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Findings in redox biology: From H2O2 to oxidative stress

  • Helmut Sies
    Correspondence
    For correspondence: Helmut Sies
    Affiliations
    Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

    Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
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Open AccessPublished:September 25, 2020DOI:https://doi.org/10.1074/jbc.X120.015651
      My interest in biological chemistry proceeded from enzymology in vitro to the study of physiological chemistry in vivo. Investigating biological redox reactions, I identified hydrogen peroxide (H2O2) as a normal constituent of aerobic life in eukaryotic cells. This finding led to developments that recognized the essential role of H2O2 in metabolic redox control. Further research included studies on GSH, toxicological aspects (the concept of “redox cycling”), biochemical pharmacology (ebselen), nutritional biochemistry and micronutrients (selenium, carotenoids, flavonoids), and the concept of “oxidative stress.” Today, we recognize that oxidative stress is two-sided. It has its positive side in physiology and health in redox signaling, “oxidative eustress,” whereas at higher intensity, there is damage to biomolecules with potentially deleterious outcome in pathophysiology and disease, “oxidative distress.” Reflecting on these developments, it is gratifying to witness the enormous progress in redox biology brought about by the science community in recent years.
      Weite Welt und breites Leben,Langer Jahre redlich Streben,Stets geforscht und stets gegründet,Nie geschlossen, oft geründet,Ältestes bewahrt mit Treue,Freundlich aufgefasstes Neue,Heitern Sinn und reine Zwecke:Nun! man kommt wohl eine Strecke.Wide world and broad life,Long years of steady work,Sustained searching, firmly foundedNever finished, often rounded,Old ideas loyally preserved,New ones amicably perceived,Cheerful mind and true intentions:Well, we've come a little ways!J. W. Goethe, Jena, May 1817
      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, cross-fertilize 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 99th 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. (Heinrich E. Steinweg, later known as Henry Steinway, was from near Seesen, where he built his very first piano in 1836. His son William donated the Steinway Park in Seesen in 1893. Influenced by this early impression, after receiving a professorship position at Düsseldorf, my first larger purchase was a Steinway; to this day, I enjoy practicing and playing on it at home.)
      In 1959, I was one of about 200 students who sailed on the Greek liner Arkadia with the Michigan Council of Churches exchange program for a year in the US, selected by the “Youth for Understanding” committee. My first destination was to live with the family of a country doctor and his wife, David P. and Marie Ward, in Pleasant Plain, Ohio, near Cincinnati, where deep conversations contributed to my interest in basic biomedical research. The second half of the exchange year was with Edward and Eleanor McBroom in Kankakee, Illinois, south of Chicago. Son of a state senator, Ed was active in politics, and I am very thankful to the McBrooms for exposing me to another realm of life, again very much “sunny side.” On weekends, we flew in a Cessna to Meigs Field in Chicago on the lakefront of Lake Michigan and listened to Oscar Peterson playing at the London House.

      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 (
      • Sies H.
      How I became a biochemist.
      )? 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.
      Figure thumbnail gr1
      Figure 1Then and now. Starting out in laboratory research (Marburg, 1963); reflecting on redox biology (Düsseldorf, 2020).
      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 then-uncharacterized 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 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 (
      • Bücher T.
      • Sies H.
      Steady state relaxation of enolase in vitro and metabolic throughput in vivo of red and white rabbit muscles.
      ). 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 slow-twitch (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 Ärzte” (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 organic-chemical 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 (
      • Krebs H.A.
      ), 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. (The Lindau Meetings of Nobel Prize Winners are still thriving, headed by Countess Bettina Bernadotte. Later I had the good fortune to be a member of the Council and its Vice President from 2005 to 2011, helping to select the young scientists from around the globe and shaping the rich scientific program for interactions with the Laureates at Lindau.)
      With an interim clinical semester in 1964–65 at the Sorbonne at Paris (Hopital Cochin, Salpetrière), I finished clinical studies at Munich and spent residency time in clinical medicine at the university hospital in Tübingen (Prof. H. E. Bock) and at small provincial hospitals. Bücher offered me a postdoc position to return to the Munich institute; he gave me liberty to choose my research topic. We organized a Mosbach Colloquium on “Inhibitors—Tools in Cell Research” in 1969 (
      • Bücher T.
      • Sies H.
      ), and we had invited Otto Warburg to attend. Warburg regretfully 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 oxygen-related 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.
      • Del Río L.A.
      ROS and RNS in plant physiology: an overview.
      ). This was perhaps one of the first of many “oxygen clubs” around the world.

      Hydrogen peroxide (H2O2) 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 (
      • Bücher T.
      • Klingenberg M.
      Wege des Wasserstoffs in der lebendigen Organisation [Pathways of hydrogen in the living organization].
      ). 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” (
      • Warburg O.
      ). 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 (
      • Brauser B.
      Ein Gerät zur höchstempfindlichen Differentialspektrophotometrie mit dem Rapidspektroskop [An apparatus for highest-sensitive differential spectrophotometry with the Rapidspektrokop].
      ,
      • Sies H.
      • Brauser B.
      Analysis of cellular electron transport systems in liver and other organs by absorbance and fluorescence techniques.
      ). 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 (
      • Sies H.
      • Brauser B.
      • Bücher T.
      On the state of mitochondria in perfused liver: action of sodium azide on respiratory carriers and respiration.
      ,
      • Sies H.
      • Brauser B.
      Interaction of mixed function oxidase with its substrates and associated redox transitions of cytochrome P-450 and pyridine nucleotides in perfused rat liver.
      ).

      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 detectable 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 H2O2 exists in the normal cell!

      Problem

      For a long time, identification and characterization of H2O2 in eukaryotes had been challenging; early attempts by Heinrich Wieland to detect H2O2 in animal metabolism had failed (
      • Wieland H.
      Über den Mechanismus der Oxidationsvorgänge. IX. [On the mechanism of oxidation processes. IX].
      ). Chance had noted that “quantitative evidence for the existence of significant amounts of… H2O2 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” (
      • Chance B.
      Enzyme-substrate compounds.
      ). Attempts by several groups to identify H2O2 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 (
      • Brill A.S.
      • Williams R.J.P.
      Primary compounds of catalase and peroxidase.
      ). 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 dual-wavelength 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 H2O2 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 (
      • Sies H.
      • Chance B.
      The steady state level of catalase compound I in isolated hemoglobin-free perfused rat liver.
      ).
      Figure thumbnail gr3
      Figure 3Hydrogen peroxide steady state in intact liver. Top, catalase Compound I, recorded by dual-wavelength spectroscopy (upward deflection, loss of Compound I). Bottom, oxygen concentration in effluent perfusate. Ar, replacing O2 by argon to induce anoxia. Methanol is infused as hydrogen donor for decomposition of Compound I in the peroxidatic reaction. From Ref.
      • Sies H.
      • Chance B.
      The steady state level of catalase compound I in isolated hemoglobin-free perfused rat liver.
      .

      Sequels

      This opened up a new direction of redox research at the Johnson Research Foundation at Philadelphia (
      • Chance B.
      • Oshino N.
      Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction.
      ,
      • Boveris A.
      • Oshino N.
      • Chance B.
      The cellular production of hydrogen peroxide.
      ). Working with Nozomu Oshino, I used steady-state titrations with methanol as hydrogen donor to quantify H2O2 generation of about 50 nmol/(min × g of liver), which corresponds to about 2.5% of oxygen uptake (
      • Oshino N.
      • Chance B.
      • Sies H.
      • Bücher T.
      The role of H2O2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors.
      ,
      • Sies H.
      • Bücher T.
      • Oshino N.
      • Chance B.
      Heme occupancy of catalase in hemoglobin-free perfused rat liver and of isolated rat liver catalase.
      ). The overall concentration of H2O2 in the liver cell was calculated to be 10 nm (
      • Chance B.
      • Sies H.
      • Boveris A.
      Hydroperoxide metabolism in mammalian organs.
      ) (see Fig. 4), with an estimated physiological range being 1–100 nm (
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ). Dean Jones further advanced analysis of H2O2 metabolism in isolated hepatocytes by spectroscopy of catalase Compound I (
      • Jones D.P.
      • Thor H.
      • Andersson B.
      • Orrenius S.
      Detoxification reactions in isolated hepatocytes: role of glutathione peroxidase, catalase, and formaldehyde dehydrogenase in reactions relating to N-demethylation by the cytochrome P-450 system.
      ). The timeline of H2O2 in chemistry and biology (Fig. 5) illustrates the development, with further milestones of the peroxiredoxins and the peroxiporins in the 20th century. A new era in H2O2 research began in the 21st century with the introduction, by Vsevolod Belousov, of the OxyR-based genetically encoded fluorescent probe, Hyper, permitting noninvasive readout of H2O2 in subcellular compartments (
      • Belousov V.V.
      • Fradkov A.F.
      • Lukyanov K.A.
      • Staroverov D.B.
      • Shakhbazov K.S.
      • Terskikh A.V.
      • Lukyanov S.
      Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.
      ,
      • Pak V.V.
      • Ezeriņa D.
      • Lyublinskaya O.G.
      • Pedre B.
      • Tyurin-Kuzmin P.A.
      • Mishina N.M.
      • Thauvin M.
      • Young D.
      • Wahni K.
      • Martínez Gache S.A.
      • Demidovich A.D.
      • Ermakova Y.G.
      • Maslova Y.D.
      • Shokhina A.G.
      • Eroglu E.
      • et al.
      Ultrasensitive genetically encoded indicator for hydrogen peroxide identifies roles for the oxidant in cell migration and mitochondrial function.
      ).
      Figure thumbnail gr4
      Figure 4General scheme of roles of catalase, GSH peroxidase, and superoxide dismutase in hydroperoxide metabolism at different subcellular locations. From Ref.
      • Chance B.
      • Sies H.
      • Boveris A.
      Hydroperoxide metabolism in mammalian organs.
      .
      Figure thumbnail gr5
      Figure 5Timeline of hydrogen peroxide in chemistry and biology. From Ref.
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      .
      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 (
      • Sies H.
      Biochemistry of the peroxisome in the liver cell.
      ).

      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+ as coenzyme of glucose-6-phosphate dehydrogenase and NAD+ as coenzyme of fermentation. The pathways of substrate hydrogen and the differences in redox state between the two systems had been described (
      • Bücher T.
      • Klingenberg M.
      Wege des Wasserstoffs in der lebendigen Organisation [Pathways of hydrogen in the living organization].
      ). Using organ spectrophotometry and fluorometry, we were able to follow the redox state of extramitochondrial NADPH during cytochrome P450-dependent drug metabolism (
      • Sies H.
      • Brauser B.
      Interaction of mixed function oxidase with its substrates and associated redox transitions of cytochrome P-450 and pyridine nucleotides in perfused rat liver.
      ). 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 (
      • Sies H.
      • Häussinger D.
      • Grosskopf M.
      Mitochondrial nicotinamide nucleotide systems: ammonium chloride responses and associated metabolic transitions in hemoglobin-free perfused rat liver.
      ,
      • Sies H.
      • Summer K.H.
      • Bücher T.
      A process requiring mitochondrial NADPH: urea formation from ammonia.
      ). 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 (
      • Häussinger D.
      • Sies H.
      • Gerok W.
      Functional hepatocyte heterogeneity in ammonia metabolism: the intercellular glutamine cycle.
      ). 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.
      • Sies H.
      Nicotinamide nucleotide compartmentation.
      ).

      GSH, selenium

      Turning back to H2O2, catalase was not the only enzyme reacting with H2O2. GSH peroxidase had been discovered (
      • Mills G.C.
      Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown.
      ), 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 (
      • Sies H.
      • Gerstenecker C.
      • Menzel H.
      • Flohé L.
      Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides.
      ). 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 (
      ) 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) (
      ). 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 (
      • Bartoli G.M.
      • Sies H.
      Reduced and oxidized glutathione efflux from liver.
      ). GSSG was released into bile, as were the GSH thioethers, the products of GSH S-transferases, also called S-conjugates (
      • Wahlländer A.
      • Sies H.
      Glutathione S-conjugate formation from 1-chloro-2,4-dinitrobenzene and biliary S-conjugate excretion in the perfused rat liver.
      ,
      • Akerboom T.P.
      • Bilzer M.
      • Sies H.
      The relationship of biliary glutathione disulfide efflux and intracellular glutathione disulfide content in perfused rat liver.
      ). In perfused rat heart, Toshihisa Ishikawa, a postdoc from Sapporo, later showed cardiac energy–dependent GSSG and S-conjugate export (
      • Ishikawa T.
      • Sies H.
      Cardiac transport of glutathione disulfide and S-conjugate: studies with isolated perfused rat heart during hydroperoxide metabolism.
      ).
      Figure thumbnail gr6
      Figure 6GSH meeting at Reisensburg Castle (near Ulm, Germany), 1978. Holding the GSH sign: Alton Meister (left) and Sir Hans Krebs (right). Many of the attendees stayed in GSH research over the years. The photo is almost a “Who's Who” in thiol research. From Ref.
      .

      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” (
      • Kappus H.
      • Sies H.
      Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation.
      ). 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 (
      • Wefers H.
      • Sies H.
      Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity.
      ,
      • Prochaska H.J.
      • Talalay P.
      • Sies H.
      Direct protective effect of NAD(P)H:quinone reductase against menadione-induced chemiluminescence of postmitochondrial fractions of mouse liver.
      ). 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) (
      • Schulz W.A.
      • Krummeck A.
      • Rösinger I.
      • Eickelmann P.
      • Neuhaus C.
      • Ebert T.
      • Schmitz-Dräger B.J.
      • Sies H.
      Increased frequency of a null-allele for NAD(P)H: quinone oxidoreductase in patients with urological malignancies.
      ).

      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 (
      • Müller A.
      • Cadenas E.
      • Graf P.
      • Sies H.
      A novel biologically active seleno-organic compound–I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen).
      ). 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.
      • Parnham M.J.
      • Sies H.
      The early research and development of ebselen.
      ). Since this time, the organoselenium field has flourished, with ebselen envisaged as a protein thiol modifier (
      • Mugesh G.
      • Du Mont W.W.
      • Sies H.
      Chemistry of biologically important synthetic organoselenium compounds.
      ,
      • Barbosa N.V.
      • Nogueira C.W.
      • Nogara P.A.
      • de Bem A.F.
      • Aschner M.
      • Rocha J.B.T.
      Organoselenium compounds as mimics of selenoproteins and thiol modifier agents.
      ). Recently, ebselen was found to most efficiently inhibit the main protease of the coronavirus, SARS-CoV-2, in a screen of >10,000 compounds (
      • Jin Z.
      • Du X.
      • Xu Y.
      • Deng Y.
      • Liu M.
      • Zhao Y.
      • Zhang B.
      • Li X.
      • Zhang L.
      • Peng C.
      • Duan Y.
      • Yu J.
      • Wang L.
      • Yang K.
      • Liu F.
      • et al.
      Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors.
      ), making it a potential lead compound for treatment of COVID-19 (
      • Sies H.
      • Parnham M.J.
      Potential therapeutic use of ebselen for COVID-19 and other respiratory viral infections.
      ). 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.
      • Sies H.
      • Parnham M.J.
      Potential therapeutic use of ebselen for COVID-19 and other respiratory viral infections.
      ). It is gratifying to see renewed interest in ebselen 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 G2 to H2 (
      • Cadenas E.
      • Sies H.
      • Nastainczyk W.
      • Ullrich V.
      Singlet oxygen formation detected by low-level chemiluminescence during enzymatic reduction of prostaglandin G2 to H2.
      ) and during peroxidation reactions in intact cells (
      • Cadenas E.
      • Wefers H.
      • Sies H.
      Low-level chemiluminescence of isolated hepatocytes.
      ). 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) (
      • Di Mascio P.
      • Wefers H.
      • Do-Thi H.P.
      • Lafleur M.V.
      • Sies H.
      Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-strand breaks.
      ). 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 (
      • Di Mascio P.
      • Devasagayam T.P.
      • Kaiser S.
      • Sies H.
      Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers.
      ), finding that lycopene, the red carotenoid in tomato, is the most efficient singlet oxygen quencher (Fig. 7A) (
      • Di Mascio P.
      • Kaiser S.
      • Sies H.
      Lycopene as the most efficient biological carotenoid singlet oxygen quencher.
      ). Lars-Oliver Klotz, a postdoc from Tübingen, investigated the role of singlet oxygen in cell signaling, with an emphasis on mitogen-activated protein kinases (p38, JNK, and ERK) (
      • Klotz L.O.
      • Pellieux C.
      • Briviba K.
      • Pierlot C.
      • Aubry J.M.
      • Sies H.
      Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.
      ,
      • Klotz L.O.
      • Kröncke K.D.
      • Sies H.
      Singlet oxygen-induced signaling effects in mammalian cells.
      ). Research on singlet oxygen and on excited carbonyls was continued in joint work with colleagues Paolo Di Mascio, Marisa Medeiros, and Etelvino Bechara in Sao Paulo and Jean Cadet in Grenoble (
      • Mano C.M.
      • Prado F.M.
      • Massari J.
      • Ronsein G.E.
      • Martinez G.R.
      • Miyamoto S.
      • Cadet J.
      • Sies H.
      • Medeiros M.H.
      • Bechara E.J.
      • Di Mascio P.
      Excited singlet molecular O2(1Δg) is generated enzymatically from excited carbonyls in the dark.
      ). 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 (
      • Wlaschek M.
      • Wenk J.
      • Brenneisen P.
      • Briviba K.
      • Schwarz A.
      • Sies H.
      • Scharffetter-Kochanek K.
      Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro.
      ,
      • Brenneisen P.
      • Wenk J.
      • Klotz L.O.
      • Wlaschek M.
      • Briviba K.
      • Krieg T.
      • Sies H.
      • Scharffetter-Kochanek K.
      Central role of ferrous/ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts.
      ), and with Jean Krutmann we found that it mediates the UV-A–induced generation of the photoaging-associated mitochondrial common deletion (
      • Berneburg M.
      • Grether-Beck S.
      • Kürten V.
      • Ruzicka T.
      • Briviba K.
      • Sies H.
      • Krutmann J.
      Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion.
      ).
      Figure thumbnail gr7
      Figure 7Translation of basic science to human health. A, singlet oxygen quenching by carotenoids (
      • Di Mascio P.
      • Kaiser S.
      • Sies H.
      Lycopene as the most efficient biological carotenoid singlet oxygen quencher.
      ). B, improvement of flow-mediated dilation of brachial artery and increase of plasma protein-nitroso compounds by flavanols from cocoa (
      • Heiss C.
      • Dejam A.
      • Kleinbongard P.
      • Schewe T.
      • Sies H.
      • Kelm M.
      Vascular effects of cocoa rich in flavan-3-ols.
      ). C, skin surface profile improvement after a high-flavanol cocoa drink (
      • Heinrich U.
      • Neukam K.
      • Tronnier H.
      • Sies H.
      • Stahl W.
      Long-term ingestion of high flavanol cocoa provides photoprotection against UV-induced erythema and improves skin condition in women.
      ). Compiled in Ref.
      • Jones D.P.
      • Radi R.
      Redox pioneer: Professor Helmut Sies.
      .

      Receptor-mediated superoxide production

      A major source of H2O2 comes from the dismutation of the superoxide anion radical (
      • McCord J.M.
      • Fridovich I.
      Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein).
      ). As “professional” immune cells, activated leukocytes release superoxide (
      • Babior B.M.
      • Kipnes R.S.
      • Curnutte J.T.
      Biological defense mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent.
      ). 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) (
      • Meier B.
      • Radeke H.H.
      • Selle S.
      • Younes M.
      • Sies H.
      • Resch K.
      • Habermehl G.G.
      Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-α.
      ).
      Figure thumbnail gr8
      Figure 8Time- and dose-dependent formation of superoxide by human fibroblasts upon stimulation with interleukin-1. From Ref.
      • Meier B.
      • Radeke H.H.
      • Selle S.
      • Younes M.
      • Sies H.
      • Resch K.
      • Habermehl G.G.
      Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-α.
      .

      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 (
      • Stahl W.
      • Sies H.
      Lycopene: a biologically important carotenoid for humans?.
      ), which was later corroborated epidemiologically for certain types of cancer. (As an aside, my contribution to the then emerging field of epidemiology 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) (
      • Sies H.
      A new parameter for sex education.
      )! This illustrates that associations can generate interest but cannot prove cause-effect relationships.) Dietary tomato paste protected against UV light–induced erythema in humans (
      • Stahl W.
      • Heinrich U.
      • Wiseman S.
      • Eichler O.
      • Sies H.
      • Tronnier H.
      Dietary tomato paste protects against ultraviolet light-induced erythema in humans.
      ,
      • Sies H.
      • Stahl W.
      Nutritional protection against skin damage from sunlight.
      ). We found that lycopene oxidation products stimulated gap-junctional intercellular communication via connexins (
      • Aust O.
      • Ale-Agha N.
      • Zhang L.
      • Wollersen H.
      • Sies H.
      • Stahl W.
      Lycopene oxidation product enhances gap junctional communication.
      ). Together with Alex Sevanian from the University of Southern California, we described the nutritionally induced oxidative responses, “postprandial oxidative stress” (
      • Sies H.
      • Stahl W.
      • Sevanian A.
      Nutritional, dietary and postprandial oxidative stress.
      ). Cristina Polidori, a postdoc from Perugia, analyzed the profile of antioxidants in plasma, with emphasis on the age-related diseases (
      • Mecocci P.
      • Polidori M.C.
      • Troiano L.
      • Cherubini A.
      • Cecchetti R.
      • Pini G.
      • Straatman M.
      • Monti D.
      • Stahl W.
      • Sies H.
      • Franceschi C.
      • Senin U.
      Plasma antioxidants and longevity: a study on healthy centenarians.
      ,
      • Polidori M.C.
      • Stahl W.
      • Eichler O.
      • Niestroj I.
      • Sies H.
      Profiles of antioxidants in human plasma.
      ). Fig. 10 shows members of the group at a laboratory workshop in 2002.
      Figure thumbnail gr9
      Figure 9Association versus cause-effect: “Storks bring babies”. From Ref.
      • Sies H.
      A new parameter for sex education.
      .
      Figure thumbnail gr10
      Figure 10Members of the Düsseldorf group at a laboratory workshop (Schloss Mickeln, 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 (
      • Arteel G.E.
      • Briviba K.
      • Sies H.
      Protection against peroxynitrite.
      ,
      • Jacob C.
      • Arteel G.E.
      • Kanda T.
      • Engman L.
      • Sies H.
      Water-soluble organotellurium compounds: catalytic protection against peroxynitrite and release of zinc from metallothionein.
      ). Flavanols isolated from cocoa beans were also found to be effective (
      • Arteel G.E.
      • Sies H.
      Protection against peroxynitrite by cocoa polyphenol oligomers.
      ). 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) (
      • Heiss C.
      • Dejam A.
      • Kleinbongard P.
      • Schewe T.
      • Sies H.
      • Kelm M.
      Vascular effects of cocoa rich in flavan-3-ols.
      ). Later, joint work with Hagen Schroeter attributed this effect to (−)-epicatechin isolated from the cocoa bean (
      • Schroeter H.
      • Heiss C.
      • Balzer J.
      • Kleinbongard P.
      • Keen C.L.
      • Hollenberg N.K.
      • Sies H.
      • Kwik-Uribe C.
      • Schmitz H.H.
      • Kelm M.
      (−)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans.
      ). High-flavanol cocoa improved the surface profile of the skin (Fig. 7C) (
      • Heinrich U.
      • Neukam K.
      • Tronnier H.
      • Sies H.
      • Stahl W.
      Long-term ingestion of high flavanol cocoa provides photoprotection against UV-induced erythema and improves skin condition in women.
      ). With Tankred Schewe we investigated the mechanisms of vascular flavanol effects in view of their anti-inflammatory action, lowering F2-isoprostanes and inhibiting 15-lipoxygenases (
      • Sies H.
      • Schewe T.
      • Heiss C.
      • Kelm M.
      Cocoa polyphenols and inflammatory mediators.
      ,
      • Schewe T.
      • Steffen Y.
      • Sies H.
      How do dietary flavanols improve vascular function? A position paper.
      ,
      • Wiswedel I.
      • Hirsch D.
      • Kropf S.
      • Gruening M.
      • Pfister E.
      • Schewe T.
      • Sies H.
      Flavanol-rich cocoa drink lowers plasma F(2)-isoprostane concentrations in humans.
      ,
      • Sadik C.D.
      • Sies H.
      • Schewe T.
      Inhibition of 15-lipoxygenases by flavonoids: structure-activity relations and mode of action.
      ).
      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 (
      • Arteel G.E.
      • Franken S.
      • Kappler J.
      • Sies H.
      Binding of selenoprotein P to heparin: characterization with surface plasmon resonance.
      ). 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 (
      • Steinbrenner H.
      • Sies H.
      Protection against reactive oxygen species by selenoproteins.
      ,
      • Steinbrenner H.
      • Speckmann B.
      • Sies H.
      Toward understanding success and failures in the use of selenium for cancer prevention.
      ). 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 (
      • Steinbrenner H.
      • Al-Quraishy S.
      • Dkhil M.A.
      • Wunderlich F.
      • Sies H.
      Dietary selenium in adjuvant therapy of viral and bacterial infections.
      ).

      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 (
      • Chance B.
      • Sies H.
      • Boveris A.
      Hydroperoxide metabolism in mammalian organs.
      ). In an attempt to conceptualize the field, I defined “oxidative stress” as “a disturbance in the prooxidant-antioxidant balance in favor of the former” (
      • Sies H.
      Oxidative stress. Introductory remarks.
      ,
      • Sies H.
      Biochemistry of oxidative stress.
      ). 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 (
      • Selye H.
      A syndrome produced by diverse nocuous agents.
      ), and the first sentence in Ref.
      • Sies H.
      Oxidative stress. Introductory remarks.
      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 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 (
      • Sies H.
      Strategies of antioxidant defense.
      ,
      • Sies H.
      Oxidative stress: oxidants and antioxidants.
      ). Also, adaptive stress responses were discovered, notably the newly discovered OxyR regulon in bacteria (
      • Christman M.F.
      • Morgan R.W.
      • Jacobson F.S.
      • Ames B.N.
      Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium.
      ), followed by the eukaryotic NF-κB, 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” (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Sies H.
      • Jones D.P.
      Oxidative stress.
      ,
      • Sies H.
      Oxidative stress: a concept in biology and medicine.
      ).
      Figure thumbnail gr11
      Figure 11Timeline showing the concepts of stress and stress responses. Mithridates VI and Paracelcus had early insights. Bernard's concept of milieu intérieur received the name “homeostasis,” and the Arndt–Schulz rule received the name “hormesis.” The 20th century brought the adaptive stress response, heat shock response, oxidative stress, OxyR, allostasis, unfolded protein response, NF-κB, HIF, and Nrf2/Keap1. From Ref.
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      , where the literature on these concepts is listed.
      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) (
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). Oxidative equivalents utilized in redox signaling target transcription factors directly or indirectly, and H2O2 emerged as the central redox signaling molecule in oxidative eustress (
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ). Aware that H2O2 is generated upon low-dose ionizing radiation, we hypothesized that it may mediate hormetic effects of low-dose ionizing radiation (
      • Sies H.
      • Feinendegen L.E.
      Radiation hormesis: the link to nanomolar hydrogen peroxide.
      ). The disturbed redox homeostasis in oxidative distress might also be the molecular link between chronic psychological work stress and coronary heart disease (
      • Siegrist J.
      • Sies H.
      Disturbed redox homeostasis in oxidative distress: a molecular link from chronic psychosocial work stress to coronary heart disease?.
      ). H2O2 signaling is part of the “redox code,” a set of principles describing the organization of redox biology (
      • Jones D.P.
      • Sies H.
      The redox code.
      ). Having followed research on H2O2 since its detection decades ago, it is satisfying to note that, as the major biologically active reactive oxygen species, H2O2 is recognized as a versatile pleiotropic physiological signaling agent, fulfilling essential functions in metabolism (
      • Sies H.
      • Jones D.P.
      Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
      ,
      • Sies H.
      Role of metabolic H2O2 generation: redox signaling and oxidative stress.
      ). 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 (
      ). From enzymology in vitro to physiological chemistry in vivo (
      • Sies H.
      From enzymology in vitro to physiological chemistry in vivo.
      ), redox biology has “come a little ways,” exposing a bright future, given the marvelous novel tools of analysis which recently have become available.
      Figure thumbnail gr12
      Figure 12Hydrogen peroxide as a physiological signaling agent: Oxidative eustress. Green and red coloring denotes predominantly beneficial or deleterious responses, respectively. At supraphysiological level (oxidative distress), stress responses are activated. An estimated 100-fold concentration gradient from extracellular to intracellular is given for rough orientation. From Ref.
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ; see also Ref.
      • Sies H.
      • Jones D.P.
      Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
      .

      Interactions with redox biologists around the globe

      Karl Popper called science an “unended quest” (
      • Popper K.
      Unended quest.
      ), which very well characterizes the research field of redox biology. Eminent scientists have discussed the creative process in science and medicine (
      ). 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 international Awardees and Fellows who visited my laboratory over the years for joint research, among them Chris Foote from UCLA; Dan Ziegler, Austin; Brian Ketterer, London; Gustav Born, London; Keith Ingold, Toronto; Fred Sundquist, UCSD; Govind Mugesh, Bangalore; Greg Bartosz, Lodz. (In later years, I served on the Alexander-von-Humboldt review and selection panels for over a decade.) I am also thankful to the Deutsche Forschungsgemeinschaft (DFG), Bonn, and the National Foundation for Cancer Research (NFCR), Bethesda, MD, USA (see below) for longstanding support.
      A wonderful description of my worldwide scientific contacts and friendship is given in a volume with over 30 contributions, initiated by the publisher, Anthony Newman, and assembled by the Editors Henry Forman and Shinya Toyokuni (
      • Forman H.J.
      • Toyokuni S.
      Tribute issue: Helmut Sies and oxidative stress: venit, vidit, vicit.
      ). The interactions with my Japanese colleagues have been written up (
      • Sies H.
      German-Japanese relationships in biochemistry: a personal perspective.
      ), and many similar close relationships still need to be formally acknowledged, with fruitful research visits by colleagues from the US (Jim Kehrer, UT Austin; Frank Meyskens, UC Irvine; Jim Thomas, Iowa State University), and interactions with colleagues from Spain (José Vina, Francisco Romero, José Estrela, Santiago Lamas), Italy (Mario Comporti, Angelo Benedetti, Alfonso Pompella, Giuseppe Poli, Fulvio Ursini), France (Jean Cadet, Alain Favier, Jean-Marie Aubry, Josiane Cillard, Ingrid Emerit), Switzerland (Wim Koppenol, Angelo Azzi), and South America (Giuseppe Cilento, Etelvino Bechara, Federico Leighton, Lionel Gil, Rafael Radi), to name a few.
      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” (
      • Moss R.W.
      ), Franklin Salisbury of Bethesda, Maryland, founded the National Foundation for Cancer Research (NFCR) to provide funds for Albert Szent-György's laboratory at Woods Hole, Massachusetts. Trevor Slater from Brunel University in the UK had developed an interest in lipid peroxidation and had established contacts to research groups in Italy and Austria. He had also agreed to serve as a project director for the NFCR. Slater, together with Robin Willson, inaugurated the Society for Free Radical Research (SFRR) in the UK in 1982. (In later years, I participated in shaping this worldwide society, serving as President of SFRR-International from 1998 to 2000.) In the US, Lester Packer at the University of California (Berkeley, CA, USA) inaugurated the Gordon Research Conference on Oxygen Radicals in 1981. In 1983, the second Gordon Research Conference on this topic took place in Ventura, California. Thereafter, a small NFCR workshop was held at Montecito, California, with Szent-György, Salisbury, and a handful of colleagues, convened by Trevor Slater and Lester Packer (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.
      Figure thumbnail gr13
      Figure 13National Foundation for Cancer Research (NFCR) Research Conference (Montecito, California, USA), 1983. Front row (from left): Harold Swartz, Lester Packer, Franklin Salisbury, Albert Szent-György, Trevor Slater, Patrick Riley, Hermann Esterbauer. Second row (from left): Keith Ingold, Bill Pryor, John Ward, Rolf Mehlhorn, Helmut Sies, Alexandre Quintanilha, Norman Krinsky, Peter Gascoyne, Les Redpath, Martyn Smith, Robin Willson.
      Figure thumbnail gr14
      Figure 14Sabbatical at University of California (Berkeley, CA, USA), 1984/1985. Left to right: Lester Packer, Bruce Ames, Helmut Sies, Martyn Smith.
      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 at the Linus Pauling Institute in Corvallis), Spain, and Italy and sponsored meetings at many other places on all continents, contributing to the development of redox biology worldwide. With Enrique Cadenas, John Maguire, Maret Traber, and Chandan Sen, I gladly served on the Board of Directors of OCC, shaping various OCC meetings together with Cesar Fraga, Patricia Oteiza, Giuseppe Poli, Juan Sastre, and Giuseppe Valacchi.
      Figure thumbnail gr15
      Figure 15Oxygen Club of California (OCC) Conference (Santa Barbara, CA, USA), 2002. Shown are Enrique Cadenas (standing) and Adjunct Professors (left to right) Lester Packer, Alberto Boveris, Helmut Sies, and Catherine Rice-Evans (University of Southern California).
      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 (
      • Sies H.
      Biological redox systems and oxidative stress.
      ). 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 academy 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.

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