Showing papers in "Mutagenesis in 2001"

Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay

Abstract: There is a need for a reliable, robust and sensitive assay for DNA repair, suitable for use with human lymphocyte samples in molecular epidemiological investigations. The comet assay (single cell alkaline gel electrophoresis) has been modified to measure the ability of a simple subcellular extract of lymphocytes to carry out the initial step of repair, i.e. incision, on a DNA substrate carrying specific lesions--namely, oxidized bases introduced by visible light in the presence of photosensitizer. The cell extract is free of non-specific nuclease activity, incising DNA only if the DNA has been treated with photosensitizer and light. The activity varies between individuals, but consistency is seen between samples from each individual taken on occasions several months apart. The lack of activity of extract from Ogg1(-) mouse cells (deficient in the glycosylase that excises 8-oxoguanine) in this assay confirms that the activity measured is predominantly excision repair of oxidized bases. This new DNA repair assay is simple, rapid and requires only small quantities of lymphocyte extract (obtainable from 10 ml blood).

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.

Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction?

Abstract: Even though the toxic effects of lead and cadmium compounds have been studied over many years, inconsistent results have been obtained about their mutagenic, clastogenic and carcinogenic properties. However, these metals are considered to be potential human carcinogens. The mechanism of metal-induced carcinogenesis is still unknown, but one possible pathway may involve the interaction of metals with DNA, either directly or indirectly. In this work we explore the capacity of lead, cadmium or a mixture of both metals to interact with acellular DNA, by employing a variant of the comet assay. Our results, using low non-cytotoxic metal concentrations (0.01, 0.1 and 1.0 microM) with the standard protocol for the acellular assay, showed an induction of DNA damage in cells of all organs studied; however, basal DNA damage was different in each organ. To confirm that we were working with pure DNA, proteinase K was added to the lysis solution. With this enriched-lysis solution we found a negative response in the induction of DNA damage in cells derived from the liver, kidney and lung of CD-1 male mice. To support the results obtained by the enriched-acellular assay, we studied the capacity of lead and cadmium (0.1 microM) to induce breaks in pooled genomic DNA in cells of the same organs, with negative results. Consistent with these findings, these metals do not induce DNA breaks in the plasmid pUSE amp+. On the whole, we did not detect direct induction of DNA strand breaks by lead acetate, cadmium chloride or a mixture of both metals, all at low non-cytotoxic concentrations. However, we found an induction of lipid peroxidation and an increase in free radical levels in the different organs of CD-1 male mice after inhalation of lead acetate (0.0068 microg/cc) or cadmium chloride (0.08 microg/cc) for 1 h, suggesting the induction of genotoxicity and carcinogenicity by indirect interactions, such as oxidative stress.

Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes

Abstract: Individual susceptibility to carcinogens, an important determinant of disease risk, is influenced by host factors such as the ability to repair DNA lesions. In order to identify subjects who are at high risk, we have developed a microgel electrophoresis assay for use in molecular epidemiological studies. The assay was validated in a pilot case-control study: Peripheral blood lymphocytes were collected from 100 patients with lung cancer and 110 control patients without cancer and from the same hospital, and stored at -80 degrees C. After thawing, phytohaemagglutinin-stimulated cells were treated with bleomycin at 20 microg/ml for 30 min and the extent of DNA damage and DNA repair capacity were determined by microgel electrophoresis. Peripheral blood lymphocytes from patients with lung cancer were significantly more sensitive to mutagens than those from controls and showed reduced DNA repair capacity (both P < 0.001). Both endpoints were independent risk factors for smoking-related lung cancer. Repeated analysis of peripheral blood lymphocytes from the same individual demonstrated good reproducibility of the assay. Cryopreservation of the lymphocytes for less than or = 12 months did not significantly affect their sensitivity. Our standardized microgel electrophoresis assay is suitable for determining individual sensitivity to mutagens and DNA repair capacity: it is sensitive and faster than cytogenetic assays, and can be applied to native and cryopreserved peripheral blood lymphocytes.

Cytogenetic analysis of Greek farmers using the micronucleus assay in peripheral lymphocytes and buccal cells

Abstract: The potential cytogenetic damage associated with pesticide use in Greek agricultural workers was evaluated using micronuclei (MN) as biomarkers in lymphocytes of peripheral blood and exfoliated cells of the buccal mucosa. In addition, the effects of pesticide exposure and other variables on the cytokinesis block proliferation index (CBPI) in lymphocytes were also evaluated. Both the exposed and control individuals were selected from Nea Makri, a village near Athens (Greece). This location was selected for its high greenhouse density. Micronuclei were analysed in 50 agricultural workers exposed to pesticides (30 men and 20 women) and in 66 non-exposed individuals that constituted the control group (41 men and 25 women). The comparison between workers and controls did not reveal any statistical significant difference in the MN frequency for either lymphocytes or buccal cells. Nevertheless, the multiple regression analysis revealed that the age and the interaction between gender and the number of X-ray examinations during the last 3 years preceding the sampling increased the number of MN in lymphocytes. Moreover, the results of the negative binomial regression analysis suggested that the level of MN in buccal cells could be reduced by the intake of fish, whilst being increased by olive oil consumption. Regarding CBPI, the value found in the exposed group was lower than in controls, the difference being statistically significant. On the other hand, CBPI was inversely associated with both age and X-ray exposure.

Chromosomal aberration and single cell gel electrophoresis (Comet) assay in the longitudinal risk assessment of occupational exposure to pesticides.

Abstract: In recent years the use of pesticides in agriculture has been increasing steadily. At present there are more than 1000 chemicals classified as pesticides. Therefore, the widespread use of pesticides and their potential genetic hazard suggests that evaluation of their genotoxicity should be extended using the newer assays now available. In the present study chromosomal aberration analysis and the alkaline single cell gel electrophoresis (Comet) assay were used to evaluate the extent of DNA damage and DNA repair in peripheral blood lymphocytes of subjects employed in pesticide production. In order to determine possible primary genotoxic effects in workers blood samples were taken after an 8 month long period of exposure to a complex mixture of pesticides. To detect the possible occurrence of DNA repair in lymphocytes of the same subjects the second blood sample was taken after an 8 month long period of absence from the pesticide exposure zone. Regardless of the period of sampling, in the exposed group statistically significantly increased numbers of aberrant cells, chromatid and chromosome breaks, acentric fragments and dicentric chromosomes compared with the controls were found. After the workers had spent 8 months out of the pesticide exposure zone the number of aberrant cells and all types of chromatid and chromosome aberrations decreased significantly compared with sampling after the high exposure period, but it still remained significantly higher in comparison with the control group. After the period of high exposure to a mixture of pesticides statistically significantly increased levels of DNA damage in the Comet assay in terms of tail length and tail moment were found. After the workers were removed from production for 8 months both Comet assay end-points decreased significantly compared with the first sampling point, but they remained increased compared with the control.

Application of the alkaline comet assay in human biomonitoring for genotoxicity: a study on Croatian medical personnel handling antineoplastic drugs.

Abstract: The alkaline comet assay was used to evaluate the genotoxicity towards peripheral blood lymphocytes of medical personnel regularly handling various antineoplastic drugs with different safety precautions. The study population consisted of 50 exposed subjects working in the oncology, pulmonology, gynaecology and haematology units of nine Croatian hospitals and 20 unexposed control subjects. Peripheral blood lymphocytes from the subjects were embedded in agarose on a microscope slide and lysed; the DNA was unwound and subjected to electrophoresis at pH 13. Staining with a fluorescent dye was used to identify cells with DNA damage, as judged by increased migration of genetic material from the cell nucleus. DNA damage was quantified by measuring the displacement between the genetic material of the nucleus and the resulting tail using an image analysis system. Three parameters were used as indicators of DNA damage: i.e. tail length, percentage of DNA in the tail and tail moment. Statistically significant differences in all three parameters were observed between the exposed and control groups. Within the exposed group, there were marked differences between individuals in the comet tail parameters. In the majority of exposed subjects an effect on DNA damage of age or duration of occupational exposure could be excluded. In the exposed group, the highest level of DNA damage was recorded in subjects who used only latex gloves in their work with antineoplastic drugs. The observed DNA damage was lower in exposed subjects who used more than one type of protective equipment and who worked in a well-ventilated safety cabinet. No statistically significant differences were found between the mean values of comet tail parameters for smoking and non-smoking subpopulations from the exposed group. In view of the results obtained, the alkaline comet assay, as a simple, rapid and sensitive method, appears to be a promising additional test for biomonitoring purposes in human populations.

Is ethanol genotoxic? A review of the published data.

Abstract: A great many studies have been carried out on the toxicology of ethanol, the majority in the context of the effects of the consumption of alcohol in beverages Published information relevant to the assessment of the possible genotoxic potential of ethanol has been reviewed and evaluated in terms of the safety of ethanol as an industrial chemical, rather than as a component of beverages The available data on ethanol from standard genotoxicity test methods are incomplete There is clear evidence that ethanol is not a bacterial or mammalian cell mutagen but in vitro assays for chromosome aberration, although mostly negative, have generally not included exogenous metabolic activation Evidence from the use of ethanol as a vehicle control suggests that it is not mutagenic or clastogenic in vitro Reported tests for chromosome aberration induction in vivo are all negative and only a minority of micronucleus tests are positive Conflicting results have been reported for the dominant lethal assay, although an inter-laboratory study performed to OECD guidelines was negative There is some evidence that ethanol induces SCE in vivo and can also act as an aneugen at high doses Many in vivo studies were designed to model alcoholism and used very high doses, sometimes for long periods Outcomes may have been affected by disturbances of metabolism giving rise to secondary effects It is concluded that there is no significant evidence that ethanol is a genotoxic hazard according to the criteria normally applied for the purpose of classification and labelling of industrial chemicals Some degree of genotoxicity may result from excessive alcohol drinking, but this is not considered relevant to any conceivable exposure obtainable by either inhalation or dermal exposure in the workplace

Automatic analysis of slides processed in the Comet assay.

Abstract: In recent years the Comet assay (or single cell gel electrophoresis assay) has been established as a rapid and sensitive method for the detection of DNA damage. For early genotoxicity screening of new chemical entities in industrial toxicology, the Comet assay is more and more used for assessment of the DNA damaging potential of a test compound. In order to increase compound screening throughput, we have established an image analysis system for fully automated measurement of microscope slides processed in the Comet assay. For the comparative investigation various cell types, such as V79 Chinese hamster cells, mouse lymphoma cells and human leukocytes, were treated with several test compounds. Using tail moment as the quantitative parameter for comet formation, we show a very high correlation between our automatic image analysis system and a commercially available, interactive system (Comet Assay II of Perceptive Instruments). The possibility of analyzing 50 samples within 1 day and the high reproducibility of results make automated image processing a powerful tool for automatic analysis of slides processed in the Comet assay.

Termini of human chromosomes display elevated rates of mitotic recombination.

Abstract: The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of Drosophila melanogaster in the wing spot test.

Abstract: The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster was employed to determine the recombinagenic and mutagenic activity of four chemicals in an in vivo eukaryotic system. Two different crosses involving the wing cell markers mwh and flr 3 were used: the standard cross and a high bioactivation cross. The high bioactivation cross is characterized by a high constitutive level of cytochromes P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Three-day-old larvae derived from both crosses were treated chronically with the oxidizing agent potassium chromate and with the three procarcinogens cyclophosphamide, p-dimethylaminoazobenzene and 9,10-dimethylanthracene. From both crosses two types of progeny were obtained: marker-heterozygous and balancer-heterozygous. The wings of both genotypes were analysed for the occurrence of single and twin spots expressing the mwh and/or flr 3 mutant phenotypes. In the marker-heterozygous genotype the spots can be due either to mitotic recombination or to mutation. In contrast, in the balancer-heterozygous genotype only mutational events lead to spot formation, all recombination events being eliminated. The oxidizing agent potassium chromate was equally and highly genotoxic in both crosses. Surprisingly, the promutagen cyclophosphamide also showed equal genotoxicity in both crosses, whereas p-dimethylaminoazobenzene was negative in the standard cross, but clearly genotoxic in the high bioactivation cross. 9,10-Dimethylanthracene showed a rather weak genotoxicity in the high bioactivation cross. Analyses of the dose-response relationships for mwh clones recorded in the two wing genotypes demonstrated that all four compounds are recombinagenic. The fraction of all genotoxic events which are due to mitotic recombination ranged from 83% (9,10-dimethylanthracene) to 99% (p-dimethylaminoazobenzene). These results demonstrate that the wing spot test in Drosophila is most suited to the detection of recombinagenic activity of genotoxic chemicals.

TP53 mutation spectrum in lung cancers and mutagenic signature of components of tobacco smoke: lessons from the IARC TP53 mutation database.

Abstract: A database of all published TP53 mutations in human cancer is maintained at the International Agency for Research on Cancer (IARC). In lung cancers, TP53 mutation patterns show an exceptionally high prevalence of G→T transversions, mostly occurring at codons demonstrated to be sites of adduction of metabolites of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, one of the major carcinogens of tobacco smoke. These observations have been challenged in a recent ‘Discussion Forum’ by T.Paschke, who claimed that a large number of discrepancies existed in the classification of smoking status between successive releases of the IARC TP53 mutation database and that no statistically significant differences could be found in G→T transversion frequencies between smoking and non-smoking lung cancer patients. In the present Letter we question the methods and the conclusions of the analysis presented by Paschke. Based on an assessment of all published data, we confirm the existence of a highly significant difference in the prevalence of G→T transversions between smoking and non-smoking lung cancer patients.

Effect of ultraviolet light, methyl methanesulfonate and ionizing radiation on the genotoxic response and apoptosis of mouse fibroblasts lacking c-Fos, p53 or both.

Abstract: c-Fos and p53 are DNA damage-inducible proteins that are involved in gene regulation, cell cycle checkpoint control and cell proliferation following exposure to genotoxic agents. To investigate comparatively the role of c-Fos and p53 in the maintenance of genomic stability and the induction of apoptosis, we generated mouse fibroblast cell lines from knockout mice deficient for either c-fos (fos -/-) or p53 (p53-/-) or for both gene products (fosp53-/-). The sensitivity of these established cell lines was compared with the corresponding wild-type cells as to the cytotoxic, clastogenic and apoptosis-inducing effects of ultraviolet (UV-C) light and methyl methanesulfonate (MMS). Additionally, we analysed the frequency of apoptosis of the cell lines after treatment with ionizing radiation (IR). We observed c-fos-/-, p53-/- and fosp53-/- cells to be more sensitive than wild-type cells with respect to cell death, as measured in a cytotoxicity (MTT) assay. Regarding apoptosis, all deficient cell lines displayed hypersensitivity to UV-C light, MMS and IR. With chromosomal aberrations as the endpoint, the sensitivity of the double-knockout cells was between wild-type and single-knockouts. The results indicate that both c-Fos and p53 play an important role in protecting fibroblasts against a broad range of genotoxic agents. The results also show that, in fibroblasts, apoptosis induced by UV-C light, MMS and IR does not require p53 and that, in this cell type, p53 rather protects against DNA damage-induced apoptotic cell death.

Spontaneous and induced chromosome damage in somatic cells of sporadic and familial Alzheimer's disease patients

Abstract: Alzheimer's disease (AD) is a neurodegenerative disorder of the elderly with a complex etiology due to the interaction between genetic and environmental factors. At least 15% of cases are inherited as an autosomal dominant mutation, but the majority are sporadic. We evaluated cytogenetic alterations, both spontaneous and chemical-induced [aluminium (Al) and griseofulvin (GF)], by means of the micronucleus (MN) test in lymphocytes or skin fibroblasts of 14 patients with sporadic and eight with familial Alzheimer's disease (FAD), respectively. The spontaneous MN frequencies of sporadic (20.8 +/- 9.2) and familial (20.7 +/- 4.6) AD patients are significantly higher than those of the respective control groups (9.0 +/- 6.8 and 6.7 +/- 3.4). In all AD patients, GF significantly increased the spontaneous MN frequency of somatic cells to a lesser extent (P < 0.05) as compared with the control group. Al treatment did not induce MN in AD patients. The results of the present study indicate that different types of somatic cells from sporadic and familial AD patients show comparable levels of spontaneous cytogenetic anomalies, and MN induction is partially reduced or lacking according to the type of chemical treatments.

Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana Ecological Disaster

Abstract: Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Donana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.

Induction of DNA-strand breaks in human peripheral blood lymphocytes and A549 lung cells by sodium dichromate: association with 8-oxo-2-deoxyguanosine formation and inter-individual variability

Abstract: Hexavalent chromium [Cr(VI)] is a genotoxic carcinogen for which inhalation is a major potential route of exposure in occupational settings. In the present study, the ability of sodium dichromate to cause DNA strand breaks in three populations of cells, human whole blood cells, isolated human peripheral blood lymphocytes and cultured A549 lung epithelial cells, was investigated. Treatment with non-cytotoxic concentrations of sodium dichromate (for 1 h) resulted in a concentration-dependent increase in the number of DNA strand breaks as measured by the Comet assay. The lowest concentrations of sodium dichromate that resulted in a statistically significant (P < 0.01) increase in the number of DNA strand breaks were 500, 50 and 10 microM, respectively, in these cells. The use of formamidopyrimidine glycosylase increased the sensitivity of detection of strand breaks in A549 cells 10-fold, suggesting a role for DNA base oxidation in the mechanism of dichromate-induced DNA strand breaks. In support of this hypothesis, immunocytochemistry indicated an elevation of 8-oxodeoxyguanosine in A549 cells treated with 10 and 500 microM sodium dichromate for 1 h. We also demonstrated 2.11- and 2.5-fold ranges in the level of control and dichromate (500 microM)-induced DNA strand breaks, respectively, in cells of whole blood within a group of healthy volunteers (n = 26). A statistically significant (P < 0.001) positive Pearson's correlation (r = 0.606) was found between control and treated levels of DNA strand breaks, suggesting that factors responsible for relatively low levels of DNA strand breaks in untreated PBL may also offer protection against the formation of dichromate-induced DNA strand breaks.

The in vivo gut micronucleus test detects clastogens and aneugens given by gavage

Abstract: A general testing battery for pharmaceuticals includes a bacterial gene mutation assay, an in vitro chromosomal aberration or a gene mutation test on mammalian cells and an in vivo test for chromosome/genome mutations. The aim of this study was to determine whether the in vivo mouse gut micronucleus assay could be a more sensitive method to detect direct clastogens and/or aneugens given orally by gavage than the in vivo bone marrow micronucleus assay (which can also detect indirect genotoxins). Two laboratories collaborated in this project, one analysing bone marrow cells and the other analysing gut cells from the same animals. The reference substances tested in this study were colchicine (COL), carbendazim (CAR), tubulazole (TUB) and griseofulvin (GRI), all known aneugens, and 1,2-dimethylhydrazine (DMH), a colon carcinogen with clastogenic activity. For all substances tested, the in vivo gut micronucleus test was as sensitive as or more sensitive than the in vivo bone marrow micronucleus assay: COL and TUB induced micronuclei in both gut and bone marrow cells; DMH, CAR and GRI induced micronuclei only in gut cells. The results show that the micronucleus test on gut cells is able to detect clastogens and aneugens given orally by gavage, some of which were not detected by the bone marrow micronucleus test.

Preliminary study of cytogenetic damage in personnel exposed to anesthetic gases

Abstract: Occupational exposure to anesthetic gases is associated with various adverse health effects. Genetic material has been shown to be a sensitive target of numerous harmful agents. The aim of this study was to examine whether chromosomal damage could serve to indicate exposure to anesthetics. A group of 43 hospital workers of three professions (anesthesiologists, technicians and operating room nurses) and 26 control subjects were examined for chromosome aberrations, sister chromatid exchanges and micronucleus frequency. The exposed groups matched in duration of exposure to anesthetics, but not in age. An equal ratio between women and men was possible in all groups except nurses. Likewise, the ratio between smokers and non-smokers was also not comparable. An increase in chromosome damage was found in all exposed groups. While the increase in sister chromatid exchange frequency was not significant, chromosome aberrations and micronucleus frequency increased significantly, showing higher rates in women. The results suggest that the micronucleus test is the most sensitive indicator of changes caused by anesthetic gases. The observed difference between sexes with respect to exposure risk call for further, targeted investigations.

The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue™ rats

Abstract: Limonene and sodium saccharin are male rat specific carcinogens giving rise to renal and bladder tumours, respectively. Both compounds give negative results in genetic toxicity assays suggesting a non-genotoxic mode of action for their carcinogenicity. The alpha 2U-globulin accumulation theory has been invoked to explain the renal carcinogenicity of limonene: the accumulation of micro masses of calcium phosphate in the bladder, coupled with a high pH environment in the male rat bladder, has been suggested to be responsible for the bladder carcinogenicity of sodium saccharin. The implication of these proposed mechanisms is that limonene and sodium saccharin will not be mutagenic to the rat kidney and bladder, respectively. This proposal has been evaluated by assessing the mutagenic potential of the two chemicals to male lacI transgenic (Big Blue) rats. Male Big Blue rats were exposed for 10 consecutive days to either limonene in diet, at a dose level in excess of that used in the original National Toxicology Program gavage carcinogenicity bioassay, or to sodium saccharin in diet at the dose known to induce bladder tumours. The multi-site rat carcinogen 4-aminobiphenyl was used as a positive control for the experiment. Limonene failed to increase the mutant frequency in the liver or kidney of the rats, and sodium saccharin failed to increase the mutant frequency in the liver or bladder of the rats. 4-Aminobiphenyl was mutagenic to all three of these tissues. These results add further support to a non-genotoxic mechanism of carcinogenic action for both limonene and sodium saccharin.

Apoptosis can be a confusing factor in in vitro clastogenic assays

Abstract: Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two muta-genicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

Heritable effects of paternal irradiation in mice on signaling protein kinase activities in F3 offspring

Abstract: We evaluated F 3 mouse offspring from paternal F 0 attenuated 137 Cs γ-irradiation (1.0 Gy) for heritable effects on gene products that can modulate cell proliferation rate and that may be markers for genomic instability. The F 3 generation was selected for evaluation as a stringent test for heritability of effects from paternal F 0 germline irradiation. Male CD1 mice were bred 6 weeks after irradiation so that the fertilizing sperm were type B spermatogonia at the time of irradiation. The resulting F 1 males were bred to CD1 females to produce F 2 four-cell embryos. The F 2 embryos with a radiation history were paired with 'control' CD1 four-cell embryos that were heterozygous for the neo transgene. These F 2 XY-XY chimeras, consisting of cells derived from both an embryo with a paternal F 0 radiation history and a control embryo, were transferred to foster mothers, raised to adulthood and bred to produce F 3 offspring. F 3 offspring were evaluated for hepatic activities of receptor tyrosine kinase, protein kinase C and MAP kinase and for protein levels of nuclear p53 and p21 waf1 . All three protein kinase activities were altered and nuclear levels of p53 and p21 waf1 protein were higher in the group of offspring that included F 3 offspring with a paternal F 0 radiation history than in littermates in the neo-positive control group. To our knowledge, this is the first observation in the descendants of paternal germline irradiation of effects on signal protein kinase activities and downstream nuclear target proteins that can influence cell proliferation rates.

Taxanes: the genetic toxicity of paclitaxel and docetaxel in somatic cells of Drosophila melanogaster.

Abstract: In this study, the taxanes, paclitaxel and docetaxel were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. These relatively new drugs are used in cancer therapy and show great promise in the treatment of a variety of cancers. Their major cellular target is the alpha,beta-tubulin dimer but, unlike other spindle poisons, they stabilize microtubules by a shift towards assembly, producing nonfunctional microtubule bundles. The Drosophila wing Somatic Mutation and Recombination Test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the standard version of SMART (with normal bioactivation) and a variant version with increased cytochrome P450-dependent biotransformation capacity. In the standard assay, docetaxel was found to be aneuploidogenic; this was effectively abolished by a high cytochrome P450-dependent detoxification capacity. This suggests, as previously reported, the involvement of this family of enzymes in the detoxification of docetaxel rather than in its activation. In contrast, paclitaxel was clearly non-genotoxic at the same (millimolar) concentrations as used for docetaxel in both crosses. The weak responsiveness of SMART assays to aneugenic compounds, the weaker ligand and assembly action of paclitaxel and the more rapid reversibility of the microtubules formed with this compound, may have caused the negative response observed in the present study.

PHA-induced cell proliferation rescues human peripheral blood lymphocytes from X-ray-induced apoptosis.

Abstract: Human peripheral blood G(0) lymphocytes were X-irradiated and allowed to recover for different periods both in the presence and absence of phytohemagglutinin (PHA). For each experimental condition the induction of apoptosis was investigated by nuclear morphology and formation of both DNA laddering and high molecular weight DNA fragments by pulsed field gel electrophoresis. The results showed that PHA cell growth stimulation could rescue peripheral blood lymphocytes (PBLs) from X-ray-induced apoptotic cell death. Instead, most X-irradiated lymphocytes held in G(0) phase, once they were committed to apoptosis, inexorably executed the process. These data indicate that the proliferative status of PBLs can influence apoptotic cell death: PHA-stimulated PBLs appear to be more radioresistant as a result of a less efficient apoptotic process. Therefore, in current tests for mutagenicity or cytotoxicity and in biodosimetrical studies the possible role of apoptosis has to be considered.

Effect of the dithiocarbamate pesticide zineb and its commercial formulation azzurro. I. Genotoxic evaluation on cultured human lymphocytes exposed in vitro

Abstract: The in vitro cytogenetic effects exerted by the dithiocarbamate fungicide zineb and one of its commercial formulations currently used in Argentina, azzurro, were studied in whole blood human lymphocyte cultures. The genotoxicity of the fungicides was measured by analysis of the frequency of chromosomal aberrations and sister chromatid exchanges (SCEs) and cell cycle progression assays. Both zineb and azzurro activities were tested within the range 0.1-100.0 μg/ml immediately after in vitro lymphocyte stimulation. Only concentrations of 50.0 and 100.0 μg/ml zineb and azzurro induced a significant increase in SCE frequency over control values. Furthermore, this genotoxicity appears to be correlated with its cytotoxicity, measured as cell cycle kinetics, since both a significant delay in cell cycle progression and a significant reduction in proliferative rate index were only observed in those cultures treated with these fungicide concentrations. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed when increasing the fungicide concentration. Moreover, only the mitotic activity statistically differed from control values when doses of zineb or azzurro <10 μg/ml were employed. For both fungicides the mitotic index reached the minimal value at doses of 100 μg/ml. Both products induced a significant dose-dependent increase in the number of abnormal cells, chromatid-type and chromosome-type aberrations as well as in the total number of aberrations in the 0.1-100.0 μg/ml dose range. Based on these results, the evaluation of zineb as a controversial genotoxic/non-genotoxic compound for human health should be reconsidered. Instead, we demonstrate that the fungicide induces large DNA alterations and should be considered as a clastogenic mutagen.

Hydrogen peroxide: effects on DNA, chromosomes, cell cycle and apoptosis induction in Fanconi's anemia cell lines

Abstract: Fanconi's anemia (FA) is an inherited autosomal recessive syndrome; cells from FA patients are very sensitive to crosslinking agents and to oxygen. Epstein-Barr virus (EBV)-transformed lymphoblasts belonging to different FA complementation groups and normal EBV-transformed lymphoblasts were studied for their response to treatment with the oxidizing agent hydrogen peroxide (H 2 O 2 ). The analysis of 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in the DNA of untreated cells showed an increased basal level of damage in cells from the complementation groups FA-C and FA-E. H 2 O 2 -induced 8-OHdG was higher in FA than in normal cell lines. The removal of 8-OHdG after H 2 O 2 treatment was significantly reduced in the cells from complementation group E. However, all FA cell lines showed a normal ability in the resealing of DNA breaks, at least soon after treatment. All cell lines were also equally efficient in the removal of damaged pyrimidines. Compared with normal cells, FA cell lines showed an increase in the baseline level of micronuclei, but not in the number of micronuclei induced by H 2 O 2 . Micronuclei in FA cells originated prevalently from chromosomal fragmentation and, at a minor extent, from chromosome loss. After H 2 O 2 treatment, FA cell lines accumulated in G 2 phase to a greater extent than normal lymphoblasts. However, reversion of mutation in FA-A and FA-C cells did not result in the correction of this phenotype. In cells evaluated for apoptosis no ladder formation was found in FA-C, FA-E and corrected FA-C cells. In conclusion, among the FA cell lines examined, only FA-E showed a defect in the repair of H 2 O 2 -induced damage. On the other hand, differences found in the cell cycle and apoptosis might be due to irreversible changes occurring in FA cell lines as a consequence of the primary defect.

Preliminary study of the genotoxic potential of homocysteine in human lymphocytes in vitro

Abstract: Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hcy concentration is also reported to correlate positively with the micronucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hcy is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hcy are genotoxic to human lymphocytes in vitro using the cytokinesis-block micronucleus assay. Eighteen lymphocyte cultures were initiated in Met-free and serum-free RPMI 1640 medium for each of four male volunteers aged 22-23 years. At 0, 24, 44 and 72 h, cultures were spiked with L-Hcy or L-Met to achieve concentrations ranging between 50 and 400 microM. The concentration of Hcy at 96 h ranged from 19.45 +/- 2.34 to 149.02 +/- 28.16 microM in Hcy cultures and 0.91 +/- 0.17 to 2.15 +/- 0.9 microM in Met cultures spiked with 50 and 400 microM of metabolite, respectively. Forty-four hours after mitogen stimulation, cytokinesis was inhibited with cytochalasin B. After 96 h, cells were transferred to microscope slides and the frequency of micronucleated-binucleate and necrotic cells was scored. Neither Hcy (P = 0.24) nor Met (P = 0.93) had an apparent dose effect on micronucleus frequency. However, when data were pooled, micronucleus frequency was moderately higher (50.1%) in Hcy- than in Met-spiked cultures (P = 0.04; paired t-test). Hcy concentration was positively correlated with necrosis (P < 0.0005; r(2)= 0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hcy-spiked cultures (P= 0.01; paired t-test). Further research is required to define more clearly the genotoxic and cytotoxic potential of homocysteine and its metabolites.

Increased formation of micronuclei after hormonal stimulation of cell proliferation in human breast cancer cells

Abstract: The carcinogenicity of sex hormones is considered to be the result of a combination of genotoxic and epigenetic modes of action. For estrogens, genotoxic activities include DNA damage by reactive metabolites and indirect genotoxicity by redox cycling and production of reactive oxygen species. Here, we present data on the induction of micronuclei in estrogen receptor-positive (MCF-7) and -negative (MDA) human breast cancer cell lines treated with estradiol to support an additional mechanism of chromosomal damage. MCF-7 cells, but not MDA cells, treated with estradiol in the picomolar concentration range showed an increase in micronucleus formation which correlated with the estradiol-induced cell proliferation. Addition of the specific estradiol-receptor antagonist hydroxytamoxifen suppressed the estradiol-induced formation of micronuclei in MCF-7 cells. Increased frequencies were also seen after normalization of the data to the number of cell divisions by additional treatment of the cells with cytochalasin B. Thus, formation of micronuclei was not due to the chromosomal damaging activity of estradiol. The induced genomic damage may be explained by a hormone-specific forcing of responsive cells through the cell cycle, thereby overriding checkpoints operating under homeostatic control of the cell cycle.

Differential chromosome behaviour in mammalian oocytes exposed to the tranquilizer diazepam in vitro.

Abstract: There are several reports demonstrating that aneugens may preferentially affect segregation of particular chromosomes in somatic cells. Much less is known on specific susceptibility of individual chromosomes to non-disjunction in mammalian meiosis in response to chemical exposures. To explore possible chromosome-specific behaviour and susceptibility to errors in chromosome segregation in mammalian oogenesis we employed spindle immunofluoresecence in combination with FISH with chromosome-specific probes to analyse congression of chromosomes X, 8 and 16 in diazepam (DZ)-treated, meiotically delayed meiosis I oocytes of the mouse. Concomitantly, we assessed the susceptibility of homologues to precociously segregate prior to anaphase I during DZ-induced meiotic arrest. About 50% of all oocytes exposed to 25 microg/ml DZ became meiotically delayed. Chromosomes failed to congress at the spindle equator in one-third of these meiosis I oocytes. The X chromosome was significantly more often located away from the spindle equator as compared with the expected random behaviour. Concomitantly, DZ exposure induced untimely segregation of homologous chromosomes of the gonosome and the autosomes in meiosis I. This occurred with similar frequencies. The observations confirm that DZ perturbs cell cycle progression, interferes with chromosome alignment, causes predivision and thus may predispose mammalian oocytes to errors in chromosome segregation. For the first time, chromosome-specific behaviour is reported in female meiosis in response to exposure to an aneugenic chemical.

Phenobarbital, oxazepam and Wyeth 14,643 cause DNA damage as measured by the Comet assay.

Abstract: Research Iriangle Park, NC, 27709, USA Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do not form DNA adducts, they induce mutations in the Big Blue transgenic mouse model. The mutations produced by these compounds were predominantly GET and G→C transversions that we suspect arose from oxidative damage to DNA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human myeloid leukemia K562 cells were treated with non-cytotoxic doses of the above compounds for 3 h, then placed on slides containing low melting point agarose. Cells were lysed, exposed to alkaline buffer, electrophoresed and analyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and apurinic/ apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort was not observed in cells exposed to phenobarbital (1 mM). However, the addition of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these studies that two of the three compounds evaluated in this study mediate their mutagenic effects through oxidative stress, but that the mechanism of DNA damage caused by phenobarbital differs from that elicited by oxazepam and Wyeth 14,643.

Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures.

Abstract: Styrene is one of the most important organic chemicals in use today. The highest human exposures to styrene take place by inhalation during the production of fibreglass-reinforced plastics. Styrene is oxidized by hepatic cytochrome P450 to styrene-7,8-oxide (SO), an epoxide that has been shown to induce chromosome aberrations, sister chromatid exchanges and micronuclei in many cell systems. In this work, the effect of SO on the expression of some genes involved in the cell cycle and apoptosis regulation in human white blood cells was studied. Lymphocyte cultures from four donors were exposed to 50 and 200 microM SO, 1% DMSO being the control. Aliquots of the cultures were taken at six different time points (30, 36, 42, 48, 60 and 72 h), total mRNA was extracted in each one of them and RT-PCR was carried out to analyze the expression of the genes p53, p21, bcl-2 and bax. Moreover, a cytokinesis block assay was performed to estimate cell proliferation kinetics by calculating the cytokinesis block proliferation index (CBPI), and to evaluate the number of cells undergoing apoptosis. Furthermore, apoptotic events were detected by the DNA fragmentation assay. In our results, a high interindividual variation in the expression of the studied genes was observed. Expression curves obtained for the four genes, together with the data from the CBPI and apoptotic cells scored, suggest that exposure to high levels of SO may induce a delay in the cell cycle, probably directed to allowing repair systems to act on the genotoxic damage produced, more than driving cells towards programmed cell death.