Vegetables and heath the Nrf2 connection

Transcription

1 Food security: The value of vegetables Warwick Crop Centre University of Warwick, Wellesbourne CV35 9EF, 11 th July 2012, Vegetables and heath the Nrf2 connection Naila Rabbani and Paul J. Thornalley Clinical Sciences Research Laboratories, Warwick Medical School, University Hospital, Coventry CV2 2DX, U.K. and Warwick Systems Biology Centre, University of Warwick, Coventry House, Coventry CV4 7AL, U.K. Warwick Medical School - CSRL Warwick Systems Biology

2 Vegetables consumed in the United Kingdom Top ten Rank Vegetable 1 Onions 2 Cucumbers 3 Peppers 4 Cauliflower 5 Cabbage 6 Lettuce 7 Tomatoes 8 Broccoli 9 Garlic 10 Mushrooms A display of fresh vegetables on a market stall in Great Yarmouth, Norfolk, England J Sainsbury plc

3 Consumption of 5 portions per day of fruit and vegetables Origin of 5-a-day: 1991: World Health Organization (WHO) recommended consuming a minimum of 400 g fruit and vegetables a day (one portion is 80g) Average number of fruit and vegetable portions consumed per day, 2004 Expected health benefits: If all nutritional guidelines were followed, an estimated 70,000 premature deaths in the UK could be avoided each year in the UK. We re getting closer Cabinet Office. Food Matters. Towards a Strategy for the 21 st Century. London: Cabinet Office, 2008

4 Expected health benefit of dietary advice Epidemiological model: DIETRON model Outcome: Approximately deaths per year would be avoided if UK dietary recommendations were met ca. 21,000 less CVD deaths, 6000 less stroke deaths and 5000 less cancer deaths > deaths avoided due to increased consumption of fruit & vegetables Reference: Scarborough et al., J Epidemiol Community Health 66, , 2012

5 Trends in vegetable consumption in the UK Source: Family Food 2010 (Figures to 2008) Approximately 160 g per day vegetables 2 of the 5-a-day (2008). Approximately 258 g per day fruit and vegetables consumed in UK 3 of the 5-a-day, 14 th in Europe - European Food Information Council (EUFIC), January 2012.

6 Health benefits of vegetable consumption Brassica vegetables and baby-leaf salad Broccoli Cabbage Cauliflower Brussel sprouts Rocket Mustard Watercress CANCER Decreased risk of cancers of the bladder, colon and lung (Cruciferous Vegetables, Isothiocyanates and Indoles, IARC WHO report, 2004) HEART DISEASE Higher intake of fruits and vegetables is associated with a reduced risk of coronary heart disease mortality risk reduction 22% with 8 portions-a-day. EPIC (Crowe et al, Eur Heart J. 32, 1235, 2011) TYPE 2 DIABETES Increasing daily intake of green leafy vegetables could significantly reduce the risk of type 2 diabetes - risk reduction 14% (Carter et al. BMJ 2010; 341, c4229)

7 Vegetable crops as a source of health beneficial compounds Brassica vegetables and baby-leaf salad Broccoli Cabbage Cauliflower Brussel sprouts Rocket Mustard Watercress Carrots, onions, garlic and tomatoes Many vegetables are relatively low in calories and fat. Sources of fibre and vitamins (vitamin C and folate B 9 ). Also a source of phytochemicals health beneficial dietary bioactives terpenoids, alkaloids, 8000 phenolic compounds and sulfur containing compounds (Goldberg, G. Plants: Diet and Health; report of a British Nutrition Foundation Task Force; Blackwell Publishing: Oxford, U.K., 2003)

8 Types of health beneficial compounds in fruits and vegetables Health-beneficial compounds (dietary bioactives) Glucosinolates (precursors of isothiocyanates) - Brassica vegetables and baby-leaf salad e.g. Glucoraphanin O CH 3 S K +- O 3 SO N S Polyphenols and flavonols - Grapes, strawberries and onions HO O OH OH OH CH 3 Myrosinase S O N C S Sulforaphane Resveratrol (from grapes) Carotenoids carrots & tomatoes Fisetin (from strawberries) Quercetin (from onions) Allyl sulfides garlic and onions Lycopene (from tomatoes) Triterpenoids citrus fruits and olives Nomilin (from bitter orange) Uvaol (from olive oil) Oleanolic acid (from olive oil and garlic)

9 Health beneficial compounds in fruits and vegetables other concepts Lifespan essentials - Bioactive compounds in fruits and vegetables are not essential nutrients for growth and development, but rather mainly non-nutrients improving health particularly in adult and later life (Holst and Williamson, Curr Opinion in Biotechnol 19, ) Bioactives contributing to healthy ageing Survival curves in Sweden over the last 140 years (Westendorp, Am J Clin Nutr 83, 404S-409S, 2006)

10 Health beneficial compounds in fruits and vegetables antioxidant paradigm There is a continuing tendency to view health effects of dietary bioactive compounds as linked inextricably and sometimes only with antioxidant activity I take lots of antioxidants. That s why I m still on the first of my nine lives Probably a too narrow approach Dietary bioactives have many other beneficial effects - antiinflammatory, anti-lipogenic, anti-metabolic stress, others

11 Health beneficial compounds in fruits and vegetables other concepts Emerging consensus for sustained metabolic and vascular health - a requirement to prevent not only oxidative damage in tissues but also to: i. Prevent and repair all types of protein and nucleotide damage, ii. Remove damaged proteins and nucleotides, iii. Efficiently metabolize and clear damaging metabolites and xenobiotic compounds, iv. Regain or reset glycolytic and lipogenic control when lost in pre-diabetes, metabolic syndrome and obesity (Thornalley, P.J, Xue, M. and Rabbani, N. Methodologies for invitro and in-vivo activity of bioactive compounds. In: Health- Promoting Properties of Fruits and Vegetables, Terry, L. Ed., CABI, Oxford, UK, , 2011).

12 Anti-stress gene response: Nuclear factor E2-related factor 2 (Nrf2) Activation: Confocal image of human endothelial cells transfected to express nrf2-gfp fusion protein Broccoli Kelch-like ECH-associated protein 1 (Keap1) - endogenous inhibitor (Yamamoto et al, Nature Cell Biology 12, , 2010) + SFN Antioxidant response element (ARE)-linked genes: 5'-ATGACTCAGCA-3' ARE (Wang et al., Human Molec Genet 16, , 2007)

13 Anti-stress gene response:

14 Anti-stress gene response: Antioxidant response element (ARE)-linked genes: TGACTCAGCA-3' 5'-A Protection against oxidative damage ARE Quinone reductase (NQO1) Glutathione synthesis and GSH-dependent enzymes (GCL, GSHRd, GSHPx and GSTs) Peroxiredoxins (PRDXs) Thioredoxin (TXN) and Thioredoxin reductase (TXNRd1) Haem oxygenase (HMOX1), Metallothionein (ME1), Ferritin (FTH1) and others Protection against glycation Aldoketo reductase (AKRd) Aldehyde dehydrogenase (AldDH) Protection against metabolic stress Glucose-6-phosphate dehydrogenase (G6PDH) Transketolase (TKT), Transaldolase (TALDO) Protection against lipogenic stress Sterol response element binding protein-1 (SREBP-1) - down regulation Fatty acid synthase (FASN) and others - down regulation Removal of damaged proteins 20S Proteasome induction (PSMA1, PSMA4, PSMB3, PSMB5, and PSMB6) Sequestosome-1/p62 (SQSTM1) autophagic removal of damaged proteins/organelles

15 Evidence emerging from synthetic activators of Nrf2: Bardoloxone methyl (BARD) Evidence from mice - Prevention of development of diabetes BARD treatment of wild-type mice decreased total body fat, plasma triglyceride and free fatty acid levels, and improved glucose tolerance and insulin sensitivity in mice fed a high fat diet fed (J Biol Chem 285, , 2010) Evidence from Nrf2 (-/-) mice Plasma cholesterol, triglycerides and hepatic lipogenic gene expression increased in Nrf2(-/-) mice, with respect to wild type controls (J Pharmacol Exp Therap 325: , 2008) Evidence from dietary bioactives: Work in progress

16 Evidence emerging from synthetic activators of Nrf2: Bardoloxone methyl Evidence from human trials of a potent synthetic activator of Nrf2 (Methyl 2-cyano-3,12-dioxo-oleana-1,9(11)dien-28-oate; Bardoloxone methyl) Reata/Abbot Pharma Bardoloxone methyl decreased vascular inflammation and improved renal function in patients with diabetic nephropathy (NEJM 365, , 2011). BEAM Trial - Bardoloxone methyl (BARD) decreased body weight. 52 week phase 2b trial, 227 patients with an egfr of 20 to 45 ml/min per 1.73 m² - randomized to 25 mg (n = 57), 75 mg (n = 57), or 150 mg (n = 56) BARD o.d. or placebo (n = 57). Baseline: mean BMI was 35.6 ± 7.4 kg/m² in BARD and 34.4 ± 8.0 kg/m² in the placebo group. OUTCOME After 52 weeks of treatment, with BARD mean weight loss was 7.7 kg with 25 mg, 9.2 kg with 75 mg, and 10.1 kg with 150 mg; and 2.4 kg with placebo.

17 Improvements of metabolic health by dietary activators of Nrf2 Selected human studies where Nrf2 activation is likely involved in benefit Broccoli sprouts decrease oxidative stress markers (Biofactors 22, 271-5, 2004; Carcinogenesis 27, , 2006) Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans (Cell Metabolism 14, , 2011)

18 Health beneficial compounds in fruits and vegetables Nrf2 activator activity Nrf2 inducers have health sustaining activities including and extending beyond antioxidants effects. Different dietary sources, with improved food processing, will likely have greater health benefit (than resveratrol) Broccoli Cabbage Rocket I take lots of Nrf2 activators. That s why I m still on the first of my nine lives and healthy Cauliflower Brussel sprouts Mustard Watercress

19 A quick word on cooking of vegetables Boiling Microwave cooking Steaming Stir fry

20 Glucosinolate content in vegetables: effect of cooking - boiling Vegetables (20 25 g) were boiled for 0, 5, 10, 20 and 30 min in 150 ml of boiling water. Vegetable Cooking water 40 ( m m ol/1 0 0 g we t we ight) (m mol/100g wet weight) Example: broccoli Glucorapha nin Time (min) 30 5 Vegetable Cooking water 4 Glucoiberi n Time (min) Boiling of the vegetables for 10 min decreased the glucosinolate level to 60-70% of the original value. Boiling for 30 min decreased glucosinolates levels further to 30-40% of the original value. Most of the lost of the glucosinolate (~90%) from the vegetables was detected in the cooking water. Therefore, prolonged boiling of Brassica vegetables produced significant losses of glucosinolates by leaching into cooking water. (Song & Thornalley, Food and Chemical Toxicology 45, , 2007)

21 Glucosinolate content in vegetables: effect of cooking - steaming Vegetables (30 36 g) were steamed for 0, 5, 10 and 20 min. Cooking time (0 min) Total glucosinolates (m mol/100 g wet weight) 60 Cooking time (5 min) 50 Cooking time (10 min) 40 Cooking time (20 min) Broccoli Brussel sprouts Caulifower Cabbage Steaming of vegetables for up to 20 min had little effect on the content of glucosinolates (Song & Thornalley, Food and Chemical Toxicology 45, , 2007)

22 Glucosinolate content in vegetables: effect of microwave cooking Vegetables (30 35 g) with 10% added water were cooked in a microwave oven for 30, 60, 120 and 180 s in a plastic bag. Cooking Cooking Cooking Cooking Cooking Total glucosinolates (m mol/100 g wet weight) time time time time time (0 s) (30 s) (60 s) (120 s) (180 s) Broccoli Brussel sprouts Caulifower Cabbage Microwave cooking of the vegetables for up to 180 s had little effect on the level of glucosinolates (Song & Thornalley, Food and Chemical Toxicology 45, , 2007)

23 Glucosinolate content in vegetables: effect of stir-fry cooking Vegetables (30 35 g) were washed, shredded coarsely and cooked immediately for 3 and 5 min Total glucosinolates (m mol/100 g wet weight) 60 Cooking time (0 min) Cooking time (3 min) 50 Cooking time (5 min) Broccoli Brussel sprouts Caulifower Cabbage Stir-fry cooking of the vegetables for up to 5 min produced minor losses (5 10%) of glucosinolate contents. (Song & Thornalley, Food and Chemical Toxicology 45, , 2007)

24 Development of screening technology for Nrf2 regulated gene expression protective of metabolic and vascular health Steps to commercialization Antioxidant response element (ARE)-linked genes: TGACTCAGCA-3' 5'-A ARE Timeline Action June 2010 File of patent on screening know-how and health beneficial effects of Glo1 inducers August 2010 UK BBSRC Follow-on-fund (FoF) grant Developing a new class of therapeutic agents for healthy ageing October 2010 FoF project initiated March 2011 Stable transfectant cell lines for reporter assays September 2011 Preliminary in-house screening results October 2011 UK Technology Strategy Board funds collaborative project with Unilever for development of novel functional foods. June 2012 TSB/Unilever collaborative project on functional food development starts May 2015 Novel functional food evaluated clinically with scientific evidence base to support health claim to EFSA

25 NQO1-ARE luciferase stable transfectant reporter cell line produced and potent activators of the Nrf2 system have been identified NQO1-ARE transcriptional response reporter assay: Add bioactives or fruit/vegetable extracts Wash cells and lyse cells with lysis buffer Develop response 6 24 h Stable transfectant NQO1-ARE luc reporter cell line Take 20 μl supernatant of luciferase assay Luminescence reporter response NQO1-ARE transcriptional response: Erucin Sulforaphane Broccoli Rocket

26 WARWICK research on Dietary activators of ARE-linked gene expression for good vascular health Protective nrf2/are transcriptional response induced by extracts of food ingredients NQO1-ARE transcriptional response (counts) Olive oil response *** ** ** ** ** *** SFN Concentration in cultute medium (% v/v) Positive control: 2 µm sulforaphane (SFN) in 0.002% DMSO. Oil dissolved 1:1 in DMSO and sterile filtered. Optimise for Nrf2 activator content by plant variety and food processing

27 Food security: The value of vegetables Summary Nrf2/ARE transcriptional system coordinates expression of a battery of protective genes to maintain metabolic and vascular health. Clinical and pre-clinical studies with synthetic Nrf2 activators suggest potent and sustained activation may help maintain good metabolic and vascular health. Fruits and vegetables are a good dietary source of Nrf2 activators more potent activatory functional foods may soon be available. Optimizing food content and bioavailability of bioactives may produce healthier foods Broccoli, cabbage, cauliflower, Brussel sprouts, rocket, tomatoes, onions, others

28 WARWICK research on Dietary activators of ARE-linked gene expression for good vascular health Research team on this project: Principal investigator: Paul J. Thornalley Co-investigators: Naila Rabbani Guy Barker (Medical School/Systems Biology) (Crop Centre) David Rand (Systems Biology) Post-doctoral researchers: Funding: BBSRC Diet & Health (DRINC) project TSB Nutrition for Life project (with Unilever) Other projects: BBSRC Follow-on-fund healthy ageing therapeutics BBSRC- Industrial CASE PhD studentship (with Unilever) Nrf2 activators and cell senescence Dr Mingzhan Xue Dr Hiroshi Momiji Technicians: Dr Maqsud Anwar and Linda Brown EU FP7 BIOCLAIMS BIOmarkers of robustness of metabolic homeostasis for nutrigenomics-derived health CLAIMS made on food