David Kitts

Phenolic compounds derived from the diet are typically sourced from cereals, fruits and vegetables and also beverages. They are documented to have biological functions that include antioxidant and anti-inflammatory activities, and can mitigate gastrointestinal disorders. This thesis focused on looking at mechanism(s) of phenolic acid induced intracellular hydrogen peroxide (H₂O₂) level and effects on intestinal epithelial cell signaling that are related to intestinal epithelial functionality. The first experiment showed that different phenolic acids varied specifically in relative stability and capacity to produce H₂O₂ when incubated in cell culture media for 24 hr. Phenolic acids varied in affinity to induce apoptosis and inhibit proliferation in non-differentiated Caco-2 cells.Subsequent studies demonstrated the effect of selective phenolic acids on extracellular/intracellular redox by controlling H₂O₂ influx activity and to relate this event to cell signaling and control of oxidative stress in differentiated Caco-2 cells. HyPer-3, a genetically encoded fluorescent H₂O₂ sensitive indicator was constructed to monitor intracellular H₂O₂ in differentiated Caco-2 cells. Results showed that extracellular generation of H₂O₂ occurred from autoxidation of phenolic acids in culture and subsequent disappearance corresponded to intracellular uptake in differentiated Caco-2 cells. Phenolic acid specific pro-oxidant activity was sufficient to activate intracellular nuclear factor-erythroid factor 2-related factor 2 (Nrf2) cell signaling; the degree of which was dependent on both the source of the phenolic acid and concentration used in culture. A Caco-2/RAW 264.7 co-culture model enabled intercellular cell signaling measurements in response to an inflammatory response by inflamed macrophage. The capacity of specific phenolic acids to regulate the inflammatory status of activated RAW 264.7 cells was dependent on the type of phenolic acid which influenced Nrf2 cell signaling, monolayer integrity, and cytokine production in Caco-2 cells. It is concluded from these experiments that phenolic acids regulate extracellular/intracellular redox due to a susceptibility for pro-oxidant activity that is specific for individual phenolic acids. The pro-oxidant activity was related to capacity to produce H₂O₂, or its cellular influx, which in turn had a role in cell signaling, cytokine production and intestinal epithelial cell functionality.

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Chlorogenic acid (CGA) is a general term used to describe the most abundant group of phenolic acids in coffee. 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA are major CGAs in coffee; but only 5-CQA has been thoroughly studied. The first objective of this thesis was to study interactions between major CGA isomers and chemical changes in coffee brew that affect antioxidant activity noted for coffee. The second overall objective was to study the potential of these six major CGA isomers in modulating oxidative stress and inflammation using a human intestinal Caco-2 cell line. The findings from Chapter 2 suggested that the other five CGA isomers (4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) together account for more than 50% of the total CGA in coffee and contributed to the antioxidant activity of coffee brew. Chapter 3 and 4 addressed the research question of whether these major CGA isomers have a modulating effect on oxidative stress and inflammation in human intestinal Caco-2 cell line. Caco-2 cells were first incubated with, or without, individual CGA isomers, followed by a phorbol 12-myristate 13-acetate plus human interferon gamma challenge. Biomarkers of oxidative stress (intracellular ROS and GSH/GSSG) and inflammation (IL-8) were measured. The results demonstrated that CGA isomers scavenged intracellular ROS in inflamed Caco-2 cells, mitigated the drop in GSH/GSSG ratio and attenuated IL-8 secretion. Dicaffeoylquinic acids (3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) had a relatively stronger capacity to evoke protection compared to caffeoylquinic acids (3-CQA, 4-CQA, and 5-CQA). To elucidate the possible mechanism underlying these actions, the effects of CGA isomers on the nuclear factor kappa B signaling pathway, mitogen-activated protein kinase cascades, and nuclear factor (erythroid-derived 2)-like 2 signaling pathway were further investigated. In conclusion, structural differences in six CGA isomers were found to correspond to differences in antioxidant and anti-inflammation activities. CGA isomers attenuate oxidative stress and inflammation in Caco-2 cells by triggering changes in redox biology parameters, lead to an up-regulation of nuclear factor kappa B signaling at very early stage, mitigation of p38 phosphorylation and up-regulation of antioxidant genes in an intermediate stage, and activation of Nrf2 signaling at a much later stage.

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The presence of Listeria spp. and L. monocytogenes (Lm) was investigated in provincially inspected food processing and retail facilities in British Columbia. Lm (n=56) was recovered in food processing environment (FPE) of dairy, meat and fish facilities, and in ready-to-eat fish products. The majority of Lm belonged to listeriosis causing serotypes 1/2a and 4b. Isolate fingerprinting revealed 14 sequence types, and 38 pulsotypes, with 66% of Lm possessing the full-length inlA, a causally linked virulence determinant. Unexpectedly, 4b serotypes more readily acquired point mutations leading to rifampicin resistance compared to other serotypes (p0.05). Lm that adapted more quickly to cold (4°C) also more often encoded a full-length inlA. No resistance to antibiotics used in listeriosis treatment was observed; however, a large proportion of isolates possessed resistance or reduced susceptibility (RSC) to ciprofloxacin (CIP, 75%) and clindamycin (CLI, 98%). When eight isolates were experimentally adapted to high concentrations of CIP, minimum inhibitory concentrations (MICs) of benzalkonium chloride (BAC) increased (n=5), gentamicin MICs remained the same (n=6) or increased 2-fold (n=2), and led to RSC to linezolid (n=1) and resistance to CLI (n=8). Tolerance to quaternary ammonium (QAC) sanitizers, BAC and E-San, was also investigated in a clinical Lm strain (08-5578) that possessed a previously uncharacterized island, LGI1. High tolerance to acid, cold, high salt conditions, and QACs was seen. Deletion of LGI1 genes lmo1851, emrE, and sel1, with putative regulatory, efflux, and adhesion functions, respectively, did not affect the acid, cold and salt tolerance, or the adhesion and invasion of TC-7 and HeLa cells. The ΔemrE mutant had impaired growth at sub-lethal concentrations of QACs, and up to three times lower MICs. No change in MICs to aminoglycosides and other antibiotics, acriflavine, and triclosan, was observed for the ΔemrE mutant, suggesting the primary role of EmrE in Lm is to increase its tolerance of QACs. Overall, findings from this research provide evidence that differences in stress survival and virulence potential exist among food chain-derived Lm. However, better understanding of the pressures occurring in FPEs that may contribute to strain persistence, and co-selection of antibiotic and sanitizer resistance mechanisms is needed.

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Vitamin (vit) E comprises 8 isoforms, of which only α-tocopherol (Toc) has been thoroughly investigated. Other vit E isoforms, particularly γ-Toc and δ-Toc, however are present in significant amounts in the North American diet. The effect of α-Toc, γ-Toc and δ-Toc in modulating oxidative status and inflammatory responses in adult-derived Caco-2 and fetal-derived FHs 74 Int intestinal cell lines were thus determined. Toc isoforms were effective antioxidants that protected against peroxyl radical-induced membrane oxidation in both cell lines in an isoform-dependent manner (δ-Toc>γ-To>α-Toc). Nevertheless, Toc isoforms exhibited differential modulation of inflammatory response in the two cell lines, in that Toc isoforms suppressed IFNγ/PMA-induced IL8 expression in Caco-2 cells, but promoted an inflammatory response in FHs 74 Int cells. Modulation of IL8 expression by Toc isoforms corresponded with an efficacy of Toc to modulate NfκB pro-inflammatory and Nrf-2 antioxidant enzyme signaling pathways. Non-α-Toc isoforms promoted Nrf-2 activation in both cell lines. Alpha-Toc and γ-Toc mitigated IFNγ/PMA-induced NfκB activation in Caco-2 cells while non-α-Toc isoforms promoted NfκB activation in FHs 74 Int cells. The pro-oxidant activity of δ-Toc corresponded to its lower ability to suppress IFNγ/PMA-induced IL8 expression and NfκB activation, but enhanced the Nrf-2 signal in Caco-2 cells. One key difference between the effect of Toc isoforms on modulation of NfκB and Nrf-2 signaling was that non-α-Toc isoforms down-regulated the gene expression of glutamate cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, in FHs 74 Int, but not in Caco-2 cells. This was supported by a reduced (P0.05) glutathione content in FHs 74 Int cells after incubation with γ-Toc and δ-Toc that was not observed in Caco-2 cells. Downregulation of the glutathione content corresponded to the finding that non-α-Toc isoforms can induce apoptosis-mediated cytotoxicity in FHs 74 Int, but not in Caco-2 cells. Taken together, Toc isoform-mediated modulation of the inflammatory response was not related to antioxidant activity, but rather was attributed to a cell-specific efficacy to modulate NfκB signaling that corresponded to depletion in glutathione content and apoptosis. Based on the current findings, non-α-Toc isoforms are biologically active forms of vit E and their effects on intestinal cells should not be overlooked.

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Maillard reaction products (MRPs) are produced when reducing sugars react with amino acids, peptides or proteins in heat-processed foods. The overall objective of this research was to isolate and identify MRPs that exhibit antioxidant and anti-inflammatory activities, from different sugar-amino acid model systems comprised of fructose, glucose or ribose, each with glycine (Fru-Gly, Glu-Gly and Rib-Gly) or lysine (Fru-Lys, Glu-Lys and Rib-Lys), respectively. The development of peroxyl radical scavenging activity was found to be positively correlated (r = 0.893-0.905, P 0.001) with MRPs derived from the intermediate-to-late stages of the reaction and influenced mostly by the type of sugar. The cytotoxicity, antioxidant and anti-inflammatory activities of MRPs were not attributed to the α-dicarbonyl compounds present in the heated mixtures. An in vitro intestinal inflammation model was established using Caco-2 cells stimulated with 8,000U/mL interferon γ (IFN-γ) and 0.1 μg/mL of phorbol myristate acetate (PMA), which induced nitric oxide (NO) through up-regulation of inducible nitric oxide synthase (iNOS) expression and increased interleukin 8 (IL-8) synthesis. The NO and IL-8 inhibitory capacity of MRPs were positively correlated (r = 0.886-0.943, P 0.05) with intracellular oxidation inhibitory activity. MRPs derived from Glu-Lys heated for 60 min showed the highest inhibitory activity for NO, IL-8, and iNOS among these six model systems and the low molecular weight ultrafiltration fraction recovered from Glu-Lys (GL60FIV, molecular weight (MW) 1kDa) was attributed to these activities. A fraction recovered from GL60FIV, termed F3, possessed the high NO, iNOS and IL-8 inhibitory activity and was further identified to contain three major sub-fractions (F3-A, F3-B and F3-C). The NO inhibitory capacity of F3-A (IC₅₀, 0.076 mmol/L) was higher (P 0.05) than that of an iNOS inhibitor, aminoguanidine (IC₅₀, 0.16mmol/L). F3-A had a MW of 191Da and λmax= 370nm. F3-B and F3-C were identified as 5-hydroxymethyl-2-furfural and 5-hydroxymethyl-2-furoic acid, respectively. F3 down-regulated the expressions of genes that were involved in both the NF-κB signaling pathway and peroxidase activities. In conclusion, MRPs isolated from sugar-amino acid model systems can exhibit antioxidative and anti-inflammatory activities that may be useful to reduce intestinal inflammation.

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