Showing papers in "Mutagenesis in 2003"

Recommendations for conducting the in vivo alkaline Comet assay

Abstract: The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.

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.

Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay.

Abstract: We have performed experiments using the WIL2-NS human B lymphoblastoid cell line and primary human lymphocytes to: (i). determine the importance of including measurements of nucleoplasmic bridges (NPB) in the cytokinesis-block micronucleus (CBMN) assay; (ii). provide evidence that NPB originate from dicentric chromosomes and centric ring chromosomes. In addition, we describe theoretical models that explain how dicentric chromosomes and centric ring chromosomes may result in the formation of NPB at anaphase. The results with WIL2-NS showed that it was possible to distinguish genotoxic effects induced by different oxidizing agents in terms of the NPB/micronucleus frequency ratio. The results with lymphocytes indicated a strong correlation: (i). between NPB, centric ring chromosomes and dicentric chromosomes in metaphases (r > 0.93, P 0.93, P < 0.0001). The dose-response curves with gamma-rays were very similar for NPB, ring chromosomes and dicentric chromosomes, as were the dose-response curves for MNi, acentric rings and fragments. However, not all acentric chromosomes and dicentric chromosomes/centric rings were converted to MNi and NPB respectively, depending on the dose of radiation. Preliminary data, using FISH, suggest that NPB often represent DNA from a structural rearrangement involving only one or two homologous chromosomes. The results from this study validate the inclusion of NPB in the CBMN assay which provides a valuable measure of chromosome breakage/rearrangement that was otherwise not available in the micronucleus assay. The CBMN assay allows NPB measurement to be achieved reliably because inhibition of cytokinesis prevents the loss of NPB that would otherwise occur if cells were allowed to divide.

Statistics of the Comet assay: a key to discriminate between genotoxic effects

Abstract: The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with delta-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA.

Evaluation of genetic damage in workers employed in pesticide production utilizing the Comet assay.

Abstract: The use of pesticides has been increasing in recent years, resulting in the need for increased production of pesticides. However, some pesticides may represent a hazard to human health, especially by causing cancer. Genotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. Therefore, in the current study the potential DNA damage associated with exposure to pesticides of Indian pesticide production workers was assessed using the single cell gel electrophoresis assay or Comet assay. Blood leukocytes of a group of 54 pesticide workers and an equal number of control subjects were examined for genotoxicity in this study. The two groups had similar mean ages and smoking prevalences. The mean comet tail length was used to measure DNA damage. The exposed workers had significantly greater mean comet tail lengths than those of controls (mean +/- SD 19.17 +/- 2.467 versus 8.938 +/- 2.889, P < 0.001). Smokers had significantly larger mean tail lengths than non-smokers (19.75 +/- 2.52 versus 18.26 +/- 2.13, P = 0.024). Analysis of covariance showed that occupational exposure (P < 0.05) and smoking (P < 0.05) had significant effects on mean tail length, whereas age and gender had no effect on DNA damage. The present study suggests that occupational exposure to pesticides and smoking can cause DNA damage. This investigation confirms the sensitivity of the Comet assay.

TP53 polymorphisms and lung cancer risk: a systematic review and meta‐analysis

Abstract: To examine the risk of lung cancer associated with the codon 72, intron 6 and intron 3 TP53 polymorphisms a meta-analysis of published case-control studies was undertaken. The principle outcome measure was the odds ratio (OR) for the risk of lung cancer using homozygosity of the 'wild-type allele' as the reference group. Data from 13 studies detailing the relationship between lung cancer and the codon 72 polymorphism of TP53 and three studies examining the intron 3 and 6 polymorphisms of TP53 were analysed. The ORs of lung cancer associated with the Pro-Pro and Pro-carrier genotypes of codon 72 were 1.18 [95% confidence interval (CI) 0.99-1.41] and 1.02 (95% CI 0.86-1.20), respectively. The ORs of lung cancer associated with homozygous and variant allele carrier genotypes of the intron 6 (MspI RFLP) polymorphism were 1.13 (95% CI 0.55-2.27) and 1.30 (95% CI 0.75-2.26) and of the intron 3 (16 bp duplication) polymorphism were 1.50 (95% CI 0.76-2.97) and 1.11 (95% CI 0.53-2.35), respectively. Although polymorphic variations in TP53 represent attractive candidate susceptibility alleles for lung cancer the results from this analysis provide little support for this hypothesis. Additional well-designed studies based on sample sizes commensurate with the detection of small genotypic risks may allow a more definitive conclusion.

Cytogenetic biomonitoring in petrol station attendants: micronucleus test in exfoliated buccal cells

Abstract: To study the effects of occupational exposure to petroleum derivates such as benzene, exfoliated buccal cells from 50 petrol station attendants and 50 age- and sex-matched control subjects were examined for micronucleus (MN) frequency. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis and karyolysis, were also evaluated. Benzene exposure was ascertained by measuring urinary phenol levels. The mean urinary phenol level of station workers was found to be significantly higher than that of control subjects (P < 0.05). Analysis of buccal cells revealed that MN and NA frequencies in petrol station workers were significantly higher than in control subjects (P < 0.01) and also significantly related to smoking habit (P < 0.01). Our findings indicate that the petrol station workers are under risk of significant cytogenetic damage.

Biomonitoring of four European populations occupationally exposed to pesticides: use of micronuclei as biomarkers

Abstract: This paper presents the results obtained within the framework of an EU research project aimed at investigating the relationship between occupational exposure to pesticides and the induction of cytogenetic damage. Populations from Greece, Spain, Poland and Hungary, all of them characterised by intensive agricultural activity, were the subject of the study. A total of 239 agricultural workers and 231 unexposed controls were examined for cytogenetic effects in lymphocytes of peripheral blood and exfoliated cells of the oral mucosa. The frequency of micronuclei (MN) was evaluated in both cell types and their relationship to different confounding factors (e.g. sex, country, smoking habit, etc.) was determined. The cytokinesis block proliferation index (CBPI) was also calculated to detect possible variations in the proliferative kinetics of lymphocytes due to pesticide exposure. The results obtained indicate that there are no increases in MN frequencies in the agricultural workers when compared with the controls for either lymphocytes or buccal cells. However, exposed individuals showed a significant decrease in CBPI when compared with controls. When the effect of the different confounding factors was evaluated, age was positively related with MN in lymphocytes and the Polish population showed a MN frequency significantly higher than those observed in the other populations. For buccal cells, the Spanish population showed a higher MN frequency, attaining significant differences in comparison with the other populations. Finally, the CBPI was found to be inversely influenced by age and Hungarian exposed men were the group that showed the lowest values.

Naringin, a citrus flavonone, protects against radiation-induced chromosome damage in mouse bone marrow.

Abstract: Free radicals are responsible for the induction of damage to the cellular DNA that leads to the formation of chromosome aberrations. Antioxidants are known to scavenge free radicals, thereby decreasing the degree of such effects. Radiation is a well-known inducer of free radicals and compounds that can scavenge free radicals may reduce radiation-induced DNA damage. Naringin, a bioflavonoid predominant in grapefruit and other citrus fruits, has been found to scavenge free radicals, therefore it may also reduce radiation-induced damage. The aim of the present study was to evaluate the radioprotective action of 2 mg/kg naringin in the bone marrow of mice exposed to different doses of (60)Co gamma-radiation by scoring the frequency of asymmetrical chromosomal aberrations. The irradiation of mice resulted in a dose-dependent elevation in the frequency of aberrant cells, acentric fragments, chromatid and chromosome breaks, dicentrics and exchanges. All these aberrations were elevated with scoring time up to 24 h post-irradiation and declined thereafter, except chromatid breaks, which were maximum at 12 h post-irradiation. Treatment of mice with 2 mg/kg body wt naringin before exposure to various doses of gamma-radiation resulted in a significant reduction in the frequencies of aberrant cells and chromosomal aberrations like acentric fragments, chromatid and chromosome breaks, centric rings, dicentrics and exchanges. The evaluation of free radical scavenging activity of naringin revealed a dose-dependent scavenging of hydroxyl, superoxide and 2,2 equal to or precedes -diphenyl-1-picryl hydrazyl radical. Naringin at 5 microM scavenged the 2,2-azino-bis-3-ethyl benzothiazoline-6-sulphonic acid cation radical very efficiently, where a 90% scavenging was observed. Our study demonstrates that naringin can protect mouse bone marrow cells against radiation-induced chromosomal damage.

Mitochondrial impairment in p53-deficient human cancer cells.

Abstract: The mechanism linking p53 inactivation to human cell malignancy remains unclear. Studies have indicated that mitochondrial dysfunction is involved in carcinogenesis. In this study we investigated the role of p53 in mitochondrial DNA (mtDNA) mutation and maintenance of proper mitochondrial function. We measured mtDNA mutation and found no difference in frequency of mutation between the p53(+/+) and p53(-/-) cell lines. However, mitochondrial cytochrome c oxidase (COX) activity was significantly diminished in p53(-/-) cells. This decrease in COX activity was attributed to decreased protein levels of the COXII subunit encoded by the mitochondrial genome and was not due to mutation in the mitochondrial COXII gene. Further investigation revealed no concomitant decrease in COXII mRNA levels in p53(-/-) cells and the stability of mRNA in p53(-/-) cells was unaffected. This study suggests that decreased COX activity is likely due to post-transcriptional regulation of the COXII subunit by p53. COX is a critical enzyme in the mitochondrial electron transport chain and reduced COX activity may affect mitochondrial structure. However, examination of mitochondrial ultrastructure revealed no obvious differences between p53(+/+) and p53(-/-) cell lines. Together, our study suggests that p53 is involved in regulation of COXII at the protein level but not at the mRNA level. p53 does not affect mtDNA mutation or mitochondrial ultrastructure.

Elevated levels of DNA-protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium.

Abstract: DNA–protein crosslinks (DPC) are a promising biomarker of exposure to hexavalent chromium, a known human carcinogen. Although trivalent chromium is considered to have much lower toxicity, the risk involved in chronic exposure is uncertain. DPC may be a useful tool in clarifying this risk, by signaling an exposure of body tissues to biologically active forms of chromium. DPC quantification was carried out in lymphocytes of a group of tannery workers exposed to trivalent chromium, a small group of manual metal arc stainless steel welders exposed to hexavalent chromium and a control group. This biomarker was compared with the frequency of micronuclei in cytokinesis blocked peripheral lymphocytes as a biomarker of cytogenetic lesions and total plasma and urine chromium levels as an index of exposure. The results indicate a significant increase in the formation of DPC in tannery workers compared with controls (0.88 0.19 versus 0.57 0.21%, P < 0.001, Mann–Whitney test) and an even higher level of DPC in welders (2.22 1.12%, P 0.03). Tanners showed a significant increase in micronucleated cells compared with controls (6.35 2.94 versus 3.58 1.69‰, P < 0.01), whereas in welders this increase was not significant (5.40 1.67‰). Urinary chromium was increased in both groups, with a greater increase observed in tanners compared with controls (2.63 1.62 versus 0.70 0.38 µg/g creatinine, P < 0.001) than in welders (1.90 0.37 µg/g creatinine, P < 0.005). Plasma chromium was also increased in both groups (tanners 2.43 2.11 µg/l, P < 0.001, welders 1.55 0.67 µg/l, P < 0.005 versus controls 0.41 0.11 µg/l). In summary, chronic occupational exposure to trivalent chromium can lead to a detectable increase in lymphocye DNA damage which correlates with a significant exposure of the cells to the metal.

Antimutagenic activity of extracts of natural substances in the Salmonella/microsome assay

Abstract: Scientific information regarding plants used in folk medicine in the form of teas and their effect on human health or on genetic material has been the subject of many different types of investigation. The antimutagenic activity of two plants Maytenus ilicifolia and Peltastes peltatus, both rich in compounds of the flavonoid and tannin groups and frequently employed in folk medicine, was studied. Antimutagenicity was determined against known mutagenic substances (4-oxide-1-nitroquinoline, sodium azide, 2-nitrofluorene, aflatoxin B 1 , 2-aminofluorene and 2-aminoanthracene), using the Salmonella/microsome assay. Infusions of P.peltatus showed high cytotoxicity and a co-mutagenic effect for induction of base pair substitution mutations with 4-oxide-1-nitroquinoline (-S9 mix). Infusions of M.ilicifolia produced similar effects for frameshift and base pair substitution mutations. With the mutagens 2-nitro-fluorene (TA98) and sodium azide (TA100) no significant enhancement effects (co-mutagenic effects) were observed and inhibition of mutagenic activity and cytotoxicity were also diminished. In assays evaluating antimutagenic activity in the presence of metabolic activation utilizing S9 mix, high and significant inhibition of aflatoxin B 1 -, 2-aminofluorene-and 2-aminoanthracene-induced mutagenicity was observed in the presence of the infusions using both TA98 and TA100 and employing doses ranging from 25 to 500 mg/plate. Seventy-five percent of the doses tested exhibited a significant or suggestive decrease in induced mutagenicity with the infusion of M.ilicifolia. With the infusion of P.peltatus significant or suggestive antimutagenic responses were observed with 50% of the doses evaluated. Complexity was clearly noted in the responses observed in the interaction of aqueous extracts of M.ilicifolia and P.peltastes with the genetic material and metabolites generated by the S9 mix played an important role in the protection of DNA.

Aspects of design and statistical analysis in the Comet assay.

Abstract: Some aspects of the statistical design and analysis of the Comet (single cell gel electrophoresis) assay have been evaluated by means of a simulation study. The tail length and tail moment were selected for the quantification of DNA migration. Results from the simulation study showed that the choice of measure to summarize the cells on each slide is extremely important in order to facilitate an efficient analysis. For tail moment, the mean of log transformed data is clearly superior to the other evaluated measures, whereas using the mean of raw data without transformation can lead to very inefficient analyses. The 90th percentile, capturing the upper tail of the distribution, performs well for the tail length, with a slight improvement obtained by applying a log transformation prior to calculations. Furthermore, the simulation study has been used to assess the appropriateness of some models for statistical analysis and to address the issue of design (i.e. number of cultures or animals in each group, number of slides per animal/ culture and number of cells scored per slide). Combining the results from the simulations with practical experience from the pharmaceutical industry, we conclude the paper by providing concise recommendations regarding the design and statistical analysis in the Comet assay.

Investigations into the concept of a threshold for topoisomerase inhibitor-induced clastogenicity

Abstract: Although the application of the concept of a threshold to risk assessment is widespread, there remains little experimental evidence for the existence of thresholds for genotoxic compounds, other than aneugens. The clastogenicity of topoisomerase inhibitors is believed to result from the transient stabilization of the topoisomerase enzyme with DNA during the catalytic cycle. This leads to the formation of a stabilized cleavage complex, which, in turn, may result in the formation of a DNA strand break. This indirect mechanism of clastogenicity is the basis for the concept of threshold for this class of drug. Using micronucleus induction in L5178Y mouse lymphoma cells as a genotoxic end-point, a three pronged approach was used to examine whether the concept of a threshold for clastogenicity could be demonstrated for topoisomerase type II inhibitors in vitro. This involved (i) the study of mechanism (TARDIS assay), (ii) hypothesis testing versus estimation (i.e. scoring up to 10,000 cells/treatment at concentrations immediately above and below the NOEL for micronucleus induction) and (iii) statistical modelling of the concentration-response curves for micronucleus induction. Several topoisomerase type II inhibitors were investigated with varying clastogenic potencies (etoposide = doxorubicin < genistein < ciprofloxacin). Pragmatic thresholds for clastogenicity in L5178Y cells were defined at 0.00236 microg/ml for etoposide, 0.00151 microg/ml for doxorubicin, 1 microg/ml for genistein and 50 microg/ml for ciprofloxacin. In addition, it was demonstrated that etoposide-induced clastogenicity was concentration and time dependent. These results, along with mechanistic data showing that all of the compounds induced concentration-dependent increases in the formation of topoisomerase II stabilized cleavage complexes, provide a weight of evidence to support a threshold concept for clastogenicity with topoisomerase II poisons.

Evaluation of the radioprotective effect of Aegle marmelos (L.) Correa in cultured human peripheral blood lymphocytes exposed to different doses of γ‐radiation: a micronucleus study

Abstract: The radioprotective effect of a hydroalcoholic extract of Aegle marmelos (AME) was evaluated in cultured human peripheral blood lymphocytes (HPBLs) by the micronucleus assay. The optimum protective dose of the extract was selected by treating HPBLs with 1.25, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 microg/ml AME before exposure to 3 Gy gamma-radiation and then evaluating the micronucleus frequency in cytokinesis blocked HPBLs. Treatment of HPBLs with different doses of AME reduced the frequency of radiation-induced micronuclei significantly, with the greatest reduction in micronucleus induction being observed for 5 microg/ml AME. Therefore, this dose of AME was considered as the optimum dose for radioprotection and further studies were carried out treating the HPBLs with 5 microg/ml AME before exposure to different doses (0, 0.5, 1, 2, 3 and 4 Gy) of gamma-radiation. The irradiation of HPBLs with different doses of gamma-radiation caused a dose-dependent increase in the frequency of lymphocytes bearing one, two and multiple micronuclei, while treatment of HPBLs with 5 microg/ml AME significantly reduced the frequency of lymphocytes bearing one, two and multiple micronuclei when compared with the irradiated control. The dose-response relationship for both groups was linear. To understand the mechanism of action of AME separate experiments were conducted to evaluate the free radical scavenging of OH, O2(-), DPPH, ABTS(+) and NO in vitro. AME was found to inhibit free radicals in a dose-dependent manner up to a dose of 200 microg/ml for the majority of radicals and plateaued thereafter. Our study demonstrates that AME at 5 microg/ml protected HPBLs against radiation-induced DNA damage and genomic instability and its radioprotective activity may be by scavenging of radiation-induced free radicals and increased oxidant status.

The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation

Abstract: The alkaline Comet assay was selected as a biomarker of exposure to evaluate the ongoing exposure to ionizing radiation of 50 medical workers occupationally exposed to ionizing radiation and 50 corresponding unexposed control subjects. The primary DNA damage was evaluated by measuring the extent of DNA migration in peripheral blood leukocytes. The inter-individual differences in DNA damage between exposed subjects were compared with their dosimeter readings and occupation. It was found that medical workers who were occupationally exposed to ionizing radiation for different periods of time showed highly significant increases in levels of DNA damage compared with controls. However, influences of the different occupational settings and doses absorbed on the levels of DNA damage, assessed by use of the Comet assay, might be excluded in the majority of subjects. Differences in comet parameters measured due to smoking and gender were not statistically significant in either exposed or control subjects. The results obtained have confirmed the usefulness of the alkaline Comet assay as an additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.

In Vitro genotoxic effects of different combinations of cobalt and metallic carbide particles

Abstract: Occupational exposure to hard metal dust, consisting of tungsten carbide (WC) and metallic cobalt particles (Co), is associated with an increased risk of lung cancer, while no increased risk was observed in workers exposed to Co alone. In vitro, in human peripheral blood mononucleated cells (PBMC), we previously demonstrated that WC-Co is more genotoxic than Co and WC alone. A possible mechanism underlying this higher genotoxicity is a specific physicochemical interaction between Co and WC particles leading to the enhanced short-term formation of active oxygen species. The aim of this study was to evaluate the in vitro genotoxicity of other combinations of Co with metal carbide particles in comparison with WC-Co. The ability of Cr(3)C(2), Mo(2)C and NbC and of their powder mixtures with Co to induce DNA strand breaks and alkali-labile sites was assessed by the alkaline Comet assay and their potential to induce chromosome(/genome) mutations by the cytokinesis-block micronucleus test on human PBMC from two donors. PBMC were treated in vitro for 15 min, 24 h after the onset of PHA stimulation. In the micronucleus test, while the metal carbides alone did not increase the micronucleus frequency, Co alone and the four tested carbide-Co mixtures induced a statistically significant concentration-dependent increase in micronucleated binucleates. In addition to WC, NbC and Cr(3)C(2) particles were able to interact with Co, producing a higher mutagenic effect than the individual metal particles. Mo(2)C particles did not display interactive mutagenicity with Co in the micronucleus test, possibly related to their small specific surface area, compactness and/or spherical shape. With the Comet assay, applied directly at the end of the treatment, less clear results, due to inter-experimental and inter-donor variation, were obtained. These data indicate that particular interaction of a metal carbide with Co leading to enhanced mutagenicity is not specific for WC.

Mitochondrial impairment is accompanied by impaired oxidative DNA repair in the nucleus

Abstract: Depletion of the mitochondrial genome is involved in several human diseases, as well as in mitochondrial diseases induced by drug therapies used in the treatment of cancer and human immunodeficiency virus. In order to identify the molecular changes underlying the pathogenesis of mitochondrial diseases, we determined the oxidative status of a human cell line following depletion of the mitochondrial genome (denoted rho0 cells). Our analysis revealed that rho0 cells contained approximately 10-fold lower levels of superoxide than parental cells (rho+), as detected by oxidation of dihydroethidium. No concurrent decrease in oxidation of hydrogen peroxide, detected using the dye dichloroflorescein diacetate, was observed in rho0 cells. Depletion of the mitochondrial genome did not affect either the expression of superoxide dismutase or its activity. However, catalase expression and its activity decreased in rho0 cells. In addition, glutathione peroxidase activity was higher in rho0 cells compared with rho+. rho0 cells showed increased lipid peroxidation, increased oxidative damage to the nuclear genome and impaired DNA repair. Our data illustrate the importance of the mitochondrial genome and its function to the cellular oxidative environment and nuclear genome instability. It also provides insights into the development of mitochondrial disease as a consequence of cancer therapy.

A comparison of folic acid and 5-methyltetrahydrofolate for prevention of DNA damage and cell death in human lymphocytes in vitro

Abstract: Folic acid (FA), the most oxidized and stable form of folate, is commonly used as a dietary supplement and in culture media. FA must be reduced and methylated to become the metabolically active form found in blood and utilized by tissues, i.e. 5-methyltetrahydrofolate (5-MeTHF). 5-MeTHF is the methyl group donor required for the conversion of homocysteine to methionine catalyzed by vitamin B(12)-dependent methionine synthase. It is hypothesized that 5-MeTHF may be more effective than FA in reducing spontaneous DNA damage and improving cell proliferation because, unlike FA, it can donate a methyl group for methionine synthesis, which is required for cell division via polyamine production and for maintenance methylation of DNA after its conversion to S-adenosylmethionine. We aimed to determine whether FA and 5-MeTHF differed in their capacity to prevent genetic damage and cell proliferation of human lymphocytes in vitro. Lymphocytes from eight female volunteers (40-48 years) were cultured in RPMI 1640 medium containing 12-120 nM FA or 5-MeTHF for 9 days. Mitogenesis was stimulated with phytohemagglutinin and the medium changed on days 3 and 6. Cytokinesis was inhibited by adding cytochalasin B on day 8 and cells were harvested and transferred to microscope slides on day 9. Chromosome damage, cell death and cytostasis was measured using the cytokinesis-block micronucleus assay in its comprehensive mode. The results showed that the frequency of micronucleated binucleate cells was significantly lower at 120 nM FA compared with 120 nM 5-MeTHF (P < 0.05), however, at 12 nM concentration both forms of folate were associated with increased frequency of micronuclei and nuclear buds relative to 120 nM (P < 0.05). Apoptosis tended to be significantly higher in 5-MeTHF cultures compared with FA cultures, however, necrosis and nuclear division were similar between cultures. We conclude that 5-MeTHF is not more efficient than FA in preventing human lymphocyte genomic instability in this in vitro system. Further research is needed to clarify the role of choline and methionine concentration and the importance of the reduced folate carrier and the folate receptor in determining the relative bioavailability of 5-MeTHF and FA with regard to genome stability.

Induction of sister chromatid exchanges by cypermethrin and carbosulfan in bone marrow cells of mice in vivo

Abstract: The public health effects of pesticides cannot be denied. However, the undesired effects of chemical pesticides have been recognized as a serious public health concern during the past decades. The present study describes the genotoxic effects of two pesticides, namely cypermethrin and carbosulfan, in a murine test system in vivo. The test parameter used was analysis of sister chromatid exchanges (SCE) in bone marrow cells. Both cypermethrin (5, 10 and 20 mg/kg) and carbosulfan (1.25, 2.5 and 5 mg/kg) induced significant increases in the frequency of SCEs (P < 0.001). However, no significant dose-response correlation could be found for either of the pesticides. Carbosulfan induced a cell cycle delay, as evidenced by an increase in average generation time accompanied by accumulation of cells in the first division cycle, but cypermethrin did not induce any such response. The present study indicates that carbosulfan has a higher potential to cause genetic alterations than cypermethrin in mice and may also pose a mutagenic risk to human beings.

Aneugenic and clastogenic effects of doxorubicin in human lymphocytes.

Abstract: Doxorubicin, a benzanthroquinone anticancer agent, was examined for its effect on micronucleus induction in cultured human lymphocytes. A statistically significant dose-dependent increase in micronucleus frequency (P<0.001) in binucleated cells was seen and an increase in the kinetochore-positive (P<0.001) and kinetochore-negative micronuclei (P<0.001) was observed. An increase was also observed in the number of necrotic cells, but the frequency of apoptotic cells remained almost constant. This confirms that doxorubicin is both clastogenic and aneugenic.

The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts.

Abstract: The involvement of the antioxidant enzymes catalase and glutathione peroxidase (both at 0.1 mg/ml) in defence against the genotoxicity of phosphamidon (80 µg/ml) and dieldrin (25 µM) was investigated in order to demonstrate that the two pesticides damage DNA through the generation of reactive oxygen species and therefore of oxidative stress. The pesticide genotoxicity was determined by the cytokinesis-block micronucleus test performed on primary mouse lung fibroblast cultures. Also, 3-aminotriazole (40 mM) and mercaptosuccinate (0.5 mM), inhibitors of catalase and glutathione peroxidase, respectively, were added to the cultures. Data indicate that catalase causes a decrease only in the damage induced by phosphamidon, while glutathione peroxidase protects against damage induced by both phosphamidon and dieldrin. Simultaneous treatment with antioxidant inhibitors and pesticides results in a decrease in micronucleus frequency and cell number, due to apoptotic death. Our results indicate that clastogenic DNA damage produced by the two pesticides is modulated by antioxidant enzymes and their inhibitors and thus could be due to oxidative stress induction.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells.

Abstract: Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms

Nutritional supplementation with antioxidants decreases chromosomal damage in humans.

Abstract: In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), beta-carotene (6 mg/day) and selenium (50 microg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage.

Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones

Abstract: The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.

UVA induces C→T transitions at methyl-CpG-associated dipyrimidine sites in mouse skin epidermis more frequently than UVB

Abstract: We studied the kinetics of mutation induction in skin epidermis and dermis of UVA-irradiated transgenic Muta mice and analyzed the sequence changes in 80 lacZ transgene mutants from the irradiated epidermis. The mutant frequency increased linearly in both the epidermis and dermis up to 240 kJ/m2 UVA, twice as efficiently in the epidermis as in the dermis, without provoking any inflammatory reactions in the exposed skin. The 83 mutations detected in the UVA-exposed epidermis were dominated by C-->T transitions (88%), found almost exclusively at dipyrimidine sites, and specified by four occurrences of CC-->TT tandem substitutions, suggesting that UV-specific photoproducts induced in DNA have a major role in the genotoxicity. No T-->G transversions, which have been considered as a UVA signature mutation, and few mutations suggesting the relevance of oxidative damage were recovered in the present study. An analysis of the bases adjacent to the mutated cytosines revealed that the 3'-cytosine of dipyrimidine sites is the preferred target of UVA-induced C-->T transition. Moreover, C-->T transitions were induced at dipyrimidine sites associated with CpG much more frequently by UVA than by UVB, forming hotspots at several of these sites. These results suggest that UVA contributes more to the formation of recurrent or hotspot mutations at methylated CpG sites in the mammalian genome than UVB, since methylation of the CpG motif is observed entirely in the lacZ transgenes and is known to enhance the formation of cyclobutane pyrimidine dimers by longer wavelength UV.

α,β‐Unsaturated carbonyl compounds: induction of oxidative DNA damage in mammalian cells

Abstract: Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to 85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals.

Lymphocyte DNA damage precedes DNA repair or cell death after orthopaedic surgery under general anaesthesia

Abstract: Anaesthetics have gained a lot of attention for their potential mutagenic/carcinogenic effects. In the present study we have investigated the genotoxicity of the inhalation anaesthetic sevoflurane on DNA of lymphocytes isolated from 20 patients undergoing orthopaedic surgery. The genotoxicity of the anaesthetic was studied by assaying DNA damage, apoptosis, DNA repair enzyme activity and GSH content in peripheral lymphocytes before, 15 min after anaesthesia and 24 h after surgery. Lymphocytes isolated 15 min after anaesthesia showed an increase in oxidized purine and pyrimidine bases without DNA strand break formation. DNA strand breaks occurred on the first post-operative day, associated with an enhancement of DNA repair activity and a decrease in GSH. Formation of strand breaks could be the consequence of DNA repair activity. In fact, at 24 h after surgery most of the oxidized DNA bases were repaired. When DNA damage was not repaired, activation of the cell cycle checkpoint protein p53 could lead to apoptosis. An altered redox status may contribute to lymphocytopenia due to an apoptotic event as a consequence of surgical trauma. The presence of apoptotic cells at 1 day after surgery could support the hypothesis that highly damaged peripheral lymphocytes are committed to undergo programmed cell death if the damage is not repaired. In conclusion, the actual risk from anaesthesia is presumably extremely small. However, these findings contribute to our understanding of the regulation of DNA damage/repair and cell death.

Induction of endoreduplication by topoisomerase II catalytic inhibitors

Abstract: The striking phenomenon of endoreduplication has long attracted attention from cytogeneticists and researchers into cell cycle enzymology and dynamics alike. Because of the variety of agents able to induce endoreduplication and the various cell types where it has been described, until now no clear or unique mechanism of induction of this phenomenon, rare in animals but otherwise quite common in plants, has been proposed. Recent years, however, have witnessed the unfolding of a number of essential physiological roles for DNA topoisomerase II, with special emphasis on its major role in mitotic chromosome segregation after DNA replication. In spite of the lack of mammalian mutants defective in topoisomerase II as compared with yeast, experiments with inhibitors of the enzyme have supported the hypothesis that this crucial untangling of daughter DNA molecules by passing an intact helix through a transient double-stranded break carried out by the enzyme, when it fails, leads to aberrant mitosis that results in endoreduplication, polyploidy and eventually cell death. Anticancer drugs that interfere with topoisomerase II can be classified into two groups. The classical poisons act by stabilizing the enzyme in the so-called cleavable complex and result in DNA damage, which represents a problem in the study of endoreduplication. The true catalytic inhibitors, which are not cleavable complex stabilizers, allow us to use doses efficient in the induction of endoreduplication while eliminating high levels of DNA and chromosome damage. This review will discuss the basic and applied aspects of this as yet scarcely explored field.

In vivo genotoxicity studies with 3-monochloropropan-1,2-diol.

Abstract: 3-Monochloropropan-1,2-diol (3-MCPD) is a contaminant of polyamine flocculants used in the production of drinking water, but more significantly for human exposure it can arise also in certain foodstuffs containing acid-hydrolysed vegetable protein. It is carcinogenic in the rat, producing tumours in males in the testes, mammary gland and the preputial gland and also kidney tumours in both sexes. It has given positive results in in vitro mutagenicity studies, but there have been no satisfactorily conducted in vivo studies in somatic cells published in the peer reviewed literature. As a result, and because of the absence of appropriate in vivo evidence, several international regulatory agencies had previously judged it prudent to assume that 3-MCPD possessed mutagenic activity in vivo and considered 3-MCPD to be a genotoxic carcinogen. We present in this paper results from two in vivo mutagenicity studies with 3-MCPD, namely a bone marrow micronucleus test in the rat and unscheduled DNA synthesis in the rat liver, both conducted in accordance with relevant OECD protocols. These studies show that 3-MCPD does not possess genotoxic activity in vivo in the tissues examined. On the basis of these findings, and along with evidence that tumours may be induced by mechanisms involving either hormonal disturbances or sustained cytotoxicity, we believe that 3-MCPD may now be considered to be carcinogenic to rodents via a non-genotoxic mechanism.