This study examines the carcinogenic effect of HZE radiation and protective effects of different types of diets against colon carcinogenesis in a rat model. The effect of HZE radiation on health state and colon cancer development was evaluated. HZE radiation was found to suppress food consumption (P<0.0001) leading to lower body weight gain of irradiated rats when compared to the non-irradiated rats (P<0.05). The animals exposed to HZE radiation were found to start dying and/or getting pathologies 11 weeks earlier and at the end of the study had morbidity/mortality rate 14.2% higher (P=0.0005) than non-irradiated rats. There was no significant effect of HZE radiation on colon cancer incidence. The effects of dietary fibers and oils on health state and colon carcinogenesis were evaluated. Morbidity/mortality was found to be delayed in rats fed with pectinbased diets when compared to cellulose-based diet, regardless of radiation treatment. Similarly, fish oil was found to beneficially affect health of the experimental animals when compared to corn oil. Ten- and twenty-week delayed morbidity/mortality for irradiated and non-irradiated groups, respectively, was observed for rats fed with fish oil-based diets when compared to corn oil-based diets. Fish oil was also found to significantly reduce colon tumor incidence and multiplicity in non-irradiated rats (P<0.05). A similar trend was observed for the irradiated animals. No significant effect of fiber on colon cancer incidence was found. Finally, the effect of diets on general health and colon cancer development was investigated. Rats fed with corn oil/cellulose diet started dying and/or getting a disease earlier than rats fed with other diets, regardless of radiation treatment. The effect of diet on colon cancer development was found to depend on radiation treatment. Thus, in the absence of radiation treatment fish oil/cellulose was found to significantly reduce tumor incidence and multiplicity when compared to corn oil/pectin diet (P<0.05). In the presence of radiation treatment fish oil/pectin was found to lower the values of tumor incidence and tumor multiplicity, though the data obtained were not significant.

Omega-3 polyunsaturated fatty acids (PUFA) and vitamin D both have anti-cancer effects through common and unique pathways. The hypothesis of this thesis is that the combination of n-3 PUFA with 1,25(OH)2D3 will inhibit breast cancer cell growth in an additive or synergistic manner. A 3X3 factorial design was used to test the combinations of five PUFA treatments (α-linoleic acid (ALA, 18:3n3), eicosapentaenoic acid (EPA, 20:5n3) and docosahexaenoic acid (DHA, 22:6n3), γ-linolenic acid (GLA, 18:3n6) and arachidonic acid (AA, 20:4n6)) with 1,25(OH)2D3 on MCF-7, MDA-MB-231, and MCF-10A cell growth, and determine any potential synergism in combination treatments. MCF-7 and MCF-10A cells responded to PUFA and 1,25(OH)2D3 treatments, but combinations provided no potential synergism. MDA-MB-231 growth was not affected by 1,25(OH)2D3, while combinations treatments involving ALA, EPA, GLA, and AA caused potentially synergistic growth inhibition. This thesis presents the novel observation that PUFA are sensitizing MDA-MB-231 cells to 1,25(OH)2D3 treatment.

Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) and curcumin modulate multiple determinants that link inflammation to cancer initiation and progression. In this dissertation, both transgenic and dietary mouse models were used to elucidate the effect of n-3 PUFA and curcumin treatment on murine intestinal inflammation. Specifically, fat-1 transgenic mice, which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, exhibited a reduced number of colonic adenocarcinomas per mouse (1.05 plus/minus 0.29 versus 2.12 plus/minus 0.51, P = 0.033), elevated apoptosis (P = 0.03), and a decrease in n-6 PUFA–derived eicosanoids compared with wild-type (wt) mice in an azoxymethane (AOM) - dextran sodium sulfate (DSS) model. Following a 2-week recovery period after 5 days of DSS exposure, colonic inflammation and ulceration scores returned to pretreatment levels only in fat-1 mice. In addition, fat-1 vs wt mice exhibited decreased (P < 0.05) levels of CD3 , CD4 T helper, and macrophage cell numbers in the colon. The ability of n-3 PUFA to favorably modulate the resolution of intestinal inflammation in fat-1 mice was linked to an enhancement (P < 0.05) in the percentage of colonic lamina propria (cLP) CD4 FoxP3 cells and a decrease in both splenic and cLP Th17 cells (0.8 vs 1.2 percent in spleen, 1.4 vs 1.7 percent in colon) (P < 0.05) in fat-1 mice compared to wt. These results suggest that the antitumorigenic effect of n-3 PUFA may be mediated via its anti-inflammatory properties. The combined effect of n-3 PUFA and curcumin on DSS induced colitis was assessed in C57BL/6 mice. Addition of fish oil (FO) and/or curcumin to a corn oil (CO) based diet increased animal mortality compared to CO alone (P < 0.05). Consistently, following 1 or 2 cycles of DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared to CO. However, compared to other diets, FO and curcumin combined feeding enhanced the resolution of chronic inflammation and suppressed (p < 0.05) a key inflammatory mediator, NF-kB, in colon mucosa. Mucosal microarray analysis revealed that dietary FO and curcumin differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.

Background: Endothelial dysfunction is a major factor in the development of atherosclerosis, thrombosis and heart disease. Evidence suggests dietary fat composition may modify cardiovascular risk, as well as surrogate markers of cardiovascular risk such as blood pressure, arterial stiffness and endothelium-dependent vasodilation. Aim: To investigate the impact of dietary fat composition on endothelial function and associated markers of vascular health. Methods: The effects of oils rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were separately investigated in a parallel-design, placebo-controlled randomised controlled trial (n=48, 6 weeks, 2.9 g/d), carried out in free-living healthy young men. Following a 2 week run-in period taking placebo capsules (olive oil), participants underwent baseline measurements of finger capillary density, endothelial progenitor cell numbers (EPC), platelet-monocyte aggregate numbers (PMA), ambulatory blood pressure (ABP), pulse wave analysis (PWA), digital volume pulse analysis (DVP), and gave blood samples for plasma lipid, glucose, insulin, nitric oxide metabolites (NOx) and isoprostanes. The same measurements were made at the study endpoint, 6 weeks. An in vitro investigation of the effects of physiologically-relevant fatty acid profiles on microvascular endothelial cell nitric oxide and prostacyclin production was also performed. Results: Neither EPA nor DHA supplementation influenced EPCs, capillary density, PMA, ABP, PWA, DVP or plasma cholesterol, triacylglycerol, glucose, insulin, NOx or isoprostanes compared to placebo. However, ambulatory night-time heart rate was increased following EPA supplementation compared to DHA. Furthermore, both EPA and DHA decreased plasma non-esterified fatty acids (NEFA) compared to placebo. The in vitro investigations suggested that the composition of circulating NEFA may differentially affect endothelial function in the microvasculature. Conclusion: Dietary EPA and DHA at relatively high doses do not improve a number of novel markers of vascular function, including microvascular function and a marker of endothelial repair in young healthy men. EPA and DHA have differing effects on heart rate during sleep, suggesting that further research is required into the possible adverse effects of higher doses of individual marine fatty acids in at-risk individuals. Further work is required to elucidate the role of physiological fatty acid profiles on endothelial function.

Studies had shown that leucine supplementation increases insulin sensitivity and it has been studied that n-3 PUFA may have an anti-inflammatory effect in adipocytes. However, the extent to which dietary sources such as leucine and/or n-3 PUFA act through PFKFB3/iPFK2 to suppress adipocyte inflammatory response has not been studied; PFKFB3/iPFK2 is a regulator that links adipocyte metabolism and inflammatory responses. In this study, the involvement of PFKFB3/iPFK2 in the effects of insulin sensitizing and anti-inflammatory effect of leucine and/or n-3 PUFA are explored using cultured 3T3-L1 adipocytes including wild-type cells, PFKFB3-control cells (iPFK2-Ctrl) and PFKFB3-knockdown cells (iPFK2-KD). In iPFK2-Ctrl cells, leucine supplementation appears to have insulin-sensitizing effects through improving p-Akt/Akt insulin signaling, but have no effect on adiponectin expression, and appear to have limited anti-inflammatory effects. n-3 PUFA supplementation appears to have limited effects on both insulin sensitizing and anti-inflammatory effects in iPFK2-Ctrl. In contrast, n-3 PUFA exhibit pro-inflammatory expression in iPFK2-KD. The results of this study support the hypothesis that PFKFB3/iPFK2 is critically involved in insulin-sensitizing effects of leucine. This role of PFKFB3/iPFK2, however, appears to be independent of anti-inflammatory responses. Given this, it is likely that PFKFB3/iPFK2 only account, in part, for the beneficial effects of leucine. n-3 PUFA stimulate PFKFB3/iPFK2 activity in wild-type adipocytes. However, PUFA do not exhibit anti-inflammatory and insulin-sensitizing effects in controls. In contrast, n3-PUFA exhibit proinflammatory effects in iPFK2-KD cells. Taken together, PFKFB3/iPFK2 is involved, at least in part, in the effects of insulin sensitization of leucine and appears to protect adipocytes from inflammatory responses, which could be exacerbated by n-3 PUFA when PFKFB3/iPFK2 is disrupted.

Un statut pauvre en Acides Gras Poly-Insaturés ω3 (AGPI ω3), favorisé par une alimentation occidentale comportant un faible ratio en ω3/ω6, semble contribuer au déclin cognitif chez les personnes âgées, mais les mécanismes cellulaires impactés sont encore mal connus. Nous avons donc étudié l’influence du statut en ω3 sur l’évolution de la neurotransmission glutamatergique et des fonctions astrocytaires au cours du vieillissement dans l’hippocampe de rats. Ces processus sont impliqués dans la formation de la mémoire et leurs dérégulations participent aux dommages cérébraux conduisant au déclin cognitif. Nous avons comparé 6 groupes de rats agés de 6 et 22 mois nourris avec un régime déficient en ω3, équilibré en ω3/ω6 ou supplémenté en ω3 (huile de poisson) : Jeunes équilibrés (JEq), déficients (JDef) ou supplémentés (JSup) et Agés équilibrés (AEq), déficients (ADef) ou supplémentés (ASup). Nous avons évalué l’efficacité synaptique et la plasticité (enregistrements électrophysiologiques), les fonctions astrocytaires (capture de glutamate et expression de la GFAP), les marqueurs neuronaux (transporteurs et récepteurs du glutamate), les capacités cognitives (Openfield et Labyrinthe de Barnes) et analysé la composition lipidique cérébrale. Les manipulations nutritionnelles d’apport en ω3 modifient efficacement l’incorporation de l’acide docosahexaénoïque (DHA, principale ω3 des membranes cellulaires) dans le cerveau (-50% deficient vs équilibré, +10% supplementé vs équilibré). Le vieillissement induit une diminution de 35% de l’efficacité synaptique en raison d’une baisse de la libération de glutamate pré-synatique, et une diminution de 30% de la capture de glutamate associé à une astrogliose conséquente (+100% GFAP). La déficience en ω3 acentue les effets du vieillissement (rats ADef vs AEq: -35% efficacité synaptique, -15% capture de glutamate, +30% GFAP). Al’inverse, la supplémentation en ω3 améliore l’efficacité synaptique (rats ASup vs AEq +25%) et semble inhiber l’astrogliose chez le rat âgé (ASup vs JEq : pas de modification de la GFAP). Les tests comportementaux montrent que le vieillissement a des effets plus marqués chez les déficients en ω3 et au contraire atténués chez les supplémentés. Nos résultats révèlent des altérations de la synapse glutamatergique de l’hippocampe au cours du vieillissement aggravées par la déficience en ω3 et atténuées par la supplémentation en ω3. Afin d’évaluer l’influence du statut en ω3 sur l’activation astrocytaire, des modèles in vitro d’astrocytes « âgés » et « activés » par des cytokines inflammatoires dont l’augmentation à bas bruit est caractéristique du vieillissement cérébral, ont été développés. / A poor ω3 polyunsaturated fatty acids (ω3 PUFA) status, favored by the low ω3/ω6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. We therefore explored the impact of ω3 status on the evolution of glutamatergic transmission and astrocytic functions in the hippocampus during ageing in rats. These processes are involved in memory formation and their dysregulation participates to the age-related brain damage leading to cognitive decline. We have compared 6 groups of rats aged 6 to 22 months fed ω3-deficient, ω3/ω6-balanced, or ω3 (fish oil) supplemented diets: Young ω3 Balanced (YB), Deficient (YD) or Supplemented (YS), and Old ω3 Balanced (OB), Deficient (OD) or Supplemented (OS) rats. We have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and GFAP expression), neuronal markers (glutamate transporters and receptors), cognitive abilities (Barnes maze and Openfield) and analyzed brain fatty acids composition. Dietary modulation of ω3 intakes efficiently modified the incorporation of docosahexaenoic acid (DHA, the main ω3 in cell membranes) in brain (-50% deficient vs balanced, +10% supplemented vs balanced). Ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (+100% GFAP). ω3 deficiency further decreased these hallmarks of ageing (OD vs OB rats: -35% synaptic efficacy, -15% glutamate uptake, +30% GFAP). On the opposite, ω3 supplementation increased synaptic efficacy (+25% OS vs OD) and seems to abolish astrogliosis (OS vs YS : no change in GFAP). Behavioural tests showed some increased effects of age in deficient rats and attenuated effects in supplemented ones. Our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in ω3 and attenuated by ω3 supplementation. In order to explore ω3 status on astrocytic activation, in vitro models of “old” astrocytes and “activated” by inflammatory cytokines which characterize the low-grade inflammation in brain aging, have been developed.

The molecular mechanisms of putative anti-inflammatory nutrients, i.e., fish oil, curcumin and limonin, were investgated with respect to CD4+ T cell function. Initially, using a DO11.10 mouse model which exhibits a transgenic T cell receptor specific to OVA 323-339 peptide, we demonstrated that dietary fish oil suppresses antigen-specific Th1 clonal expansion in vivo. Following immunization, the accumulation of adoptively transferred transgenic cells in wild type recipient mouse lymph nodes was suppressed. In addition, cell division analysis by carboxyfluorescein succinimidyl ester (CFSE) revealed that both total cell number in lymph nodes as well as cell division were decreased by fish oil. Since n-3 polyunsaturated fatty acids (PUFA), active long chain fatty acids in fish oil, elicit favorable effects on a variety of cell types, e.g., anti-tumor effect on colonocytes, amelioration of coronary heart disease and anti-inflammatory effects involving T cells, B cells, dendritic cells and macrophages, we postulated that a fundamental mechanism of action may explain the multiple effects observed. In a series of experiments described herein, we demonstrated that n-3 PUFA alters the formation/location of membrane subdomains, referenced to as lipid rafts. Specifically, lipid raft formation at the immunological synapse (IS) in CD4+ T cells was suppressed following membrane enrichment with n-3 PUFA. The alteration of lipid rafts down-regulated the localization of select signaling proteins, including F-actin, PKC and PLC-1, and phosphorylation of PLC-1 at the IS. Consequently, CD4+ T cell proliferation was suppressed as assessed by CFSE analysis and radioactive thymidine incorporation. Phytochemicals have been used for chemopreventive and chemotherapeutic purposes. We examined the putative anti-inflammatory effects of curcumin (1%) and limonin (0.02%) with respect to CD4+ T cell function. Dietary curcumin and limonin suppressed NF-B activation in CD4+ T cells. In addition, CD4+ T cell proliferation was modulated by 2% curcumin. We further investigated the combined therapeutic potential of phytochemicals and fish oil, containing n-3 PUFA. Interestingly, fish oil and limonin together significantly (P<0.05) suppressed T cell proliferation, whereas feeding either fish oil or limonin alone showed little effect. In summary, our data indicated that dietary fish oil alters proximal signaling of T cells by perturbing lipid raft formation. Curcumin and limoin are capable of suppressing NF-B in T cells, thereby exhibiting a synergistic effect when combined with fish oil. Further studies are required to elucidate the relationship of dietary dose of active compoments with respect to mechanism of actions.

Adipose tissue represents an important endocrine organ that secretes a multitude of adipokines known to mediate inflammation, lipid metabolism, and insulin sensitivity in peripheral tissues such as skeletal muscle. Specifically, adiponectin stimulates skeletal muscle fatty acid oxidation and is associated with improvements in insulin response. Long-chain n-3 polyunsaturated fatty acids (PUFA) are well known for their anti-inflammatory and insulin-sensitizing properties, and their dietary consumption is associated with a more favourable circulating adipokine profile, including increased adiponectin. However, whether n-3 PUFA can directly stimulate adiponectin secretion from human adipocytes, as well as the underlying mechanisms involved, is unknown. In contrast to n-3 PUFA, diets high in saturated fatty acids (SFA) are thought to decrease adiponectin and increase pro-inflammatory adipokines, as well as blunt skeletal muscle response to both adiponectin and insulin, possibly via activation of inflammatory pathways. The role of n-3 PUFA in mediating the communication between adipose tissue and skeletal muscle, as well as preventing SFA-induced impairments in skeletal muscle function, has yet to be examined. In this thesis, it was found that long-chain n-3 PUFA increase adiponectin secretion from human adipocytes via a peroxisome proliferator-activated receptor gamma-dependent mechanism. The effects of n-3 PUFA on adiponectin secretion were additive when combined with the thiazolidinedione, rosiglitazone. Secondly, incorporation of n-3 PUFA into a high SFA diet prevented impairments in adiponectin response and both prevented and restored impairments in insulin response in rodent skeletal muscle. Interestingly, these findings were paralleled by prevention of SFA-induced increases in toll-like receptor 4 expression by n-3 PUFA, suggesting inflammatory changes may be involved. Finally, dietary n-3 PUFA and SFA modulated the secretion of adipose tissue-derived factors from visceral rodent adipose tissue and subsequent exposure of isolated skeletal muscle to such factors induced acute changes in inflammatory gene expression without affecting insulin sensitivity. Together, the findings in this thesis suggest that n-3 PUFA modulate adipokine secretion from adipose tissue and that adipose-derived factors mediate skeletal muscle inflammation and response to adiponectin and insulin. Ultimately, this work highlights the importance of considering n-3 PUFA as a therapeutic strategy in the prevention and treatment of obesity and related pathologies.

Dysregulation of adipokines in obese adipose tissue contributes to inflammation and insulin resistance. Fatty acids and lipopolysaccharide (LPS) can modulate adipokine secretion, however, less is known about their effects in combination. Long-chain n-3 polyunsaturated fatty acids (PUFA) exert anti-inflammatory effects and less is known about other n-3 and n-6 PUFA, which are more prevalent in the typical diet. Co-incubation of 3T3-L1 adipocytes with LPS and long-chain n-3 PUFA decreased LPS-induced secreted MCP-1 protein. n-6 PUFA arachidonic acid and LPS synergistically increased MCP-1 and IL-6 secreted proteins. Plant-derived PUFA were relatively neutral stimuli. mRNA expression results suggest potential roles for G protein-coupled receptor 120 and toll-like receptor 2 in mediating the effects of long-chain n-3 PUFA and arachidonic acid, respectively. Overall, this thesis suggests that both n-3 and n-6 PUFA are important factors to consider in the development of nutritional strategies for improving adipose tissue inflammation associated with obesity. / NSERC CGS, Ontario Graduate Scholarship

Breast cancer molecular subtypes, based on expression of estrogen, progesterone and human epidermal growth factor 2 receptors, alter prognosis and treatment options. &alpha;-linolenic acid (ALA) is a complementary therapy, however its effectiveness across breast cancer types and estrogen environments is unclear. This research determined the effect of ALA on growth, apoptosis, fatty acid profile, and gene changes in four breast cancer cell lines with varying receptor expression with or without (&plusmn;) estradiol (E2). ALA (50-200uM) &plusmn; E2 reduced growth in all cell lines. 75&mu;M ALA +E2 increased phospholipid % ALA in all cell lines and induced apoptosis in cell lines lacking the three receptors. Cellular % ALA was positively associated with apoptosis and inversely associated with cell growth. ALA altered expression of cell cycle, apoptosis and signal transduction genes. In conclusion, ALA incorporates into breast cancer cells, reduces growth and induces apoptosis regardless of receptor status or E2 level.