- Title
- In vitro and in vivo approaches to the examination of folate-related nutritional genetics in health and disease
- Creator
- Choi, Jeong-Hwa
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - PhD (Food Science)
- Description
- Folic acid has been the focus of extensive research since it plays an essential role in the human body as a major coenzyme in one-carbon metabolism. This metabolic role facilitates both methionine and DNA synthesis. It has been suggested that increased folate intake, and hence blood status, provides benefits in maintaining health. However, a growing body of literature now reports differing opinions with respect to the level of folic acid intake, the type of vitamer and pathoaetiological outcomes. Part of this controversy lies in the study of cancer aetiology, particularly in the context of adenomatous polyp (AP) occurrence. AP is an antecedent of colorectal cancer. As part of the present study an in vivo model was therefore developed in order to re-examine the role of folate in carcinogenesis, taking into account both genetic variants and environmental factors. To obtain further evidence, the biological characteristics of folate vitamers in promoting cancer cell proliferation were examined using an in vitro model. 202 individuals were recruited and placed into two groups depending upon whether they had been diagnosed with AP or were control subjects. The blood folate, thiol levels, dietary folate intake, including synthetic and natural forms, and intake of nutrients related to one-carbon metabolism (pyridoxine, riboflavin, cobalamin, niacin, methionine and vitamin C) of each subject were evaluated. Sixteen major genetic variants in folate metabolism were also determined as potential risk factors in AP aetiology: MTHFR C677T, A1208C, G1793A, SHMT C1420T, TS 1496del6, TSER 2R3R, TS 3RG>C, GCPII C1561T, CBS 844ins66, DHFR 19bp del , RCF G80A, CTH G1364T, IVS 10-430 C>T, BHMT G595A, MS A2756G and MSR A66G. Statistical analysis has provided evidence to suggest that AP aetiology depends upon a range of interactions between genes and nutrition. The following factors were found to be associated with an increased risk of developing AP: RBC folate level (p for females = 0.0021 and males = 0.004), nutritional intake (p for methylfolate intake below the median = 0.0189 and cobalamin intake above median = 0.0402) and genetic variants (p for CBS 844ins68 = 0.035, TS 1496del6 = 0.048, MSR A66G = 0.025 and MTHFR C677T- CBS 844ins66 = 0.0403). Although no direct correlation was found between PteGlu intake and the occurrence of AP, genetic variants predicted a differential AP risk depending on total dietary folate intake level (in subjects with total dietary folate intake above the RDI: p for MTHFR A1298C= 0.026, MTHFR C677T-A1298C-G1793A=0.016, TS 1496del6 = 0.024, DHFR 19bp del = 0.032, and, in subjects with total dietary folate intake below the RDI: MTHFR A1298C2= 0.011, RFC G80A-GCPII C1561T3= 0.033). Additionally, vitamin C was also associated with occurrence of AP when examined by RCF G80A and GCPII C1561T genotype (p for RFC G80A AA = 0.0473, GCP II C1561T CT= 0.0276 and RFC G80A-GCPII C1561T AA/CC = 0.0026). An in vitro model utilizing both colon (Caco-2) and breast cancer (MCF 7) cell lines was developed. The cells were cultured for 8 days. There was found to be a differential growth response depending upon which folyl vitamer was used as a source of one-carbon units, its concentration and the presence/absence of a folate protecting antioxidant. At a relatively low concentration (≤ 1μg/mL) of PteGlu no significant difference in Caco-2 cell growth was observed, but the cells showed a differential growth pattern at higher concentrations (50μg/mL and 500μg/mL). Fully reduced, labile 5-CH3H3PteGlu with no added antioxidant inhibited Caco-2 cell growth in a dose-dependent manner, but this inhibition was altered by the presence of an antioxidant. The addition of 10μg/mL of GSH resulted in a similar, but enhanced, inhibition of Caco-2 cell growth to that of 5-CH3H4PteGlu in the absence of any additional antioxidant. By contrast, 5-CH3H4PteGlu in the presence of ascorbic acid (10μg/mL) exhibited a differential change depending on time of incubation and vitamin concentration. Partially oxidised 5-CH3H2PteGlu increased cell growth in both cell lines, providing evidence that 5-CH3H2PteGlu could potentially enter the one-carbon metabolic pool in this in vitro model. The photo-oxidative degradation products of 5-CH3H2PteGlu did not show a significant effect on growth in Caco-2 cells. Findings from the current study provide new information on the physico-chemical characteristics of folate vitamers and their role in cell proliferation. The interactions between these vitamers and nutrients related to methyl group metabolism and genetic variants collectively illuminate the multifactorial nature of AP aetiology. In terms of gene-gene, gene-nutrient and nutrient-nutrient interactions, this study has generated valuable new information about the molecular mechanisms that play a role in AP and their response to the nutritional environment.
- Subject
- cancer; folic acid; in vitro; in vivo
- Identifier
- http://hdl.handle.net/1959.13/1041655
- Identifier
- uon:13941
- Rights
- Copyright 2014 Jeong-Hwa Choi
- Language
- eng
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