Variability in cytogenetic adaptive response of cultured human lymphocytes to mitomycin C, bleomycin, quinacrine dihydrochloride, Co60 gamma-rays and hyperthermia.

Adaptive response (AR) is a well-documented phenomenon by which cells or organisms exposed to low dose of a genotoxicant become less sensitive to subsequent high-dose exposure to the same or another genotoxicant. AR, if induced can modify the efficacy leading to drug or radio-resistance, during anti-neoplastic drug or radiation treatment. Contradictions exist in AR induction by different genotoxicants with respect to the biomarkers, time schedules, and inter-individual variability, reflecting the complexity of AR in eukaryotic cells. In order to further ascertain these factors, AR induced by anti-neoplastic agents mitomycin C (MMC), bleomycin (BLM) and chemosterilant quinacrine dihydrochloride was examined in different donors and time schedules using cytogenetic biomarkers chromosome aberrations, sister chromatid exchanges and micronuclei (MN). BLM- and hyperthermia (HT)-induced cross-resistance to gamma rays and MMC/BLM, respectively, was also studied. Difference between MMC- and BLM-induced protective effects in biomarkers examined in the same donors was noticed. Adaptation to BLM and HT showed cross-resistance to chromosome damage induction by gamma rays and BLM/MMC, respectively. Cell cycle analysis indicated that adaptation is not caused by change in the rate of cell proliferation after challenge dose. MN as a chromosomal biomarker in large-scale population studies on AR is advocated, based on similar AR induced in all donors by MMC/BLM and rapid assessment in binucleated cells. Influence of certain genotypes on chromosomal biomarkers used in AR studies and role of AR in radiation and chemotherapy need to be further deciphered.

Transmission of gamma-ray-induced unstable chromosomal aberrations through successive mitotic divisions in human lymphocytes in vitro.

Transmission of unstable chromosomal aberrations through successive mitotic divisions M1-M4 was analysed in 3 Gy gamma-ray-irradiated G0 human lymphocytes in vitro, harvested at 50, 72 and 96 h. Bromodeoxyuridine (BrdU) incorporation and subsequently the fluorescence plus Giemsa (FPG) method allowed the cell cycle status of each cell scored to be ascertained. Higher dicentric frequencies were observed in cells within the same post-irradiation division derived from extended culture times, indicating either a delay in cell cycle progression of aberrant cells or different lymphocyte sub-populations. The yield of complete dicentrics decreased by a factor of two in passing from M1 to M2 and showed further reductions of 26 and 44%, respectively, in moving from M2 to M3 and from M3 to M4. In the case of conventionally stained metaphases, wherein the cell cycle status does not enter the picture, the dicentric frequencies showed a reduction in yield of 39.6% at 72 h and 52.1% at 96 h compared with 50 h, because the cells analysed comprise a mixture of metaphases in different cell cycles. In the FPG stained first division metaphases, 92-100% of dicentrics analysed at 50, 72 and 96 h and in the conventionally stained metaphases, 90-94, 72-84 and 54-80% of dicentrics analysed at 50, 72 and 96 h respectively were complete dicentrics (with fragments). In the M1, M2, M3 and M4 metaphases analysed, 92-100, 50-89, 20-70 and 10-50% of dicentrics, respectively, were complete dicentrics and 55-75, 53-68, 43-57 and 36-50% excess acentrics, respectively, were seen in cells with complete dicentrics. Interindividual variation was observed in cell cycle kinetics, radiation-induced chromosomal aberrations and micronucleus frequencies. A salient feature of the delayed effects was the induction of giant cells and the mirror dicentrics observed in them. The reduction in dicentric frequencies due to either cell cycle delay or cell death in successive generations is aided by the multiplicity of aberrations. Bridge-breakage-fusion (BBF) events mediated by dicentric chromosomes may also be instrumental in the perpetuation of chromosomal instability.

Ascorbic acid potentiates mitomycin C-induced micronuclei and sister chromatid exchanges in human peripheral blood lymphocytes in vitro.

Vitamin C (l-ascorbic acid), an effective free radical scavenger present as ascorbate in most biological systems, is one of the most extensively studied antioxidant vitamins. Vitamin C acts as either a free radical scavenger or a pro-oxidant producing hydrogen peroxide and free radicals. The modulatory effect of L-ascorbic acid (AA) on Mitomycin C (MMC) induced chromosome damage has been evaluated in human peripheral blood lymphocytes in vitro. The effect of L-ascorbic acid, 200 microg/ml as 1- and 2-h pretreatment on the frequencies of the biomarkers micronuclei (MN), sister chromatid exchanges (SCEs), and chromosome aberrations (CA) induced by mitomycin C 0.1 and 0.2 microg/ml has been studied. AA pretreatment caused a statistically significant increase in MMC-induced MN and SCE frequencies for all treatment groups, but did not show an increase in induced chromosome aberrations compared to MMC treatment alone. Cell division delays caused by MMC was reversed in the presence of AA. Interindividual variability in MMC as well as AA plus MMC-induced MN, SCE, and CA frequencies were evident. Ascorbic acid potentiated MMC-induced chromosome damage in human lymphocytes in vitro. The potentiation observed has to be viewed in the light of metal ion catalysed autooxidation of AA in oxygenated media and the existence of an antioxidant system in vivo that inactivates oxyradicals before their interaction with DNA.

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.

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.

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.

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 per thousand and the interquartile range was between 3 and 12 per thousand. 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.

Quinacrine dihydrochloride, the non-surgical female sterilant induces dicentrics, rings, and marker chromosomes in human peripheral blood lymphocytes treated in vitro: a preliminary report.

During the last decade, quinacrine dihydrochloride (QDH) has been promoted for clinical trials as a much needed non-surgical female sterilant, largely in the Third World. Recently, however, these human trials have come under severe criticism due to lack of adequate evidence of biological safety of QDH, particularly of its genotoxicity in mammalian systems. In the present study, the cytogenetic analysis of QDH-treated human lymphocytes, grown as whole blood cultures in vitro, surprisingly showed a wide range of chromosomal aberrations. At a concentration of 3.0 and 6.0 microg/ml in culture, QDH was cytotoxic, as shown by the very few analyzable metaphases that could be observed. G(0) lymphocytes, treated with 0. 6 microg/ml QDH, exhibited chromosome aberrations including dicentrics, ring configurations, translocations, inversions, and marker chromosomes. Near haploid, polyploid, and endoreduplicated cells were also observed. All the rings appeared to be formed as a result of telomere fusion/association. Twenty percent of the dicentrics observed also indicated telomere fusion/association in the D and G groups of chromosomes. Overall, a frequent involvement of chromosomes 1, 2, and 3 in both unstable and stable chromosome rearrangements was also observed. Exposure of 72-h cultures to 0.45 microg/ml QDH at 69 h resulted in an accumulation of C-metaphases, suggesting that probably QDH behaves as a mitotic spindle inhibitor. The G(2) lymphocytes from two donors exposed to 0.6, 1.5 or 3.0 microg/ml of QDH showed no increase in chromatid aberrations in two donors. However, QDH at 0.6 microg/ml increased the frequency of micronucleated binucleate cells. No increase in sister chromatid exchanges was observed at this concentration. Though preliminary, these observations demonstrate the chromosome damaging ability of QDH in human lymphocytes treated in vitro. Surprisingly, like ionizing radiation, QDH acted by an S-phase-independent mechanism, unlike most of the chemical mutagens. These results warrant detailed investigations on the cytogenetic effects of QDH in vitro, as well as among women exposed to this agent during clinical trials for non-surgical sterilization. The interesting cytogenetic profile of QDH deserves to be pursued and the underlying mechanisms, in particular, the DNA topoisomerase II inhibitory effect, if any, needs to be elucidated.

Heterogeneity in chemical mutagen-induced chromosome damage after G2 phase exposure to bleomycin, ara-C and gentian violet in cultured lymphocytes of beta-thalassaemia traits.

Chemical mutagen-induced chromosome damage was analysed in cultured peripheral blood lymphocytes from beta-thalassaemia traits and healthy individuals. This was promoted by the fact that beta-thalassaemia trait is present in 1-17% of different population groups in India. To study mutagen-induced chromosome instability, G2 lymphocytes were exposed to bleomycin, ara-C or gentian violet in 48-h cultures. Spontaneous chromosome aberration frequencies in lymphocytes from beta-thalassaemia traits were found to be in the normal range. In all three clastogen-treated lymphocytes from beta-thalassaemia traits, there is a degree of hypersensitivity, when the results are averaged over a number of individuals, but some individuals overlap within the normal range. The heterogeneity in chemical mutagen sensitivity observed in beta-thalassaemia traits is discussed in terms of the oxidative damage consequent on the genetic and biochemical features peculiar to the beta-thalassaemia trait cell.

Heterogeneity of chromosome damage in beta-thalassaemia traits. An evaluation of spontaneous and gamma-ray-induced micronuclei and chromosome aberrations in lymphocytes in vitro after G0 and G2 phase irradiation.

This study is an attempt to evaluate the chromosomal radiosensitivity of beta-thalassaemia traits compared with healthy individuals from the general population, necessitated by the fact that beta-thalassaemia trait is present in 1-17% of different population groups in India and the chances of encountering them in radiation and chemical related industries do exist. Spontaneous chromosome aberration frequencies in peripheral blood lymphocytes from beta-thalassaemia traits were found to be in the normal range, whereas significantly higher frequencies of micronuclei (MN) were observed in thalassaemia traits. Based on MN frequency at 2 Gy, beta-thalassaemia traits fall into two distinct categories. A hypersensitive group with significant increase in radiation-induced MN over the control group, and a second group with MN frequency slightly above normal individuals. Even when compared with the fitted data at 2 Gy obtained from the pooled results of extensive dose-response investigations from 0.5-5 Gy gamma-rays with normal donors for MN, dicentrics and total aberrations, the difference between the means of MN frequencies in beta-thalassaemia traits and normals is significant. MN have a higher control level, a higher value for the alpha component, and much lower value for the beta component, when compared with total aberrations. Both in 2-Gy irradiated G0 lymphocytes as well as 0.5 and 1-Gy irradiated G2 lymphocytes, there is a certain degree of hypersensitivity when the results are meaned over a number of individuals, but some individuals overlap within the normal range. In G0 irradiated lymphocytes from beta-thalassaemia traits, the dicentric chromosome is the only aberration category in which the yield is seen to rise. The heterogeneity in chromosomal radiosensitivity observed in beta-thalassaemia traits is discussed in terms of the oxidative damage consequent to the genetic and biochemical features peculiar to the beta-thalassaemia trait cell. The data presented herein may have wider implications in radiation protection.

Induction of chromosomal aberrations in fish Boleophthalmus dissumieri after exposure in vivo to mitomycin C and heavy metals mercury, selenium and chromium.

The possibilities were explored of using fish as a cytogenetic model in vivo for the detection of potential mutagens. Boleophthalmus dussumieri (2n = 46, fairly large acrocentric chromosomes), an edible mud-skipper and a widely occurring Goby along the Bombay coast, was chosen as the test species after screening 20 species of fish locally available. I.m. injections of mitomycin C in the dose range of 0.5-2.0 mg/kg body weight resulted in a significant increase in the frequency of aberrations per metaphase compared with the control. A dose-response effect was also evident. The types of aberration observed included chromatid and isochromatid breaks, fragments, rings, exchanges and unclassified markers. A significant increase in the number of gaps was also observed. Clastogenic effects of metals such as Hg, Se and Cr in the form of phenyl mercuric acetate, selenium dioxide and sodium dichromate following direct (i.m. injections) and indirect (dissolved in the aquarial water) exposures were studied. A marked enhancement was noticed in the aberration frequency at most of the dose levels tested. Spontaneous chromosomal aberrations in this species were rather rare and occurred at a rate close to zero. If developed along proper lines, fish could be a useful biological model for studying the teratogenic, carcinogenic and mutagenic effects of environmental chemicals.