Publisable summary. Project description. Main objectives. Updated summary

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1 Publisable summary Project description The overall aim of this project is to investigate the protective action of agents with potential use as functional food constituents with respect to cancer, diabetes and cardiovascular disease. The burden posed by these age related diseases on public health is enormous. Even a modest decrease of these ailments caused by consumption of appropriate functional foods translates into millions of lives saved. Based on animal experiments a number of agents have shown promise in preventing cancer as well as diabetes. However, proof of the utility of specific dietary ingredients as well as the utility of various biomarkers for determination of protection against these diseases in humans is incomplete. Main objectives The overall objective for this project is to select food ingredients beneficial with respect to (i) genotoxicity/carcinogenicity as well as (ii) diabetes type 2 and associated cardiovascular complications (CVD). Some of these agents may prove suitable for inclusion in functional foods. Updated summary The above mentioned diseases take decades to develop, and within this project the potential positive impact of protective agents will, therefore, be monitored by means of predictive molecular and cytogenetic biomarkers. The strategy for the investigations is by using a decision-based tier approach. Initially, studies are conducted in cultured cells as well as in rodent models with respect to induction of DNA lesions, chromosome aberrations, tumours as well as biomarkers for the development of diabetes type 2, diabetic retinopathy and atherosclerosis. Finally, testing of the protective efficacy of agents selected on basis of the experimental systems will be carried out in intervention studies in humans exposed to grilled foods and cooking fumes, exposures that normally contain carcinogens at significant levels. Overview of the results of the candidate compounds that are investigated for protection against genotoxic/carcinogenic agents. The overall results of the part that concerns cancer is summarized in table 1 below. Although selectively biased, it suggests that the strategy with tier decisions has been successful up to now, although all data has not yet being generated and evaluated. It was found that the compounds that were positive in the in vitro and in vivo experimental assays and the most promising could also be identified as positive in the human intervention studies. The intervention study in India, in which participant 1 and 3 take part, has not yet started. Decisions have been taken at the annual meeting in India in February 2013 that next compounds for the intervention in Serbia will be Gentiana extract and lycopene. In Sweden intervention studies has started and with lycopene the final 5 th protective agents for testing will be Gentiana extract. The Indian participants have not started yet but discussed three options; blueberry, chlorophyllin and Gentiana

2 extract for their intervention studies. The interventions studies are delayed due to difficulties in recruiting volunteers. Table 1. Overview of the results of the candidate compounds that are investigated for protection against diabetes type 2 and cardiovascular diseases (CVD). Experiments with different cell systems demonstrated that among a number of dietary supplements carnosine and coenzyme Q (CoQ) improve the pathological metabolic responses operating in the presence of high glucose and hypoxia, cardinal events in diabetic conditions. Using diabetic animal model systems it was found that in kidney carnosinase activity is greatly increased, leading to decrease of carnosine and anserine concentrations, resulting in disturbances of several basic functions of the kidney. Carnosine treatment largely prevented the pathological modifications. In diabetes wound healing is poor but upon treatment with carnosine the wound healing process in db/db mice is significantly improved. Prolonged treatment of db/db mice with CoQ during six months is to a large extent counteracting the neuronal dysfunctions, indicating a real possibility for treatment of this serious complication in diabetic patients. A large number of parameters were analyzed when patients with typ1 and 2 diabetes received CoQ supplement. Analysis of blood detected a decrease of both total LDL and oxidized LDL and in type 1diabetes a decrease of the HbA1c. Some of the cytokines and adhesion factors responded positively for the treatment. NK-cell activation was also observed and there was a decreased production of cytotoxic cytokines. Reduction of 8-hydroxy-deoxyguanosine in the plasma upon treatment with CoQ

3 indicates the efficiency to counteracting DNA-oxidation. It is known that oxidative stress is a major factor during development of diabetes and diabetic complications and for this reason several markers were employed to follow changes during supplementation. Plasma glutaredoxin activity was significantly up regulated in both type 1 and 2 diabetic patients. After 12-weeks treatment with CoQ glutaredoxin activity is greatly decreased demonstrating improvement of the antioxidative defence. The results suggest a further extended treatment of diabetic patients with CoQ is necessary. Human trials using carnosine and blueberry drinks are also in progress analyzing biochemical markers for diabetes type 2 and CVD. Achieved results Cancer summary for each WP WP1: Based on an extensive literature review, the following 15 candidate chemoprotective agents were selected for consideration to be test: astaxanthin, Astralagus polysaccharides, blueberry extract, butylated hydroxyanisole, carnosine, chlorophyllin, co enzyme Q10, ellagic acid, extract from Gentiana lutea, lycopene extract, silibinin, teaphenon E, 6 gingerol, resveratrol and turmeric. Agents finally selected for investigation according to updated list during duration of the project and at meetings were the following 13; astaxanthine, BHA, blueberry, chlorophyllin, CoQ10, ellagic acid, Gentiana lutea extract (isolated and characterized under this WP), 6-gingerol, lycopene, resveratrol, silibinin, theaphenone/egcg and tumeric. These agents, with the exception of the Gentiana extract, were purchased from different sources to be distributed from the same batch to all partners. The purity and other chemical properties were recorded and taken into consideration (WP1). WP2: For screening of the possible protection from these agents against the mutagenic effects of dibenzopyrene and its metabolite dibenzopyrene diolepoxide, as well as the methylating agent N- methylnitrosourea, the DRAG (Detection of Repairable Adducts based on Growth Inhibition) assay was initially used in metabolically competent HepG2 human cancer cells. However, the DRAG assay was found to be less suited for this purpose, and was replaced by the classical hprt mutation assay. For further testing in vivo of antigenotoxic effects, the results indicated astaxanthine, blueberry extract, chlorophyllin, CoQ10, ellagic acid, Gentiana extract, 6-gingerol and theaphenone to be the most promising candidates. For induction of micronuclei in HepG2 cells by MNNG and DMBA, 5 protected compounds were selected on the basis of the screening, chlorophyllin, ellagic acid, blueberry, Gentiana lutea extract and 6-gingerol. The results on micronuclei induction in vitro in HepG2 cells were considered protective when the reduction in the frequency of micronuclei was not associated with a significant increase of cytostasis. All 5 compounds showed protective effect against MNNG induced DNA damage. Only chlorophyllin and 6-gingerol were found to be protective against DMBA. WP3: An assay for induction of micronuclei by 7,12 dimethylbenz(a)anthracene and N methyl N nitro N nitrosoguanidine in Swiss albino mice was set up, and dose response relationships for these carcinogens established. The protective effect of blueberry, chlorophyllin and ellagic acid on the induction of micronuclei in bone marrow given a single dose DMBA and MNNG was selected for investigation on the basis of results retrieved in WP2. All three agents were protective against MNNGinduced micronuclei, but only blueberry against DMBA. WP4 and 5: Reported by the Indian participants to DBB, India.

4 WP6: Standard operating procedures (SOPs) for taking samples of blood, buccal cells and urine from human subjects have been successfully established, and the appropriate permits obtained for the Vinča Institute of Nuclear Sciences in Belgrade, the site for conducting this study. WP7: Optimal conditions for the grilling of pork and Serbian sausages (ċevapčiċi) have been established, and adequate analytical procedures tested (GC/MS, UPLC). The PAH profiles from the grilled meat samples were almost identical with that obtained under the 6 th Fp project Dietary exposure to PAH and DNA damages (DIEPHY; FOOD-CT ), with a total concentration of 28 individually determined PAH in the range 1100 to 3200 μg/kg, and with benzo[a]pyrene levels between 53 and 158 μg/kg. WP8: A total of 48 persons have been recruited from the staff of the Vinča Institute. The dietary regime involves daily ingestion of 200g grilled meats during 4 days, with sampling of blood on day 1 and 5, whereas urine samples are taken on days 1, 2, 3, 5 and 11. After a wash-out period of 10 days, the same dietary regime is employed together with ingestion of the putative protective agent. Statistical evaluation of data have shown statistical significant difference between baseline level of lymphocyte micronuclei and the level after consuming the grilled meat with high content of PAHs (p<0.01). Pretreatment with Chlorophyllin (CHL) restored the MN level on the baseline value, and no further incensement of MN was observed while grilled meat was consumed. Comet Tail Intensity has shown statistically significant difference between baseline level and level after PAHs ingestion (p<0.04) in entire group of volunteers. Supplementation with CHL insignificantly reduce the level of Comet Tail Intensity (p<0.50), and no further difference in the Comet Tail Intensity was found. Statistical difference in the yield of micronuclei in buccal cells was independent of any treatment (p<0.6; p<0.55 respectively). Correlative analysis (Spearman Rank Order Correlations) showed negative, statistically insignificant correlation between incidence of MN and Comet Tail Intensity in all analyzed points. For the polycyclic aromatic hydrocarbon (PAH) biomarker analysis in urine the well-established GC-MS method at BIU has been used. The analysis of the excretion of phenolic PAH exposure markers indicate that overall a substantial direct or indirect inhibition of cytochrome P450 including CYP1A1, 1A2, and 1B1 competent for PAH metabolism and/or phase 2 enzymes such as UDPglucuronosyltransferase and sulfotransferases occurs in vivo by BB supplementation. Analysis of PAH-metabolites in urine and 8-OxodG, vitamin E and Q10 in blood has started (Tasks 8.4-6) but evaluation has not been performed. WP9: The measurement of Hb adducts is based on detachment of adducts to N-terminal amino acids with Edman reagents and analysis by mass spectrometric techniques. Acrylonitrile is marker of tobacco smoking. Acrylamide is a marker of tobacco smoking and also of intake of certain types of food. Glycidamide reflects metabolic activation of acrylamide. The initial work showed that additional developmental work had to be done concerning the analytical method(s), which has delayed the analysis of samples from the intervention studies. The first problem is now solved. The step in the analytical procedure where the second problem arises is identified, and a way to adjust for the sidereaction is now being tested out. These developments would allow reproducible quantification of both acrylonitrile and acrylamide adducts in smokers. Ca. 70 blood samples from the intervention studies with smokers with/without intake of antioxidants have been pre-prepared for further work-up. To optimize the work the Hb adduct levels of studied compounds will be quantified in all samples at one occasion when all samples are collected.

5 Preliminary data on chromosomal damage indicates that astaxanthine, blueberry extract and chlorophyllin exhibit protective effects. The process of the analysis of 8-oxo-dG in blood of donors is going on and we have preliminary results of an protective effect of blueberry, lycopene (taken as tomato extract), chlorophyllin and astaxantine. Urinary PAH biomarker analysis in Swedish smoker and the influence of antioxidants have started during the period. The determination of urinary cotinine levels is presently undertaken which is required for a better estimate of the hitherto only self-reported smoking status. The measurement of urinary cotinine levels is ongoing work and will allow a more objective estimate of the smoking status than of the self-reported numbers of smoked cigarettes. At present the influence of antioxidants including chlorophyllin, astaxanthin and lycopene on the urinary excretion levels of 3-HPMA is investigated in selected smokers. Hitherto identified life style factors were intake of vitamins and alcohol. It should be emphasized that the multivariate analysis did not include several of the endpoints, e.g., blood levels of vitamin E, Co-Q10 and 8-OxodG and PAH metabolites in urine. Therefore, the final evaluation of the influence of life-style factors cannot be performed. This WP is delayed because of difficulties to recruit smoking volunteers. Diabetes and CVD summary for each WP WP10, 11 and 12: Reported by the Indian participants to DBB, India. WP13: Experiments with different cell systems demonstrated that among a number of dietary supplements carnosine and coenzyme Q (CoQ) improve the pathological metabolic responses operating in the presence of high glucose and hypoxia, cardinal events in diabetic conditions. Using diabetic animal model systems it was found that in kidney carnosinase activity is greatly increased, leading to decrease of carnosine and anserine concentrations, resulting in disturbances of several basic functions of the kidney. Carnosine treatment largely prevented the pathological modifications. In diabetes wound healing is poor but upon treatment with carnosine the wound healing process in db/db mice is significantly improved. The mitochondrial function in diabetic kidney is damaged by uncoupled respiration causing lowered ATP production. Upon CoQ treatment of db/db mice the respiration was restored to normal values. Diabetic neuropathy is a common complication in human type 1 and type 2 diabetes, leading to non-healing wounds and amputation. Serious disturbances of sensory functions and reduction of neuronal loss was found in the diabetic models. Prolonged treatment of db/db mice with CoQ during six months is to a large extent counteracting the neuronal dysfunctions, indicating a real possibility for treatment of this serious complication in diabetic patients. WP14: A large number of parameters were analyzed when patients with typ1 and 2 diabetes received CoQ supplement. Analysis of blood detected a decrease of both total LDL and oxidized LDL and in type 1diabetes a decrease of the HbA1c. Some of the cytokines and adhesion factors responded positively for the treatment. NK-cell activation was also observed and there was a decreased production of cytotoxic cytokines. Reduction of 8-hydroxy-deoxyguanosine in the plasma upon treatment with CoQ indicates the efficiency to counteracting DNA-oxidation. It is known that oxidative stress is a major factor during development of diabetes and diabetic complications and for this reason several markers were employed to follow changes during supplementation. Plasma glutaredoxin activity was significantly up regulated in both type 1 and 2 diabetic patients. After 12- weeks treatment with CoQ glutaredoxin activity is greatly decreased demonstrating improvement of

6 the antioxidative defence. The results suggest a further extended treatment of diabetic patients with CoQ is necessary. Human trials using carnosine and blueberry drinks are also in progress. Potential impact - The challenge posed by this project is to enhance the health benefit from diets beyond the traditional nutrients it contains by means of addition of bioactive ingredients. This goal has partly being achieved by the step-wise approach providing robust scientific evidence for selecting active ingredients appropriate for inclusion in functional foods. - The research will also include mechanistic aspects, in as much as the impact of protective agents on carcinogen uptake and metabolism in humans is included, as well as the mode of action of bioactive ingredients on the prevention of diabetic ocular complications. Publications already exist concerning mechanistical insight in diabetes complications. - Although there has been remarkable progress in our understanding of the processes that lead to neoplasia, the mechanisms underlying chemoprevention are, in general, little understood. However, initiation - i.e. by trapping DNA reactive metabolites, modulation of the expression of phase I and I metabolism enzymes, as well as suppression of the promotion and progression stages, represent suggested main modes of action. Numerous studies in vitro have reported interaction with a number of components of the intracellular signal-transduction pathways, and the significance of these findings for the situation in vivo will be assessed in this project. - This project will provide concrete evidence of active principles likely to exert antimutagenic/anticarcinogenic action in humans. - The above goal will be achieved by verifying the utility of various biomarkers in human cohorts. - This project will provide concrete evidence of active substances and the principles to which they are likely to exert protective action against diabetes and associated cardiovascular diseases (CVD) in humans. - Several substances that are chemoprotective with respect to cancer initiation and oxidative stress, are expected to have preventive effects for other types of age related diseases like atherosclerosis and diabetes.