Optimized noninvasive procedures to measure DNA damage in comet assay.

The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human biomonitoring studies, but lymphocytes collected from the mouth offer a potentially attractive, noninvasive alternative. The aim of the current study was to develop a buccal cell lymphocyte comet assay procedure. Cells were collected from mouthwash of three healthy volunteers and tested individually. The comet assay was performed under different pH and times of alkaline treatment, electrophoresis run times and hydrogen peroxide concentrations. Optimal conditions for buccal lymphocytes in comet assay were found to be pH >13 for unwinding and electrophoresis buffers, 10-min alkaline unwinding treatment and 20-min electrophoresis run time. We successfully utilized our optimized assay conditions to demonstrate the genoprotective activity of quercetin. This newly established procedure offers an alternative noninvasive sampling method for the investigation of DNA protection and/or damaging effect.

Photoprotective potential of Cordyceps polysaccharides against ultraviolet B radiation-induced DNA damage to human skin cells.

BACKGROUND: Ultraviolet (UV) radiation causes DNA damage resulting in photoageing and skin cancer. UVB (290-320 nm) interacts directly with DNA, inducing two major photoproducts: cyclobutane-pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts. Cordyceps sinensis (Berk.) Sacc. is a medicinal fungus with reported anticancer and cytoprotective effects. OBJECTIVES: To investigate genoprotective effects of polysaccharide-rich Cordyceps mycelial components against UVB-induced damage in normal human fibroblast cells. METHODS: Cultured human fibroblasts (BJ cells) were treated for 30 min and, separately, for 24 h with hot water extract of Cordyceps fungal mycelia or exopolysaccharides. Cells were washed, irradiated with UVB (302 nm), and immediately lysed, after which DNA damage, as strand breaks, was measured using an enzyme-assisted comet assay that detects CPDs. RESULTS: DNA damage in UVB-irradiated cells was significantly lowered (P < 0·01) with Cordyceps pretreatment. Similar results were seen with 30 min and 24 h pretreatment. Specifically, and in comparison with irradiated cells with no Cordyceps pretreatment, there was a 27% reduction in CPDs in irradiated cells with 24 h pretreatment with 200 µg mL(-1) of the hot water Cordyceps extract, and a 34% reduction with 24 h pretreatment with 200 µg mL(-1) of the exopolysaccharide extract. CONCLUSIONS: Clear evidence of protection against UVB-induced CPDs was seen with Cordyceps mycelial extracts. Results indicate that Cordyceps may offer photoprotection and lower the risk of basal cell carcinoma, the main skin cancer caused by CPDs. Further study is needed to identify protective mechanisms.

Synthetic colourings of some snack foods consumed by primary school children aged 8-9 years in Hong Kong.

An HPLC method with photodiode array detection was used for the quantification of 11 synthetic dyes in 87 snack food products commonly consumed by children in Hong Kong, China. Dietary exposure to synthetic colours was estimated using food-frequency questionnaire data obtained from 142 primary school children aged 8-9 years in three districts of Hong Kong. Dietary exposure to synthetic colours for an average primary school student was considerably lower than the threshold for acceptable daily intake (ADI) for their ages, except for sunset yellow FCF. Data obtained showed that the average daily intake of sunset yellow FCF (E110) was 51% over the ADI threshold in 9-year-old boys. The higher intakes of sunset yellow FCF were mainly due to the high consumption of soft drinks and desserts such as jellies, which have high concentrations of this synthetic colour additive.

Dietary-related profile of older persons in the Chinese community: an exploratory study.

BACKGROUND: Nutrition plays an important role throughout the life span. It is the interaction of nutrition and health that form part of the aging process. Nutrition affects the maintenance of physiological and biological process of aging, also, the risk of development of acute and chronic diseases. OBJECTIVE: To examine dietary related behaviors and lifestyle factors among non-institutionalized older persons in a local Chinese community. DESIGN, SETTING AND PARTICIPANTS: It was a cross-sectional qualitative descriptive design. A convenience sample of 36 older persons (mean +/- SD age, 75 +/- 7.8 years) in a community center were approached and invited to complete a questionnaire regarding their dietary-related profile and the self-perceived nutritional and health status. RESULTS: Results showed that 40% (n=14) of the older persons lived alone and ate alone on a regular basis, taking few fruit and vegetables per day, inadequate fluid and no dairy or bean curd products, and 48% (n=17) were overweight or obese. The self-perceived nutritional status correlated directly with perceived health status, which was high. CLINICAL RELEVANCE: The clinical relevance of this study is highlighted by the far from optimal dietary behaviors among this group of older persons. Nurses and health care providers working in the community should provide education on healthy diet and nutrition-related health problems, especially to older persons, for health maintenance and disease prevention.

Inter-relationships between DNA damage, ascorbic acid and glycaemic control in Type 2 diabetes mellitus.

AIMS: The onset of complications in Type 2 diabetes mellitus (DM) patients cannot be predicted in individuals. Evidence suggests a link between complications and hyperglycaemia, oxidative stress and antioxidants, but causality is unclear. This study investigated baseline (entry) fasting plasma ascorbic acid, lymphocytic DNA damage and glycaemic control in Type 2 DM as part of a long-term study, the aim of which is to explore a biomarker profiling approach to identify and improve outcome in high-risk subjects. METHODS: A cross-sectional study, in which DNA damage, glycated haemoglobin (HbA(1c)), fasting plasma glucose (FPG) and ascorbic acid (AA) were measured on fasting blood samples collected from 427 Type 2 DM subjects. RESULTS: DNA damage was significantly (P < 0.0001) and directly correlated to both FPG (r = 0.540) and HbA(1c) (r = 0.282), and was significantly (P < 0.0001), independently and inversely correlated to plasma AA (r = -0.449). In those subjects with both poor glycaemic control and low AA (< 48 microm, the overall mean value for the study group), DNA damage was significantly (P < 0.005) higher compared with those subjects with a similar degree of hyperglycaemia but with AA above the mean. CONCLUSIONS: The novel finding of a significant inverse relationship between plasma AA and DNA damage in Type 2 DM indicates that poorly controlled diabetic subjects might benefit from increased dietary vitamin C. The data also have important implications for biomarker profiling to identify those subjects who might benefit most from intensive therapy. Longer-term follow-up is underway.

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Vitamins C and E: acute interactive effects on biomarkers of antioxidant defence and oxidative stress.

Oxidative stress is implicated in the aetiology of many diseases; however, most supplementation trials with antioxidant micronutrients have not shown expected beneficial effects. This randomized, double-blinded, placebo-controlled study evaluated acute effects (at 90, 180min and 24h [fasting] post-ingestion) of single doses of Vitamins C (500mg) and E (400IU), alone and in combination, on biomarkers of plasma antioxidant status, lipid peroxidation and lymphocyte DNA damage in 12 healthy, consenting volunteers. Plasma ascorbic acid increased significantly (P < 0.01) within 2h of ingestion of Vitamin C, and alpha-tocopherol was significantly (P < 0.01) higher at 24h post-ingestion Vitamin E. The pattern of response was not significantly different whether Vitamin C (or Vitamin E) was taken alone or in combination, indicating no augmentation of response to one by co-ingestion of the other vitamin. No significant changes were seen in plasma FRAP in the group overall (although increases (P < 0.05) were seen at 90 and 180min post-ingestion in women after Vitamin C ingestion) or in MDA across treatments, and no evidence of increased DNA damage, or of DNA protection, was seen at any time point after Vitamin C and/or E ingestion. In conclusion, the data from this first controlled study of acute effects of single doses of Vitamin C and/or E show no evidence of either a protective or deleterious effect on DNA damage, resistance of DNA to oxidant challenge, or lipid peroxidation. No evidence of a synergistic or cooperative interaction between Vitamins C and E was seen, but further study is needed to determine possible interactive effects in a staggered supplementation cycle, and study of subjects under increased oxidative stress or with marginal antioxidant status would be useful. It would be of interest also to study the effects of these vitamins ingested with, or in, whole food, to determine if they are directly protective at doses above the minimum required to prevent deficiency, if combinations with other food components are needed for effective protection, or if Vitamins C and E are largely surrogate biomarkers of a 'healthy' diet, but are not the key protective agents.

Hydrogen peroxide in urine as a potential biomarker of whole body oxidative stress.

The level of hydrogen peroxide (H2O2) in urine has been suggested as a potential biomarker of whole body oxidative stress, but issues of stability, reproducibility and biological variation have not been investigated to date. In this study, we used a refined protocol, which demonstrated improved sensitivity and precision, to determine the stability of H2O2 in urine, and to measure its concentration in apparently healthy subjects. We also investigated intra-individual variation within and between days. Results showed that H2O2 in urine is stable for up to 48 h at 4 degrees C, however, storage of urine at room temperature was associated with up to 50% increase in H2O2 concentration over a few hours. Total H2O2 in freshly voided urine from 55 healthy, fasting subjects ranged from 0.84 to 5.71 microM, or 90-1164 micromol H2O2/mol creatinine. Intra-individual variation was wide. Even when concentration corrected and collected at the same time of day, 2- to 3-fold variation was seen over 4 consecutive days, and over the course of a single day the creatinine-corrected H2O2 also varied significantly. We suggest that this large biological variation limits the usefulness of urine H2O2 as a biomarker of oxidative stress, the exception being when the effects of disease, therapy or diet induce very large changes in its concentration.

No effect of a high-fat diet on promotion of sex hormone-induced prostate and mammary carcinogenesis in the Noble rat model.

Results of international correlation and migrant studies suggest that dietary fat promotes carcinogenesis in hormone-sensitive sites, but this is disputed. In the present study, we used a Noble rat model of sex hormone-induced cancers to examine the effect of a high-fat diet on the incidence and latency of prostate and mammary cancer in male (n 139) and female (n 72) animals respectively. We also measured alpha-tocopherol levels in female breast tissue to determine whether a high intake of polyunsaturated fatty acids depletes antioxidant defence in target tissues, providing a possible potentiating mechanism for carcinogenesis. Results showed a very high incidence of hormone-induced adenocarcinomas of prostate and mammary gland, irrespective of diet. There was no difference in the pattern of carcinogenesis in different prostatic locations, weight of the prostate, or weight gain between male rats on the high-fat diet compared with the control (standard, low-fat) diet. In female rats, the incidence of mammary cancer and the body-weight gain were the same in both dietary groups, and breast alpha-tocopherol was also unaffected by dietary fat intake. Our present results are supportive of recent cohort studies that reported no significant association between intake of fat and the development of human prostate and breast cancer, and do not support a role for dietary fat in promoting sex hormone-induced prostate and mammary carcinogenesis.

Glycohaemoglobin measurement: methodological differences in relation to interference by urea.

The aim of this study was to investigate the agreement between a cation-exchange HPLC method and a boronate affinity method of measuring glycohaemoglobin (HbA1c), with particular reference to the effect of elevated urea concentration. HbA1c was measured by both methods in samples from 75 subjects who were classified as diabetic with normal (n=36) or abnormal (n=12) renal function, and non-diabetic with normal (n=8) or abnormal (n=19) renal function. Urea was found to cause a clinically significant interference in the HPLC method at a level > or =17.0 mmol/l. Each increase of 1 mmol/l urea in serum was associated with an absolute increase of 0.04% in the HbA1c value as measured by the HPLC method. The boronate affinity method for HbA1c did not appear to be affected by elevated urea concentration. There was significant correlation (r=0.97, p<0.001) between HbA1c results obtained by the two methods, however, results obtained by the boronate affinity method were generally lower. The discrepancy between results obtained by the two methods was particularly marked in uraemic samples from diabetic subjects, as the HPLC/boronate affinity difference increased as the HbA1c increased and also as the urea concentration increased. Results indicate that blood from diabetic patients with renal failure may give erroneously high HbA1c values by HPLC. Results also highlight the importance of choosing appropriate clinical samples and statistical techniques when evaluating or comparing test methods.

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure.

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.