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M Fenech

Publications and source records attributed to M Fenech.

At least 37 records · Page 2Linked to original sources
PubMed2001

The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro.

We performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid deficiency on primary human lymphocytes. Lymphocytes were cultured in medium containing 12-120 nM folic acid for 9 days in a novel cytokinesis-block micronucleus (CBMN) assay system (n = 20). Besides identifying optimal folic acid concentrations for in vitro genomic stability, we tested the hypothesis that lymphocytes from individuals homozygous for the C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism (TTs, n = 10) are protected against chromosome damage relative to controls (CCs, n = 10) under conditions of folic acid deficiency. This hypothesis is based on the assumption that reduced MTHFR activity in TT lymphocytes causes a diversion of 5,10-methylene tetrahydrofolate toward thymidine synthesis, which minimizes uracil-induced double-stranded DNA breakage. Cells were scored for micronuclei, apoptosis, necrosis, nucleoplasmic bridges, and nuclear budding. The latter two endpoints are indicative of chromosome rearrangements and gene amplification, respectively, and to the best of our knowledge, this is the first report of their association with folic acid concentration. Folic acid concentration correlated significantly (P < 0.0001) and negatively (r, -0.63 to -0.74) with all markers of chromosome damage, which were minimized at 60-120 nM folic acid, much greater than concentrations assumed "normal," but not necessarily optimal in plasma. Two-way ANOVA revealed no effect of the MTHFR genotype on any of the endpoints. Results show that the C677T polymorphism does not affect the ability of a cell to resist chromosome damage induced by folic acid deficiency in this in vitro system.

Analysis of Variance↗
PubMed2000

Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis produced by a number of cyanobacterial species, the most common being Cylindrospermopsis raciborskii. CYN contains a uracil moiety attached to a sulphated guanidino moiety, suggesting that it may have carcinogenic activity. This report describes the use of the WIL2-NS lymphoblastoid cell-line in the well-validated cytokinesis-block micronucleus (CBMN) assay to test this hypothesis. Centromeres (CENs) were identified in micronuclei (MNi) of binucleated cells (BNCs) by fluorescent in situ hybridisation of alpha centromeric DNA sequence repeats. The results indicate that CYN induced a significant increase in the frequency of MNi in BNCs exposed to 6 and 10microg/ml, and a significant increase in CEN-positive MNi at all concentrations of CYN tested (1, 3, 6, and 10microg/ml). However, despite this apparently greater sensitivity of WIL2-NS cells to induction of CEN-positive MNi at low CYN concentrations, at the higher concentrations the magnitude of the increase in CEN-positive MNi did not account for the greater increase in MNi in BNCs, indicating that both CEN-positive and CEN-negative MNi were induced. This suggests that CYN acts to induce cytogenetic damage via two mechanisms, one at the level of the DNA to induce strand breaks, the other at the level of kinetochore/spindle function to induce loss of whole chromosomes (aneuploidy). C. raciborskii occurs in a number of human drinking water sources worldwide and so these findings may have important public health implications.

Alkaloids↗
PubMed2000

The in vitro micronucleus technique.

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.

Humans↗
PubMed2000

Report from the In Vitro Micronucleus Assay Working Group.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.

Chromosome Aberrations↗
PubMed2000

Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.

We have developed a method that can detect the DNA-damaging and cytotoxic effects of physiological levels of reactive oxygen species (ROS) and activated human neutrophils. This was achieved using WIL2-NS cells, a human B lymphoblastoid cell line, as target cells and the cytokinesis-block micronucleus (CBMN) assay. With this method, we observed a 4- and a 30-fold increase in the frequency of micronucleated binucleated cells (MNed BNC) when cells were exposed to 10 and 30 microM hydrogen peroxide, for 1 h, respectively. A dose-dependent increase in the frequency of MNed BNC was also detected when cells were exposed to hypoxanthine (HX)/xanthine oxidase (XO), a superoxide generating system: a 50-fold increase in the frequency of MNed BNC was observed at the highest XO dose (12.5 mU/ml). In this CBMN assay, nucleoplasmic bridges (NPB) in BNC and necrotic cells were also readily detected, especially at the higher exposure doses of hydrogen peroxide or HX/XO. When WIL2-NS cells were exposed to neutrophils stimulated with phorbol 12-myristate acetate (PMA) for 1 h, the frequencies of MNed BNC in WIL2-NS cells increased in a dose-dependent manner (30-fold increase at 100 nM PMA) and with an increasing neutrophil:WIL2-NS co-culture ratio. The frequencies of MNed BNC were closely related to the production of ROS, especially hydrogen peroxide, by the neutrophils. Differentiated HL60 cells (DMSO-treated HL60) also produced ROS in response to PMA. In this case, we used a 'Transwell' system to expose WIL2-NS cells to DMSO-treated HL60 cells, because direct contact with DMSO-treated HL60 cells impaired cell division in WIL2-NS target cells. Exposure to PMA-stimulated DMSO-treated HL60 cells resulted in a PMA dose-dependent increase in the frequency of MNed BNC in WIL2-NS cells. MNed BNC frequencies were positively correlated with NPB (r = 0.61-0.93) and necrosis (r = 0.55-0.86) and negatively correlated with nuclear division index (r = -0.72 to -0. 91) in all of the above experiments. These results suggest that the CBMN assay using WIL2-NS cells is a sensitive assay system to examine ROS-induced chromosomal damage and necrosis by activated human neutrophils.

B-Lymphocytes↗
PubMed2000

A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division.

A mathematical model is described that predicts the effect of altered nuclear/cell division kinetics and cytotoxicity on micronucleus expression in vitro when the micronucleus assay is performed without discriminating between cells that have divided once and cells that have not divided after genotoxic insult. The model is based on the probabilities of: (i) a viable cell completing nuclear division; (ii) micronucleus expression in a cell that completes nuclear division after genotoxic insult; (iii) a cell not dividing and surviving as a mononuclear cell; (iv) a cell dying by necrosis or apoptosis. The model predicts: (i) false negative results for relatively weak chromosome damaging agents that also inhibit nuclear division, if micronuclei are scored in mononucleated cells without discriminating between divided and non-divided cells; (ii) this tendency for a false negative result when scoring micronuclei without discriminating between non-divided and once-divided mononuclear cells increases with cell lines and culture conditions that do not result in optimal rates of nuclear division (i.e. >90% of dividing cells); (iii) the absolute increment in micronucleus frequency in binucleated cells is at least 2-fold greater than that observed in mononucleated cells when nuclear division is not inhibited and this difference increases with increasing nuclear inhibition. The number of dead cells does not influence the micronucleus frequency if only viable cells are considered when determining the micronucleus frequency ratio. The results from this model suggest that the micronucleus assay when performed by scoring mononucleated cells, without restricting the score to those cells that have divided once after genotoxic insult, is prone to produce false negative results and, therefore, cannot be considered reliable or conclusive. Scoring of micronuclei in cytokinesis-blocked binucleated cells is predicted by the model to provide consistent results under all culture conditions and based on these theoretical results should be considered the preferred choice.

Animals↗
PubMed1999

The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans.

The International Collaborative Project on Micronucleus Frequency in Human Populations (HUMN) was organized to collect data on micronucleus (MN) frequencies in different human populations and different cell types. The test procedures considered by this project are assays using human lymphocytes (cytokinesis-block method), exfoliated epithelial cells, and other cell types. Data (including descriptions of the populations monitored, detailed test protocols, and test results) are being obtained from a large number of laboratories throughout the world and are being entered into a unified database. The information will be used to: (1) determine the extent of variation of 'normal' values for different laboratories and the influence of other factors potentially affecting baseline MN frequency, e.g., age, gender and life-style; (2) provide information on the effect of experimental protocol variations on MN frequency measurements; (3) design and test optimal protocols for the different cell types; and (4) determine the extent to which MN frequency is a valid biomarker of ageing and risk for diseases such as cancer.

Cell Division↗
PubMed1999

Micronucleus frequency in human lymphocytes is related to plasma vitamin B12 and homocysteine.

In a series of studies, we have been able to confirm that the micronucleus index in cytokinesis-blocked lymphocytes is significantly negatively correlated with plasma vitamin B12 (B12) concentration and significantly positively correlated with plasma homocysteine (HC). Furthermore we have shown in a randomised double-blind placebo-controlled dietary intervention study that intake of 3.5 times the RDI of folic acid and B12 significantly reduces the micronucleus index only in those with above average levels of micronucleus frequency. Micronucleus frequency is minimised when plasma HC is below 7.5 micromol/l and plasma B12 is above 300 pmol/l. Therefore, it is important to take account of the effect of B12 and HC when using the micronucleus assay for human biomonitoring studies.

Adolescent↗
PubMed1999

Effect of vitamin C supplementation on chromosome damage, apoptosis and necrosis ex vivo.

We investigated whether high dose vitamin C influenced the viability of human lymphocytes in plasma, in the presence or absence of hydrogen peroxide (512 microM) by scoring necrotic, apoptotic and micronucleated cells using the cytokinesis-block micronucleus assay. The in vitro results showed that vitamin C (0.57-2.27 mM) on its own had no effect on the above parameters. However, in the presence of hydrogen peroxide vitamin C significantly reduced the number of dividing cells and apoptosis, and increased necrosis and micronucleated cells. A double-blind placebo controlled intervention, with a cross-over, involving 11 male subjects, aged 20-40 years, was performed to determine whether high plasma vitamin C concentration resulting from vitamin C supplementation promotes or protects against genetic damage and cell death ex vivo. Venous blood samples were collected before and after an anti-oxidant-poor diet which reduced plasma vitamin C concentrations by 15% (P < 0.05), and was followed with a 2 g vitamin C supplement, which raised plasma concentrations by 115 and 125% (0.12 mM) after 2 and 4 h, respectively (P < 0.05). Plasma collected post-vitamin C ingestion did not alter micronucleus expression or apoptosis in control or hydrogen peroxide-treated lymphocytes, but it moderately increased necrosis (P < 0.08). Analysis of combined data showed that necrotic cell frequency correlated positively with micronucleated cell frequency (r = 0.66, P < 0.0001) and negatively with apoptotic cell frequency (r = -0.81, P < 0.0001). Overall, vitamin C supplementation did not appear to cause DNA damage under normal physiological conditions nor did it protect cells against hydrogen peroxide-induced toxicity.

Adult↗
PubMed1999

Aleurone flour is a rich source of bioavailable folate in humans.

With the use of novel milling technology, it has become commercially viable to isolate the aleurone layer of cells from wheat grain and to prepare a novel flour from this fraction that has a natural folate concentration of approximately 500 microgram/100 g. The aim of this study was to determine the relative bioavailability of natural folate from aleurone flour when ingested as a cereal. Using a series of randomized, short-term intervention trials with a cross-over involving eight men and eight women aged between 29 and 50 y, we compared the increment of plasma folate following ingestion of 1) 100 g wheat bran cereal (low folate control), 2) 100 g aleurone cereal, and 3) a tablet containing 500 microgram folic acid taken together with 100 g wheat bran cereal (high folate control). Folate absorption was measured by estimating the area under the plasma folate concentration versus time curve. The extent of increase in plasma folate over the 7-hour period following ingestion of aleurone cereal was more than fourfold greater than that observed following the wheat bran cereal (P < 0.0001) and not different from that observed following the 500 microgram folic acid tablet taken with wheat bran cereal. Differences were significant when data for males and females were analyzed separately (P < 0.001). This study has shown that cereal made from wheat aleurone flour is a good source of bioavailable, natural folate.

Absorption↗
PubMed1999

Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide.

A method is described for the inclusion of apoptotic and necrotic cells in the cell counts obtained in the cytokinesis-block micronucleus (CBMN) assay, which is conventionally used solely for the assessment of chromosome breakage, chromosome loss and frequency of dividing cells. The morphological criteria for the recognition and discrimination between necrotic, apoptotic and viable cells are described. Using this comprehensive method we have evaluated the cytotoxic and genotoxic effects of hydrogen peroxide (0-100 microM) in lymphocytes exposed in RPMI 1640 medium. The results obtained indicated significant (P < 0.05) correlations between hydrogen peroxide concentration and the frequency of micronucleated cells (r = 0.39), necrotic cells (r = 0.73), apoptotic cells (r = -0.26) and binucleated cells (r = -0.55). Almost similar results were obtained using the cytosine arabinoside modification of the CBMN assay, which enables excision-repaired sites to be converted to micronuclei. Some of the above end-points were significantly (P < 0.05) correlated with each other (necrosis and apoptosis, R = -0.39; necrosis and micronucleated cell frequency, R = 0.46; necrosis and binucleated cells, R = -0.78; apoptosis and binucleated cells, R = 0.32). It was therefore necessary to use multiple regression analysis to identify the main event induced by hydrogen peroxide, which was necrosis (beta = 0.57, P = 0.0001) and not micronucleus formation (beta = 0. 15, P = 0.1332). Using an ELISA assay we showed that hydrogen peroxide did not induce 8-hydroxydeoxyguanosine. Our data show that the proposed comprehensive test system may provide a better procedure for classifying potential toxic chemicals and enable discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects. The integration of apoptosis and necrosis into the micronucleus assay may also be of practical use in radiosensitivity studies.

Adult↗
PubMed1998

Chromosomal damage rate, aging, and diet.

Chromosomal damage as measured by frequency of translocations, acentric fragments, telomere shortening, nondisjunction, chromosome loss, aneuploidy, and micronucleus formation has been shown to increase progressively with age. Using the cytokinesis-block micronucleus technique, which provides an efficient measure of chromosomal breakage and loss, we have been able to show that aging can explain at least 25% of the variation in chromosomal damage rate in lymphocytes from both males and females. We have also performed cross-sectional and placebo-controlled intervention studies to determine the relationship between the micronucleus (MN) frequency in lymphocytes and diet, and blood status for vitamins C, E, B12, and folic acid. Our studies have shown that MN frequency in the 41- to 60-year age group is significantly lower in vegetarians when compared to nonvegetarians, but the reverse was true in males aged between 20 and 40 years. This was accounted for by a deficient/low B12 status in vegetarian males; there was no difference in the MN frequency of vegetarian and nonvegetarian subjects aged between 61 and 90 years. Results from this study also showed significant negative correlations of MN frequency with folic acid and vitamin B12 but not with vitamin C or vitamin E. In separate studies on healthy men aged 50-70, we have verified the significant negative correlation between vitamin B12 status in plasma and MN frequency (r = -0.315, p = 0.013) in subjects who were not vitamin B12 deficient and observed a significant positive correlation between MN frequency and homocysteine status (r = 0.414, p = 0.0086) in those men who were not vitamin B12 and/or folate deficient. These data suggest that MN frequency is minimized when plasma B12 is above 300 pmol/L and plasma homocysteine is below 7.5 mumol/L. Double-blind placebo-controlled intervention studies conducted over four months have shown that above RDI intake of vitamin E (30 x RDI) or folic acid (10 x RDI) did not produce a significant reduction in MN frequency in men aged 50-70 years. In the latter case plasma homocysteine was reduced from a mean value of 9.33 mumol/L to 8.51 mumol/L, a level that does not correspond with minimization of MN frequency. We have also tested the hypothesis that moderate wine drinking can protect against the DNA-damaging effect of hydrogen peroxide and found that there was a strong ex vivo inhibition (> 70%) of hydrogen peroxide-induced MN frequency by plasma samples from blood collected one hour after consumption of red or white wine, as compared to plasma samples collected immediately before wine consumption (p = 0.0008). However, only samples following red wine consumption produced a significant reduction in baseline MN frequency. The above results suggest that chromosome damage can be modulated, under selected circumstances, by diverse dietary factors.

Adult↗
PubMed1998

Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes-a biomarker for DNA damage in human populations.

The cytokinesis-block micronucleus (CBMN) assay has been adopted by numerous laboratories as a means for rapidly assessing base-line chromosome damage (breakage and loss) in human populations. However, the appropriate implementation of this assay requires a thorough understanding of both experimental variables and biological factors that can have impact on micronucleus (MN) frequency. The paper describes, with the help of experimental data the from the author's laboratory as well as other data, the impact of these variables. With regards to experimental variables, the scoring of micronuclei on slides by different technicians has been identified as an important factor; however, the use of different culture media, namely RPMI 1640 and McCoy's medium, did not have a significant effect on base-line frequencies. The paper also describes results showing that the MN index in cytokinesis-blocked cells, measured once every three months over a 12-month period for 53 healthy subjects, remains constant and the data measured on these occasions were significantly and positively correlated (R=0.477 to 0.684, P<0. 0001) with each other thus indicating the reliability and intra-individual variability of the assay over time. Inter-individual variation for males and female subjects has been estimated for each decade of age between 20 and 80 years; the difference between the 25th and 75th percentile of MN frequency varied between 1.4 fold and 2.3 fold and the minimum and maximum values for MN frequency varied by a factor of 4.7 and 12.5 depending on the age group. Age and gender are the most important demographic variables impacting on the MN index with MN frequencies in females being greater than those in males by a factor of 1.2 to 1.6 depending on the age group. For both sexes, MN frequency was significantly and positively correlated with age (R=0.62 in males and R=0.65 in females) and the slope of the regression line in males was 0.314 (P<0.0001) and in females it was 0.517 (P<0.0001). The main dietary factors influencing the MN index in subjects who are not folate deficient are plasma B12 (R=-0.315, P=0.0127) and plasma homocysteine (R=0.415, P=0.0086). In addition, it was proposed that the MN index is likely to be influenced by the propensity of an individual's cells to undergo apoptosis when damaged so that one might expect the MN frequency to be negatively correlated with apoptotic rate although this has yet to be tested. The above indicates the importance of maintaining an international network of scientists working with the CBMN assay to ensure appropriate quality control and for the development of standard experimental and documentation protocols. The human micronucleus (HUMN) project launched in 1997 is briefly described and proposed as the vehicle for these activities.

Adult↗
PubMed1998

Folate, vitamin B12, homocysteine status and DNA damage in young Australian adults.

We performed a cross-sectional study (n = 49 males, 57 females) and a randomized double-blind placebo-controlled dietary intervention study (n = 31/32 per group) to determine the effect of folate and vitamin B12 (B12) on DNA damage (micronucleus formation and DNA methylation) and plasma homocysteine (HC) in young Australian adults aged 18-32 years. None of the volunteers were folate deficient (i.e. red blood cell folate <136 nmol/l) and only 4.4% (all females) were vitamin B12 deficient (i.e. serum vitamin B12 <150 pmol/l). The cross-sectional study showed that (i) the frequency of micronucleated cells (MNCs) was positively correlated with plasma HC in males (R = 0.293, P < 0.05) and (ii) in females MNC frequency was negatively correlated with serum vitamin B12 (R = -0.359, P < 0.01) but (iii) there was no significant correlation between micronucleus index and folate status. The results also showed that the level of unmethylated CpG (DNA) was not significantly related to vitamin B12 or folate status. The dietary intervention involved supplementation with 3.5x the recommended dietary intake (RDI) of folate and vitamin B12 in wheat bran cereal for three months followed by ten times the RDI of these vitamins via tablets for a further three months. In the supplemented group, MNC frequency was significantly reduced during the intervention by 25.4% in those subjects with initial MNC frequency in the high 50th percentile but there was no change in those subjects in the low 50th percentile for initial MNC frequency. The reduction in MNC frequency was significantly correlated with serum vitamin B12 (R = -0.49, P < 0.0005) and plasma HC (R = 0.39, P < 0.006), but was not significantly related to red blood cell folate. DNA methylation status was not altered in the supplemented group. The greatest decrease in plasma HC (by 37%) during the intervention was observed in those subjects in the supplemented group with initial plasma HC in the high 50th percentile, and correlated significantly with increases in red blood cell folate (R = -0.64, P < 0.0001) but not with serum vitamin B12. The results from this study suggest that (i) MNC frequency is minimized when plasma HC is below 7.5 micromol/l and serum vitamin B12 is above 300 pmol/l and (ii) dietary supplement intake of 700 microg folic acid and 7 microg vitamin B12 is sufficient to minimize MNC frequency and plasma HC. Thus, it appears that elevated plasma HC, a risk factor for cardiovascular disease, may also be a risk factor for chromosome damage.

Adult↗
PubMed1997

The advantages and disadvantages of the cytokinesis-block micronucleus method.

There is current interest in adopting the micronucleus test instead of metaphase analysis of chromosomes to assess the in vitro genotoxic potential of chemical and physical agents. In this paper, the advantages and disadvantages of the cytokinesis-block micronucleus technique relative to metaphase analysis of chromosomes and the conventional micronucleus technique that does not distinguish between dividing and non-dividing cells are discussed. Furthermore, additional features, such as the measurement of non-disjunction, excision-repairable DNA lesions and HPRT variants and possibilities for further improving the application of the cytokinesis-block method are proposed.

Animals↗
PubMed1997

Vitamin-E supplements and their effect on vitamin-E status in blood and genetic damage rate in peripheral blood lymphocytes.

A placebo-controlled double-blind intervention trial was performed using 60 male volunteers aged between 50 and 70 years to test the hypothesis that intake of d-alpha-tocopherol (VITE) above the recommended dietary intake (RDI) level (10.0 mg or 14.9 IU VITE) can protect against DNA damage in human peripheral blood lymphocytes. The intervention consisted of two phases, each of 8 weeks duration: during the initial phase the VITE supplement was 5 x RDI (provided in cereal) and during the second phase the VITE supplement was 30 x RDI (provided in capsules). Blood samples were collected before the initial phase, between phases and at the end of the second phase; the level of VITE was measured in plasma using HPLC and genetic damage rate in peripheral blood lymphocytes was measured using the cytokinesis-block micronucleus assay. The study has shown that cereal supplementation is an effective route for an above RDI intake of VITE; a 5 x RDI supplement resulted in a 22% elevation in the plasma VITE status. By comparison the use of 30 x RDI supplementation with capsules as the route of delivery resulted in an 89% increment in plasma VITE status. The increased VITE status during the intervention, however, had no significant impact on the spontaneous genetic damage rate in human lymphocytes. There was also no correlation between baseline genetic damage frequency and VITE status. However, a 32% (P < 0.007) decrease in the micronucleus index was recorded in both the control and VITE-supplemented groups during the course of the study which could have been due either to seasonal effects or other common components in the diet such as the carrier used for the VITE in the cereal and the capsules. The study has identified a small proportion (3.4%) of apparently healthy individuals who are abnormally sensitive to oxidative stress by hydrogen peroxide and demonstrated that VITE supplementation did not attenuate the impact of the oxidative challenge on genetic damage rate. The above data suggest that supplements in cereal are a viable route for delivering VITE and that supplementation with VITE is unlikely to affect chromosome damage occurring spontaneously or as a result of exposure to oxidative radicals.

Aged↗
PubMed1997

Moderate wine consumption protects against hydrogen peroxide-induced DNA damage.

We have tested the hypothesis that moderate wine drinking can protect somatic cells against the DNA-damaging effect of hydrogen peroxide which is an endogenous source of reactive oxygen metabolites. In this preliminary investigation, four male volunteers were placed on a plant-polyphenol-free (PPF) diet to ensure that the wine provided was the only main source of plant phenolic compounds. After 48 h on the PPF diet the volunteers were required to consume 300 ml of red or white wine and blood samples collected 1, 3, 8 and 24 h post-consumption while still on a PPF diet. Plasma was isolated from the blood samples and stored frozen for subsequent assays. In the subsequent assays, fresh lymphocytes from each donor were incubated in their corresponding plasma from the various intervention time-points for 30 min. The capacity of the plasma to prevent damage to DNA in lymphocytes by hydrogen peroxide was assessed using the cytokinesis-block micronucleus technique. The data from this preliminary investigation indicated that there was a strong inhibition (>70%) of hydrogen peroxide-induced micronucleated cells by the plasma samples from the blood collected 1 h after consumption of wine as compared to plasma samples from blood immediately before the consumption of wine. This protective effect was apparent for both red and white wine although statistical significance (P = 0.0068) was achieved only in the white wine intervention. A higher degree of statistical significance (P = 0.0008) was achieved when the data for samples following the consumption of red and white wine were combined. There was no difference in the hydrogen-peroxide-induced micronucleated cell frequency when comparing results immediately before starting on the PPF diet, before consumption of wine, 8 h after or 24 h after wine consumption. The hydrogen peroxide-induced micronucleated cell frequency in cells incubated with plasma from blood collected 3 h after wine consumption was intermediate to that observed for plasma after 1 and 8 h after wine intake. The protective effect of plasma against DNA damage cannot be readily explained by the red wine content of phenolic compounds because results for red wine were similar to those for white wine even though white wine had a much lower level of total polyphenols. A possible explanation could be that alcohol, glycerol and ascorbate in wine together with specific wine phenolic compounds that are also equally present in red and white wine (e.g. hydroxycinnamates) may have contributed to the observed protection of nuclear material from hydrogen peroxide-derived reactive oxygen metabolites. This explanation is supported by data from in vitro experiments showing that incubation of lymphocytes either with alcohol or wine stripped of phenolic compounds resulted in a statistically significant (P < 0.05) dose-related reduction (up to 87% reduction) in hydrogen peroxide-induced micronucleated cell frequency.

Adult↗
PubMed1997

A more comprehensive application of the micronucleus technique for biomonitoring of genetic damage rates in human populations--experiences from the Chernobyl catastrophe.

The current method for scoring micronuclei as a measure of genetic damage rate in peripheral blood cells is to enumerate this end point in cytokinesis-blocked binucleated cultured lymphocytes. However, one can expect that, due to chronic exposure to genotoxins or inherent genetic instability, micronuclei may be expressed continually in vivo in dividing cell populations such as the progenitor cell lineages leading to mature lymphocytes or erythrocytes. Consequently, micronuclei may already be expressed in peripheral blood lymphocytes prior to culture. In view of these considerations, we have performed a study in children living in regions of Belarus that are contaminated by radionuclides from the Chernobyl disaster and compared their micronucleus frequency in erythrocytes, nondivided lymphocytes, and cultured cytokinesis-blocked binucleated lymphocytes to that of controls living in noncontaminated areas. Preliminary data presented in this paper indicate a significant two- to fourfold increase in micronucleus expression (P < 0.05) in exposed children relative to controls in erythrocytes or peripheral blood lymphocytes in blood smears as well as in mononuclear and cytokinesis-blocked binucleated lymphocytes in cultures. The measurement of micronuclei in nondivided mononuclear lymphocytes represents chromosomal damage expressed during in vivo divisions. The micronuclei in binucleated cultured cells represent micronuclei expressed ex-vivo and may include micronuclei already present in a cell prior to tissue culture. These preliminary data suggest that a different spectrum and level of damage may be observed in nondivided mononuclear lymphocytes, binucleated lymphocytes, and erythrocytes and that a combination of these approaches may provide a more comprehensive assessment of the extent of genetic damage induced by chronic exposure to radionuclides or other genotoxins in haematopoietic tissue.

Adolescent↗