Nicholas Vaughan

Comet assay in human biomonitoring studies: Reliability, validation, and applications

The comet assay (single‐cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies. For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion‐specific endonuclease. Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra‐ and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers. We applied the assay in investigations of human disease and occupational exposure of factory workers. Environ. Mol. Mutagen. 30:139–146, 1997.

Effect of increased consumption of whole-grain foods on blood pressure and other cardiovascular risk markers in healthy middle-aged persons: a randomized controlled trial

BACKGROUND Three daily portions of whole-grain foods could lower cardiovascular disease risk, but a comprehensive intervention trial was needed to confirm this recommendation. OBJECTIVES We aimed to assess the effects of consumption of 3 daily portions of whole-grain foods (provided as only wheat or a mixture of wheat and oats) on markers of cardiovascular disease risk in relatively high-risk individuals. DESIGN This was a randomized controlled dietary trial in middle-aged healthy individuals. After a 4-wk run-in period with a refined diet, we randomly allocated volunteers to a control (refined diet), wheat, or wheat + oats group for 12 wk. The primary outcome was a reduction of cardiovascular disease risk factors by dietary intervention with whole grains, which included lipid and inflammatory marker concentrations, insulin sensitivity, and blood pressure. RESULTS We recruited a total of 233 volunteers; 24 volunteers withdrew, and 3 volunteers were excluded. Systolic blood pressure and pulse pressure were significantly reduced by 6 and 3 mm Hg, respectively, in the whole-grain foods groups compared with the control group. Systemic markers of cardiovascular disease risk remained unchanged apart from cholesterol concentrations, which decreased slightly but significantly in the refined group. CONCLUSIONS Daily consumption of 3 portions of whole-grain foods can significantly reduce cardiovascular disease risk in middle-aged people mainly through blood pressure-lowering mechanisms. The observed decrease in systolic blood pressure could decrease the incidence of coronary artery disease and stroke by ≥15% and 25%, respectively. This trial was registered at clinicaltrials.gov as ISRCTN27657880.

Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers.

We have previously reported that supplementation with folic acid (1.2 mg day−1 for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P<0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P<0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P<0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people.

Effect of a tomato-rich diet on markers of cardiovascular disease risk in moderately overweight, disease-free, middle-aged adults: a randomized controlled trial

BACKGROUND Cardiovascular disease (CVD) is a major cause of mortality in the United Kingdom. Epidemiologic studies suggest that consumption of tomato-based foods may lower CVD risk. Such potential benefits have been ascribed in part to high concentrations of lycopene in the tomatoes. However, these findings have not yet been validated by comprehensive intervention trials. OBJECTIVE The aim of this study was to conduct a single-blind, randomized controlled intervention trial with healthy middle-aged volunteers to assess whether the consumption of tomato-based foods affects recognized biomarkers of CVD risk. DESIGN After a 4-wk run-in period with a low-tomato diet, 225 volunteers (94 men and 131 women) aged 40-65 y were randomly assigned into 1 of 3 dietary intervention groups and asked to consume a control diet (low in tomato-based foods), a high-tomato-based diet, or a control diet supplemented with lycopene capsules (10 mg/d) for 12 wk. Blood samples were collected at baseline, at 6 wk, and after the intervention and were analyzed for carotenoid and lipid profiles and inflammatory markers. Blood pressure, weight, and arterial stiffness were also measured. Dietary intake was also determined during the intervention. RESULTS None of the systemic markers (inflammatory markers, markers of insulin resistance and sensitivity) changed significantly after the dietary intervention. Moreover, lipid concentrations and arterial stiffness were also unaffected by the interventions. CONCLUSION These data indicate that a relatively high daily consumption of tomato-based products (equivalent to 32-50 mg lycopene/d) or lycopene supplements (10 mg/d) is ineffective at reducing conventional CVD risk markers in moderately overweight, healthy, middle-aged individuals. This trial was registered at isrctn.org as ISRCTN34203810.

Measurement of 8-Oxo-deoxyguanosine in Lymphocytes, Cultured Cells, and Tissue Samples by HPLC with Electrochemical Detection

8-Oxoguanine is one of the most studied base oxidation products found in DNA. It has potential biological significance, because if present in DNA that is replicating, it can lead to incorporation of adenine rather than cytosine in the daughter strand. Thus it is considered as a premutagenic lesion. It occurs as a result of attack by reactive oxygen species released during the inflammatory response, and in small but significant amounts during normal respiration. The hydroxyl (OH) radical (arising from H(2)O(2) by the transition metal ion-catalyzed Fenton reaction within the nucleus) is most likely responsible for the formation of 8-oxoguanine. Analytical methods-gas chromatography with mass spectrometric detection (GC-MS) and high-performance liquid chromatography (HPLC) -were developed for quantitation of oxidized bases produced in experimental studies of radiation and chemical damage to DNA, and these methods were naturally also applied to the measurement of background levels of oxidized bases in cellular DNA (1). With GC-MS, very high levels of 8-oxoguanine have been reported, typically between 10 and 100 for every 10(5) normal guanines. It has recently been recognized that spurious oxidation of DNA readily occurs during isolation and hydrolysis of DNA, and derivatization of the bases for analysis. HPLC, normally applied to measurement of the nucleoside, 8-oxo-deoxyguanosine (8-oxo-dG), has generally given values below those obtained with GC-MS; but with HPLC, too, oxidation artefacts have been identified.