Factors affecting the incidence of genotoxicity biomarkers in peripheral blood lymphocytes: impact on design of biomonitoring studies.

A review of risk factors affecting background rates of micronuclei and chromosomal aberration (CA) formation in peripheral blood lymphocytes (PBLs) was undertaken with a view to aiding the interpretation of genotoxicity biomonitoring studies. Both endogenous factors and those due to methodological variation were evaluated. Background variation of other indices of genotoxicity in PBLs (specifically 8-hydroxy-deoxyguanosine and comet assays) were also considered as these data likely reflect overlapping causes of DNA damage and may provide some indicators for future research areas. A number of host risk factors, namely age, gender, smoking, vitamin B(12) and folate status, were identified for which there is strong or sufficient evidence that they impact on background levels of genotoxicity biomarkers. Evaluation of these factors should be routinely included in genotoxicity biomonitoring studies. Although data on the influence of smoking is somewhat inconsistent, because of its known association with cancer and DNA damage, it is also classified as a high-risk factor. A number of other factors were identified for which there is weak or insufficient evidence including alcohol consumption, disease conditions and infections, physical exercise, body mass index and genotype. The review shows that the evaluation of biomonitoring studies of genotoxicity is complex and there is a need to improve study designs by setting an a priori hypothesis, collecting good exposure data and stratifying groups appropriately, using appropriate power calculations before initiating biomonitoring studies, and collecting information on appropriate risk factors. There is a need for further collaborative work and the establishment of centres of excellence on genotoxicity biomonitoring. If these measures are achieved, then it would be possible to use the data from biomonitoring studies in risk assessments to derive risk management measures.

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title: Chromosomal aberrations and cancer risk: results of a cohort study from Central Europe.
abstract: A high level of chromosomal aberrations in peripheral blood lymphocytes may be an early marker of cancer risk, but data on risk of specific cancers and types of chromosomal aberrations (chromosome type and chromatid type) are limited. A total of 6,430 healthy individuals from nine laboratories in Croatia, Hungary, Lithuania, Poland, and Slovakia, included in chromosomal aberration surveys performed during 1978-2002, were followed up for cancer incidence or mortality for an average of 8.5 years; 200 cancer cases were observed. Compared with that for the low-tertile level of chromosomal aberrations, the relative risks of cancer for the medium and high tertiles were 1.78 (95% confidence interval: 1.19, 2.67) and 1.81 (95% confidence interval: 1.20, 2.73), respectively. The relative risk for chromosome-type aberrations above versus below the median was 1.50 (95% confidence interval: 1.12, 2.01), while that for chromatid-type aberrations was 0.97 (95% confidence interval: 0.72, 1.31). The analyses of risk of specific cancers were limited by small numbers, but the association was stronger for stomach cancer. This study confirms the previously reported association between level of chromosomal aberrations and cancer risk and provides novel information on the type of aberrations more strongly predictive of cancer risk and on the types of cancer more strongly predicted by chromosomal aberrations.

title: An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.
abstract: The frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) is extensively used as a biomarker of chromosomal damage and genome stability in human populations. Much theoretical evidence has been accumulated supporting the causal role of MN induction in cancer development, although prospective cohort studies are needed to validate MN as a cancer risk biomarker. A total of 6718 subjects from of 10 countries, screened in 20 laboratories for MN frequency between 1980 and 2002 in ad hoc studies or routine cytogenetic surveillance, were selected from the database of the HUman MicroNucleus (HUMN) international collaborative project and followed up for cancer incidence or mortality. To standardize for the inter-laboratory variability subjects were classified according to the percentiles of MN distribution within each laboratory as low, medium or high frequency. A significant increase of all cancers incidence was found for subjects in the groups with medium (RR=1.84; 95% CI: 1.28-2.66) and high MN frequency (RR=1.53; 1.04-2.25). The same groups also showed a decreased cancer-free survival, i.e. P=0.001 and P=0.025, respectively. This association was present in all national cohorts and for all major cancer sites, especially urogenital (RR=2.80; 1.17-6.73) and gastro-intestinal cancers (RR=1.74; 1.01-4.71). The results from the present study provide preliminary evidence that MN frequency in PBL is a predictive biomarker of cancer risk within a population of healthy subjects. The current wide-spread use of the MN assay provides a valuable opportunity to apply this assay in the planning and validation of cancer surveillance and prevention programs.

title: Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. European Study Group on Cytogenetic Biomarkers and Health.
abstract: An increased risk of cancer in healthy individuals with high levels of chromosomal aberrations (CAs) in peripheral blood lymphocytes has been described in recent epidemiological studies. This association did not appear to be modified by sex, age, country, or time since CA test, whereas the role played by exposure to carcinogens is still uncertain because of the requisite information concerning occupation and lifestyle was lacking. We evaluated in the present study whether CAs predicted cancer because they were the result of past exposure to carcinogens or because they were an intermediate end point in the pathway leading to disease. A nested case-control study was performed on 93 incident cancer cases and 62 deceased cancer cases coming from two prospective cohort studies performed in Nordic countries (Denmark, Finland, Norway, and Sweden) and Italy. For each case, four controls matched by country, sex, year of birth, and year of CA test were randomly selected. Occupational exposure and smoking habit were assessed by a collaborative group of occupational hygienists. Logistic regression models indicated a statistically significant increase in risk for subjects with a high level of CAs compared to those with a low level in the Nordic cohort (odds ratio, 2.35; 95% confidence interval, 1.31-4.23) and in the Italian cohort (odds ratio, 2.66; 95% confidence interval, 1.26-5.62). These estimates were not affected by the inclusion of occupational exposure level and smoking habit in the regression model. The risk for high versus low levels of CAs was similar in subjects heavily exposed to carcinogens and in those who had never, to their knowledge, been exposed to any major carcinogenic agent during their lifetime, supporting the idea that chromosome damage itself is involved in the pathway to cancer. The results have important ramifications for the understanding of the role played by sporadic chromosome damage for the origin of neoplasia-associated CAs.

title: Impact of Types of Lymphocyte Chromosomal Aberrations on Human Cancer Risk
abstract: The frequency of cells with structural chromosomal aberrations (CAs) in peripheral blood lymphocytes is the first genotoxicity biomarker that has shown an association with cancer risk. CAs are usually divided into chromosome-type (CSAs) and chromatid-type aberrations (CTAs), with different mechanisms of formation. From a mechanistic point of view, it is of interest to clarify whether the cancer predictivity of CAs is different with respect to CSAs and CTAs. We report here cancer risk for cytogenetically tested, healthy subjects with respect to frequency of CAs, CSAs, and CTAs in peripheral blood lymphocytes, using Nordic (1981 subjects with CA data, 1871 subjects with CSA/CTA data) and Italian (1573 subjects with CA data, 877 subjects with CTA/CSA data) cohorts, with a median follow-up of 17 years. High levels of CAs at test were clearly associated with increased total cancer incidence in the Nordic cohorts and increased total cancer mortality in the Italian cohort. In the Nordic cohorts, significantly elevated cancer risks were observed for subjects with both high CSAs and high CTAs at test, and these variables showed equally strong cancer predictivity. The results of the Italian cohort did not indicate any clear-cut difference in cancer predictivity between the CSA and CTA biomarkers. There was no significant effect modification by age at test, gender, country, or time since test. The results suggest that both DNA double-strand breaks and other initial DNA lesions responsible for CSAs and CTAs are associated with cancer risk.

title: Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH).
abstract: Chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronuclei (MN) in peripheral blood lymphocytes have for decades been used as cytogenetic biomarkers to survey genotoxic risks in the work environment. The conceptual basis for this application has been the idea that increased cytogenetic damage reflects an enhanced cancer risk. Nordic and Italian cohorts have been established to evaluate this hypothesis, and analyses presented previously have shown a positive trend between CA frequency and increased cancer risk. We now report on a pooled analysis of updated data for 3541 subjects examined for CAs, 2703 for SCEs, and 1496 for MN. To standardize for interlaboratory variation, the results for the various cytogenetic end points were trichotomized on the basis of the absolute value distribution within each laboratory as "low" (1-33 percentile), "medium" (34-66 percentile), or "high" (67-100 percentile). In the Nordic cohort, there was an elevated standardized incidence ratio (SMR) for all cancer among subjects with high CA frequency [1.53; 95% confidence interval (CI), 1.13-2.05] but not for those with medium or low CA frequency. In the Italian cohort, a SMR in cancer of 2.01 (95% CI, 1.35-2.89) was obtained for those with a high CA frequency level, whereas the SMRs for those with medium or low did not noticeably differ from unity. Cox's proportional hazards models gave no evidence that the effect of CAs on total cancer incidence/mortality was modified by gender, age at test, or time since test. No association was seen between the SCEs or the MN frequencies and subsequent cancer incidence/mortality. The present study further supports our previous observation on the cancer predictivity of the CA biomarker, which seems to be independent of age at test, gender, and time since test. The risk patterns were similar within each national cohort. This result suggests that the frequency of CAs in peripheral blood lymphocytes is a relevant biomarker for cancer risk in humans, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility.

title: HUman MicroNucleus project: international database comparison for results with the cytokinesis‐block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei
abstract: Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis‐block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin‐B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5‰ and the interquartile range was between 3 and 12‰. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14–24%). Statistical analyses were performed using random‐effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.  2001

title: Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.
abstract: One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.

title: Effect of smoking habit on the frequency of micronuclei in human lymphocytes: results from the Human MicroNucleus project.
abstract: The effect of tobacco smoking on the frequency of micronuclei (MN) in human lymphocytes has been the object of many population studies. In most reports, the results were unexpectedly negative, and in many instances smokers had lower frequencies of MN than non-smokers. A pooled re-analysis of 24 databases from the HUMN international collaborative project has been performed with the aim of understanding the impact of smoking habits on MN frequency. The complete database included 5710 subjects, with 3501 non-smokers, 1409 current smokers, and 800 former smokers, among subjects in occupational and environmental surveys. The overall result of the re-analysis confirmed the small decrease of MN frequencies in current smokers (frequency ratio (FR) = 0.97, 95% confidence interval (CI) = 0.93-1.01) and in former smokers (FR = 0.96, 95% CI = 0.91-1.01), when compared to non-smokers. MN frequency was not influenced by the number of cigarettes smoked per day among subjects occupationally exposed to genotoxic agents, whereas a typical U-shaped curve is observed for non-exposed smokers, showing a significant increase of MN frequency in individuals smoking 30 cigarettes or more per day (FR = 1.59, 95% CI = 1.35-1.88). This analysis confirmed that smokers do not experience an overall increase in MN frequency, although when the interaction with occupational exposure is taken into account, heavy smokers were the only group showing a significant increase in genotoxic damage as measured by the micronucleus assay in lymphocytes. From these results some general recommendations for the design of biomonitoring studies involving smokers can be formulated. Quantitative data about smoking habit should always be collected because, in the absence of such data, the simple comparison of smokers versus non-smokers could be misleading. The sub-group of heavy smokers (> or =30 cigarettes per day) should be specifically evaluated whenever it is large enough to satisfy statistical requirements. The presence of an interaction between smoking habit and occupational exposure to genotoxic agents should be always tested.

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

title: Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes.
abstract: Intra- and interindividual variations of baseline frequencies of cytogenetic end points in lymphocytes of human populations have been reported by various authors. Personal characteristics seem to account for a significant proportion of this variability. Several studies investigating the role of age as a confounding factor in cytogenetic biomonitoring found an age-related increase of micronucleus (MN) frequency, whereas contradictory results were reported for chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). We have quantitatively evaluated the effect of age on SCE, CA, and MN through the analysis of a population sample that included data from several biomonitoring studies performed over the last few decades in 12 Italian laboratories. The large size of the data set, i.e., more than 2000 tests for each end point, allowed us to estimate the independent effect of age, taking into account other covariates, such as sex, smoking habits, occupational exposure, and inter- and intralaboratory variability. A greater frequency of the mean standardized values by increasing of age was observed for all of the end points. A leveling off was evident in the last age classes in the trend of MN frequencies. Frequency ratios (FRs), which express the increase of the cytogenetic damage with respect to the first age classes, i.e., 1-19 years, were estimated using Poisson regression analysis after adjustment for the potential confounding factors and confirmed the increasing trend by age class for all three end points. The most dramatic increase was observed for MN, with a FR that approaches the value of 2 at the age class 50-59 (FR, 1.97; 95% confidence interval, 1.43-2.71) and remains substantially unchanged thereafter. The trend of FRs for CA is more homogeneous, with a constant rise even in the older classes, whereas the frequency of SCE increases with age to a lesser extent, reaching a plateau in the age class 40-49 and the maximum value of FR in the age class over 70 (FR, 1.14; 95% confidence interval, 1.07-1.23). In conclusion, our results point to an age-related increase of the chromosome damage in lymphocytes and emphasize the need to take into account the potential confounding effect of this variable in the design of biomonitoring studies based on chromosome damage.

title: On the frequency of chromosome exchanges in a control population measured by chromosome painting.
abstract: Chromosome painting has been shown to be a valid and rapid method for quantifying structural chromosome rearrangements in human lymphocytes. The method is particularly useful for detecting stable aberrations which are difficult and expensive to quantify with classical methods. The inherent stability of translocations has enabled them to be used as a biodosimeter for chronic and temporally displaced exposure to radiation. Translocations may also be useful for quantifying chronic exposure to other environmental agents which may result in an accumulation of cytogenetic damage with age. Most exposures are chronic and occur at low rates, and conventional cytogenetic methods such as dicentric analysis are not expected to be informative. To understand the extent to which age and lifestyle factors impact the frequency of stable aberrations, we have performed chromosome painting on metaphase-arrested lymphocytes cultured from 47 healthy adults ranging in age from 19 to 77 years, and from umbilical cord blood obtained from eight healthy full-term infants. All subjects had previously been screened to eliminate those who had received significant occupational or accidental exposure to radiation or chemicals, and none had received chemo- or radiotherapy. Due to the infrequent occurrence of stable aberrations in peripheral lymphocytes, we analyzed the equivalent of more than 1100 metaphase cells from each of these 55 people. An average of one cell in 130 (0.77%) was observed to have a translocation or a stable insertion. A significant relationship between stable aberrations and the square of the age is apparent (R2 = 0.69, Y = 0.0615 + 0.000304 age2; p < 0.00001). These results support the hypothesis that stable aberrations accumulate with time, and are likely to integrate adverse environmental exposure.

title: The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people.
abstract: Peripheral blood lymphocytes from unirradiated control subjects and workers exposed within permitted limits to gamma-radiation, have been examined for the incidence of dicentric and acentric chromosome aberrations. The results are compared with a review of data published elsewhere. Background levels show inter-laboratory variation and possible reasons for this are discussed. By combining the present data with those from the literature the spontaneous incidence of dicentric aberrations in approx. 0.55 X 10(-3) and for acentrics is 3.7 X 10(-3). In occupationally exposed subjects a significantly higher incidence of aberrations was found. When allowance was made for the turnover of lymphocytes for the period over which each man had worked with radiation a linear dose--effect relationship was apparent. The incidence of dicentrics was 2.22 +/- 0.94 X 10(-4) rad-1 and for all unstable aberrations 8.24 +/- 2.8 X 10(-4) rad-1. These are in reasonable agreement with dose--response data obtained in vitro.

title: Influence of sex on cytogenetic end points: evidence from a large human sample and review of the literature.
abstract: The planning and evaluation of human cytogenetic studies should contemplate various confounders and effect modifiers, among these, sex and sex-related factors. The association between this variable and cytogenetic damage has been extensively studied, but conclusive evidence has thus far not been reached, especially for the most recent assays, such as the micronucleus test (MN). In the attempt to quantitatively estimate the sex effect on sister chromatid exchange (SCE), chromosomal aberration (CA), and MN in peripheral blood lymphocytes, we reanalyzed the original data sets of several biomonitoring studies performed over the last decades in 10 Italian laboratories. This approach yielded a very large database, namely 2140, 2495, and 2131 subjects screened for SCE, CA, and MN, respectively. Differences between sexes were expressed in terms of relative risk (RR) of females versus males, after adjustment for age, smoking habits, occupation exposure and inter- and intralaboratory variation. No difference between sexes was found for the frequency of SCE [RR = 1.01; 95% confidence interval (CI) = 0.99-1.03] and CA (RR = 1.00; 95% CI = 0.92-1.08) even if the CI of the RR for SCE includes the 3% excess in females frequently reported by the literature. Conversely, a 29% overall increase of the MN rate in females was observed in the whole data set (RR = 1.29; 95% CI = 1.20-1.38). Different trends by age of the MN rate are described in the two sexes, focusing on the peak observed in females in the menopausal period and on the subsequent decrease.

title: Choosing Metaphases for Biological Dosimetry by Fluorescence In Situ Hybridization (FISH)
abstract: A., Maznik, N., Moquet, J., Hone, P., Vinnikov, V., Lloyd, D. and Cox, R. Choosing Metaphases for Biological Dosimetry by Fluorescence In Situ Hybridization (FISH).  (2002). Data are presented for a subset of lymphocytes characterized by FISH as missing painted chromosomal material. These lymphocytes occur in both control and irradiated subjects. These cells have a much greater frequency of one-way translocations than cells in which all of the painted chromosomal material is present. Their presence contributes to interindividual variability in control translocation yields. These cells do not appear to be more prevalent in persons exposed to high radiation doses. It is suggested that their exclusion when selecting cells for analysis may improve the sensitivity of FISH as a biological dosimeter at low doses. Mechanisms for the production of these one-way translocations in vivo are also discussed, with a proposal that their variable frequency in individuals may be consistent with exposure to chemical clastogens.

title: Inter-rater agreement on chromosome 5 breakage in FISH-based mutagen sensitivity assays (MSAs).
abstract: In chromosome breakage assays, validated, universal criteria for selection of cells and classification of chromosome aberrations may enhance their utility for cancer susceptibility screening. To standardize a fluorescence in situ hybridization (FISH) modification of the mutagen sensitivity assay (MSA), scoring criteria were evaluated by web-based validation. Two hundred digital FISH images were assigned random identification numbers. With this set of images, criteria for inclusion of cells and measurement of the frequency of abnormal cells were evaluated by eight observers, all of whom had five or more years of experience. Observers included doctoral and MS/BS level cytogeneticists, and were drawn from a randomized pool of 54 volunteers. Questions addressed were: (1) how uniformly were criteria applied to analysis of a standard digital FISH image set and (2) did concordance vary with educational level? These data suggest inter-rater agreement within a factor of 2 for average breakage frequency, but revealed greater variability in cell selection. These results aid in estimating the components of assay variance due to definitions, technical parameters and biological variables.

title: Human population studies with cytogenetic biomarkers: Review of the literature and future prospectives
abstract: Cytogenetic biomarkers are by far the most frequently used endpoints in human population studies. Their sensitivity for measuring exposure to genotoxic agents and their role as early predictors of cancer risk have contributed to this success. In this article, we present an overview of the last 25 years of population studies with cytogenetic biomarkers, describing the evolution of this research and addressing the most promising innovations for the future. The evaluation has been restricted to the most popular assays, i.e., chromosomal aberrations (CAs) and micronucleus (MN), which are considered to be causally related to early stages of chronic diseases, especially cancer, and may therefore play a major role in prevention. An extensive literature search covering the period 1 January 1980 to 31 December 2003 was performed using the Medline/PubMed database. A total of 833 population studies using CAs and 434 using matched MN inclusion criteria were included in the analysis. We report the distribution of selected papers by year of publication, country, language, agents investigated, and methods employed. The state of the art and future prospects regarding cytogenetic techniques and epidemiologic and statistical methods are discussed. The role of susceptibility and its potential impact on genotoxic damage are discussed with special attention to the effect of major genetic polymorphisms on the baseline frequency of CAs and micronuclei.  2005.

title: Measurement of micronuclei in lymphocytes.
abstract: The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.

title: A more comprehensive application of the micronucleus technique for biomonitoring of genetic damage rates in human populations—experiences from the Chernobyl catastrophe
abstract: 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.  1997.

title: Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes.
abstract: Human biomonitoring of early genetic effects requires accurate, sensitive and, if possible, easy and not too time-consuming methodologies to assess mutations. One of the most promising methodologies at the present time is the cytokinesis block micronucleus (MN) assay (CBMN), which detects both chromosome breakage and chromosome loss in once-divided binucleated (BN) cells. Many studies have been published with this methodology, but before its extensive application is recommended, it is necessary to evaluate its strengths and limitations. Recently, Fenech et al. reviewed the advantages of the CBMN assay for biomonitoring purposes. However, up to now information present in mononucleated (MONO) cells has rarely been taken into account, although it might be complementary to that assessed in BN cells. Indeed, MONO cells should indicate damage which was present in vivo before the start of culture and BN cells may contain pre-existing micronuclei (MNi) plus lesions which are expressed as MNi during in vitro culture. To address this question, the objectives of this paper were as follows. (i) To situate the CBMN assay in a historical and mechanistic perspective. (ii) To consider whether impaired mitotic capacity in vitro may be responsible for false negative biomonitoring studies if MN in MONO cells are not taken into account in the CBMN test. The following factors were considered: division delay for repair and mitotic block, in vitro apoptosis and necrosis of damaged cells, mitotic slippage and correlation between MN expression in vitro versus in vivo. (iii) To analyse the factors which may cause a negative result in the CBMN assay in biomonitoring when exposure to specific genotoxins is evident. The specific effects of aneugens and of adaptive responses to chronic low level exposure were examined. (iv) To compare the sensitivity of MONO and BN cells in relation to the genotoxic mechanism. (v) To propose an adequate sampling scheme to study MN in both MONO and BN cells. It was concluded that a more comprehensive assessment of DNA damage may be achieved if the CBMN assay includes measures of: (i) MNi in MONO cells; (ii) MNi in BN cells; (iii) apoptotic cells; (iv) necrotic cells. It is probable that the 24 h post-phytohaemagglutinin time point may be the optimal time to assess the frequency of MNi in MONO cells, apoptotic cells and necrotic cells. It is also practical to include these measures when scoring MNi in BN cells after cytokinesis block.

title: Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes.
abstract: Micronuclei are formed from chromosomes and chromosomal fragments that lag behind in anaphase and are left outside daughter nuclei in telophase. They may also be derived from broken anaphase bridges. Nuclear buds, micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds) of normal human cells in general. Our results suggest that nuclear buds and micronuclei have partly different mechanistic origin. Interstitial DNA without centromere or telomere label was clearly more prevalent in nuclear buds (43%) than in micronuclei (13%). DNA with only telomere label or with both centromere and telomere label was more frequent in micronuclei (62% and 22%, respectively) than in nuclear buds (44% and 10%, respectively). Folate deprivation especially increased the frequency of nuclear buds and micronuclei harboring telomeric DNA and nuclear buds harboring interstitial DNA but also buds and micronuclei with both centromeric and telomeric DNA. According to the model we propose, that micronuclei in binucleate lymphocytes primarily derive from lagging chromosomes and terminal acentric fragments during mitosis. Most nuclear buds, however, are suggested to originate from interstitial or terminal acentric fragments, possibly representing nuclear membrane entrapment of DNA that has been left in cytoplasm after nuclear division or excess DNA that is being extruded from the nucleus.

title: Chromosomal biomarkers of genomic instability relevant to cancer.
abstract: It is generally acknowledged that a crucial event in the initiation and evolution of cancer is the acquisition of a genomic instability phenotype. This review focuses on mechanisms of chromosomal instability including aneuploidy, chromosome rearrangement and breakage-fusion-bridge cycles. The role of micronutrient deficiency, such as folate deficiency, in the causation of chromosomal instability is briefly reviewed and the concept of recommended dietary allowances for genomic stability is introduced. In addition, the techniques for measuring the various chromosomal instability events are discussed with a focus on the cytokinesis-block micronucleus assay as an almost complete system for measuring these various genetic mishaps.

title: Cytokinesis-block micronucleus assay evolves into a "cytome" assay of chromosomal instability, mitotic dysfunction and cell death.
abstract: The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r=0.75-0.77, P<0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a "cytome" method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations.

title: Cytokinesis-block micronucleus cytome assay
abstract: The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. Further information regarding mechanisms leading to MNi, NPBs and NBUDs formation is obtained using centromere and/or telomere probes. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.

title: What do human micronuclei contain?
abstract: As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.

title: Micronuclei frequency in children exposed to environmental mutagens: a review.
abstract: Cytogenetic monitoring has been traditionally used for the surveillance of populations exposed to genotoxic agents. In recent years sensitivity problems emerged in surveys of populations exposed to low levels of mutagens, and therefore alternative approaches have been explored. Biomonitoring studies in children are a promising field, since because of evident differences in the uptake, metabolism, distribution and excretion of mutagens this population seems to be more susceptible than adults. Further, the effect of major confounders such as cigarettes smoking, occupation, life-style, and dietary factors plays a minor role. Among cytogenetic assays, the micronucleus assay (MN) has several advantages and is increasingly used. A review was then carried out to synthesize the published data on the occurrence of MN in children and adolescents (age range 0-18 years), and to assess the impact of genotoxic exposure on MN frequency. Overall, 20 papers from international literature and 8 Russian papers were included. An effect of age was found within this age range, while the influence of gender on MN frequency was irrelevant. These results were confirmed by the re-analysis of data for 448 children selected from the HUMN database. An effect of chronic and infectious diseases on MN levels has been reported by various authors. Most studies describing the effect of exposure to genotoxic agents (ionizing radiation, chemicals, drugs, environmental tobacco smoke) found an increase of MN in exposed children. The limited number of published papers indicates that the conduct of properly designed studies on the effect of environmental pollutants in children may be difficult. This review confirmed the usefulness of MN assay in biomonitoring studies conducted in children, revealing that in many circumstances investigating children increases the sensitivity of the study, even with low dose exposures.

title: Children's exposure to environmental pollutants and biomarkers of genetic damage. II. Results of a comprehensive literature search and meta-analysis.
abstract: The present review is based on findings from 178 publications retrieved through an extensive search of the MedLine/PubMed database for a 25 years time period (1980-2004) and 10 manually identified papers. Among the cytogenetic biomarkers that are frequently used in field studies, chromosome aberrations (CA) and micronuclei (MN) but not sister chromatid exchanges (SCE) were found consistently increased in children exposed to environmental pollutants. Meta-analysis of the studies reporting SCE in cord blood showed similar levels of SCE in exposed and in non-exposed newborns. Exposure to airborne pollutants, soil and drinking water contaminants, mostly increased CA and, to a lesser extent, MN levels in children. The effect of exposure to airborne urban pollutants was consistently reported by field studies measuring DNA, albumin and hemoglobin adducts. Prenatal (in utero) and postnatal exposure (environmental tobacco smoke, ETS) to tobacco smoke compounds were associated with increased frequencies of DNA and hemoglobin adducts and CA. The limited number of field studies measuring DNA fragmentation (Comet assay), hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the glycophorinA (GPA) mutation frequency in environmentally exposed children precluded a meaningful evaluation of the usefulness of these assays. Meta-analyses performed in children exposed to ETS and in newborns exposed in utero to their mothers' smoke showed 1.3 and 7 times higher levels of hemoglobin adducts compared to referent subjects, respectively. These increases are consistent with the epidemiological evidence of higher lung cancer risks reported in adults who had never smoked and were exposed to ETS during childhood and with 7-15 times higher lung cancer risks reported in smokers than in non-smokers. Higher levels of PAH-DNA adducts were found in fetal than in maternal tissue, suggesting a specific susceptibility of the fetus to this class of ubiquitous environmental pollutants. According to these findings, future research and biomonitoring programs on children would greatly benefit from the inclusion of selected biomarkers that could provide biologically based evidence for the identification of intervention priorities in environmental health.

title: Biomarkers of intermediate endpoints in environmental and occupational health.
abstract: The use of biomarkers in environmental and occupational health is increasing due to increasing demands on information about health risks from unfavourable exposures. Biomarkers provide information about individual loads. Biomarkers of intermediate endpoints benefit in comparison with biomarkers of exposure from the fact that they are closer to the adverse outcome in the pathway from exposure to health effects and may provide powerful information for intervention. Some biomarkers are specific, e.g., DNA and protein adducts, while others are unspecific like the cytogenetic biomarkers of chromosomal aberrations (CA), sister chromatid exchanges and micronuclei (MN). The validation of biomarkers includes measurements of sensitivity and specificity of biomarkers and round robin tests to ensure reproducible protocols within different laboratories. The predictive value of biomarkers with respect to adverse health effect from the result of the measurement has been performed for the cytogenetic biomarkers showing a predictive value of high levels of CA and increased risk of cancer. The use of CA in future studies is, however, limited by the laborious and sensitive procedure of the test and lack of trained cytogeneticists. Less time consuming, but robust biomarkers, sensitive to environmental exposures are suggested. From the selection of developed biomarkers, the comet assay is highly sensitive to lifestyle exposures, often confounding the output, while MN in lymphocytes seem promising with respect to laboratory and health effect (cancer) validity. Also, new biomarkers exploiting the new 'omics' technologies are being developed. A number of ethical issues arise from the use of biomarkers with a predictive value aiming at respecting the autonomy of the study person in participation (only upon written informed consent and with obligations of withdrawal at any time), access to personal information (right to know and right not to know the study result) and securing proper data management (data protection to avoid misuse in employment, insurance, loaning and learning opportunities).

title: Use of primary blood cells for the assessment of exposure to occupational genotoxicants in human biomonitoring studies.
abstract: The Comet assay is an often used approach for the assessment of genetic damage in primary cells of exposed populations. In the majority of these studies lymphocytes are used. Therefore, we reviewed human biomonitoring studies of occupational exposure using the Comet assay with lymphocytes. We also tried to elucidate the strengths of the studies, which were that (i) data could be obtained in a fast and cost-effective manner, (ii) the ease at which these cells can be collected and (iii) a remarkable concordance between Comet assay and cytogenetic assays. However, the analysis also revealed some shortcomings: (i) the low number of study participants, (ii) the bias in the distribution of gender, (iii) lack of qualitative and quantitative exposure data, (iv) omission to consider differences in physical activity and diet between control and exposed groups, (v) lack of uniformity in the Comet assay procedures, and (vi) controversy in the sensitivity of Comet assay since it picked up DNA damage caused by agents which were found to be weak genotoxicants or non-genotoxicants in other tests, but gave inconsistent results with known mutagens/carcinogens such as cigarette smoke.

title: 8-Hydroxy-2′-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures
abstract: Oxidative DNA damage is considered to play an important role in pathophysiological processes, ageing and cancer. So far major interest has been on measuring 8-hydroxy-2′-deoxyguanosine (8-OHdG), the preferred methods relying on HPLC or GC-mass spectrometry. The high biological relevance of 8-OHdG is due to its ability to induce G→T transversions, which are among the most frequent somatic mutations found in human cancers. Effects of workplace exposures on the level of white blood cell 8-OHdG or urinary 8-OHdG have been reported with controversial results. Exposures examined include asbestos, azo-dyes, benzene, fine particulate matter (PM2.5), glassworks, polycyclic aromatic hydrocarbons (PAHs), rubber manufacturing, silica, metals, styrene, toluene and xylenes. The available data indicate that there is still a lack of well established dose-response relations between occupational or environmental exposures and the induction of 8-OHdG. Smoking has been most consistently identified as a confounder for 8-OHdG, but various occupational studies did not reveal higher levels of 8-OHdG in smokers. Despite the conflicting results, the reported studies show promise for 8-OHdG as a biomarker of oxidative stress associated with chemical exposure. However, there are critical aspects related to the analytical challenge, artifactual production of 8-OHdG, inter- and intra-individual variation, confounding factors and inter-laboratory differences, implying that further work is needed to reach a consensus on the background level of 8-OHdG.

title: 'Spontaneous' genetic damage in man: evaluation of interindividual variability, relationship among markers of damage, and influence of nutritional status.
abstract: The 'spontaneous' frequency of genetic damage (normal background) and the possible relationship of this damage to nutritional variables in humans were investigated in 22 subjects using several indices of genetic damage. The subjects were chosen, out of 122 initially analyzed, for being at the extremes of the highest and lowest values of one index of genetic damage, the frequency of micronucleated erythrocytes in peripheral blood. This index reflects chromosomal damage and loss in bone marrow erythropoietic cells. The assay for micronuclei is convenient but is restricted to splenectomized individuals because the human spleen removes micronucleated cells. The initial 122 subjects were splenectomized, but all were normal and healthy at the time of this study and none had a previous history of neoplastic disease. Factors investigated were stability of micronucleus frequency as a function of time, correlations among multiple markers of genetic damage, and influence on damage indices of nutritional variables, including blood levels of folate, B12 and antioxidant vitamins. Among different individuals, the range of values was 10-fold or more in the erythrocyte micronucleus, glycophorin A, plasma ascorbate and urinary 8-hydroxydeoxyguanosine (oxo8dG) assays, was approximately 6-fold in the lymphocyte micronucleus assay, and was 2-fold in the lymphocyte sister chromatid exchange (SCE) assay. Red blood cell folate and plasma folate, B12 and alpha-tocopherol values varied by up to 10-fold among individuals. Micronucleus frequencies in erythrocytes and peripheral blood lymphocytes ranged from < 0.3 to 16.9/1000 in mature red blood cells, < 1 to 33/1000 in reticulocytes, and 2.5 to 15/1000 in binucleate lymphocytes. Frequencies of glycophorin A variant erythrocytes ranged from 5.6 to 77.3 x 10(6) N/0 cells and 3.2 to 16.2 x 10(6) N/N cells, and oxo8dG excretion varied from 32 to 397 pmol/kg/day. Although a wide range of values was observed in each genetic endpoint, the extreme values for various endpoints of genetic damage were not observed in the same individuals. The frequency of micronucleated erythrocytes varied over time within individuals and indicated that individuals with the highest levels of damage exhibit greater variability than those with lower levels. In some subjects, frequencies of micronucleated erythrocytes changed dramatically over an interval of 2-3 years: four subjects with initial micronucleated reticulocyte frequencies of 20.4, 5.9, 6.4 and 33/1000 changed to 2.5, 20.5, 18.5 and 12/1000, respectively. Among more than 150 individuals we have studied, including the 64 individuals studied by Everson et al.  and Smith et al. [(1990) ], the seven individuals with the highest observed frequencies of micronucleated erythrocytes all had exceptionally low values of plasma folate, red cell folate, or plasma B12, suggesting that folate and B12 status are the major determinants of the types of damage that lead to spontaneous micronucleus formation in erythrocytic cells.

title: A battery of DNA effect biomarkers to evaluate environmental exposure of Flemish adolescents
abstract: The present paper deals with the evaluation of a battery of genotoxicity biomarkers in healthy Flemish adolescents and their relation with common pollutants occurring in their life environment. DNA damage as reflected by the comet assay appeared to be most sensitive to ozone (partial r2 = 0.102, p < 0.00001), and to a lesser extent to ortho‐cresol (partial r2 = 0.055; p = 0.001) and 1‐hydroxy‐pyrene (1‐OH‐pyrene, partial r2 = 0.031; p = 0.013). 8‐hydroxy‐deoxyguanosine (8‐OHdG) was only related to ortho‐cresol (r2 = 0.069; p < 0.007). Interestingly, the comet assay results and urinary 8‐OHdG concentrations were positively correlated with a Pearson r = 0.21 (p = 0.003, N = 200). Logistic regression models revealed significant relations between chromatid breaks and 1‐OH‐pyrene (relative risk (RR): 1.58; p = 0.008), and t,t‐muconic acid (RR: 1.71; p = 0.014). There was no correlation between micronucleus formation or occurrence of chromosomal or chromatid breaks on the one hand and comet or 8‐OHdG results on the other hand. Thus, in this study the comet assay on whole blood samples and urine 8‐OHdG measurements especially appeared sensitive biomarkers for assessing the genetic effects of environmental pollutants to which adolescents may be exposed.

title: Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test.
abstract: The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.

title: Cigarette smoke alters human vitamin E requirements.
abstract: Vitamin E is a lipophilic chain-breaking antioxidant that prevents lipid peroxidation. Although cigarette smoke is a potent source of oxidative stress that depletes vitamin E in vitro, it is unclear whether it has a similar effect in vivo, particularly in humans. Therefore, this review will discuss the role of cigarette smoke on gamma-tocopherol (gamma-T) nitration, its effect on alpha-tocopherol (alpha-T) biokinetics in smokers, and the changes in the synthesis, plasma concentrations, and urinary excretion of the vitamin E metabolite (CEHC; carboxy-ethyl-hydroxy-chroman). Last, the possibility of CEHC as a biomarker of vitamin E status will be assessed as will the question whether smokers have increased dietary requirements of vitamin E.

title: Vitamin C and human health--a review of recent data relevant to human requirements.
abstract: The recent scientific literature indicates that beyond merely protecting against scurvy vitamin C contributes to many aspects of human health. The main areas of research reviewed include: 1. Vitamin C requirements of smokers. The data indicate that the vitamin C requirement of smokers is higher by at least 60 mg per day (up to 140 mg per day) than that of nonsmokers. 2. Important functions of the body, such as immune response, pulmonary function, and iron absorption are related to vitamin C intakes. Daily vitamin C intake of at least 150-200 mg per day enhance these functions. 3. Vitamin C may play critical roles in the prevention of CHD, cancer and cataract. Based on the available data, vitamin C intakes of at least 80-120 mg per day are associated with lowering the risk of these chronic diseases. 4. The literature documents that these and much higher intake levels of vitamin C are safe.

title: Influence of one bout of intensive running on lymphocyte micronucleus frequencies in endurance-trained and untrained men.
abstract: Exercise induced chromosomal damage was evaluated in trained and untrained subjects, who performed treadmill running at 85% of maximal oxygen uptake for 30 min. The subjects had their peripheral blood taken before, immediately after and 30 min after the running test for the analysis of lymphocyte chromosomal damage that was evaluated by micronucleus assay. The blood samples were also subjected to X-ray irradiation in vitro to examine the modification of exercise induced chromosomal damage by a secondarily induced oxidative stress. Spontaneous chromosomal damage in lymphocytes did not significantly increase at least until 30 min after the running both in the trained and untrained subjects. However, the X-ray-induced chromosomal damage was significantly enhanced at 30 min after the running in the untrained group, but not in the trained group. The ratio of X-ray-induced/spontaneous chromosomal damage also tended to increase after the running only in the untrained group. These preliminary results suggest that intensive exercise induced very slight chromosomal damage only in the untrained group, which could be intensified by the secondarily induced oxidative stress.

title: Exhaustive physical exercise increases frequency of micronuclei.
abstract: Micronucleus (MN) tests were performed on PHA-stimulated blood lymphocytes of six healthy volunteers before and after two exhaustive sprints, causing lactate concentrations in the peripheral blood between 9.6 and 12.4 mmol/l. The number of micronuclei was significantly increased after 24-48 h in all six subjects. The mean of the total group increased from 37 MN per 3000 binucleated cells before exercise to 56 and 55 MN 24 and 48 h after exercise, respectively. These differences were also significant. These results indicate that exhaustive physical exercise causes severe mutations at the chromosome level in blood lymphocytes.

title: Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection.
abstract: Chronic hepatitis C virus (HCV) infection is associated with an increased production of reactive oxygen species within the liver that are responsible for the oxidation of intracellular macromolecules. To ascertain whether the increased risk of hepatocellular carcinoma in individuals with chronic HCV infection is related to an accumulation of oxidative DNA damage, the 8-hydroxydeoxyguanosine (8-OHdG) content in the DNA of liver tissue and leukocytes of 87 individuals with HCV- or HBV-related liver disease and of 10 healthy controls was measured. Serum levels of thiobarbituric acid reactive substances (TBARS) were also assessed as an index of lipid peroxidation.



The 8-OHdG content in the circulating leukocytes correlated with that of liver tissue (r = 0.618, p < .0004). HCV patients had the highest median 8-OHdG levels (p < .0004). 8-OHdG leukocyte levels in HCV patients were higher than in HBV patients (p < .04) and they significantly correlated with the clinical diagnosis (p < .025), the serum ferritin levels (p < .05), and the amount of liver steatosis (p < .001). No correlation was found with age, gender, history of drinking or smoking, ALT or GGT levels, ESR, alpha-1, or gamma-globulin level and Ishak score. TBARS levels were significantly higher in cirrhotics than in noncirrhotics (p < .01).



The 8-OHdG level in circulating leukocytes is a reliable marker of oxidative stress occurring in the liver of individuals with chronic HCV infection. DNA oxidative damage appears to be an early and unique event in the natural history of HCV-related hepatitis. This injury increases the risk of genomic damage and may be one of the important factors involved in the carcinogenic process in cases of HCV-related chronic liver disease.

title: Relationship between levels of DNA damage in lymphocytes and histopathological severity of chronic hepatitis C and various clinical forms of hepatitis B
abstract: A significant proportion of cancer is attributable to DNA damage caused by chronic infection and inflammation. Because both hepatitis B and C viruses (HBV and HCV, respectively) cause chronic infection and inflammatory disease, the aim of the present study was to investigate whether there is a difference in peripheral DNA damage in patients with chronic HCV compared with patients with chronic HBV; and whether there is an association in the level of peripheral DNA damage with a natural history of HBV infection.

title: Chromosomal damage and ageing: effect on micronuclei frequency in peripheral blood lymphocytes.
abstract: Instability in the organization and expression of the genetic material has been hypothesized as the basic mechanism of ageing.



To quantify the effect of ageing on chromosomal damage as measured by spontaneous micronuclei (MN) frequency in peripheral blood lymphocytes.



Analysis of a large population sample from two laboratories applying the cytokinesis-block technique and a third using traditional interphase analysis. The age-related effect on baseline level of micronuclei frequency and on cell proliferation measures was further investigated in a study of peripheral blood samples from healthy subjects.



There was an increase of MN frequency with age. The regression lines showed a positive slope and were statistically significant (P< 0.01) with a steeper trend for cytochalasin B-treated samples. An inverse correlation with age was detected for the percentage of binucleated cells in laboratories using cytochalasin B. This study confirms the increase of basal level of MN with age. A decrease by age in proliferation efficiency measured by the percentage of binucleated cells suggests an interference of age-related factors on cell division.



There is an increase in MN frequency with increasing age.

title: Sources of variability in the human lymphocyte micronucleus assay: A population‐based study
abstract: The cytokinesis block method was used to examine the intraclass correlation coefficient of the human lymphocyte micronucleus assay, sources of variability, and practical issues regarding the number of samples per subject. Twenty samples of 100 binucleate cells from a single phlebotomy per subject were analyzed (n = 112), using methods to evaluate variance components. The results showed marked intraindividual (sampling error) variation greater than interindividual variation, and no between‐group contribution to the total variance. The intraclass correlation was 41.6%, indicating that slightly greater than half of the total variation in micronucleus outcomes was due to error variance (i.e., 58.4%). After adjusting for age, the intraclass correlation coefficient decreased trivially from 41.6% to 39.8%. There was a strong differential gender effect, favoring a greater micronuclei frequency in women. In conclusion, the data suggest that most of the variability in our data set for the micronucleus assay was due to sampling error; a strong differential gender effect favoring females was also verified. Equally important, in terms of practical applications, our analysis of the appropriate number of samples per subject revealed that scoring greater than 1,000 cells (10 determinations per subject) yielded no substantial improvement in statistical sensitivity, compared to the traditional 20 determinations. We suggest that more attention should be directed toward improving the assay's utility, while reducing sampling error.

title: Effect of vitamin-antioxidant micronutrients on the frequency of spontaneous and in vitro gamma-ray-induced micronuclei in lymphocytes of donors: the age factor.
abstract: The effect of prolonged consumption of a vitamin-antioxidant mixture (VAM) on the frequency of spontaneous and in vitro gamma-radiation-induced micronuclei (MN) in peripheral blood lymphocytes in donors of various ages was investigated. Three groups of donors were recruited: (i) 56-83 years old (35 subjects), (ii) 23-30 years old (13 subjects), and (iii) 63-82 years old (12 subjects). Blood was sampled every 4 months for one year in all donors of the three groups. After the first sampling of blood, the donors of groups (i) and (ii) took VAM containing the vitamins A, C, E, as well as beta-carotene, folic acid, and rutin daily for 4 months. After the second blood sampling, the intake of VAM was terminated. The third blood sample was taken 4 months after termination of VAM intake. A part of the blood was exposed to gamma-radiation and the frequency of spontaneous and induced MN in lymphocytes was assayed. The analyses showed that the frequency of spontaneous and in vitro gamma-ray-induced MN in aged donors was significantly higher than that in young donors. No seasonal variations in MN frequency were observed in human lymphocytes during one year. Aged donors showed a statistically significant decrease in spontaneous MN in lymphocytes after a 4 month period of consumption of VAM. The intake of VAM by both aged and young donors promoted a decrease in MN induced lymphocytes in vitro by gamma-radiation. The results of our observations enable the suggestion that consumption of VAM favours a decrease in the chromosome damage produced by endogenous and exogenous factors in human lymphocytes.

title: Low intake of calcium, folate, nicotinic acid, vitamin E, retinol, beta-carotene and high intake of pantothenic acid, biotin and riboflavin are significantly associated with increased genome instability--results from a dietary intake and micronucleus index survey in South Australia.
abstract: The aim of this study was to determine the association between dietary intake, determined using a food frequency questionnaire, and genome damage in lymphocytes measured using the micronucleus (MN) assay. The study, performed on 190 healthy individuals (mean age 47.8 years, 46% males), also examined whether a supplementation with beta-carotene, vitamins C and E along with zinc (ACEZn), in a randomized trial for 6 months, improves genome stability. Multivariate analysis of baseline data showed that (1) the highest tertile of intake of vitamin E, retinol, folic acid, nicotinic acid (preformed) and calcium is associated with significant reductions in MN frequency, i.e. -28, -31, -33, -46 and -49%, respectively (P < 0.005) relative to the lowest tertile of intake and (2) the highest tertile of intake of riboflavin, pantothenic acid and biotin was associated with significant increases in MN frequency, i.e. +36% (P = 0.054), +51% (P = 0.021), and +65% (P = 0.001), respectively, relative to the lowest tertile of intake. Mid-tertile beta-carotene intake was associated with an 18% reduction in MN frequency (P = 0.038); however, the highest tertile of intake (>6400 microg/day) resulted in an 18% increment in MN frequency. Supplementation with ACEZn significantly reduced the MN index by 13% (P = 0.038). The study also showed interactive additive effects such as the protective effect of increased calcium intake (-46%) and the exacerbating effect of riboflavin (+42%) on increased genome damage caused by low folate intake. The results from this study illustrate the strong impact of a wide variety of micronutrients and their interactions on genome health, depending on the level of intake.

title: Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma
abstract: Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-α-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.

title: Sex chromosomes, micronuclei and aging in women
abstract: Several studies on aneuploidy and aging have shown a significant increase in the loss of chromosomes in both males and females with age. Others have observed a significant increase in micronucleus formation in lymphocytes with age. The objectives of this investigation were to determine the relationship between sex chromosome loss and increased micronucleus frequencies with age, to establish sex chromosome loss frequencies unbiased by cellular survival factors or slide preparation, and to determine the effect of smoking on sex chromosome loss. Blood samples were obtained from 8 newborn females and 38 adult females ranging in age from 19 to 77. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Two thousand binucleated cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. Slides were then hybridized with a 2.0 kb centromeric X chromosome-specific probe labeled with biotinylated dUTP, and detected with fluorescein-conjugated avidin. All micronucleated cells were relocated and their X chromosome status was determined. We found the X chromosome to be present in 72.2% of the micronuclei scored; additionally our results show a significant increase with age in the number of micronuclei containing an X chromosome.

title: Cytogenetic effects in children and mothers exposed to air pollution assessed by the frequency of micronuclei and fluorescence in situ hybridization (FISH): a family pilot study in the Czech Republic.
abstract: A family pilot study was conducted in the Czech Republic to test the hypothesis that exposure to air pollution with particulate matter (PM) in children results in detectable effects indicated by a number of biomarkers of exposure and early effects. The frequency of micronuclei (MN) in peripheral blood lymphocytes (PBLs) was analysed to assess the cytogenetic effects in children and mothers living in two different areas. From each area two groups of children from a total of 24 families (mean age: 6.0+/-0.6 and 9.0+/-1.2 years) in a total of 47 children and 19 mothers (mean age: 33.6+/-3.9 years) participated. Chromosome aberrations determined with fluorescence in situ hybridization (FISH) painting for chromosomes #1 and #4 were analysed in 39 children and 20 parents. Teplice, a mining district, in Northern Bohemia was selected for the analyses of the effects in a population exposed to high levels of air pollution, especially during winter, and compared with a population from the rural area of Prachatice in Southern Bohemia. Significant higher frequencies of MN were found in the younger children living in the Teplice area as compared with those living in the Prachatice area (7.0+/-2.3 per thousand versus 4.9+/-2.0 per thousand, p=0.04). Higher levels of MN were also measured in the older children and the mothers from the Teplice area (9.2+/-3.7 per thousand versus 6.6+/-4.4 per thousand) and (12.6+/-3.4 per thousand versus 10.1+/-4.0 per thousand). The increased MN frequency may be associated with elevated carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) concentration of the PM(2.5) measured in the ambient Teplice air, but other factors like genotoxic compounds from the diet or protective effect of micronutrients, which was not addressed in this pilot study, may also differ between the two areas. MN frequencies were found to increase with age in children. Lower MN frequency was found in boys as compared to girls. The result of the FISH analyses showed a low number of individuals with detectable levels of aberrations and no significant increases in genomic frequency of stable chromosome exchanges (F(G)/100) were found in children or parents from the Teplice area in comparison with those from the Prachatice area. The family pilot study indicates that MN is a valuable and sensitive biomarker for early biological effect in children and adults living in two different areas characterised with significant exposure differences in c-PAHs concentrations during winter.

title: The role of folic acid and Vitamin B12 in genomic stability of human cells.
abstract: Folic acid plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. This activity is compromised when Vitamin B12 (B12) concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation and DNA hypomethylation. In vivo studies show that Vitamin B12 deficiency and elevated plasma homocysteine are significantly correlated with increased micronucleus formation. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is >227nmol/l. Intervention studies in humans show: (a) that DNA hypomethylation, chromosome breaks, uracil misincorporation and micronucleus formation are minimised when red cell folate concentration is >700nmol/l folate; and (b) micronucleus formation is minimised when plasma concentration of Vitamin B12 is >300pmol/l and plasma homocysteine is <7.5micromol/l. These concentrations are achievable at intake levels in excess of current RDIs i.e. more than 200-400microgram folic acid per day and more than 2microgram Vitamin B12 per day. A placebo-controlled study with a dose-response suggests that based on the micronucleus index in lymphocytes, an RDI level of 700microgram/day for folic acid and 7microgram/day for Vitamin B12 would be appropriate for genomic stability in young adults. Dietary intakes above the current RDI may be particularly important in those with extreme defects in the absorption and metabolism of these Vitamins, for which ageing is a contributing factor.

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

title: The impact of folate status and folic acid supplementation on the micronucleus frequency in human erythrocytes.
abstract: Folic acid has a well-documented stabilising effect on chromosomes. A correlation between folate status and chromosome stability in humans has been reported in studies that were restricted to certain subpopulations, e.g., folate-deficient persons. The goal of the present investigation was to clarify if there also is a correlation between folate status and chromosome stability among individuals without any folate deficiency. The method used here is the recently developed flow cytometry-based micronucleus assay in human transferrin-positive reticulocytes (MN-Trf-Ret). In a blood sample, separation of the very young reticulocytes from the mature erythrocytes makes this micronucleus assay possible. This investigation comprises three studies (cross-sectional, giving baseline data), two of which are connected to an intervention study. In the three cross-sectional studies (total number of subjects, 99) the frequency of MN-Trf-Ret (fMN-Trf-Ret) was measured and compared with the serum folate status. In two of the studies also serum homocysteine and Vitamin B12 were measured and compared with the baseline fMN-Trf-Ret. Combining the results from the three cross-sectional studies, a negative correlation between folate status and fMN-Trf-Ret was obtained (p<0.05). The goal of the intervention studies was to clarify if different nutritional supplementations had any effect on the fMN-Trf-Ret and the cell proliferation (percentage polychromatic erythrocytes, PCE). Each of the two studies involved two groups, one placebo and one supplemented group. In one of the studies the supplementation was folic acid, 1000 microg/day during 1 week (n=30, both sexes); in the other intervention study, folic acid (800 microg/day), B12 (20 microg/day) and B6 (4 mg/day) were taken during 1 week (n=29, both sexes). No significant difference in %PCE or fMN-Trf-Ret between the two groups was found in either of the two intervention studies.

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

title: IARC Monographs on the evaluation of carcinogenic risks to humans; dry cleaning, some chlorinated solvents and other industrial chemicals.
abstract: Mohamed Abdallah, University of Birmingham, United Kingdom Geza Benke, Monash University, Australia Mark F. Cesta, National Institute of Environmental Health Sciences, USA Melissa Friesen, National Cancer Institute, USA Dori Germolec, National Institute of Environmental Health Sciences, USA Keith Houck, U.S. Environmental Protection Agency, USA Gaku Ichihara, Tokyo University of Science, Japan Charles William Jameson, CWJ Consulting, LLC, USA Jun Kanno, Japan Organization of Occupational Health and Safety, Japan Hans Kromhout, Utrecht University, The Netherlands M. Matilde Marques, University of Lisbon Igor Pogribny, National Center for Toxicological Research, USA Consolato Maria Sergi, University of Alberta, Canada Camilla Svendsen, Norwegian Institute of Public Health, Norway

title: The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations.
abstract: The development of the cytokinesis-block (CB) technique has transformed the human-lymphocyte micronucleus assay (MN) into a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in (a) patients undergoing fractionated partial-body radiotherapy and (b) rodents exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring we performed extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various life-style factors. We have also developed a new variation to the CBMN assay that permits the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (UV, 254 nm) radiation and approximately 40-fold for methylnitrosourea. Consequently the CBMN assay can now be used to measure not only whole chromosome loss or chromosome breaks but also excision-repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should ensure its successful application in monitoring exposed populations as well as in identifying mutagen-sensitive individuals within a population.

title: The frequency of chromosome aberrations in a control population.
abstract: Chromosome aberration frequencies in a group of new entrants and non-radiation workers from this establishment have been studied using both block-staining and G-banding techniques. Increased levels of chromosome exchanges were found in those with a previous history of occupational or medical exposure to potential clastogens and in smokers. The possibility of an age effect was suggested. The study emphasizes the problems encountered in obtaining suitable control levels for comparison with occupational studies of exposure to clastogens.

title: Chromosomal aberrations in peripheral lymphocytes from healthy subjects as detected in first cell division.
abstract: Baseline frequencies of chromosomal aberrations were analysed in human peripheral lymphocytes and the influence of age, sex and smoking habits was considered. From 53 healthy subjects (29 males, 24 females) 54,689 exclusively first division cells (M1) were scored. The frequencies of chromosome aberrations per 1000 cells were 1.15 +/- 0.15 dicentrics (dic), 2.6 +/- 0.3 excess acentric fragments (ace) and 7.0 +/- 0.6 chromatid breaks (crb). An age dependency could only be established for ace. Between males and females no differences in any of the aberration types were observed. For heavy smokers (> 30 cigarettes per day) a significant increase was only found for dic (2.5 +/- 0.6 per 1000 cells). Dicentric frequency was compared with background levels of other studies in which results were reported also from exclusively M1 cells. Despite cell cycle control, differences between laboratories can be observed which may be partly influenced by environmental conditions. But on the other hand the mean frequency of dic (excluding heavy smokers) of 0.95 per 1000 cells reported here is consistent for more than one decade. Since such a consistency of the mean frequency of dic is reported also from another laboratory, the conclusion is drawn that especially for the detection of low-level exposures, each laboratory should establish its own base line data, otherwise, the interpretation of the findings is dependent on the selected background level from the literature.

title: The effect of smoking on the frequencies of asymmetrical and symmetrical chromosome exchanges in human lymphocytes.
abstract: Cytogenetic analysis was performed on blood lymphocyte cultures from 12 moderate smokers (15-20 cigarettes/day) and 12 non-smokers. An increase in dicentrics was observed in the smokers using block-stained material but this was not significant. Analysis of banded material, enabling both symmetrical and asymmetrical aberrations to be scored, revealed a significant increase in total aberrations thus emphasizing the need to identify all chromosome rearrangements when examining the effects of low-level chronic exposures to clastogens.

title: Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test.
abstract: The present study was undertaken to contribute to the characterization of the degree of variability in baseline damage in white blood cells from control population, and to investigate how this variability is associated with external and internal factors. Altogether 170 healthy volunteers, randomly selected from the general population of the Republic of Croatia, participated in the study. Two sensitive tests: the alkaline comet assay and the chromosome aberration test were applied to study the background levels of DNA damage in their white blood cells. The results point to inter-individual differences, indicating different genome sensitivity. As revealed by both assays, the background levels of DNA damage were mostly influenced by smoking habit as well as medical exposure (especially to diagnostic X-rays). Sex and age of subjects did not significantly influence the values of DNA damage recorded in the white blood cells. Although higher levels of DNA damage were recorded in blood samples collected during winter and autumn, they were mostly influenced by medicinal exposure and smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.

title: The effect of smoking on DNA effects in the comet assay: a meta-analysis.
abstract: The comet assay (alkaline single-cell gel electrophoresis, SCG or SCGE) is frequently used in biomonitoring to detect genotoxic effects in humans exposed at the workplace or in their environment. Because of its ready accessibility, blood is most frequently used in such studies. Many studies investigated cigarette smoking either as a genotoxic exposure itself or as a potential confounding factor in occupational studies. However, although smoking is considered to be a relevant exposure towards various genotoxins, conflicting results have been reported in the comet assay studies. The actual reasons for this discrepancy are not known. To further evaluate evidence for smoking-related DNA effects in the comet assay, we now used a meta-analysis approach based on a literature search. We identified 38 studies from 37 publications which were suited for a formal meta-analysis based on the standardized mean difference (SMD) between the study groups. The evaluation of these 38 studies indicated higher levels of DNA damage in smokers than in non-smokers [under a random effects model, SMD = 0.55, 95% confidence interval = (0.16-0.93)]. Subdividing these studies into studies investigating the effect of smoking as a genotoxic exposure (Type A studies, n = 12) and studies investigating smoking as a potential confounder in occupational studies (Type B, n = 26) indicated a significant difference only in Type A studies but not in Type B studies. Furthermore, studies using image analysis or image length measurements (n = 23) only indicated a tendency for a genotoxic effect of smoking, whereas studies using an arbitrary score (n = 15) found a significantly higher level of DNA damage in smokers.

title: Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress.
abstract: The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.

title: The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors.
abstract: Within the last decade, the comet assay has been used with increasing popularity to investigate the level of DNA damage in terms of strand breaks and alkaline labile sites in biomonitoring studies. The assay is easily performed on WBCs and has been included in a wide range of biomonitoring studies of occupational exposures encompassing styrene, vinyl chloride, 1,3-butadiene, pesticides, hair dyes, antineoplastic agents, organic solvents, sewage and waste materials, wood dust, and ionizing radiation. Eleven of the occupational studies were positive, whereas seven were negative. Notably, the negative studies appeared to have less power than the positive studies. Also, there were poor dose-response relationships in many of the biomonitoring studies. Many factors have been reported to produce effects by the comet assay, e.g., age, air pollution exposure, diet, exercise, gender, infection, residential radon exposure, smoking, and season. Until now, the use of the comet assay has been hampered by the uncertainty of the influence of confounding factors. We argue that none of the confounding factors are unequivocally positive in the majority of the studies. We recommend that age, gender, and smoking status be used as criteria for the selection of populations and that data on exercise, diet, and recent infections be registered before blood sampling. Samples from exposed and unexposed populations should be collected at the same time to avoid seasonal variation. In general, the comet assay is considered a suitable and fast test for DNA-damaging potential in biomonitoring studies.

title: Gene-environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes.
abstract: Ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome p4502E1 (CYP2E1) and catalase. This metabolite is then detoxified by aldehyde dehydrogenase 2 (ALDH2), a key enzyme in the elimination of acetaldehyde, via further oxidation to acetic acid. The toxic effects of acetaldehyde are well documented and may be partially mediated by genotoxic damage. In the present study, we investigated the effects of alcohol-drinking behavior and genetic polymorphisms in two different genes (ALDH2 and CYP2E1) on the micronuclei (MN) frequency in 248 healthy Japanese men. Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient type) was significantly associated with an increased MN frequency in subjects drinking more than three times/wk, while habitual drinkers with wild-type CYP2E1 also had a significantly increased MN frequency. Furthermore, when the subjects were divided into eight groups according to their drinking frequency and genotypes of ALDH2 and CYP2E1, we found that habitual drinkers with homozygous CYP2E1*1/*1 and heterozygous ALDH2*1/*2 or homozygous ALDH2*2/*2 showed the highest mean MN frequency. In the present study, we found clear associations among ALDH2 and CYP2E1 gene polymorphisms, alcohol-drinking behavior and genotoxic effects in a healthy Japanese population. Therefore, analysis of the polymorphisms of alcohol-metabolizing enzymes may lead to elucidation of the mechanism(s) for individual susceptibilities to the toxicity of ethanol metabolites.

title: Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers.
abstract: Alcohol abuse is a serious health problem, leading to life-threatening damage to most of the important organ systems. Genotoxic damage is used as an early effect indicator in the surveillance of human exposure to genotoxic substances. Intra- and inter-individual variations of baseline frequencies of micronuclei (MN) in peripheral blood lymphocytes of human populations have been reported previously. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impact of specific genetic variants on MN frequencies has not yet been clarified. In 42 healthy Japanese non-smoking men, we investigated the relationship between the MN frequency levels and genetic polymorphisms in three different genes: aldehyde dehydrogenase 2 (ALDH2), X-ray repair cross-complementing group 1 (XRCC1) and excision repair cross-complementing group 2 (ERCC2). Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient-type) was significantly associated with increased MN frequency levels in subjects with drinking more than three times per week, whereas the XRCC1 and ERCC2 variants seemed to be unrelated to the MN frequency. The ALDH2-deficient habitual drinkers had an average MN frequency of 5.88+/-0.58 (+/- S.E.) compared with 3.20 +/- 0.80 in the ALDH2-proficient habitual drinkers (P<0.05). The ALDH2-proficient non-habitual drinkers had the lowest MN frequency (1.56 +/- 0.41). Furthermore, subjects with highest levels of mean MN frequency, who consumed more than 100g of alcohol per week and more than three times per week, had A2 genotype of ALDH2. A significant odds ratio (12.25, P<0.05) for the MN frequency levels above the 50th percentile value was observed for the ALDH2-deficient individuals versus the ALDH2-proficient individuals after adjustment for several confounders. These results strongly suggest that human early genotoxic effect studies based on the cytogenetic markers of MN should take into account both the individual ALDH2 polymorphism and the potential confounding effect of the drinking behavior.

title: Association of ALDH2 polymorphism with sensitivity to acetaldehyde-induced micronuclei and facial flushing after alcohol intake.
abstract: To investigate whether sensitivity to the induction of micronuclei by acetaldehyde is associated with genetic polymorphisms of the aldehyde dehydrogenase-2 (ALDH2) gene, cytokinesis-block micronucleus (CBMN) assays were performed on peripheral lymphocytes from 47 healthy human subjects exposed to acetaldehyde in vitro. Facial flushing following alcohol intake was analyzed to determine if it was correlated with ALDH2 gene polymorphisms. The frequencies of the ALDH2 genotypes ALDH2(1)/ALDH2(1), ALDH2(1)/ALDH2(2), and ALDH2(2)/ALDH2(2) were 66.0, 27.7, and 6.4%, respectively, in the 47 subjects. Therefore, 34% of the studied subjects carried the mutant allele ALDH2(2), which is associated with the lack of enzyme activity. The frequency of micronuclei induced by acetaldehyde increased in a dose-dependent manner with the largest increase seen in subjects that were homozygous for the ALDH2(2) allele. A significant association was observed between the ALDH2 genotype and alcohol-induced facial flushing. Average alcohol consumption of the study subjects was also associated with the ALDH2 genotype. The frequency of heavy drinking was significantly higher among subjects with the ALDH2(1)/ALDH2(1) genotype than among subjects with the ALDH2(2) allele (ALDH2(1)/ALDH2(2) and ALDH2(2)/ALDH2(2) genotypes). Alcohol-induced facial flushing was also associated with an increased frequency of micronuclei in lymphocytes treated with acetaldehyde. The results suggest that the ALDH2 genotype is significantly associated with acetaldehyde-induced micronuclei and alcohol-induced facial flushing.

title: Genetic polymorphisms and micronucleus formation: a review of the literature.
abstract: The formation of micronuclei (MN) is extensively used in molecular epidemiology as a biomarker of chromosomal damage, genome instability, and eventually of cancer risk. The occurrence of MN represents an integrated response to chromosome-instability phenotypes and altered cellular viabilities caused by genetic defects and/or exogenous exposures to genotoxic agents. The present article reviews human population studies addressing the relationship between genetic polymorphisms and MN formation, and provides insight into how genetic variants could modulate the effect of environmental exposures to genotoxic agents, host factors (gender, age), lifestyle characteristics (smoking, alcohol, folate), and diseases (coronary artery disease, cancer). Seventy-two studies measuring MN frequency either in peripheral blood lymphocytes or exfoliated cells were retrieved after an extensive search of the MedLine/PubMed database. The effect of genetic polymorphisms on MN formation is complex, influenced to a different extent by several polymorphisms of proteins or enzymes involved in xenobiotic metabolism, DNA repair proteins, and folate-metabolism enzymes. This heterogeneity reflects the presence of multiple external and internal exposures, and the large number of chromosomal alterations eventually resulting in MN formation. Polymorphisms of EPHX, GSTT1, and GSTM1 are of special importance in modulating the frequency of chromosomal damage in individuals exposed to genotoxic agents and in unexposed populations. Variants of ALDH2 genes are consistently associated with MN formation induced by alcohol drinking. Carriers of BRCA1 and BRCA2 mutations (with or without breast cancer) show enhanced sensitivity to clastogens. Some evidence further suggests that DNA repair (XRCC1 and XRCC3) and folate-metabolism genes (MTHFR) also influence MN formation. As some of the findings are based on relatively small numbers of subjects, larger scale studies are required that include scoring of additional endpoints (e.g., MN in combination with fluorescent in situ hybridization, analysis of nucleoplasmic bridges and nuclear buds), and address gene-gene interactions.

title: Exposure to genotoxic agents, host factors, and lifestyle influence the number of centromeric signals in micronuclei: a pooled re-analysis.
abstract: We pooled data from three biomonitoring studies using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes in combination with fluorescence in situ hybridization. Centromere-positive micronuclei (C+MN) were classified in two groups: those containing one centromere (C1+MN) and those with two or more (Cx+MN). The three studies evaluated untreated cancer patients, welders, and pathologists/anatomists exposed to formaldehyde. The total number of subjects included in the pooled re-analysis was 113. A higher frequency of C+MN was observed in cancer patients and exposed workers, who showed significant differences from controls in all studies. C1+MN were particularly increased in the group of pathologists/anatomists, who showed a 3.29 times higher frequency than controls (95% CI: 2.04-5.30). A borderline increase in Cx+MN was observed in welders when compared to the corresponding control group (FR: 1.31; 95% CI: 0.99-1.74). An evident effect of gender was found, with significantly increased frequencies of all endpoints measuring aneuploidy in females (C+MN, C1+MN, and Cx+MN). Alcohol consumption had a significant effect on total MN frequency and particularly on C+MN and C1+MN. In conclusion, scoring the number of centromeric signals in the micronucleus assay provides additional information about the mechanism of action of various genotoxic agents, and the role of confounding factors may be more specifically accounted for. Indeed, C+MN could be efficiently used in biomonitoring studies as an independent biomarker of exposure and early biological effect. The use of centromeric signals allows the identification of two further endpoints, representing two alternative pathways of chromosome loss, i.e., impaired chromosome migration, leading to increased C1+MN frequency, and centrosome amplification, possibly leading to Cx+MN with two or more centromeric signals.

title: Indicators of genetic damage in alcoholics: reversibility after alcohol abstinence.
abstract: Although a primary carcinogenic role of alcohol is not proven, alcohol abuse is associated with an increased risk of cancer of the upper airways, esophagus and liver.



Chromosome aberrations and the presence of micronuclei that reflect two types of genetic damage were analyzed in peripheral blood lymphocytes from 11 alcoholic patients who were heavy smokers and in a fair state of general nutrition and 9 subjects who had abstained from alcohol for at least a year. Ten heavy smokers were studied as healthy controls.



Chromosome aberrations and micronuclei in alcoholics showed significantly elevated frequencies compared to the control groups, while in alcohol abstainers the values of all two parameters were similar to the values of the control subjects.



These results show that long-term alcohol intake could induce genetic alterations in synergy with tobacco smoke. Interestingly, this damaging action is reversed by abstinence. Our results call for a further effort to find an eventual diagnostic role for these early genetic damage markers.

title: Biomarkers to assess the genetic damage induced by alcohol abuse in human lymphocytes.
abstract: Alcohol abuse is a major risk factor for cancer of the upper alimentary tract, the upper respiratory tract, and liver. Chromosome damage is used as early effect biomarker in the surveillance of human exposure to genotoxic carcinogens. In the present study, two genetic markers, namely chromosome aberrations (CAs) and micronuclei (MN), were used to evaluate genetic damage in peripheral lymphocytes from 20 alcoholics, 20 abstinent alcoholics, and 20 controls. Composition of the three groups was fairly similar as regards sex, age and smoking habits. A highly significant increase was observed in the frequencies of CA and MN in lymphocytes of alcoholics as compared both with controls and abstinent alcoholics. However, no correlation was found between the length of alcohol abuse and the frequencies of either biomarkers in alcoholics. CA and MN frequencies in abstinent alcoholics were similar than those in controls. Our data indicate that CA and MN can be two useful biomarkers to assess genetic damage associated with alcohol abuse. They could be included in programs for cancer prevention in alcoholics. Abstinence appears to normalize the frequency of both MN and CA. This could offer therapists another tool to help alcoholics change their lifestyle.

title: A comparison of lymphocyte micronuclei and plasma micronutrients in vegetarians and non-vegetarians.
abstract: We performed a biochemical and cytogenetic epidemiological study to establish if there are significant differences between vegetarians (V) and non-vegetarians (NV) in their peripheral blood lymphocyte micronucleus (MN) index, which is a measure of chromosome damage rate. The levels of plasma vitamin C (VIT-C), vitamin E (VIT-E), vitamin B12 (B12) and folic acid were also analysed to assess if differences in chromosome damage rates were associated with these potentially antimutagenic micronutrients. Volunteers were classified as either 'vegetarian' if they had abstained from eating any flesh foods for at least 3 years prior to the study or 'non-vegetarian' if they consumed meat or meat products at least 5 days/week for at least 3 years before participation in the study. The volunteers in the study consisted of 47 male and 79 female V and 66 male and 72 female NV, all of whom were non-smokers for at least 3 years prior to the study. The age of the volunteers varied between 20 and 89 years. There was no significant difference in the slope of the age-related increase in MN index of V and NV of either sex. However, the MN index was significantly lower in NV males in the age group 20-40 years and significantly lower for V males in the 41-60 years age group. No difference between the MN index of older males was detectable and there also was no difference in the MN index of V and NV females across all age groups. V were generally found to have significantly higher plasma levels of VIT-C and folic acid, significantly lower levels of B12, and similar levels of VIT-E when compared with NV. VIT-C correlated positively with MN index in young males, but the reverse was true for B12. In young females folate and B12 appeared to correlate negatively with MN index. VIT-E had no apparent impact on MN index. These data suggest that the level of folate and B12 may be more important than VIT-C or VIT-E in minimizing chromosome damage rates in human lymphocytes. Overall, the data from this study do not support the hypothesis that V have a lower genetic damage rate than NV.

title: Does a vegetarian diet influence
genomic stability?
abstract: The vegetarian lifestyle is supposedly healthy, and
differences between vegetarians and non-vegetarians in
biomarkers related to diseases such as cancer might be
expected.To investigate the possible role of different diets in
maintaining genomic stability.The vegetarian group, consisting of 24 volunteers (13
women and 11 men), were matched for age and sex with 24
volunteers (12 women and 12 men) with a traditional dietary
habit.Among vegetarians there were 13 lacto-ovo-vegetarians (8
women, 5 men) with average length of vegetarian diet 10.8 years
(ranging from 5 to 26) and 11 lacto-vegetarians (5 women, 6 men)
with average length of vegetarian diet 8.2 years (ranging from 3
to15). All volunteers were nonsmokers, non-consumers of alcohol
and had similar education and patterns of physical activity.
Chromosome aberrations, micronuclei and DNA damage (strand
breaks, oxidised bases and
H2O2-sensitivity)
were examined in peripheral blood lymphocytes of vegetarians and
non-vegetarians. Plasma antioxidant status was assessed with the
FRAP assay.We did not find any differences in percentage of cells
with chromosome aberrations or in the frequency of micronuclei
between vegetarians and non-vegetarians or between lacto-ovo and
lacto-vegetarians. There was no statistically significant
difference in total antioxidant capacity between the groups. The
group with traditional dietary habits had significantly higher
levels of oxidative DNA damage (strand breaks and oxidised
purines, P = 0.005) compared with vegetarians. A significant
positive correlation between age and oxidative DNA damage (net
FPG-sensitive sites) was found in non-vegetarians, while there
was an opposite trend towards a negative association in
vegetarians. On the other hand chromosome aberrations correlated
with age in vegetarians (r = 0.48, P = 0.017) but not in
non-vegetarians.Our results indicate that a vegetarian diet can lead to a
slight decrease in oxidative DNA damage in lymphocytes, but
other markers of genetic stability are not affected. The lowest
level of DNA damage was found in lymphocytes of
lactovegetarians, (especially oxidised pyrimidines, P = 0.0017),
suggesting that this diet provides some protection against
oxidative stress.

title: The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians.
abstract: A vegetarian diet results in higher intake of vitamins and micronutrients, which - although providing antioxidant defence - may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, beta-carotene, B(12), folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and beta-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B(12) were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and beta-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P<0.001). There were no significant differences in serum levels of folic acid and Vitamin B(12) between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r=0.62, P=0.001, N=24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P=0.007), as did vegetarian women compared with non-vegetarian women (P=0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r=0.62, P<0.001, N=48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N=48, P<0.001).

title: Nutritional and lifestyle determinants of DNA oxidative damage: a study in a Mediterranean population.
abstract: In order to evaluate dietary and lifestyle determinants of oxidative DNA damage we used a modification of the 'comet assay' (single cell alkaline gel electrophoresis), with the fpg enzyme (formamidopyrimidine DNA glycosilase), to measure the basal level of DNA oxidation in peripheral lymphocytes donated by 71 healthy adults living in Florence, Italy. Detailed information about dietary and lifestyle habits was collected by two validated and standardized questionnaires; we also measured plasma concentrations of selected micro-nutrients (six carotenoids, retinol, alpha- and gamma-tocopherol). DNA damage, measured as percent DNA migrated in the comet tail (mean 4.67%, interquartile range 2.36-6.62%), was not associated with gender, age, weight, body mass index, physical activity or smoking history. A positive correlation with height and period of blood sampling emerged: DNA damage tended to be higher among taller subjects (P = 0.02) and in samples obtained in summer months (P = 0.02). Multivariate analyses showed a positive association with coffee (P = 0.01) and tomato consumption (P = 0.05). Instead, the consumption of cruciferous vegetables tended to be negatively associated with oxidative damage (P = 0.09). Furthermore, a positive non-significant association between the consumption of total vegetables and fresh fruit and DNA damage emerged (P = 0.08 and P = 0.10, respectively). The estimated intake of simple sugars showed a strong positive association with oxidative DNA damage (P = 0.01), while vitamin E showed a borderline positive association (P = 0.06). The plasma levels of several micro-nutrients did not appear to influence DNA damage. Our results, although based on a relatively small group of subjects, indicate that individual dietary and lifestyle habits only modestly affect the levels of lymphocyte DNA oxidation and suggest that specific dietary patterns, rich in fresh fruit and vegetables, are not clearly related to decreased oxidative damage in peripheral lymphocytes in a Mediterranean population.

title: Importance of diet in protection against oxidative damage.
abstract: In humans, epidemiological evidence suggests that increased consumption of fruits and vegetables can substantially enhance the protection against many common types of cancer.



A molecular epidemiological study in 3 Slovak factories producing asbestos, glass fibres and rockwool was conducted. Altogether 388 subjects (239 exposed, 148 controls) were investigated. Food frequency questionnaire was used to ascertain nutrient intake and compared to plasma levels of selected micronutrients, as well as to markers of oxidative stress (MDA, oxidative DNA damage and DNA repair) and antioxidant protection. We found a negative correlation between MDA concentrations and consumption of fruits (p=0.05) and vegetables (p=0.05) in all control subjects. Intake of fruits (p=0.05), vegetables (p=0.01), milk (p=0.01) and cereals (p=0.05) inversely correlated with oxidative DNA damage (net FPG) in all subjects investigated. There was a negative correlation between the intake of fruits (p=0.05) and vegetables (p=0.01) in all exposed subjects.



Our results suggest that well balanced food consumption with higher fruits and vegetables intake has a protective effect against oxidative damage.

title: Diet and Biomarkers of Oxidative Damage in Women Previously Treated for Breast Cancer
abstract: This study sought to evaluate the relationship between dietary intake of fat, polyunsaturated fat, saturated fat, arachidonic acid, and selected dietary antioxidants and levels of oxidative damage as measured by urinary levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-epi-prostaglandin F2α (8-iso-PGF2α) in women previously treated for breast cancer. Two hundred two study subjects participating in the Women's Healthy Eating and Living (WHEL) study were included in this ancillary study. Dietary intakes and concentrations of urinary 8-OHdG and 8-iso-PGF2α were measured at baseline and 12 mo in the 179 women included in the analytical cohort. Study subjects demonstrated a significant reduction in dietary total, polyunsaturated, and saturated fat intake and a significant increase in vitamins E and C and β-carotene intake from baseline to 12 mo. Linear mixed-models analysis using baseline and Year 1 data indicated that vitamin E intake was inversely associated with both 8-OHdG and 8-iso-PGF2α. 8-Iso-PGF2α is increased with increased body mass index (BMI) and polyunsaturated fatty acid (PUFA) intake, indicating an increase in lipid peroxidation with greater BMI and higher PUFA intake. 8-OHdG was inversely related to age but positively related to arachidonic acid, indicating an increase in DNA damage with higher intake of arachidonic acid (meat). The results of this nested case-controlled study provide potential mechanisms by which a high fruit and vegetable, low-fat diet might reduce the recurrence rate of or early-stage breast cancer.

title: Associations of Antioxidant Nutrients and Oxidative DNA Damage in Healthy African-American and White Adults
abstract: High antioxidant intake has been shown to reduce cancer risk and may also mitigate the effects of oxidative DNA damage, which is hypothesized to be causally linked to carcinogenesis. This study examined potential racial differences in (a) dietary intakes and plasma concentrations of vitamin C, vitamin E, and carotenoids and oxidative DNA damage and (b) associations between plasma antioxidants and oxidative DNA damage. Data were from a cross-sectional study of 164 generally healthy nonsmoking African-Americans and Whites in North Carolina, ages 20 to 45 years, equally distributed by race and sex. Participants completed a demographic and health questionnaire, four 24-h dietary recalls, and a dietary supplement inventory; had height and weight measured; and provided a semifasting blood sample. African-Americans had statistically significantly lower plasma concentrations of vitamin E, α-carotene, β-carotene, and lutein + zeaxanthin than Whites, as well as lower self-reported intake of most antioxidants. Levels of oxidative DNA damage, measured using the alkaline comet assay, were lower in African-Americans than Whites. An inverse association between lycopene and oxidative DNA damage (r = −0.20; P = 0.03) was found in the combined study population after adjusting for sex, age, body mass index, passive smoke exposure, physical activity, education, income, and alcohol intake. There was also a positive association of vitamin E with oxidative DNA damage in the total population (r = 0.21; P = 0.02) and in African-American men (r = 0.63; P = 0.01) after adjusting for covariates. This study is among the first to examine these associations in a sample of healthy adults with an adequate representation of African-Americans.

title: Antioxidant supplementation reduces inter-individual variation in markers of oxidative damage
abstract: The aim of this study was to examine the effect of antioxidant supplementation on oxidative damage and chromosome stability in middle-aged men, smokers and non-smokers. A total of 124 men aged 48±6 years from Bratislava and from the rural population near Bratislava were investigated; 64 men (22 smokers and 42 non-smokers) were supplemented for 12 weeks with antioxidants, while 60 (25 smokers and 35 non-smokers) were given placebo. The daily antioxidant supplementation consisted of vitamin C (100 mg), vitamin E (100 mg), β-carotene (6 mg), and selenium (50 μg). Samples of blood were taken on two occasions: At the beginning and at the end of the supplementation trial. Concentrations of dietary antioxidants, ferric reducing ability, malondialdehyde as an indicator of lipid peroxidation in plasma, micronuclei and chromosome aberrations in lymphocytes were measured. Antioxidant supplementation significantly increased the levels of vitamin C, β-carotene, α-tocopherol and selenium in plasma. The overall antioxidant status of plasma measured as ferric reducing ability of plasma (FRAP) increased significantly (p<0.001) after antioxidant supplementation as well. The increase in antioxidant parameters after supplementation were consistently more pronounced in non-smokers than in smokers. There was a significant decrease of malondialdehyde concentration in the non-smokers, while in smokers the decrease of malondialdehyde concentration was not significant. Antioxidant supplementation did not affect the proportion of lymphocytes with micronuclei or the total number of micronuclei; however, there was a significant positive correlation (p<0.001) between the malondialdehyde concentration at the beginning of the supplementation trial and the difference in number of cells with micronuclei before and after the supplementation. The percent of cells with chromosome aberrations decreased significantly after antioxidant supplementation in smokers. These results indicate that a combined antioxidant supplementation (a) is effective even at very moderate doses; (b) significantly diminishes oxidative damage to lipids when it is high initially; and (c) is effective in decreasing chromosomal instability in lymphocytes of middle-aged men.

title: Vitamin C and genomic stability.
abstract: Vitamin C, a water-soluble glucose derivative, has considerable antioxidant activity in vitro, in part because of its ease of oxidation and because the semidehydroascorbate radical derived from it is of low reactivity. Vitamin C in vivo is an essential cofactor for a range of enzymes involved in diverse metabolic pathways, but much recent literature has focused on its antioxidant effects. Consumption of foods rich in Vitamin C (fruits and vegetables) is associated with decreased risk of cardiovascular disease, of many types of cancer and possibly of neurodegenerative disease, but the extent to which Vitamin C contributes to these effects is uncertain. Data using biomarkers of oxidative damage to DNA bases have given no compelling evidence to date that ascorbate supplements can decrease the levels of oxidative DNA damage in vivo, except perhaps in subjects with very low Vitamin C intakes. Similarly, there is no conclusive evidence from studies of strand breaks, micronuclei, or chromosomal aberrations for a protective effect of Vitamin C. There is limited evidence that supplements of Vitamin C might have beneficial effects in disorders of vascular function, and that diet-derived Vitamin C may decrease gastric cancer incidence in certain populations, but it is not clear whether it is the antioxidant or other properties of ascorbate that are responsible for these two actions.

title: Vitamin E and genome stability.
abstract: Free radicals and reactive oxygen species (ROS) which are generated continuously cause mutagenic alterations resulting in cancer, aging and abnormalities in the nervous system. Accumulating evidence indicates that Vitamin E, the most potent lipid peroxyl radical scavenger, may reduce free radical induced chromosomal damages through inhibition of free radical formation, and activation of endonuclease that can be triggered by intracellular oxidative stress, and by increasing the rate of removal of damaged DNA. Although some studies suggest a potential usefulness of Vitamin E in the prevention of mutagenic effects caused by genotoxic free radicals, other studies report no effects. Thus the data are not conclusive enough to be used as a basis to change the current recommended dietary allowances (RDA). Future research should address molecular mechanisms underlying the protective effects of Vitamin E and develop appropriate biologically relevant biomarkers of DNA damage to further help in determining the dietary levels of Vitamin E needed to protect the genetic pool from internally and externally induced DNA damages.

title: Recommended dietary allowances (RDAs) for genomic stability.
abstract: Diet as a key factor in determining genomic stability is more important than previously imagined because we now know it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis. Current recommended dietary allowances for vitamins and minerals are based largely on the prevention of diseases of deficiency such as scurvy in the case of Vitamin C. Because diseases of development, degenerative disease and ageing itself are partly caused by damage to DNA, it seems logical that we should focus better our attention on defining optimal requirements of key minerals and vitamins for preventing damage to both nuclear and mitochondrial DNA. To date our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganised. Appropriately designed placebo, controlled trials are required to define recommended dietary allowances for genomic stability. Recently, it has been shown that above RDA intakes of folic acid and Vitamin B12 are required to reduce the micronucleus index in humans by 25%. In the future, clinical trials with a defined wider array of complementary DNA damage end-points would be necessary. That there is a need for an international collaborative group to establish RDAs for genomic stability is self-evident and this paper is a call for such a process to begin.

title: Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients
abstract: It is well established that oxidative stress plays a key role in the degenerative neuronal death and progression of Alzheimer's disease (AD), although it is not clear if it is the primary triggering event in the pathogenesis of this disorder. Mild cognitive impairment (MCI) is a clinical condition between normal aging and AD, characterized by a memory deficit without loss of general cognitive and functional abilities. We performed this study by a comet assay analysis to evaluate the level of primary and oxidative DNA damage in two groups of MCI and AD patients, compared to healthy controls. Data showed a significantly higher level of primary DNA damage in leukocytes of AD and also of MCI patients compared to control individuals (average: 2.09+/-0.79 and 2.47+/-1.01, respectively for AD and MCI, versus 1.04+/-0.31 in controls). Moreover, the amount of oxidised DNA bases (both purines and pyrimidines) was significatively higher in the two groups of patients (AD and MCI) compared to controls. Our results give a further indication that oxidative stress, at least at the DNA level, is an earlier event in the pathogenesis of AD.

title: Relationship between DNA damage, total antioxidant capacity and coronary artery disease.
abstract: It has been known that there was a relation between the levels of DNA damage and the severity of the coronary artery disease (CAD). However, little is known about association of DNA damage with total antioxidant capacity (TAC) and CAD. The aim of this study is to evaluate the relationship between DNA damage, TAC and CAD.



We used the comet assay to measure DNA damage from 53 patients with angiographically documented CAD and 42 patients with angiographically documented normal coronary vessel. The extent and severity of CAD was calculated to Gensini score index. TAC of plasma was determined using a novel automated measurement method.



Mean values of DNA damage were significantly higher in CAD patients than in the control group (p<0.001). There was a positive correlation between Gensini score index and DNA damage (r=0.590, p<0.001). Additionally, significantly positive correlations between score of DNA damage, and diabetes, smoking, obesity and hyperlipidemia were found (p<0.05). There was also a negative correlation between TAC and DNA damage (r=-0.711, p<0.001). The DNA damage was significantly higher in diabetic, smoker, hyperlipidemic and obese individuals than those without these conditions (p=0.001, p=0.006, p=0.001, p=0.004, respectively).



These data indicate that the level of DNA damage is increased and TAC level is decreased in CAD. DNA damage is correlated with the severity of the CAD, and levels of TAC.

title: Chromosomal aberrations and micronuclei in lymphocytes of patients before and after exposure to anti-tuberculosis drugs.
abstract: To evaluate the genetic damage induced by tuberculosis infection and also by a combination of therapeutic exposure to anti-tuberculosis (anti-TB) drugs (isoniazid + rifampicin + pyrazinamide + ethambutol or streptomycin) we have considered chromosome aberrations (CA) and micronuclei (MN) in binucleate peripheral blood lymphocytes from 36 patients diagnosed with pulmonary tuberculosis prior to receiving anti-TB drugs in the first phase and in the second phase after 6 months of therapy with anti-TB drugs. The same cytogenetic study has also been performed on 36 healthy individual controls. It was shown that the frequencies of both CA and micronucleated binucleate cells increased significantly after therapy with anti-TB drugs as compared with TB patients and controls. In addition, we observed that both cytogenetic markers (CA and MN) in patients before treatment with anti-TB drugs were significantly (P: < 0.05) higher than controls. No relationship was found between the frequency of MN and donor age. Thus, apart from drug effects, infection with tuberculosis is associated with increased CA and MN.

title: Analysis of Mitomycin C‐induced micronuclei in lymphocytes from malnourished infected children
abstract: The purpose of this study was to determine if peripheral blood lymphocytes from malnourished children with gastrointestinal or respiratory bacterial infection show increased frequencies of Mitomycin C (MMC)‐induced micronuclei as compared to well‐nourished, infected children. The results indicate that cells from malnourished, infected children had greater chromosome damage. This may indicate that such children would be more susceptible to environmental damage and malignant transformation.

title: Increased frequency of micronuclei in peripheral blood lymphocytes of subjects infected with Helicobacter pylori.
abstract: Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori and the development of gastric carcinoma and mucosa-associated lymphoid tissue lymphomas in humans. The cytokinesis-block micronucleus assay was performed on peripheral blood lymphocytes of H. pylori-infected patients in order to investigate the possible induction of genotoxic damage. The study group consisted of 70 infected subjects including 33 women and 37 men, and 66 healthy controls (37 females and 29 males). Our results indicate that in the infected group the overall frequency of binucleated micronucleated cells (BNMN) per 1000 cells was higher (17.65+/-1.55) than in the controls (7.39+/-0.66), this difference being statistically significant. No differences were found between the infected and control groups regarding the cytokinesis-block proliferation index (CBPI). When the effect of different counfounding factors was evaluated, mutivariate statistical analysis revealed that age and alcohol consumption modulated the frequency of BNMN in infected people, and the interaction between alcohol use-smoking-infection also affected the BNMN frequency in H. pylori patients. Our results indicate that infection by H. pylori is associated with an increased level of cytogenetic damage in the cells of the host.

title: Helicobacter pylori causes DNA damage in gastric epithelial cells.
abstract: Helicobacter pylori infection has been considered as a risk factor for gastric carcinoma. Strong evidence exists that reactive oxygen species (ROS) play an important role in carcinogenesis, and in vivo investigations have shown increased synthesis of ROS in the gastric mucosa of H.pylori-infected patients. In the present study the direct effects of H.pylori on ROS and DNA synthesis, induction of apoptosis and DNA repair were investigated in the gastric epithelial cell lines AGS and HM02. Incubation of gastric cells with H.pylori extract induced the synthesis of ROS, diminished the levels of reduced glutathione (GSH), induced DNA fragmentation and increased DNA synthesis in gastric cells. Poly(ADP-ribose) formation was increased in gastric cells exposed to H.pylori extract. FACS analysis of gastric cells exposed to H.pylori extract did not reveal any change in the percentage of cells in the G(2)/M phase of the cell cycle. The radical scavengers MnTBAP (a cell permeable superoxide dismutase mimic), ebselen (a GSH peroxidase mimic) and high doses of catalase completely blocked H.pylori extract-induced elevation in DNA synthesis. Our results indicate that H.pylori extract directly induces the synthesis of ROS in gastric epithelial cells and causes DNA damage.

title: Viral induction of site‐specific chromosome damage
abstract: The advent of advanced cell culture and cytogenetics techniques in the 1950s opened a new avenue for research on the pathogenic interactions between animal viruses and their hosts. Studies of many viruses revealed their ability to nonspecifically induce cytogenetic damage to their host cell's chromosomes. However, only three viruses, the oncogenic adenoviruses, herpes simplex virus (HSV) and human cytomegalovirus (HCMV), have been found to cause non‐random, site‐specific chromosomal damage. Adenovirus (Ad) type 12 induces fragility at four distinct loci (RNU1, RNU2, RN5S and PSU1) in many different types of human cells. A common feature of these loci is that they contain a repeated array of transcriptionally active genes encoding small structural RNAs. Site‐specific induction of breaks also requires the virally encoded E1B protein of Mr 55 000 and the C‐terminus of the cellular p53 protein. Analysis of the induction of damage by HSV and HCMV necessitates consideration of several factors, including the strain of virus used, the timing of infection, the type of cell used, and the multiplicity of infection. Both HSV strains 1 and 2 are cytotoxic, although the former seems to be more proficient at inducing damage. At early times post infection, HSV induces breaks and specific uncoiling of the centromeres of chromosomes 1, 9 and 16. This is followed at later times by a more complete severing of all of the chromosomes, termed pulverisation. Damage by HSV requires viral entry and de novo viral protein synthesis, with immediate early viral proteins responsible for the induction of breaks and uncoiling and early gene products (most likely nucleases) involved in the extensive pulverisation seen later. HCMV has been studied primarily in permissive human fibroblasts. Its ability to induce specific damage in chromosome 1 at two loci, 1q21 and 1q42, was only recently revealed as the cells must be in S‐phase when they are infected for the breaks to be observed. In contrast to adenovirus and HSV, HCMV induction of specific breakage requires only viral entry into the cell and not de novo viral protein expression. This latter point may be a factor in its ability to cause damage in the developing fetal brain, where the most severe clinical manifest ations of congenital infection are observed.

title: DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study.
abstract: Oxidative DNA damage, identifiable in the formation of 8-hydroxydeoxyguanosine (8-OHdG), is relevant in the mutagenesis/carcinogenesis process. The aim of this study was to assess 8-OHdG levels in patients with hepatitis C virus (HCV) infection in relation to extent of liver damage and HCV genotype.



8-OHdG levels were measured in DNA from circulating leukocytes of 110 anti-HCV positive subjects belonging to the population of the Dionysos study, subgrouped in: 50 anti-HCV+ with persistently normal ALT, 48 with chronic hepatitis and 12 with cirrhosis. Twenty normal subjects served as Controls. 8-OHdG levels were assayed by HPLC/electrochemical detector.



8-OHdG levels rose (P < 0.00001) from Controls to HCV+; chronic hepatitis and cirrhosis were associated with a further increase (P < 0.02 versus HCV+). Genotype 1 was associated with higher levels of 8-OHdG (P < 0.04). Multiple logistic regression analysis showed that, after correction for potential confoundings, 8-OHdG levels correlated (P < 0.02) with presence and extent of liver damage.



An accumulation of 8-OHdG in circulating leukocytes is a reliable marker of the extent of liver damage in HCV+ patients and is present in particular in genotype 1 infection. This genomic damage may contribute to liver carcinogenesis by causing persistent DNA changes.

title: Differential mutagen sensitivity of peripheral blood lymphocytes from smokers and nonsmokers: Effect of human cytomegalovirus infection
abstract: We used the mutagen sensitivity assay to test the hypothesis that human cytomegalovirus (HCMV) infection modifies the sensitivity of cells to genetic damage from genotoxic agents. Chromosome aberration (CA) frequency in peripheral blood lymphocytes (PBLs) from 20 smokers who were matched with 20 nonsmokers by age (± 5 years), sex, and ethnicity was evaluated following in vitro exposure to bleomycin and/or HCMV infection. Bleomycin induced significant (P < 0.05) concentration‐dependent increases in the frequency of aberrant cells, chromatid‐type damage (breaks), and chromosome‐type aberrations (deletions, rearrangements) in PBLs. The baseline (background) CA frequency was similar in both smokers and nonsmokers. Significantly higher frequencies of aberrant cells (P < 0.05) were observed in PBLs from smokers compared to nonsmokers at all bleomycin concentrations tested (10, 30 and 100 μg/ml). Infection of PBLs with HCMV induced a significant (P < 0.05) twofold increase in the frequency of CA (primarily chromatid breaks) in PBLs, regardless of the smoking status. PBLs from smokers and nonsmokers infected with HCMV prior to challenge with bleomycin demonstrated significant (P < 0.05) concentration‐dependent increases in the levels of aberrant cells, chromatid‐type damage (breaks), and chromosome‐type aberrations (deletions, rearrangements) compared to noninfected cells challenged with bleomycin. The frequency of induced CA was consistently higher for PBLs derived from smokers relative to nonsmokers (P = 0.06 and 0.002). These data indicate that, individually, both smoking and HCMV infection significantly enhance the sensitivity of PBLs to bleomycin‐induced genetic damage. More importantly, the data also suggest that smoking and HCMV infection interact synergistically to enhance the sensitivity of PBLs to such damage.  2004.

title: Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1
abstract: Vpr, an accessory gene of HIV‐1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT‐23, in which Vpr expression is controlled by the tetracycline‐responsive promoter. Treatment of MIT‐23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT‐23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy‐terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS‐related malignancies.—Shimura, M., Tanaka, Y., Nakamura, S., Minemoto, Y., Yamashita, K., Hatake, K., Takaku, F., Ishizaka, Y. Micronuclei formation and aneuploidyinduced by Vpr, an accessory gene of human immunodeficiency virus type 1. FASEB J. 13, 621–637 (1999)

title: Prolonged exercise does not cause lymphocyte DNA damage or increased apoptosis in well-trained endurance athletes
abstract: Recent research has demonstrated that lymphocyte apoptosis sensitivity appears to be related to training status and exercise intensity. This work investigated the effect of prolonged, submaximal treadmill running on percentage (%) apoptosis, % necrosis and DNA strand breaks in lymphocytes and related these to changes in total lymphocyte and blood cortisol concentrations in well-trained runners. Venous blood samples (n = 14) were taken immediately before (PRE), immediately after (IPE) and 3 h after (3PE) 2.5 h of treadmill running at 75% of VO2 max from eight well-trained male endurance athletes (age 34.2 ± 2.44 years) and analysed for cellular content and serum cortisol concentrations. Lymphocytes were isolated from whole blood and % apoptotic and necrotic cell were detected by flow cytometry using Annexin V-FITC and propidium iodide uptake. DNA strand breaks were measured by single-cell gel electrophoresis. Despite a significant (P < 0.001) exercise-induced increase in mean serum cortisol concentrations and reduction in lymphocyte counts, the mean % Annexin-V positive cells (13.3 ± 6.78 in PRE, 11.3 ± 5.51 in IPE and 12.8 ± 6.75 in 3PE samples) were not significantly different at the three time-points (P > 0.05). Mean DNA strand breaks in the lymphocytes also did not change significantly (P > 0.05) rising from 25.7 ± 2.16 to 26.9 ± 1.89 and 27.1 ± 1.38 μm in IPE and 3PE samples, respectively. The exercise-induced changes in total blood lymphocyte counts and cortisol concentrations did not result in a significant change in % apoptotic lymphocytes or DNA strand breaks in the endurance-trained athletes during this prolonged, submaximal exercise.

title: Does physical activity induce DNA damage?
abstract: The single cell gel electrophoresis (SCG) assay (comet assay) is a sensitive technique for detecting the presence of DNA strand-breaks and alkali-labile damage in individual cells. This technique was used to study peripheral blood cells from three volunteers after physical activity. The test subjects had to run on a treadmill and were checked for blood pressure and ECG, lactate concentration and creatine kinase activity. Blood was taken before and several times during and after the run. In a first multiple step test, the volunteers ran as long as possible with increasing speed. In a second test they had to run for 45 min with a fixed individual speed which was defined to ensure an aerobic metabolism. In the first test, the white blood cells of all subjects showed increased DNA migration in the SCG assay. The effect was seen 6 h after the end of the exercise and reached its maximum 24 h later. After 72 h, DNA migration decreased to about control level. The distribution of DNA migration among cells clearly demonstrated that the majority of white blood cells exhibited increased DNA migration and that the effect was not only due to a small fraction of damaged cells. From the same blood samples, blood cultures were set up to study a possible effect on the frequency of sister chromatid exchanges (SCE), another indicator for genotoxic effects. However, there was no significant increase in SCE in any of the cultures. In the second exercise, during aerobic metabolism, the effect on DNA migration was not seen.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Effects of intensive endurance exercise on DNA damage in leucocytes.
abstract: It has been shown that highly intensive anaerobic exercise induces DNA damage in leucocytes (LEU). The present study was designed to investigate whether intensive endurance exercise is capable of inducing comparable effects.



A prospective study.



Twelve men (aged 27.3 +/- 4.1 years) who undertook a regular training of different extent (running volume 45 +/- 25 km.week-1) volunteered in the study.



The subjects competed in a half marathon (HM) of 21.1 km, 93.0 +/- 10.4 min.



Blood was taken at rest, 1 and 24 hrs after HM for determination of creatine kinase, neutrophil (PMN), lymphocyte and monocyte counts. DNA damage in LEU at rest and 24 hrs after HM was quantified using the single cell gel-electrophoresis (SCG) assay.



PMN increased from 2.81 +/- 0.69 to 13.13 +/- 2.91 1 hrs after HM (p < 0.01) and returned to 3.26 +/- 0.47 10(9) cells.l-1 by 24 hr recovery. DNA migration (image length, IL) reflecting the extent of DNA damage was elevated significantly in 10 of 12 subjects one day after HM. IL rose from 32.7 +/- 2.2 to 40.7 +/- 3.9 microns (p < 0.01). Correlation analysis revealed a relationship between DNA migration 24 hrs after HM and PMN count 1 hr post-exercise (r = 0.67, p < 0.05).



The results confirm the hypothesis that DNA damage in LEU occurs after intensive endurance exercise. We suppose our observation of exercise-induced DNA damage in LEU is affected by reactive oxygen species which are released from PMN. It is quite unclear whether DNA damage in LEU is causal involved in exercise-induced modifications of the immune system.

title: Reduction of 8-hydroxyguanine in human leukocyte DNA by physical exercise.
abstract: We investigated the effect of physical exercise on the level of 8-hydroxyguanine (8-OH-Gua), a form of oxidative DNA damage, and its repair activity in human peripheral leukocytes. Whole blood samples were collected by venipuncture from 21 healthy male volunteers (10 trained athletes and 13 untrained men), aged 19-50 years, both before and after physical exercise. Trained athletes showed a lower level of 8-OH-Gua (2.4+/-0.5/10(6) Gua, p = 0.0032) before exercise when compared to that of untrained men (6.2+/-3.5). The mean levels of 8-OH-Gua of untrained subjects decreased significantly (p = 0.0057) from 6.2+/-3.5/10(6) Gua (mean+/-SD/10(6) Gua) to 3.3+/-1.4/10(6) Gua after physical exercise. On the other hand, the mean levels of repair activity of untrained subjects significantly increased after exercise (p = 0.0093) from 0.037+/-0.024 (mean DNA cleavage ratio+/-SD) to 0.056+/-0.036. In the trained athletes 8-OH-Gua level and its repair activity were not changed before and after the exercise. We also observed inter-individual differences in 8-OH-Gua levels and its repair activities. These results suggest that physical exercise causes both rapid and long-range reduction of oxidative DNA damage in human leukocytes, with individually different efficiencies.

title: Life Style and Urinary 8‐Hydroxydeoxyguanosine, a Marker of Oxidative DNA Damage: Effects of Exercise, Working Conditions, Meat Intake, Body Mass Index, and Smoking
abstract: The urinary levels of 8‐hydroxydeoxyguanosine (8‐OH‐dG), a marker of oxidative DNA damage, of 318 healthy men aged 18‐58 were measured with high resolution by a newly developed automated high‐pressure liquid chromatography (HPLC) system coupled to an electrochemical detector (ECD). The mean 8‐OH‐dG level (μMg/g creatinine) was 4.12±1.73 (SD). An eleven‐fold inter‐individual variation was observed. The accuracy of the measurement estimated from the recovery of an added 8‐OH‐dG standard was 90‐98%. By univariate analysis, it was found that moderate physical exercise (P=0.0023) and high body mass index (BMI) (P=0.0032) reduced the 8‐OH‐dG level, while physical labor (P=0.0097), smoking (P=0.032), and low meat intake (less than once/ week) (P=0.041) increased its level. Based on a multi‐regression analysis of the log‐transformed values, moderate physical exercise (P=0.0039), high BMI (P=0.0099), and age (P=0.021) showed significant reducing effects on the 8‐OH‐dG level, while low meat intake (P=0.010), smoking (P=0.013), and day‐night shift work (P=0.044) increased its level. These results suggest that many types of life‐style factors that either generate or scavenge oxygen radicals may affect the level of oxidative DNA damage of each individual.

title: DNA damage in lymphocytes of Indian rickshaw pullers as measured by the alkaline comet assay
abstract: Rickshaw pullers (RPs) engage in strenuous physical activity and are exposed to the air pollutants found in urban environments. Air pollutants and the reactive oxygen species generated by the physical activity both potentially can damage DNA. In the present study, the Comet assay, a sensitive tool for measuring DNA damage in single cells, was used to study genomic DNA damage in lymphocytes of Indian RPs. The study evaluated DNA damage in 118 healthy male volunteers, including 63 RPs whose work demanded high levels of physical activity for 7–9 hr/day, and 55 controls matched for age, habits, socio‐economic status, and exposure to air pollution. A significant increase was found for the mean Olive tail moment (arbitrary units) among the RPs (4.13 ± 0.11; P < 0.001) in comparison with the controls (3.21 ± 0.10). Likewise, comet tail length (μm) (RPs: 58.98 ± 1.01 vs. controls: 52.38 ± 1.24) and tail DNA (%) (RPs: 13.52 ± 0.31 vs. controls: 10.04 ± 0.24) were also significantly higher for RPs compared with those of their matched controls (both, P < 0.001). To our knowledge, this is the first demonstration that physical activity due to occupation can produce DNA damage in peripheral lymphocytes.  2006.

title: Lack of correlation between environmental or biological indicators of benzene exposure at parts per billion levels and micronuclei induction.
abstract: Despite growing concern for possible carcinogenic effects associated with environmental benzene exposure in the general population, few studies exist at parts per billion (ppb) levels. We investigated the existence of a relationship between airborne/biological measurements of benzene exposure (i.e., personal/area sampling and unmodified urinary benzene/trans,trans-muconic acid; t,t-MA) and micronuclei induction (cytochalasin B technique) among exposed chemical laboratory workers (n=47) and traffic wardens (n=15). Although urinary t,t-MA (106.9+/-123.17 microg/L(urine)) correlated (R(2)=0.37) with urinary benzene (0.66+/-0.99 microg/L(urine)), neither biological measurement correlated with environmental benzene exposure (14.04+/-9.71 microg/m(3); 4.39+/-3.03ppb), suggesting that, at ppb level (1ppb=3.2 microg/m(3)), airborne benzene constitutes a fraction of the total intake. Traffic wardens and laboratory workers had comparable numbers of micronuclei (4.70+/-2.63 versus 5.76+/-3.11; n.s.), similar to levels recorded in the general population. With univariate/multivariate analysis, no association was found between micronuclei induction and air/urinary benzene exposure variables. Notably, among the personal characteristics examined (including age, gender, smoking, drinking, etc.), high body mass index correlated with micronuclei induction while, among females, use of hormonal medication was associated with less micronuclei. Thus the present study provides no evidence that ppb levels of environmental benzene exposure appreciably affect micronuclei incidence (against the background of other relevant factors). However, this should not be taken as an argument against efforts aiming to reduce environmental benzene pollution.

title: Leanness, Smoking, and Enhanced Oxidative DNA Damage
abstract: An increased risk of some forms of cancer, including lung cancer, among lean individuals has been consistent; however, there is a paucity of biological evidence supporting this relation. Subjects analyzed were 177 healthy Japanese workers who participated in a lifestyle intervention study. The levels of urinary 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, were measured using an automated high-pressure liquid chromatography and urinary creatinine levels were adjusted for before statistical analysis. A clear inverse association was found between body mass index (BMI) and 8-OHdG levels among smokers [Pearson correlation coefficient (r) = −0.48], and the association did not materially change after adjustment for potential confounding factors. In contrast, no apparent relation was observed between BMI and 8-OHdG levels among nonsmokers (r = −0.12), although lean nonsmokers had a slightly higher mean of 8-OHdG levels compared with nonlean nonsmokers. The interaction of smoking and BMI reached statistical significance (P = 0.04). Leanness may enhance oxidative DNA damage induced by smoking and thus serve as a marker of host susceptibility to smoking-related cancers.

title: Body mass index and oxidative DNA damage: A longitudinal study
abstract: Leanness has been shown to be related to an increased risk of some cancer forms, including lung cancer. However, biological evidence supporting a causal link between leanness and carcinogenesis is limited. The authors investigated longitudinally the association between body mass index (BMI) and levels of urinary 8‐hydroxydeoxyguanosine (8‐OHdG), a marker of oxidative DNA damage, using data from 174 healthy employees who participated in a lifestyle intervention study. 8‐OHdG levels were measured using automated high‐performance liquid chromatography and adjusted for urinary creatinine levels. Analysis of repeated measurements using a random effects model detected a statistically significant inverse association between BMI and 8‐OHdG levels (P = 0.003); one unit decrease in BMI was associated with a 2.7% (95% confidence interval 0.9–4.4) increase in 8‐OHdG levels. The association was pronounced among men consuming less than 20 cigarettes per day (8.8% increase per unit decrease in BMI) and among non‐smoking men (3.7% increase). The results based on a longitudinal observation suggest that weight loss is associated with increased oxidative DNA damage, a state presumably related to an increased risk of cancer.

title: Effect of 6-month calorie restriction on biomarkers of longevity, metabolic adaptation, and oxidative stress in overweight individuals: a randomized controlled trial.
abstract: Prolonged calorie restriction increases life span in rodents. Whether prolonged calorie restriction affects biomarkers of longevity or markers of oxidative stress, or reduces metabolic rate beyond that expected from reduced metabolic mass, has not been investigated in humans.



To examine the effects of 6 months of calorie restriction, with or without exercise, in overweight, nonobese (body mass index, 25 to <30) men and women.



Randomized controlled trial of healthy, sedentary men and women (N = 48) conducted between March 2002 and August 2004 at a research center in Baton Rouge, La.



Participants were randomized to 1 of 4 groups for 6 months: control (weight maintenance diet); calorie restriction (25% calorie restriction of baseline energy requirements); calorie restriction with exercise (12.5% calorie restriction plus 12.5% increase in energy expenditure by structured exercise); very low-calorie diet (890 kcal/d until 15% weight reduction, followed by a weight maintenance diet).



Body composition; dehydroepiandrosterone sulfate (DHEAS), glucose, and insulin levels; protein carbonyls; DNA damage; 24-hour energy expenditure; and core body temperature.



Mean (SEM) weight change at 6 months in the 4 groups was as follows: controls, -1.0% (1.1%); calorie restriction, -10.4% (0.9%); calorie restriction with exercise, -10.0% (0.8%); and very low-calorie diet, -13.9% (0.7%). At 6 months, fasting insulin levels were significantly reduced from baseline in the intervention groups (all P<.01), whereas DHEAS and glucose levels were unchanged. Core body temperature was reduced in the calorie restriction and calorie restriction with exercise groups (both P<.05). After adjustment for changes in body composition, sedentary 24-hour energy expenditure was unchanged in controls, but decreased in the calorie restriction (-135 kcal/d [42 kcal/d]), calorie restriction with exercise (-117 kcal/d [52 kcal/d]), and very low-calorie diet (-125 kcal/d [35 kcal/d]) groups (all P<.008). These "metabolic adaptations" (~ 6% more than expected based on loss of metabolic mass) were statistically different from controls (P<.05). Protein carbonyl concentrations were not changed from baseline to month 6 in any group, whereas DNA damage was also reduced from baseline in all intervention groups (P <.005).



Our findings suggest that 2 biomarkers of longevity (fasting insulin level and body temperature) are decreased by prolonged calorie restriction in humans and support the theory that metabolic rate is reduced beyond the level expected from reduced metabolic body mass. Studies of longer duration are required to determine if calorie restriction attenuates the aging process in humans.

title: Weight increase and overweight are associated with DNA oxidative damage in skeletal muscle.
abstract: Weight maintenance within normal standards is recommended for prevention of conditions associated with oxidative injury. To compare oxidative damage in a post mitotic tissue, between adults differing in long-term energy balance.



During hernia surgery, a sample of skeletal muscle was obtained in 17 non-obese adults. Subjects were divided into two groups according to their self-reported weight change: weight maintainers (WM) reported <4kg increase, and weight gainers (WG) reported >5kg increment. Muscle immunohistochemistry for 8-hydroxy-deoxyguanosine (8OHdG), 4-Hydroxy-2-nonenal (4HNE), and TNF-alpha, as markers of oxidative injury and inflammation, were performed. As known positive controls for oxidative injury, we included 10 elderly subjects (66-101yr). Anthropometric measures and blood samples for clinical laboratory and serum cytokines (TNF-alpha and IL-6) were obtained.



8OHdG was higher in WG compared with WM (149.1+/-16.2 versus 117.8+/-29.5, P=0.03), and was associated with anthropometric indicators of fat accumulation. 4HNE was similar in WG compared with WM (10.9+/-7.6 versus 9.8+/-6.3) but noticeably higher in elderly subjects (21.5+/-15.3, P=0.059). TNF-alpha protein in WG was higher compared with WM (114.0+/-41.7 versus 70.1+/-23.3, P=0.025), and was associated with weight increase.



Moderate self-reported weight increase, and body fat accumulation, suggesting long-term positive energy balance is associated with muscle DNA oxidative injury and inflammation.

title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: a study of adolescents in a high risk region for hepatocellular carcinoma in China.
abstract: To study the oxidative DNA damage to adolescents of hepatocellular carcinoma (HCC) families in Guangxi Zhuang Autonomous Region, China.



Peripheral leukocytes' DNA 7, 8-dihydro-8-oxoguanine (8-oxoG) and repair enzyme hOGG1 were quantified by flow-cytometry. hOGG1-Cys326Ser single nucleotide polymorphism (SNP) was distinguished by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) assay.



There was a positive correlation between 8-oxoG and repair enzyme hOGG1 expression (P<0.001). HCC children (n=21) in Fusui county had a higher level of hOGG1 (P<0.01) and a lower level of 8-oxoG (P<0.05) than the controls (n=63) in Nanning city. Children in Nanning exposed to passive-smoking had a higher hOGG1 expression (P<0.05) than the non-exposers. 8-oxoG and hOGG1 were negatively correlated with body mass index, while hOGG1 was positively correlated with age. There was a peak of 8-oxoG level nearby the 12 year point. Individuals with the hOGG1 326Ser allele had a significantly marginal higher concentration of leukocyte 8-oxoG level than hOGG1 326Cys allele.



This is the first report using flow-cytometry to simultaneously quantify both the DNA oxidative damage and its repairing enzyme hOGG1. The results provide new insights towards a better understanding of the mechanisms of oxidative stress in a population highly susceptible to hepatocarcinogenesis.

title: Urinary 8-Hydroxydeoxyguanosine in Belarussian Children Relates to Urban Living Rather Than Radiation Dose After the Chernobyl Accident: A Pilot Study
abstract: As a result of the Chernobyl accident in 1986, exposure to radioactive cesium is still a concern in the contaminated regions of Belarus. We tested the hypothesis that long-term radiation exposure from the Chernobyl accident might increase the urinary excretion of the oxidative stress marker, 8-hydroxydeoxyguanosine (8-OHdG), in Belarussian children. Urinary 8-OHdG was determined in two groups of children (—n = 31 and n = 46) —living in contaminated and uncontaminated areas of Belarus, respectively (the majority of the unexposed children lived in the capital Minsk). The children from the contaminated areas had a significantly higher annual summary effective dose but significantly lower urinary 8-OHdG levels than the children from the uncontaminated areas. Unexpectedly, children living in uncontaminated urban areas had significantly higher urinary 8-OHdG levels than children living in uncontaminated rural areas. There was no statistically significant effect of sex or body mass index on urinary 8-OHdG, but there was a weak significant inverse correlation to age as well as to the annual summary effective dose. These findings suggest that radiation from the Chernobyl accident is now a less important contributor to oxidative stress in Belarussian children than urban living.

title: Methylenetetrahydrofolate reductase gene C677T polymorphism, homocysteine, vitamin B12, and DNA damage in coronary artery disease
abstract: Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutrional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients (n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8±5.2 vs 13.7±2.2 and 12.9±1.9 µmol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6±1.2, 22.0±4.7, 19.3±3.9 µmol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4±0.7, 11.1±2.0, 14.2±1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7±2.4 vs 8.9±1.7 and 9.9±0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration (r=–0.343; P=0.019) and positively with plasma Hcy (r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.

title: Genetic polymorphisms in folate and homocysteine metabolism as risk factors for DNA damage
abstract: Epidemiological studies indicated a role for polymorphisms in genes of folate and homocysteine (Hcy) metabolism in the etiology of neurodegenerative disease, congenital defects and coronary artery disease (CAD). This study investigated the effect of several polymorphisms [C677 T, A1298C of methylenetetrahydrofolate reductase (MTHFR) and A66G of methionine synthase reductase (MTRR) genes] on Hcy levels and DNA damage in 68 patients who underwent coronary angiography. Plasma Hcy concentrations were higher in patients with multivessel disease with respect to monovessel disease and no-CAD patients (19.4±2.6 vs 11.6±1.2 and 13.7±1.4 μmol/l, respectively; P=0.03). 677TT patients had higher Hcy levels than those with 677CC or 677CT genotypes (26.2±4.3 vs 13.1±1.4 and 13.0±1.4 μmol/l, respectively; P=0.0006). No significant associations were found between A1298C and A66G polymorphisms and plasma Hcy levels. Among patients with 677CC genotype, 66GG individuals tended to have higher levels of Hcy than 66AA homozygotes (14.5±1.9 vs 8.9±0.7 μmol/l, P=0.06). Multivessel disease patients showed an increased frequency of DNA damage, measured by the micronucleus (MN) frequency, as compared to monovessel disease and no-CAD subjects (12.5±1.1 vs 8.5±0.8 and 8.2±0.9, respectively; P=0.006). The MN were positively correlated with Hcy levels (r=0.33, P=0.006) and were significantly higher in subjects with the 677TT genotype compared with the 677CC or 677CT genotypes (14.4±2.0 vs 8.8±1.2 and 9.5±0.7, respectively; P=0.006). A1298C and A66G polymorphisms had no effect on MN frequency. However, among 677TT patients, 66GG subjects tended to have higher levels of MN than those 66AG and 66AA (18.2±3.6 vs 13.8±4.0 and 10.3±1.7, respectively; P=NS). Our results indicate that genetic instability may be associated with increased risk for multiple Hcy-related diseases.

title: Influence of DNA repair polymorphisms on biomarkers of genotoxic damage in peripheral lymphocytes of healthy subjects.
abstract: DNA repair polymorphisms may represent susceptibility factors affecting DNA integrity, and possibly cancer risk, in human population. In order to elucidate the influence of a few widely studied DNA repair polymorphisms on individual levels of DNA damage and their possible interaction with lifestyle and environmental exposures, 171 subjects from a well-characterized human population enrolled in a previous study on genetic effects of air pollution were genotyped for the XRCC1 Arg280His and Arg399Glu, XRCC3 Thr241Met and ERCC2 Lys751Gln polymorphisms. The association between DNA repair genotype, alone or in combination with metabolic genotype, on the levels of SCE, micronuclei and tail moment values in peripheral lymphocytes was evaluated. A significant influence of the ERCC2 genotype on SCE frequency was observed. Subjects with ERCC2 751 Gln/Gln genotype had significantly higher risk of high (above the median) SCE/cell with respect to Lys/Lys referents (OR 4.55, 95% CI 1.48-13.99). A non-significantly elevated OR was also observed in Gln/Lys heterozygotes, suggesting a gene dosage effect. When subjects were categorized by smoking habits and professional exposure, the variant ERCC2 751 Gln/Gln genotype was associated with elevated SCE rates in non-smokers and in exposed subjects, but not in smokers. The results of this study support the hypothesis that some DNA repair polymorphisms exert a modifying effect on individual levels of DNA damage in healthy subjects, possibly also modulating cancer risk.

title: Influence of glutathione S-transferase polymorphisms on genotoxic effects induced by tobacco smoke.
abstract: Genotoxicity of tobacco smoke has long been investigated and tobacco smoke is considered to be one of the principal human carcinogens. Although its role in DNA-damage induction and cancer development has been documented, the mechanisms by which this happens are not well understood. Many chemical constituents of tobacco smoke are enzymatically metabolized by phase-I and phase-II enzymes, but modifications in coding and regulating sequences of these genes could influence their ability to detoxify these compounds. In this work, we studied several enzymes involved in the metabolism of xenobiotics, viz. the glutathione S-transferases (GST) M1, T1, P1 and A1, with respect to their influence on the genotoxic effects induced by cigarette smoking. We assessed the genotoxic effects of tobacco smoke on peripheral blood lymphocytes of 72 healthy caucasians by use of the chromosomal aberration (CA) assay and the micronucleus (MN) test. Genotypes of GST M1, T1, P1 and A1 were determined by means of the polymerase chain reaction and methods based on restriction fragment length polymorphism (RFLP). We found that smoke and gender are the two variables that most influence the DNA damage. In particular, we observed that female smokers seem to be more sensitive than male smokers, having a significantly higher frequency of CAs. Moreover, a significant increase in frequency of micronuclei in bi-nucleated cells (BNMN) was found in smokers, but not in non-smokers. This increase seems to be influenced not only by age and gender, but also by genetic constitution. Subjects carrying GSTM1-null genotype seemed to have an higher susceptibility to DNA damage induced by tobacco smoke than GSTM1-positive ones. When considering a combination of GST genotypes, we found a lower BNMN frequency in subjects with GSTP1 variant allele plus GSTM1-positive genotypes, while the most damaged cells are found in subjects bearing GSTM1-null plus GSTP1-wild type. Our results suggest that investigation of the association between several gene polymorphisms and important endpoints of DNA damage could contribute to better understanding the role of gene-gene interaction.

title: Chromosomal changes: induction, detection methods and applicability in human biomonitoring.
abstract: The objective of this state of the art paper is to review the mechanisms of induction, the fate, the methodology, the sensitivity/specificity and predictivity of two major cytogenetic endpoints applied for genotoxicity studies and biomonitoring purposes: chromosome aberrations and micronuclei. Chromosomal aberrations (CAs) are changes in normal chromosome structure or number that can occur spontaneously or as a result of chemical/radiation treatment. Structural CAs in peripheral blood lymphocytes (PBLs), as assessed by the chromosome aberration (CA) assay, have been used for over 30 years in occupational and environmental settings as a biomarker of early effects of genotoxic carcinogens. A high frequency of structural CAs in lymphocytes (reporter tissue) is predictive of increased cancer risk, irrespective of the cause of the initial CA increase. Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. The cytokinesis-block micronucleus (CBMN) assay is the most extensively used method for measuring MN in human lymphocytes, and can be considered as a "cytome" assay covering cell proliferation, cell death and chromosomal changes. The key advantages of the CBMN assay lie in its ability to detect both clastogenic and aneugenic events and to identify cells which divided once in culture. Evaluation of the mechanistic origin of individual MN by centromere and kinetochore identification contributes to the high sensitivity of the method. A number of findings support the hypothesis of a predictive association between the frequency of MN in cytokinesis-blocked lymphocytes and cancer development. Recent advances in fluorescence in situ hybridization (FISH) and microarray technologies are modifying the nature of cytogenetics, allowing chromosome and gene identification on metaphase as well as in interphase. Automated scoring by flow cytometry and/or image analysis will enhance their applicability.

title: HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures.
abstract: Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.

title: Scoring variability of micronuclei in binucleated human lymphocytes in a case-control study.
abstract: The micronucleus test in binucleated lymphocytes is a sensitive standard assay for biomonitoring, mutagenicity testing and to assess radiosensitivity of blood donors. The results vary between laboratories and scorers which led to the definition of international scoring criteria. We used these criteria in a case-control study, but nevertheless observed large differences between the seven scorers on the level of descriptive analysis. Therefore, we used the repeat measurements (267 in 98 blood donors) from this dataset (354 measurements in 185 blood donors) to analyse scoring variability in the setting of a case-control study. The variability was assessed by analysis of variance, which revealed the storage time of the blood samples, the blood donors including their disease status, and the scorers as sources of variation in the entire dataset. In addition, the coefficient of variation (CV) of the measurements was determined (overall: CV = 24.3%). After stepwise removal of biological and experimental variation by normalizations, the CV dropped to 6.8% on average, which may reflect the 'pure counting error'. The scorer-specific CVs were between 5.5 and 9.5%. The differences between the scorers suggested by the raw data were neither related to the scorer-specific CV nor to their experience. Instead, we observed a general decline of the micronuclei frequencies towards the end of the study for all scorers. This could not be related to a change in experimental conditions or in the defined scoring criteria. An explanation could be an unintended and unrecognized change of scoring criteria. Since the change in the results did not occur in automated counting we suggest to use either reference slides in longer-lasting studies or automated counting by image analysis.

title: Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytes.
abstract: The frequency of micronuclei in binucleated lymphocytes (cytochalasin B assay) may serve as a biological dosimeter after radiation exposure. The automation of the micronucleus assay in binucleated human lymphocytes has been considerably advanced in recent years. In our studies for this purpose the detection of binucleated cells (BNCs) and the scoring of micronuclei (MN) was divided into two parts. First, detection of BNCs was feasible with low microscopic magnification (x 100). The positions of classified BNCs were stored. Second, after an automatic change of microscope objective, the stored BNCs were automatically analyzed in sequence at high microscopic magnification (x 630) for occurrence of MN. For both phases of image analysis we used empirical methods based on mathematical morphology. The system is able to recognize nearly 65% of BNCs with false positive decisions of 6% and about 75% of the MN with false positive decisions of 7%.

title: Automated scoring of micronuclei in binucleated human lymphocytes.
abstract: Manual and automatic scoring of micronuclei (MN) in binucleated human lymphocytes (BNC) were compared after irradiation of whole blood samples. The blood samples were irradiated with X-ray doses (1, 2 or 3 Gy) and stained with Giemsa. The preparation technique was optimized in such a way that acceptable conditions (cell density, contrast) were obtained for both scoring procedures. To estimate the quality of automatic micronucleus detection, two researchers who had different experience in scoring MN (6 months and 5 years) analysed the samples independently from each other. Automatic scoring was carried out with a digital image analysis system and the recognition procedure was divided into two parts. The BNC positions were detected with low microscope magnification (100x), and the recognition of micronuclei within the cytoplasm of the classified BNC was carried out at high magnification (630x). A fuzzy logic classification system as well as two different segmentation steps (preclassification and postclassification) made it possible that about 94% of all automatically recognized BNC were classified correctly). On the other hand, the classification system was optimized in such a way that false positive decisions were minimized (95% of automatically recognized micronuclei were classified correctly). Failure to recognize micronuclei (8.5%-25% false negatives) was mainly due to extremely small micronuclei, poor contrast with respect to the cytoplasm, and aggregation of micronuclei especially at higher doses.

title: Scoring of radiation-induced micronuclei in cytokinesis-blocked human lymphocytes by automated image analysis.
abstract: The micronucleus assay in human lymphocytes is, at present, frequently used to assess chromosomal damage caused by ionizing radiation or mutagens. Manual scoring of micronuclei (MN) by trained personnel is very time-consuming, tiring work, and the results depend on subjective interpretation of scoring criteria. More objective scoring can be accomplished only if the test can be automated. Furthermore, an automated system allows scoring of large numbers of cells, thereby increasing the statistical significance of the results. This is of special importance for screening programs for low doses of chromosome-damaging agents. In this paper, the first results of our effort to automate the micronucleus assay with an image-analysis system are represented. The method we used is described in detail, and the results are compared to those of other groups. Our system is able to detect 88% of the binucleated lymphocytes on the slides. The procedure consists of a fully automated localization of binucleated cells and counting of the MN within these cells, followed by a simple and fast manual operation in which the false positives are removed. Preliminary measurements for blood samples irradiated with a dose of 1 Gy X-rays indicate that the automated system can find 89% +/- 12% of the micronuclei within the binucleated cells compared to a manual screening.

title: International reference study on the identification and scoring of human chromosome aberrations. Results of a WHO comparative study.
abstract: Chromosome aberrations in human peripheral blood are recognized parameters of cellular damage and are used as indicators of exposure to ionizing radiation and certain chemicals. However, significant interlaboratory variability exists in the results reported from different laboratories. The primary objective of the present study was to examine problems associated with the identification and analysis of chromosome aberrations. Significant interlaboratory variability was found to exist in the analysis of human chromosome spreads for induced interphase aberrations, apparently owing in part to differences in the selection and rejection of spreads for scoring and also in the recognition and classification of various types of aberrations. These differences are reflected in the dose-response relationships for aberrations as well as for damaged spreads. For damaged spreads the scoring variability appears to be relatively small. It is inferred that factors additional to differences in scoring may play a significant role in the large variation in the reported dose-response relationships both for ionizing radiation and for chemically induced aberrations.

title: Sex chromosome loss, micronuclei, sister chromatid exchange and aging: a study including 16 centenarians.
abstract: In the present study we analysed the possible effect of age, sex and smoking on the mean values of micronucleus (MN) and sister chromatid exchange (SCE) frequencies on peripheral blood obtained from 38 subjects ranging in age from 16 to 63 years and 16 centenarians. The mean number of binucleated cells with micronuclei varied in function of age and sex (as demonstrated by the analysis of covariance (F=13.13; P<0.001), particularly evident was the increment observed in women with increasing age (interaction age/sex: F=5.53; P<0.05). Smoking habits had no effects on MN frequency (F=0.36; P>0.05). Sex (F=4.18; P<0.05) and smoking habits (F=14.64; P<0.001) influenced significantly SCE per cell frequencies, but age had no effects on them (F=2.45; P>0.05). The age-associated increase of sex chromosome loss was studied using fluorescence in situ hybridisation (FISH) on interphase nuclei. The loss of Y signals was observed in approximately 10% of interphase cells from the centenarians males, that is six times more often than in the younger control men (approximately 1.6%). The frequency of X signal loss (approximately 1.7%) in young women was similar to that observed in male controls of the same age but the incidence of the X chromosome aneuploidy in centenarian females was appreciably higher (approximately 22%) than that found for the Y chromosome in males. These results were correlated with the data on MN formation and a positive correlation between the percentage of aneuploid cells (FISH) and MN values was observed (r=0.50; P<0.05).

title: Aneuploidy and ageing: sex chromosome exclusion into micronuclei
abstract: In lymphocyte cultures, the number of aneuploid cell nuclei increases with proband age mainly because of the loss of sex chromosomes. Since one possible cause of aneuploidy in cell nuclei is chromosomal lag at anaphase, with subsequent chromosome loss via micronucleus formation, we scored 5000 interphase nuclei from ten female and ten male probands for associated micronuclei. Whereas, in young (< 10 years) probands, an average of 0.15% interphase nuclei exhibited micronuclei, the frequency rose to 0.46% in older probands (> 70 years). In situ hybridizations with X-specific and Y-specific DNA probes were carried out, and the signal distribution in ten nuclei with associated micronuclei was documented for each donor. Our results indicate that the exclusion of sex chromosomes into micronuclei doubles during a human life, from 11% in young probands to 20% in old donors.

title: Genome health nutrigenomics and nutrigenetics--diagnosis and nutritional treatment of genome damage on an individual basis.
abstract: The term nutrigenomics refers to the effect of diet on gene expression. The term nutrigenetics refers to the impact of inherited traits on the response to a specific dietary pattern, functional food or supplement on a specific health outcome. The specific fields of genome health nutrigenomics and genome health nutrigenetics are emerging as important new research areas because it is becoming increasingly evident that (a) risk for developmental and degenerative disease increases with DNA damage which in turn is dependent on nutritional status and (b) optimal concentration of micronutrients for prevention of genome damage is also dependent on genetic polymorphisms that alter function of genes involved directly or indirectly in uptake and metabolism of micronutrients required for DNA repair and DNA replication. Development of dietary patterns, functional foods and supplements that are designed to improve genome health maintenance in humans with specific genetic backgrounds may provide an important contribution to a new optimum health strategy based on the diagnosis and individualised nutritional treatment of genome instability i.e. Genome Health Clinics.