and : how to assess a risk or a prevention? Theoretical consideration and application to SOD-Gliadin.

Professor Xavier Leverve Laboratory of Fundamental and Applied Bioenergetics INSERM U884 and University Joseph-Fourier Grenoble, France Tel: +334 76 51 43 86, Fax: +334 76 51 42 18 [email protected]

I) Introduction evidenced in normal biological conditions Since the earliest beginning of Life, living and detection of oxidative stress is mostly organisms are continuously exposed to a achieved by assessing the result of this major risk of oxidative damage, which is process: i.e. through the detection of mostly related to oxygen. Other sources, damaged (oxidized) molecules (DNA, such as light, radiations or chemicals, are lipids, proteins etc.). On the other hand, the also involved. The extreme toxicity of balance between pro and oxygen is related to its unique capacity of activities is so finely tuned that any generating free radicals, which have long increase in antioxidant defense can be been recognized as harmful compounds on viewed either as the development of a good biological molecules. Actually, Life protection against oxidative stress or as an appeared before oxygen, since its early adapted response to previous oxidative emergence resulted from a long period of damage. Hence an increase in the microorganism and plant photosynthesis. antioxidant defense indicates actually the The increase in oxygen partial tension in result of an exposure to higher oxidative earth atmosphere was extremely conditions. Therefore, assessing deleterious for all biological molecules and antioxidant capacity cannot be obtained by the massive contemporary vanishing of using a single parameter, whatever its living species was related to oxygen intrinsic value and it is probably more toxicity. Among several antioxidant informative to perform a dynamic strategies developed though the process of assessment to a response of a given stress Evolution, mitochondrion ancestors played than a basal biological compound probably a major role in adapting Life to determination. survive to such massive oxidative stress. II) Metabolic basis of oxidative stress. Because of this long and mandatory A free radical is an atom or a molecule, metabolic evolution, a highly complex which contains one (or more) unpaired interaction between life (mitochondrial electron(s) (single-electron) on its outer ATP synthesis) and death (oxidative layer. When generated from oxygen, free toxicity) has been developed. The most radicals are also called Reactive Oxygen harmful free radicals or (ROS, Figure 1). Different species (ROS) have in common a very mechanisms of production occur at the short half-life indicating that they can very level of (i) mitochondrial respiratory chain, quickly exchange electrons with any kind (ii) NADPH oxidase, (iii) multienzymatic of biological molecule. Therefore, these complex cytochrome P450, (iv) xanthine molecules are difficult to be directly oxidase, and also (v) NO synthesis from arginine (Figure 2). In addition, metallic several natural compounds act as ions such as iron and copper are very “antioxidant”, this is mostly due to their potent catalysts leading to ROS production property of changing from oxidized to (hydroxyl radical) from hydrogen peroxide reduced status representing a first line of (Fenton’s reaction). Several members of defense, this action must be followed by the ROS family are involved (i) in cellular the endogenous antioxidant pathway sensing and signaling pathways, (ii) in the represented by the key antioxidant regulation of the vascular tone, (iii) in the enzymes GPX and catalase. immune response, (iv) in the fight against Numerous evidences involve oxidative exogenous organisms and (v) in the stress in several pathological disorders and destruction of several exogenous or it has been recognized as a factor of endogenous molecules. morbidity in acutely ill patients. Indeed, However, ROS (mostly hydroxyl radical: oxidative stress alters cellular homeostasis OH° and peroxonitrite: ONOO-) are also by ischemia/reperfusion or by activation of extremely harmful for the biological ROS production from immune cells. In molecules, explaining the major danger of such acutely ill patients, antioxidant oxidative stress for any living organism. If capacity is often depressed after a few days such toxicity can be developed against all due to a high rate of consumption and/or of biological molecules, some are especially losses in urine of several key components, sensitive to this peculiar stress: DNA, in fluid drainage or through the burned membrane-phospholipids, lipoproteins and skin. However, some of these patients are proteins. Polyunsaturated fatty acids are also frequently previously depleted, mostly particularly sensitive targets for oxidative because of preexisting chronic or sub-acute damage, due to the presence of double- disease, thus favoring the risk of oxidative bounds. However, monounsaturated fatty damage. If several experimental and acids, such as oleic acid, appear protective clinical studies have already pointed out against membrane-related ROS damage. the potential benefits of antioxidant The mechanisms of defense either prevent therapy, it was quite disappointing in ROS generation or scavenge these toxic others and the question of antioxidant compounds. The scavenging pathway supplement is not completely solved. includes (i) oxidoreduction of several Hence, further studies are needed to assess “protecting” compounds such as vitamins the clinical conditions associated with an E, C and glutathione and (ii) enzymatic unequivocal beneficial effect, but it is catalysis (glutathione peroxidase, catalase). highly probable that a better approach of It is very important to understand this two oxidative stress management in acutely ill steps mechanism. First, a “lightening patients will permit to improve their conductor”-like process allowing the outcome. avoidance of the harmful consequence of Assessing the efficacy of a given oxidizing a biological compound thanks to antioxidant component needs to consider a quicker oxidation of a given metabolite not only its mechanisms of action (known alternately oxidized and reduced. or supposed) but also the resulting effect in Secondly, a succession of enzymatic vivo, beyond its action. As an example, processes (glutathione peroxidase (GPX) vitamin supplements are given to correct a and reductase (GPR) or catalase) allowing potential vitamin deficiency and/or to a final reduction leads to an actual increase the vitamin storage in the body. scavenging process (Figure 2). Indeed, if This could be assessed in principle, although storage compartments are not determination (SOD ASSAY KIT-WST). It easily accessible and often only plasma or provides similar results and the red cell are considered, but this does not equivalence between the two imply an actual efficacy on the whole spectrophotometric methods have been pathway in the clinical situation. Even demonstrated (Zhou & Prognon). worse, opposite effects have been reported. Therefore, it is possible to assess the Indeed, considering the complexity of the enzymatic activity of the final product, i.e. entire antioxidant pathway, an activation of after manufacturing, by either method. ROS scavenging rate would require a 2) Of course it is not possible to assess this coordinated effect of many different steps. activity in the digestive tract: i.e. after its This opens the question of the consequence absorption. Therefore, an unknown beyond the target of any intervention. remains concerning the form and the When SOD-gliadin, as natural oral intimate mechanism in the gut and antioxidant, is used in the prevention of assessing its activity in vivo relies on the oxidative stress, different levels of determination of some appreciable determination must be considered in the consequences. Strikingly contrasting with attempt of assessing its efficacity, each of vitamins and some other metabolites them being a complement of the others. supplemented in the food, it is of course SOD is a fragile enzyme and therefore not possible to assume here that SOD, an determination of its enzymatic activity is a exogenous and heterologous protein, given perquisite, if considering its action is orally, may replenish one or more of the obtained through its activity. This can be various endogenous pools of this enzyme. performed by a classic spectrophotometric Hence, assessing endogenous SOD- enzymatic assay (Xanthine-/XOD-NBT), activity(ies) with oral treatment with SOD- which can be performed on each batch of gliadin reflect no less, but no more, than the raw material (Extramel®). This first other indicators of the global activity of assessment of enzymatic activity is then key-enzymes and intermediates involved in used as reference for assessing this the ROS-scavenging pathway. Any change enzymatic activity in the final product in one or more of the steps and/or (SOD-gliadin). Assessment of the activity intermediates of this pathway indicates a in the final product is routinely achieved global response of the pathway to a by gel-electrophoresis. S u p e r o x i d e signaling event consecutive to the oral dismutase (SOD) is electrophoresed on supplement. A very similar mechanism polyacrylamide gel. SOD is then localized follows the physiological adaptation to an by soaking the gel in nitro-blue tetrazolium increased oxidative pressure: hyperoxia, (NBT), followed by an immersion in a tobacco, alcohol, etc. have been shown to solution containing TEMED and activate the scavenging pathway as a RIBOFLAVIN. Gel is then illuminated to response to the ROS aggression. As stated °- induce superoxide radical (O2 ) generation above, activation of the antioxidant thanks to the photo-oxidation of riboflavin (scavenging) pathway indicates an in the presence of Temed and Oxygen. exposure to an enhanced risk. This has a While illuminated, the gel becomes uniformly blue except at the spots were very important consequence that is in SOD is present. SOD activity is principle the effect of oral administration appreciated by the achromatic zone present of SOD-gliadin is very similar to that of an on the gel. A commercial kit is now exposure to an increased risk of oxidative available permitting spectrophotometric stress, only the signal responsible for the response is different! The parameters of purposes (RPE). Hence the choice in the antioxidant pathway, which are practice is to assess the result of oxidative accessible and susceptible to a modulation, stress by assessing damaged molecules: involve enzymatic activities such as: Mn- MDA, TBARS, di-ene conjugated SOD, Cu,Zn-SOD, GPX, GPR, catalase metabolites (pentane or ethane) and lipids and ostly reduced and oxidized (isoprostanes). Each of these compounds glutathione. The enzymes are endogenous has specific advantages and drawbacks, proteins under transcriptional regulation. isoprostanes being often proposed as a The “pro versus antioxidant equilibrium” good marker. Again, interpreting these data is the matter of a delicate balance since is delicate! Indeed, an increase in these besides their potential harmful effects, oxidized metabolites indicates clearly an ROS are intracellular signaling molecules oxidative stress, it does not inform really if and an adequate level of intracellular the phenomenon is acute (signal leading to “oxidative stress” should be maintained. an adapted response) or prolonged and Accordingly, the level of the antioxidant potentially harmful. enzyme activity is finely tuned to the Among the endogenous molecules actual exposure to oxidative stress: “too susceptible to peroxidation, the DNA is much or too low are both unsuitable”. very prominent. Assessment of DNA Again it is important to stress the difficulty damage can be performed as described in in interpreting the results of these the comet assay (electrophoresis of DNA). enzymatic determinations. Chronic This test is a good marker of oxidative depletion of antioxidant elements provided stress at the nucleus level and antioxidant by lack in the diet (vitamins, minerals and capacity, the lesions depending on both. few other compounds) might lead to a Interestingly the presence of DNA breaks depression of the global transcription also indicates the capacity of the leading to low enzyme activities, but this endogenous repairing machinery. Hence it situation might also indicates a very is a global assessment of the situation and moderate exposure to oxidative stress. The a good marker when clearly abnormal. The difference between the two situations value of this test has been amplified by depends on the levels of the dietary exposing cells (lymphocytes) to a “antioxidant elements” low with chronic calibrated exogenous oxidative stress ex depletion and high the case of an vivo, allowing determining the capacity of equilibrated diet with low exposure to these cells to buffer the exogenous oxidative stress. challenge. Hence it is possible to Conversely high level of defense determine the basal situation and the potentially indicates an adapted response to capability of response to acute and sever oxidative stress exposure, the question challenge. being to know if the initial oxidative stress The final level for assessing the effect of were harmful or simply a temporary signal. an antioxidant therapy on oxidative stress This could be determined by measuring the is to measure directly the pathological damage of endogenous compounds by effects and their consequences. With SOD- oxidative stress. As it was stated above, the gliadin, several abnormal events, in both very short half-life of ROS renders these animal and human works, have been molecules not very accessible for a routine shown to respond to the oral supplement. use in clinical practice although several Such effect was demonstrated with the analytical methods useful for research consequence of exposure to hyperbaric oxygen, where the damaged DNA was significantly less after SOD-gliadin as compared to controls, validating then the whole concept from oral vegetal extract to cellular antioxidant defense. A significant effect has also shown in several animal models (mices, rats, pigs) with an effect on tumor development, insulin sensibility and oxidative stress related to sepsis. Finally, it was recently shown in a prospective study that this supplement was also responsible for an improvement of lipid profile and atheromatous lesions after long-term supplementation. As conclusion, it is important to underline the fact that a simple appreciation of oxidative stress is not pertinent since a similar result on several parameters might be due to different if not opposed situation. The best criteria, and the most difficult to evidenced, is an effect in vivo on a function clearly related to oxidative stress. References Bailey DM, Raman S, McEneny J et al: Vitamin C prophylaxis promotes oxidative lipid damage during surgical ischemia-reperfusion. Free Radic Biol Med 2006, 40(4):591-600 Cloarec M, Caillard P, Provost JC et al: GliSODin, a vegetal sod with gliadin, as preventative agent vs. atherosclerosis, as confirmed with carotid ultrasound-B imaging. Allerg Immunol (Paris) 2007, 39(2):45-50 Dojindo Molecular Technologies I: SOD Assay Kit-WST. wwwdojindocom/tm 2006 Kick J, Hauser B, Bracht H et al: Effects of a cantaloupe melon extract/wheat gliadin biopolymer during aortic cross-clamping. Intensive Care Med 2007, 33(4):694-702 Ling X, Cota-Gomez A, Flores NC et al: Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice. Free Radic Biol Med 2005, 39(8):1089-1098 Muth CM, Glenz Y, Klaus M et al: Influence of an orally effective SOD on hyperbaric oxygen-related cell damage. Free Radic Res 2004, 38(9):927-932 Naito Y, Akagiri S, Uchiyama K et al: Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice. Biofactors 2005, 23(2):85-95 Nelson SK, Bose SK, Grunwald GK et al: The induction of human and catalase in vivo: a fundamentally new approach to antioxidant therapy. Free Radic Biol Med 2006, 40(2):341-347 Okada F, Shionoya H, Kobayashi M et al: Prevention of inflammation-mediated acquisition of metastatic properties of benign mouse fibrosarcoma cells by administration of an orally available superoxide dismutase. Br J Cancer 2006, 94(6):854-862 Zhou JY, Prognon P: Raw material enzymatic activity determination: a specific case for validation and comparison of analytical methods--the example of superoxide dismutase (SOD). J Pharm Biomed Anal 2006, 40(5):1143-1148 Figure 1

Figure 2