|
|||||||||||||||||||
 |
 |
||||||||||||||||||
|
|||||||||||||||||||
|
|||||||||||||||||||
|
 |
|
||||||||||||||
|
||||||||||||||||
Program of 3rd HUMN workshop:
Although the HUMN workshop focused on the application of the micronucleus (MN) techniques to measure in vivo chromosome damage, it also involved discussion relating to comparison and validation of techniques, which would be of interest to researchers involved with in vitro genotoxicity testing. |
||||||||||||||||
|
||||||||||||||||
|
Abstracts 1. Brief overview of the objectives of the HUman MicroNucleus (HUMN) project. Errol Zeiger, Chapel Hill, NC, USA The International Collaborative Project on Micronucleus Frequency in Human populations (HUMN) was launched in 1997, because of interest in micronuclei (MN) in human lymphocytes and exfoliated epithelial cells as biomonitors for exposure to toxic substances, and as a potential predictor of adverse health effects. The availability of MN data from a variety of human populations, and the possibility of combining those data, would provide a powerful tool for the evaluation of MN frequencies for public health and epidemiological studies. Laboratories that had performed MN studies in human lymphocytes were invited to participate. Twenty-five laboratories, from 16 countries, yielded a database of nearly 7000 subjects. The cytokinesis block method for determining MN was used for all subjects. The lymphocyte MN frequencies from each laboratory were analyzed, and the extent of variation of "normal" values for different laboratories and populations was determined. This combined database also provides information on methodological (e.g., cell culture; protocol) and personal (e.g., age, sex, smoking) variables that may affect the baseline MN frequency. An interlaboratory study to measure intra- and inter-laboratory slide scoring variability has been completed and the results are being analyzed. The results of this study will be used to develop criteria against which laboratories can evaluate their performance, and to develop more appropriate statistical procedures for analyzing MN results. A prospective study linking the accumulated MN data from each laboratory is being considered. The international database would provide a powerful cohort in person-years that could be used to determine associations with cancer and aging. 2. New developments in the lymphocyte micronucleus assay - the effect of micronutrients, genotype and the importance of nucleoplasmic bridges and nuclear buds Michael Fenech1, Keizo Umegaki2, Julie Turner1, Philip Thomas1, Jimmy Crott3. 1 CSIRO Health Sciences and Nutrition, Adelaide, Australia 2 National Institute of Health and Nutrition, Shinjuku, Tokyo, Japan 3 Dept. of Physiology, University of Adelaide, Adelaide, Australia The cytokinesis-block micronucleus (CBMN) assay is an established method for studying chromosome breakage and loss in human cells both in vitro and in vivo. However, its use in the study of genomic stability could be improved by a better understanding of the impact of (a) micronutrients (b) genetic background and (c) of other associated important nuclear events indicative of chromosome rearrangement (ie nucleoplasmic bridges) and gene amplification (nuclear buds). Over the past two years we have verified that folic acid, vitamin B12 and plasma homocysteine are important determinants of micronucleus frequency in vitro or in vivo. We also determined the extent to which polymorphisms in genes involved in folic acid/homocysteine metabolism (ie methylenetetrahydrofolate reductase and methionine synthase) impact on base-line micronucleus frequency. At the same time we have shown that inclusion of nucleoplasmic bridges in the CBMN assay provides an opportunity to fingerprint the genotoxic effects of different sources of oxidative stress depending on the micronucleus/nucleoplasmic bridge ratio. Micronuclei may also arise from extrusion of amplified genes via a nuclear budding process. Our recent studies in folate deficient cells have shown that all indeces ie micronuclei, nucleoplasmic bridges and nuclear buds are significantly increased in a dose-dependent manner suggesting a multitude of genetic events caused by a single micronutrient deficiency. The CBMN assay has now been validated as a method that can be used to comprehensively measure chromosome breakage, chromosome loss, chromosome rearrangement, gene amplification, necrosis, apoptosis and cytostatic effects. 3. Results from the world-wide data base comparison of MN frequency in human lymphocytes Stefano Bonassi, Department of Environmental Epidemiology, National Cancer Research Institute, Largo R. Benzi, 10 - 16132, Genoa, Italy. 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 assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, host factors, and lifestyle on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values 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 and in criteria for scoring micronuclei. The overall median MNC frequency in non-exposed (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% C.I.: 14-24%). No increase of risk was found for current smokers (RR=0.96; 95% Confidence Interval (CI)=0.95-0.98) and former smokers (RR=0.95; CI=0.94-0.98), which conversely showed a small decrease. MN frequency increased with the amount of cigarettes/day when the analysis was restricted to non-occupationally exposed subjects. The best statistical model applied to this database explained 75% of the total variance, with the largest contribution attributable to laboratory methods. 4. Molecular correlation of micronucleus formation and carcinogenesis in human cells Wushou P. Chang1,2 and Mon-hsiung Tsai2 1 Institute of Environmental Health Sciences, National Yangming University Medical School 2 Institute of Public Health, NYMU; Taipei, Taiwan Micronucleus formation has been shown to be derived from DNA double strand breaks and chromosomal breakage. The increased micronucleus frequencies in human subjects is now under intense evaluation for its association for cancer risk. Meanwhile, the mechanistic correlation between micronucleus formation and carcinogenesis deserves more studies. In a follow-up study in a human population with previous chronic low-dose rate environmental g-irradiation, cytochalasin-B micronucleus frequencies (CBMN) have been conducted continuously. The frequencies of micronucleus were observed significantly increased in an dose-dependent correlation. 14 subjects were further observed for the micronucleus frequencies more than 3 years since relocating to non-radioactive environment, while the T-lymphocyte micronuclei were labeled with total mouse anti-human P53monoclonal antibody Pab 1801 and counterstained with propidium iodide. The frequencies of CBMN were 8.4+3.1 (0/00) and not significantly different from 14 non-exposed subjects (8.7+3.4 0/00). However, the p53 staining positive CBMN (TP53+MN; 3.7+1.8) and p53 positive rate on the micronuclei (TP53+/total MN; 43.7+14.8%) were significantly higher than those of the reference subjects (2.7+0.9 0/00 and 31.4+5.9%, respectively; p values <0.05). Merging results of these 28 subjects and analyzed by linear correlation, the TP53+/total MN and TP53+/total MN were both shown with significant association with cumulative dose of radiation exposure (p values <0.05). This increase in TP53+/total MN and TP53+/total MN were further shown in a parallel study with human T-lymphocytes irradiated in vitro. The above results suggest that residual DNA damage could remain in human lymphocytes post previous DNA damage and carrying higher amounts of p53 proteins in the cytoplasm concomitantly, while the micronucleus frequencies were not shown significantly elevated. These residual DNA damage can play as genetic instability in somatic cells and eventually lead to carcinogenesis. Moreover, increased micronucleus frequencies in human subjects may play as a surrogate for cancer risk prediction in the future (supported by the National Science Council and partly by the National Health Research Institute, Taiwan). 6. Inclusion of Micronuclei in Non-divided Mononuclear Lymphocytes and Necrosis/Apoptosis May Provide a More Comprehensive Cytokinesis-Block Micronucleus Assay for Biomonitoring Purposes *Micheline KIRSCH-VOLDERS1 and Michael FENECH2 1 Vrije Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Pleinlaan 2, 1050 Brussel, Belgium 2 CSIRO Health Sciences and Nutrition, PO Box 10041, Gouger Street, Adelaide, BS, SA, Australia 5000 Abbreviations BN = binucleated; MONO = mononucleated; MN = micronucleus, MNi = micronuclei ; MNed = micronucleated ; MNBN = binucleated cell containing a micronucleus ; MNMONO = mononucleated cell containing a micronucleus. 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 MN assay (CBMN) which detects both chromosome breakage and chromosome loss in once-divided binucleated (BN) cells. Many studies are published with this methodology, but before its extensive application is recommended, it is necessary to evaluate its strengths and limitations. Recently, Fenech et al. (1999) reviewed the advantages of the cytokinesis block MN assay for biomonitoring purposes. However, up to now, information present in the MONO cells was rarely taken into account, although it might be quite complementary to that assessed in BN cells. Indeed, MONO cells should indicate damage which was present in vivo before the start of the culture and BN cells may contain preexisting MNi plus lesions which are expressed as MNi during in vitro culture. To address this question, the objectives of this paper were: 1) to situate the cytokinesis-block MN assay in a historical and mechanistic perspective; 2) 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; 3) 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; 4) to compare the sensitivity of MONO and BN cells in relation to the genotoxic mechanism; 5) 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 (a) MNi in MONO cells; (b) MNi in BN cells; (c) apoptotic cells and (d) necrotic cells. It is probable that the 24 hours post-PHA 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. 7. The use of the micronucleus assay in predicting cancer risk and response to radiotherapy. David Scott, James Barber, Richard Papworth, Wayne Burrill, Nick Slevin, Steve Roberts. Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, UK. The mean yield of gamma-ray-induced micronuclei (MN) in lymphocytes of 130 breast cancer patients was significantly greater than that of 68 healthy controls. Using the 90th percentile of controls as the cutoff point between a normal and a sensitive response, 27% of patients were sensitive. Similar results were seen for 49 patients with head and neck cancer, particularly early-onset cases, compared with 31 controls. We suggest that enhanced sensitivity in this assay indicates an inherited predisposition to cancer, mediated through low penetrance genes. Support for this hypothesis comes from our demonstration that 22 first degree relatives of 11 sensitive breast cancer cases had a mean MN yield significantly higher than that of 68 unrelated controls. We propose that a substantial proportion of patients with breast or head and neck cancer have an inherited defect in the ability to process DNA damage of the types induced by ionising radiation, from exogenous or endogenous sources. In a prospective study of 116 breast cancer patients, MN tested prior to radiotherapy, 6 showed severe acute skin reactions, but their mean MN yields were no higher than those of normally-reacting cases. On the other hand, 9 breast and 4 head and neck cancer patients who had shown delayed adverse normal tissue reactions had higher yields than 61 normal reactors, on the borderline of statistical significance. Studies of larger numbers of severe reactors will be required to determine the value of this assay in predicting response to therapy. |
||||||||||||||||
|
||||||||||||||||
 |
 |
| The HUman MicroNucleus Project | |