground in clinical enzymology and had helped found the scientific basis of the Eppendorf Co. at Hamburg as well as the Boehringer Biochemicals Co. at Tutzing. His group developed ingenious methods in analysis, including what is known as the "Eppendorf cup" and the microliter pipette system with the disposable tip, now worldwide standard laboratory equipment. I approached Professor Bücher to ask whether he would accept me as a doctoral

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Findings in redox biology: From H 2 O 2 to oxidative stress student, and I was given a chance to spend time in his laboratory in Marburg over the summer months ( Fig. 1), staying in a room next to the laboratory (and the rats). If there is something like "epigenetics" in shaping scientific behavior and outlook, these weeks had an imprint: a whole group of enthusiastic young researchers, guided by a fascinated and fascinating professor, working together on a broad range of topics unified by the main theme of cell physiology. So, when Bücher moved to Munich, he brought with him some associates from Marburg, and I was given the chance to start my own experimental work, setting up a laboratory according to what I had picked up during the few weeks at Marburg. What an opportunity, at age 21! With Bücher as my thesis advisor, I worked on near-equilibrium states and steady-state kinetics: the concept that living cells operate at steady state near thermodynamic equilibrium, not at thermodynamic equilibrium (2). Glycolytic enzymes are present at a high activity level compared with the metabolic flux through the glycolysis pathway, and the deviation of substrate/product ratio from thermodynamic equilibrium should be relatively small. However, in fast-twitch (red) and slowtwitch (white) muscle, there should be a detectable difference of this deviation because of the differences in glycolytic flux, particularly upon electrical stimulation of the muscle. I chose enolase as a suitable enzyme to study this phenomenon, using the UV absorbance of phosphoenolpyruvate as a direct readout of enzyme activity (and hence needing to wash quartz cuvettes in concentrated chromosulfuric acid a few hundred times a day). What did I learn from this sometimes tedious exercise? (i) Try to have a direct (in a sense, noninvasive) readout, (ii) do as many runs in a day as necessary to complete a set, so as to avoid novel calibration headaches on another day, (iii) plot results while you still remember what they are, (iv) learn to live with a constant level of frustration and enjoy the fascination of research (this later was what I told my own students as a definition of experimental science). I would like to offer a few words on the stimulating scientific atmosphere at Munich. I had moved to the Max-Kade-House, a student dormitory, which had been donated by the German-American philanthropist Max Kade. Werner Heisenberg had his home across the street, bordering the park, Englischer Garten. He had his daily walk through the park to his nearby Max Planck Institute of Physics and Astrophysics, and I first chatted with him on a walk along the park. In 1964, Heisenberg was the patron of a meeting of the "Deutsche Gesellschaft für Naturforscher und € Arzte" (German Society of Scientists and Physicians) at Weimar, at the other side of the Iron Curtain, then East Germany; I was among the few students from the West to be able to participate. At Munich, there was no formal study of biochemistry yet, so parallel to my experimental thesis work, I enrolled in an autodidactic fashion in the organicchemical laboratory course and in the biochemical colloquium by Feodor Lynen (Nobel laureate for his work on fatty acid metabolism) and courses of electronics and biomathematics. Also, there were great seminars on behavioral physiology by Konrad Lorenz at nearby Seewiesen and on circadian rhythm by Jürgen Aschoff at Erling-Andechs. This brief description of the scientific atmosphere at the time may illustrate how it boosted interest and fostered curiosity.
In 1963, Bert and I had an exciting time, participating as students in the 13th Nobel Prize Winners Meeting at Lindau, on Lake Constance. It was fascinating to meet and talk to eminent scientists, after a grandiose opening by Count Lennart Bernadotte on the Isle of Mainau. Great biochemists were in attendance: Hugo Theorell from Stockholm, talking on ethanol combustion in the liver; Severo Ochoa from New York, on the chemical basis of heredity; Otto Warburg from Berlin, who was in a way my "scientific grandfather-to-be," on the chemistry of photosynthesis; Sir MacFarlane Burnet from Melbourne on the role of thymus in immunity; and last but certainly not least, Sir Hans Krebs from Oxford, who in later years would become a close personal friend and mentor (3), on the regulation of cell metabolism. Wonderful physicists and chemists were also present and available for a brief chat, notably Max Born from Bad Pyrmont and Otto Hahn from Göttingen. It is, of course, difficult to assess what the direct personal exposure to such "Olympic" figures in science does to young students and their own future outlook, but I strongly believe it is a most positive event.  REFLECTIONS: Findings in redox biology: From H 2 O 2 to oxidative stress declined, saying: "I have already attended a meeting this semester"! Otto Wieland and Benno Hess organized informal meetings at Hochhausen Castle on the Neckar river, inviting a few young biochemists for lectures with no slides, just chalk and blackboard, with intense discussion. The castle did not have enough rooms, and I recall sharing a double-room with Detlev Riesner (he later founded the bioanalytics company Qiagen after his move to Düsseldorf).
Several groups in Munich became interested in oxygenrelated topics, ranging from medicine to biochemistry, toxicology, nutrition research, botany, and radiation chemistry. We decided to meet for cross-discipline discussions and in 1977 founded the "Münchner Sauerstoffclub" (Munich Oxygen Club) at an appropriate place: the Max-Emanuel Brewery (briefly described in Ref. 5). This was perhaps one of the first of many "oxygen clubs" around the world.

Hydrogen peroxide (H 2 O 2 ) as a normal constituent of aerobic metabolism
How did this topic become of interest? Focusing here on my background mindset, the answer is 2-fold: a prepared mind and curiosity, plus some serendipity.

Prepared mind
Bücher and Klingenberg had published a masterpiece article in Angewandte Chemie on the organization of living cells in 1958 (6). Their work remains central to cell physiology still today. Otto Warburg had noted already in 1928 that one should ". . ..study enzymes under the most natural conditions of action, in the living cell itself. From the standpoint of preparative chemistry they may be looked upon as being of the utmost impurity. However, if one finds reactants that selectively react with the enzymes, the rest of the cell interferes as little as does the glass wall of a test tube in which a chemical reaction is carried out" (7). Bücher's group had developed the experimental system of the isolated perfused rat liver, maintaining its normal metabolism at physiological capacity for hours. Importantly, organ spectrophotometry made it possible to monitor a noninvasive readout of ongoing metabolic processes in the intact organ. Bolko Brauser, a congenial biophysicist and senior assistant in the laboratory, had adapted a rapid-scanning spectrophotometer, the "Rapidspektroskop," for supersensitive differential spectrophotometry (8,9). I joined Brauser, and we investigated the redox state of mitochondrial cytochromes and cytochrome P450, the cellular heme proteins with a prominent absorbance band in the blue spectral region, the Soret band (10,11).

Curiosity
Given the opportunity to examine cellular physiology noninvasively with very high sensitivity at all the wavelengths from blue to red and beyond, I wondered whether other heme proteins could be analyzed. Catalase and heme peroxidases came to mind. How to "pin them down," how to follow their action? Mitochondrial cytochromes and cytochrome P450 were detect-able by their redox transitions (e.g. becoming reduced when oxygen became limiting in hypoxia or anoxia). Would it be possible to detect catalase Compound I as distinct from catalase, as one could distinguish reduced from oxidized cytochrome P450? This would provide proof that H 2 O 2 exists in the normal cell!

Problem
For a long time, identification and characterization of H 2 O 2 in eukaryotes had been challenging; early attempts by Heinrich Wieland to detect H 2 O 2 in animal metabolism had failed (12). Chance had noted that "quantitative evidence for the existence of significant amounts of. . . H 2 O 2 in tissue is lacking, since catalase, by virtue of its peculiar capacity for catalatic reactions. . . literally 'destroys the evidence' of free hydrogen peroxide in the cell" (13). Attempts by several groups to identify H 2 O 2 in intact cells by monitoring the Soret band of catalase Compound I had remained futile, largely because of scattering artifacts and low signal-to-noise ratios.

Solution
The breakthrough came by employing noninvasive organ spectrophotometry in the near-IR region, the spectral area where light scattering is far lower than at the Soret band spectral region. Catalase Compound I has a charge-transfer band with an absorbance peak at 660 nm in the difference spectrum with catalase (14). So I remember one late evening in 1969 in the basement laboratory in Munich at the Rapidspektroskop when I decided to take a look at the near-IR, using the dualwavelength difference between 660 and 640 nm to cancel out noise and then infuse ethanol, a known hydrogen donor for the peroxidatic reaction of catalase, at low concentration to the hemoglobin-free perfused rat liver. Hooray, it worked! There was a swift deflection, and after I stopped the ethanol infusion, the signal returned to the original level. The signal also responded in normoxia-anoxia transitions, and the two transitions were not additive (Fig. 3). A steady-state level of catalase Compound I was identified. This proved the existence of normal H 2 O 2 production in the intact eukaryotic cell, a slightly heretical thought at the time. After discussing it with Britton Chance on one of his visits to Munich and completing appropriate control experiments (e.g. the complete absence of the deflections shown in Fig. 3 when the animals had been pretreated with the catalase inhibitor 3-amino-1,2,4-triazole), I published jointly with Chance in the then-new FEBS Letters (15).

Sequels
This opened up a new direction of redox research at the Johnson Research Foundation at Philadelphia (16, 17). Working with Nozomu Oshino, I used steady-state titrations with methanol as hydrogen donor to quantify H 2 O 2 generation of about 50 nmol/(min 3 g of liver), which corresponds to about 2.5% of oxygen uptake (18,19). The overall concentration of H 2 O 2 in the liver cell was calculated to be 10 nM (20) (23,24).
In 1971, I submitted my "Habilitation" thesis to become recognized as "Privatdozent," an independent member of the University. Entitled The peroxisome in the hepatocyte: catalase Compound I in hemoglobin-free perfused rat liver, it was published in Angewandte Chemie in 1974 (25).

Nicotinamide adenine dinucleotides, NADPH and NADH
In 1935-1936, Warburg had discovered the nicotinamide adenine dinucleotides (then called pyridine nucleotides, DPNH and TPNH) as coenzymes of dehydrogenases: NADP 1 as coenzyme of glucose-6-phosphate dehydrogenase and NAD 1 as coenzyme of fermentation. The pathways of substrate hydrogen and the differences in redox state between the two systems had been described (6). Using organ spectrophotometry and fluorometry, we were able to follow the redox state of extramitochondrial NADPH during cytochrome P450-dependent drug metabolism (11). These were the early days in the drug metabolism field, and at a workshop in Konstanz in 1968, I met Lars Ernster and Sten Orrenius from Stockholm, forming the basis for a lifelong friendship and many scientific interactions. We found that mitochondrial NADPH was oxidized during ammonia metabolism (26,27). Work with Dieter Häussinger, one of my early doctoral students, led to the identification of flux regulation of glutamine synthase and glutaminase and to the description of cell heterogeneity, or zonation, in the liver (28). These observations stimulated research in experimental hepatology; Dieter became a prominent clinician and hepatologist, and he also founded an Institute of Tropical Medicine in Ethiopia. Metabolic compartmentation became amenable for research, and we continued to investigate nicotinamide nucleotide compartmentation (summarized in Ref. 29).

GSH, selenium
Turning back to H 2 O 2 , catalase was not the only enzyme reacting with H 2 O 2 . GSH peroxidase had been discovered (30), and Leopold Flohé at Tübingen had started working on its enzymology. In collaborative work, we showed in perfused liver that hydroperoxides indeed led to oxidation of GSH to the disulfide GSSG, that the reaction utilized NADPH, and that GSSG was released from the liver (31). Albrecht Wendel, a doctoral student of Flohé's, joined our endeavor, and we had great times in our GSH research. Two international conferences on GSH testify to this, one at an exquisite villa at Tübingen (32) and the other at Reisensburg Castle near Ulm, with Alton Meister and Sir Hans Krebs attending, as well as all of us junior scientists who were starting in research (Fig. 6) (33). Gianna Bartoli, a postdoc from Rome, found that GSH release from the liver exceeded GSSG release by about 10-fold, opening the field of interorgan relationships in GSH metabolism (34). GSSG was released into bile, as were the GSH thioethers, the products of GSH S-transferases, also called S-conjugates (35,36). In perfused rat heart, Toshihisa Ishikawa, a postdoc from Sapporo, later showed cardiac energy-dependent GSSG and S-conjugate export (37).

Düsseldorf
As it went in German academia, a time came to wander off. In contrast to today's tenure track career pathway, one needed to be recruited from another university to become Full Professor and Chairman, rather than "moving up the ladder" internally. In 1978, the chair at the Institute for Physiological Chemistry at the University of Düsseldorf was announced, and I applied. My first visit to that campus had been during the 1974 meeting of the German Society for Biological Chemistry, to speak in a session chaired by Sir Hans Krebs, related to glutamine metabolism. The talk had ended and, in his low voice, Sir Hans said that he recalled having done a similar experiment in 1935! This taught me a lesson to go back to read the literature more deeply. (Nowadays, we may miss important literature if we depend solely on a superficial search by an easy click on PubMed).

Redox cycling
A variety of compounds, such as quinones, iron chelates, and aromatic nitro compounds, can undergo one-electron reduction at the expense of NADPH, followed by autoxidation. Together with Hermann Kappus, we introduced the concept of "redox cycling" (38). Several anticancer agents, and also some mutagens, operate on this principle. We examined the protection by NADPH:quinone oxidoreductase, which reduces the quinone by two-electron reduction (39,40). The resulting hydroquinone then is available for glucuronidation, thus circumventing redox cycling. The absence of an active NADPH:quinone oxidoreductase (null-allele) is associated with increased cancer incidence (e.g. in urological malignancies) (41).

Ebselen
Having worked on the selenoenzyme, GSH peroxidase, a fortunate contact brought us into the field of organoselenium compounds. Erich Graf, the head of research and development at a pharmaceutical company, Nattermann & Cie., at nearby Cologne, asked us to examine an organic selenium compound first synthesized in 1928, 2-phenyl-1,2-benzoisoselenazol-3 (2H)-one, which had shown anti-inflammatory activity in their assays. In the in vitro assay for GSH peroxidase activity, we found that the selenium compound exhibited enzyme-like activity, whereas the sulfur analog was inactive (42). The compound received the name ebselen, and we published several papers together. As collaborators from academia and industry, we were awarded the Galenus Prize in 1990 (see Ref. 43). Since this time, the organoselenium field has flourished, with ebselen envisaged as a protein thiol modifier (44,45). Recently, ebselen was found to most efficiently inhibit the main protease of the coronavirus, SARS-CoV-2, in a screen of .10,000 compounds (46), making it a potential lead compound for treatment of COVID-19 (47). It is also being studied in clinical trials as a lithium mimetic in bipolar disorder and as a lead drug for hearing loss (see Ref. 47). It is gratifying to see renewed interest in ebse-len in the clinical setting after our basic research from decades ago.

Singlet molecular oxygen
Enrique Cadenas, originally from Buenos Aires, Argentina, joined our group in 1981 as an Alexander-von-Humboldt Fellow, then coming from the Johnson Research Foundation at Philadelphia. He used a photon counter to set up measurement of low-level chemiluminescence emitted by electronically excited reactive oxygen species. This enabled the detection of the formation of singlet molecular oxygen by its dimol emission in the enzymatic reduction of prostaglandin G 2 to H 2 (48) and during peroxidation reactions in intact cells (49). Together with Paolo di Mascio, a doctoral student from Brussels, we used a thermolabile endoperoxide to generate singlet oxygen and analyzed its reactions with biological targets (e.g. the formation of single-strand breaks in plasmid and bacteriophage DNA) (50). Using a germanium diode to monitor the monomol emission of singlet oxygen at 1,270 nm, we examined the quenching of singlet oxygen by carotenoids, tocopherols, and thiols (51), finding that lycopene, the red carotenoid in tomato, is the most efficient singlet oxygen quencher (Fig. 7A) (52). Lars-Oliver Klotz,  In work with Karin Scharffetter-Kochanek and Peter Brenneisen, we found that singlet oxygen is an early intermediate in induction of interstitial collagenase by UV radiation in skin fibroblasts (59,60), and with Jean Krutmann we found that it mediates the UV-A-induced generation of the photoagingassociated mitochondrial common deletion (61).

Receptor-mediated superoxide production
A major source of H 2 O 2 comes from the dismutation of the superoxide anion radical (62). As "professional" immune cells, activated leukocytes release superoxide (63). With Beate Meier we found that normal fibroblasts also release superoxide under the control of cytokines, an early observation in the field of redox signaling (Fig. 8) (64).

Nutritional biochemistry: Carotenoids, flavonoids, selenium
Due to their polyene structure, carotenoids quench singlet molecular oxygen particularly well. Wilhelm Stahl joined the laboratory in 1990 as a senior postdoc with experience in industry and started in-depth analysis of nutritional biochemical properties of carotenoids. We established lycopene as a biologically important carotenoid in humans (65), which was later corroborated epidemiologically for certain types of cancer. (As an aside, my contribution to the then emerging field of epidemiol-ogy was the nearly perfect association (r = 0.982) of the number of newborn babies with the number of brooding storks, showing what every child in Germany knows: storks bring babies ( Fig. 9) (66)! This illustrates that associations can generate interest but cannot prove cause-effect relationships.) Dietary tomato paste protected against UV light-induced erythema in humans (67,68). We found that lycopene oxidation products stimulated gap-junctional intercellular communication via connexins (69). Together with Alex Sevanian from the University of Southern California, we described the nutritionally induced oxidative responses, "postprandial oxidative stress" (70). Cristina Polidori, a postdoc from Perugia, analyzed the profile of antioxidants in plasma, with emphasis on the agerelated diseases (71,72). Fig. 10 shows members of the group at a laboratory workshop in 2002.
Gavin Arteel, a postdoc from Ron Thurman's group at the University of North Carolina (Chapel Hill, NC, USA), together with Karlis Briviba and Claus Jacob, worked on protection against peroxynitrite by selenium and tellurium compounds (73,74). Flavanols isolated from cocoa beans were also found to be effective (75). This led to a longstanding interaction with the cardiology department: Christian Heiss, a joint doctoral student with Malte Kelm from cardiology, found that cocoa flavanols had clear-cut positive vascular effects in human volunteers, demonstrated by an increase in flow-mediated dilation of the brachial artery (as a marker of vascular health) and of circulating plasma protein-nitroso compounds (Fig. 7B) (53). Later, joint work with Hagen Schroeter attributed this effect to (2)-epicatechin isolated from the cocoa bean (76). Highflavanol cocoa improved the surface profile of the skin (Fig.  7C) (54). With Tankred Schewe we investigated the mechanisms of vascular flavanol effects in view of their anti- inflammatory action, lowering F 2 -isoprostanes and inhibiting 15-lipoxygenases (77)(78)(79)(80).
Selenoprotein P, when released by the liver, circulates in the bloodstream and is taken up via receptors by peripheral organs to provide its selenocysteine residues for subsequent synthesis of other selenoproteins. We found that selenoprotein P also covers glycoproteins at the surface of cells by analyzing heparin binding in surface plasmon resonance experiments (81). Together with Holger Steinbrenner, we investigated the mechanisms by which selenoproteins protect against reactive oxygen species, working toward understanding success and failure in the use of selenium for cancer prevention (82,83). We also described the role of dietary selenium as an adjuvant therapy for viral and bacterial infections, a topic of current interest during the SARS-CoV-2 pandemic (84).

The concept of oxidative stress: Eustress and distress
In the early 1980s, research had rapidly progressed from the already comprehensive knowledge in redox reactions that had been presented in our extensive review (20). In an attempt to conceptualize the field, I defined "oxidative stress" as "a disturbance in the prooxidant-antioxidant balance in favor of the former" (85,86). The underlying concept is that living organisms operate at steady state in an open metabolic system, maintaining a balance of in-flow and out-flow, or homeostasis. The origin of the basic principle of stress and stress responses dated back to Selye in 1936 (87), and the first sentence in Ref. 85 was to state that, "as a biochemist, one may wonder whether Selye's term should be stressed as it is in the present context." The experimental evidence supported the thesis that the favorable aspects of aerobic life are also linked to the potentially Figure 7. Translation of basic science to human health. A, singlet oxygen quenching by carotenoids (52). B, improvement of flow-mediated dilation of brachial artery and increase of plasma protein-nitroso compounds by flavanols from cocoa (53). C, skin surface profile improvement after a high-flavanol cocoa drink (54). Compiled in Ref. 55. dangerous oxygen-linked processes as diverse as inflammation, aging, carcinogenesis, drug action and toxicity, defense against invading organisms, and more. Strategies of antioxidant defense were becoming better-known (88,89). Also, adaptive stress responses were discovered, notably the newly discovered OxyR regulon in bacteria (90), followed by the eukaryotic NF-kB, HIF, and Nrf2/Keap1 response systems (Fig. 11). Advances in understanding redox regulation, redox sensing, and redox signaling led us to update the concept: oxidative stress is defined as "an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage" (91)(92)(93).
It follows from this definition that oxidative stress can range from mild physiological maintenance challenge, called oxidative eustress, to toxic oxidative burden which damages biomolecules, oxidative distress (Fig. 12) (21,91). Oxidative equivalents utilized in redox signaling target transcription factors directly or indirectly, and H 2 O 2 emerged as the central redox signaling molecule in oxidative eustress (21). Aware that H 2 O 2 is generated upon low-dose ionizing radiation, we hypothesized that it may mediate hormetic effects of low-dose ionizing radiation (95). The disturbed redox homeostasis in oxidative distress might also be the molecular link between chronic psychological work stress and coronary heart disease (96). H 2 O 2 signaling is part of the "redox code," a set of principles describing the organization of redox biology (97). Having followed research on H 2 O 2 since its detection decades ago, it is satisfying to note that, as the major biologically active reactive oxygen species, H 2 O 2 is recognized as a versatile pleiotropic physiological signaling agent, fulfilling essential functions in metabolism (94,98). These are considered to form a basis for a future "redox medicine." It was a pleasure to convene experts to present current knowledge in this field in a book, entitled Oxidative Stress: Eustress and Distress (99). From enzymology in vitro to physiological chemistry in vivo (100), redox biology has "come a little ways," exposing a bright future, given the marvelous novel tools of analysis which recently have become available.
Interactions with redox biologists around the globe Karl Popper called science an "unended quest" (101), which very well characterizes the research field of redox biology. Eminent scientists have discussed the creative process in science and medicine (102). I was very fortunate to have witnessed, and be part of, the development of this global activity. Visiting scientists came from all continents to join my group, first in Munich, then in Düsseldorf. Here, I would like to express my appreciation and thanks to the Alexander-von-Humboldt Foundation for longstanding support of numerous   Most of these relationships stemmed from personal interactions at scientific meetings.
The colleagues organizing such events deserve immense credit. I just want to mention one line of development, which influenced my own path in science considerably. In the early 1970s, the heyday of "free radicals" (105) (Fig. 13). Fortunately, I was one of them (again, "sunny side"), which was consequential in two respects: (i) it established my relationship with NFCR (motto: "Laboratory without Walls"), resulting in research funding in 1984, which lasted throughout the years until 2016, for which I am most thankful; and (ii) it led to a sabbatical at Berkeley in 1984/1985 with Bruce Ames, Lester Packer, and Martyn Smith (Fig. 14). This was truly "sunny side," as I also met my future wife, Nancy, who then was completing her Ph.D. work at the Berkeley neuroendocrinology laboratory of Paola Timiras.
Lester Packer, with his gregarious personality and extraordinary enthusiasm, attracted colleagues from around the world for workshops in the redox field, an activity that grew into the founding of the "Oxygen Club of California" (OCC), which held biennial meetings in California (Fig. 15), Oregon (with Balz Frei In 1998, the Institute of Medicine of the National Academies held a meeting of the Panel of Dietary Antioxidants (Chair, Norman Krinsky) in Washington, D.C., at which I gave a survey lecture. In the audience there was Harold Schmitz, whom I had known from his dissertation work on carotenoids with John Erdman at Champaign-Urbana. Working by this time at the Hackettstown, New Jersey, facility of Mars, Inc., Schmitz had recently embarked on identifying procyanidins and flavonoids in cocoa. This was the starting point of our long-term joint research in nutritional biochemistry, which led to the clinical studies in cardiovascular medicine, referred to above (Fig. 7B).
In acquiring seniority in science, one takes on responsibilities to serve the next generation (106). I did so in several ways. I took active part in the Academy of Sciences and Arts in our state, Northrhine-Westphalia, serving as its President from 2002 to 2005. One project was to introduce the "Junges Kolleg" (Young College), where young scientists with outstanding achievements are elected to join as junior members of the acad-emy and are provided with funds and no-strings-attached freedom of research activities. By now, many of them have become successful professors in their respective fields.
After stepping down as professor and chairman at the Institute of Biochemistry and Molecular Biology at Heinrich-Heine-University Düsseldorf in 2008, as an emeritus professor I had the good fortune to be informally appointed as research professor by our rector and as senior scientist at the Leibniz Research Institute for Environmental Medicine, positions that I hold to this day. Also, I served for a few years as visiting professor of biology and biochemistry at the College of Science of King Saud University in Riyadh, Saudi Arabia. As a council member of the Starck Foundation in Germany, I have helped support Jewish students with university scholarships. A really unique and wonderful characteristic of science is the continuing progress into the previously unexplored-it never stops ("unended quest," mentioned above), and so I am very content observing the advances in redox biology from my current vantage point (Fig. 1).

Appreciation
Happenstance and serendipity play a role, but what one needs to be particularly thankful for is the initial trust and guidance by family, teachers, mentors, and colleagues, starting in early childhood all the way through to university, academia, and beyond. I was lucky to have had caring parents and an intact home, stimulating foster parents overseas, and, of pivotal importance, academic mentors. I can only hope that during my own career as a university professor and research scientist, I was able to generate the feeling of enthusiasm and support in my students and associates to help their creativity and development unfold in science. I thank Andreas Reichert, my successor, for his collegiality and friendship. I would like to pay special tribute to a close personal friend and mentor who had much to do with my own path in the world of science: Gustav V. R. Born. I was fortunate to know this superbly generous scientist who helped shape scientific careers in all parts of the world, including many in Germany. I also would like to acknowledge my children, Alexander, Caroline, Katharina, and Audrey, for their contribution to our happy family. Special thanks go to my wife, Nancy, for her warmth and continuous understanding, help, and support.