Effect of increased fruit and vegetable intake on phytophenolic levels in humans and the impact on antioxidant capacity, DNA damage and protein expression

The importance of a fruit and vegetable-rich diet on human health and for the prevention of chronic diseases is widely accepted and is associated with a lower risk of mortality from all causes (Schatzer, et al., 2010; Wang, et al., 2014). Phytophenolics in fruit and vegetables have been proposed as compounds that contribute to the beneficial effects of a high fruit and vegetable consumption (del Rio, et al., 2013). Diets rich in fruit and vegetables are considered to protect against colon cancer (Chan and Giovannucci, 2010). The research carried out in this PhD project addressed the hypothesis that taxonomically diverse and readily accessible fruit and vegetables containing phytophenols would positively influence biomarkers of antioxidant capacity, endogenous and oxidative DNA damage and protein expression linked to resistance to colon carcinogenesis. Forty four men and women (39-58 y; BMI 18-30 kgm-2 ) with a habitually low fruit and vegetable intake were randomised by age and sex into two groups, control and intervention. The control group (n=23) maintained their habitual diet (up to 160 g of fruit and vegetables/day) and the intervention group (n=21) consumed an additional 500-600 g of fruit and vegetables/day for 12 weeks that delivered between 750 mg and 850 mg phytophenolics daily. The in vivo phytophenolic metabolite profiles and the concentration range of these metabolites within plasma and in the human colon were measured by targeted LC-MS. Antioxidant plasma nutrient status was measured by HPLC. The antioxidant capacity of plasma, faecal waters and individual phytophenols were measured by FRAP and HORAC assays. Endogenous and oxidative DNA damage in lymphocytes from the volunteers and in normal human colon cells, exposed ex vivo either to faecal waters or individual phytophenols, were measured using the comet assay. The cytotoxicity of human faecal waters towards human colon cells in vitro was measured using Trypan Blue and MTT assays. The effect of intervention with fruit and vegetables on global protein expression in cultured normal human colon cells exposed to faecal waters from the volunteers, and the associations between those compounds significantly increased in response to intervention, was assessed using proteomic analysis. Intervention with a high fruit and vegetable diet significantly increased eight phytophenolic colonic metabolites (chlorogenic acid, ellagic acid, epicatechin, homovanillic acid, isorhamnetin, naringenin, phloretin and veratraldehyde) in human faecal waters at concentrations ranging from 0.002-3.364 mM. No similar increases were observed in plasma from the volunteers. Plasma vitamin C, a-carotene, ß-carotene and lutein/zeaxanthin were significantly increased (20-60% depending on the nutrient) during the intervention, whereas retinol, a-tocopherol, ?-tocopherol remained unchanged. No significant functional change was observed on antioxidant enzyme activity (SOD, CAT and GPx), antioxidant capacity or oxidative DNA damage in isolated lymphocytes. Moreover, there was no significant functional change in faecal water antioxidant capacity or in cytotoxicity and genotoxicity in normal human colon cells in vitro exposed to faecal waters from the volunteers. Nine proteins (14-3-3 protein epsilon, elongation factor 1-ß, elongation factor 1-?, glycine-tRNA ligase, hypoxia up-regulated protein 1, myosin regulatory light chain 12A pyruvate kinase, prefoldin subunit 2, and WD repeat-containing protein 1) were significantly altered in human colonocytes following exposure to faecal waters from volunteers consuming a sustained high fruit and vegetable diet. Furthermore, significant associations were observed between those phytophenols that were found to have increased on the intervention in faecal waters and several proteins (such as isovaleryl-CoA dehydrogenase, nucleolin and a-centractin), that are associated with the carcinogenic processes by affecting mechanisms related to tissue stability and integrity, cell signalling and redox regulation. Individual phenolics, identified in faecal waters from the human study showed significant antioxidant potential and were able to modify DNA damage in human colonocytes in vitro, when tested at their in vivo concentrations. In summary, increased fruit and vegetable intake did not have a major effect on most plasma or faecal water biomarkers measured. This study revealed, for the first time, that high fruit and vegetable consumption modulated global protein expression in normal human colon cells exposed to faecal waters in culture. The project has shown that individual phytophenols at physiological concentrations have antioxidant capacity and the ability to modulate endogenous and oxidative DNA damage.