BIOMARKERS for Oxidative Stress in Veterinary Medicine, Part 1

1. BIOMARKERS for Oxidative Stress in Veterinary Medicine, Part 1 W. Jean Dodds, DVM & Denis M. Callewaert, PhD AHVMA 2016 Columbus, OH

2. Cellular Oxidative Stress • Gene Expression • Upregulation of antioxidant synthesis occurs to help restore oxidant/antioxidant balance (cell redox) • Adaptive Response • Involves changes in gene expression which result in elevated antioxidant defenses • However, inappropriate use of antioxidants may severely blunt the adaptive response (Mandelker, JAHVMA , 41:22-24, Winter 2016)

3. Cellular Oxidative Stress • Cell Injury • Physiologic reaction draws immune cells and molecules to sites of cell injury • May be transient or irreversible, leading to cell death by necrosis or apoptosis • Immediate biologic mediators are prostaglandins, leukotrienes, serotonins, histamine, platelet-activating factor, and other enzymes • Chronic or sustained release mediators are tumor necrosis factor-alpha, acute phase proteins, interferons, adhesion molecules and colony-stimulating factors

4. Cellular Oxidative Stress • Signaling Pathways • Moderate amounts of ROS (or RNS) are important regulatory mediators in the cell signaling processes • Mitochondrial cell respiration generates a high rate of ROS during energy production; this creates more oxidative stress • Body responds through complex cell signaling pathways to restore oxidant /antioxidant balance • This physiologic mechanism drives vital cellular functions (Mandelker, JAHVMA , 41:22-24, Winter 2016)

5. Cellular Oxidative Stress • Antioxidant Enzymes Primary Antioxidants • Three: Catalase, Glutathione peroxidase, and Superoxide dismutase • Located within the RNS of cells • Under extreme stress, system can be overwhelmed & free radicals build up • Free radicals are released into the blood and tissues leading to tissue and organ destruction and a variety of diseases Secondary Antioxidants • Include Vitamins E & C, selenium, zinc, co-enzyme Q10, alpha -lipoic acid, and bioflavonoids (carotenoids, polyphenols) • Assist cells in removing free radicals (ROS/ RNS) • Need to be replenished by diet and supplements (Mandelker, JAHVMA , 41:22-24, Winter 2016)

6. Cellular Oxidative Stress • Cell Injury in Impaired Renal Function • Dogs with impaired renal function of various causes had markedly higher expression of 3 cytokines and 5-lipoxygenase compared to healthy dogs • Elevated cytokines included: interleukins 1-alpha and 1-beta, and transforming growth factor-beta • These elevated markers are part of the cellular inflammatory response to both acute kidney injury and chronic kidney disease (Nentwig et al, AJVR, 77:218-224, 2016)

7. Prevention & Risk AssessmentA Paradigm Shift in Medicine •••

8. Traditional Medicine: Focus on Diagnosis ofDiseases • Lots of specific tests (glucose, PSA, cholesterol) • Variable accuracy in diagnosis (e.g. PSA ~ 60% accurate) • Most don’t detect until disease has progressed significantly • Most don’t evaluate risk of contracting • The diagnostics industry • Paradigm: slow to adopt new tests • Focus = diagnostics, disease-related tests • low cost, high volume • The market • Ordered by clinicians • Regulated by FDA • Reimbursable by 3rd party insurers

9. Major Causes of Death (US 2005) • Heart disease: 652,091 • Cancer: 559,312 • Stroke (cerebrovascular diseases): 143,579 • Chronic lower respiratory diseases:130,933 • Accidents (unintentional injuries): 117,809 • Diabetes: 75,119 • Alzheimer's disease: 71,599 • Influenza/Pneumonia: 63,001

10. What Causes Diseases? A better understanding • Role of Genetics - estimated to explain only ~ 5% of all cancers • Breast cancer - BRCa1 and BRCa2 mutations in 5 - 10% of cases • Colon Cancer - 5.29% of men and women will be diagnosed with cancer of the colon. Familial Adenomatous Polyposis (FAP), is the most clearly identified genetic condition associated with colon cancer. Yet, the incidence is only 1 in 7,000 to 1 in 22,000 live births. • Cardiovascular Disease - Several known hereditary “risk factors” (involving maybe 50 genes), including hypertension, hypercholesterolemia with ~ 50% hereditable risk. • Diabetes - one in every three individuals has diabetes (based on fasting blood sugar), with approximately one third of diabetics that remain undiagnosed. Genetic components known but complex. • So ---- • Genetics plays a role in several major diseases but, except for some specific genes (e.g. BrCa1), not the main factor.

11. What Causes Diseases? A better understanding • Environment, Diet & Exercise • U.S. Cancer incidence by county - more in industrial areas • Estimated that 20 - 60% of cancers in US are related to diet & nutritional factors • Asians living in the US • 5-fold increase in cardiovascular disease • Huge increase in risk of colorectal cancer • Decreased risk of stomach cancer • Why the increase in some diseases (e. g allergies, autoimmune diseases?) • Diet & lifestyle ? • Carcinogens and atomic fallout? • Is it the beef? • Several studies have focused on high fat diets as a major risk factor • Few have considered the reduced fish intake - until recently • Associated 47% reduction in risk of colorectal cancer with statin use (as opposed to other lipid-lowering drugs). Poynter, JN., et al. New Eng J Med, May 26, 2005; 352:2184-92.

12. What Causes Diseases? A better understanding • Exercise vs obesity • Clear associations between obesity and diabetes, cancer and CVD • Exercise turns on several genes that are protective. • Many studies in US and the world have consistently found adults who increase their physical activity either in intensity, duration, or frequency can reduce their risk of developing colon cancer by 30-40 %, relative to those who are sedentary, with the greatest reduction in risk among those who are most active. • Physically active women are at lower risk of developing breast cancer than inactive women; however the amount of risk reduction achieved through physical activity varies widely (between 20-80 %). • Endometrial cancer about 20 studies suggest an inverse association between physical activity and endometrial cancer incidence. These studies suggest that women who are physically active have a 20 - 40 % reduced risk of endometrial cancer

13. From Reactive to Proactive: Preventive Medicine • New interest in preventative medicine • Traditional diagnostics probe for disease, not health • Diet, lifestyle, exercise, and nutritional supplements impact health • Should “health” only be assessed by the absence of disease? • Personal approaches • Diet & Exercise • New ways to check health • Not just blood pressure, cholesterol level, mammogram • Tests for risk indicators - altered by genetics, diet & lifestyle • Oxidative stress & Antioxidant activity • Inflammatory status • Several well established tests for these indicators • They don’t diagnose specific diseases • They don’t fit current diagnostic industry / FDA / insurance paradigms • Currently not readily accessible to clinicians

15. Free Radicals and Oxidative Stress • Definition • Free radicals are highly reactive molecules containing “unpaired electrons” • Major examples: Hydroxyl (-OH), peroxyl (-OOH) and superoxide, O3-, and peroxynitrite HONO- • Byproducts of normal oxidative metabolism • The body has many ways to inactivate and eliminate • If not eliminated quickly, or if they are made to fast, they can react with lipids, proteins and DNA - causing damage. • Free radical damage • Involved in many diseases (CVD, cancer, diabetes…) • Several studies show that free radicals play a key role in aging

16. Antioxidants - in the body and in the diet • Natural antioxidants • Enzymes • Catalase, superoxide dismutase, glutathione reductase and many more • Small molecules: • Glutathione, uric acid • Vitamins C and E • Dietary antioxidants • Supplemental vitamin E and/or selenium has no effect at low doses • Lots of other supplements in your diet - and in your pet’s diet • Flavonoids in various fruits and vegetables (ORAC index) • Exercise and antioxidant activity • Increases level of natural antioxidant defenses • But only to a point as over exercising leads to oxidative stress

17. Example: Vitamin E & BioMarkers of Oxidative Stress

18. Example: Vitamin E & BioMarkers of Oxidative Stress

19. Nutraceutical Efficacy • Big business - but is it needed? Does it do anything? • Global nutraceutical market over $230 billion/year, growing at > 6%/year • 70% of adult Americans take nutritional supplements • No test to determine if they do anything (good or bad) • Increasing FDA scrutiny is a huge unmet market need • Available methods • Measure antioxidant activity before consumption • BUT is the antioxidant adsorbed by the body? Beneficial?

20. Inflammation: The fire within • What is inflammation? • Physiological response to injury, infection, malignancy • Classis signs include fever, redness, swelling • Many molecules and cells involved (e.g. cytokines, eicosanoids) • Acute Inflammation necessary for to protect the body • Chronic inflammation associated with many diseases such as • Cardiovascular Diseases • Inflammatory bowel disease • Many types of cancer • Macular degeneration • Anti-inflammatory agents can prevent or slow disease progression • Monitoring for inflammation may help promote wellness

21. Statins: Cholesterol vs Inflammation? • Cholesterol level • Relatively meaningless by itself • LDL (bad) vs HDL (good) cholesterol levels more useful • Many other factors influence CVD (e.g. platelet aggregation) • Statins reduce cholesterol synthesis • Dietary uptake also important • Statins and inflammation • C Reactive Protein (CRP) = a liver protein made during inflammation • Very recent study: tested Crestor on • People with high cholesterol + low CRP • People with low cholesterol + high CRP • Crestor reduced CRP level AND reduced heart disease by ~50% • So additional effect of statins = reduction in inflammation • May soon see prescriptions for CVD prevention based on CRP

22. Aspirin - as a miracle drug • Well known • Aspirin (and other NSAIDs) block the function of Cox-1 and Cox-2 • Cox-1 involved in platelet aggregation, so aspirin can reduce tendency of blood to clot • Less well known • Some individuals have high sensitivity to NSAIDs with gastric bleeding, which causes ~ 16,000 deaths in US/year • Cox-2 inhibitors reduce GI problems, but can (e.g. VIOXX) increase the risk of blood clots • Needs to be well known • Regular NSAID use reduces the incidence of colon cancer by about 50% and appears to also reduce risks of other cancers • NSAID use also appears to slow onset of Alzheimer’s disease

23. Inflammation, Cox Inhibition & Cancer • VIOXX • A good Cox-2 inhibitor for people with aspirin sensitivity and severe arthritis • But Merck wanted it approved as a cancer preventative • Higher dose (7x) caused unacceptable number of deaths • So, withdrawn entirely from the market • Is Cox-2 the key? • Cox-2 is made in high levels during inflammation • Cox-2 present in colon in high levels • Inflammatory bowel disease • Colon cancer • Is this why NSAIDs reduce colon cancer risk ? • Not known, but other targets being investigated

24. Cancer Chemoprevention • NSAIDs • Well documented - if not widely known • Aspirin, coated aspirin, ibuprofen, naproxen • NOT tylenol (acetaminophen) • Nutraceuticals - some data • Curcumin, Soy isoflavones, resveratrol, and others • Genes turned on by cancer chemopreventatives • NSAIDs and nutraceuticals activate some genes • NAG-1 = NSAID activated Gene • Activated by all NSAIDs and by resveratrol, soy, ECG, etc • Released into the blood (a blood test for health ?) • Causes cancer cells to commit suicide • Also elevated in blood in prostate cancer, CVD??

25. Fish Oil - Antioxidant or Anti-inflammatory ? • Omega-3 fatty acids • Found in fish, flax and some other plants • Popular nutraceutical • Long thought to act just as scavengers for free radicals • Fish vs meat and Japanese Americans • Japanese who eat a western diet have higher CVD and colon cancer • Long thought to involve unsaturated fatty acids in meat • Arachidonic acid is substrate for COX-1 and COX-2 • Association of COX with CVD and cancer • But maybe is due to less fish in the diet • DHA (one of the omega 3 fatty acids) is converted by Cox-2 to Neuoprotectin D1 - helps reduce stroke damage and may have other beneficial effects • DHA is actually better substrate for Cox-2 than arachidonic acid • Just beginning to be understood

26. Tests to Assess Human & Animal Wellness • Available Now • Good tests for antioxidant activity & for oxidative damage • In development • Measured in serum , saliva, urine • Oxidized proteins as indicators of tissue-specific damage • Inflammatory status biomarker(s) • Cancer resistance indicator(s) • Further down the road = metabolic profiling • Simultaneous rapid analysis of >1000 molecules in urine to yield “metabolic fingerprints” for health or disease • Goal = individualized wellness/disease profiling

27. Human & Animal Wellness Assessment - -Some Applications • Optimizing physical fitness • Well established: training enhances antioxidant systems • Overtraining - rapid increase in oxidative stress • Key targets: professional athletes and military; performance dogs • Community-based wellness testing • Complemented by blood pressure, and other current simple tests • Evaluate impact of diet, lifestyle, etc. on genetic background • Can provide feedback to assist development of healthy lifestyles

28. Day 3 0.8 300 200 500 400 100 Sneak preview: Metabolic Profiling DB-Wax 1.6 The effect of a simple diet change on the human urine metabolome A 0.8 1.6 DB-210 High protein diet (>500 g/day)for > 3 weeks [stable baseline profile] 0.8 DB-Wax 1.6 A B 0.8 Day 5 1.6 DB-210 Low protein (<50 g/day) high carbohydrate diet B Primary RT (s)