Showing papers in "Mutation Research-genetic Toxicology and Environmental Mutagenesis in 2000"

Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test.

Abstract: In this work, we have measured both micronuclei and other nuclear abnormalities in renal erythrocytes from European minnow Phoxinus phoxinus and mollie Poecilia latipinna, with the aim to contribute to the standardisation of the micronucleus test for fish species. Intraperitoneal injections of colchicine (10 mg/kg), cyclophosphamide (40 mg/kg), or mitomycin C (20 mg/kg) for 24 h induced diverse nuclear abnormalities in minnow erythrocytes, therefore nuclear abnormalities should be added to micronuclei as genotoxicity indicators in fish micronucleus tests. The adequacy of administration protocols based on intraperitoneal injections has been evaluated by injecting saline solution to both species: single or double injections have not induced neither micronuclei nor other nuclear abnormalities in any case. Finally, the differential sensitivity of both species to toxic heavy metals was evaluated by exposing individuals of both species to different doses (0.17, 1.7, 2x1.7, and 3.4 mg/kg) of cadmium and mercury for 24 h; we concluded that the mollie is sensitive to both metals whereas the minnow is not sensitive to mercury.

Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin

Abstract: Cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis produced by a number of cyanobacterial species, the most common being Cylindrospermopsis raciborskii . CYN contains a uracil moiety attached to a sulphated guanidino moiety, suggesting that it may have carcinogenic activity. This report describes the use of the WIL2-NS lymphoblastoid cell-line in the well-validated cytokinesis-block micronucleus (CBMN) assay to test this hypothesis. Centromeres (CENs) were identified in micronuclei (MNi) of binucleated cells (BNCs) by fluorescent in situ hybridisation of alpha centromeric DNA sequence repeats. The results indicate that CYN induced a significant increase in the frequency of MNi in BNCs exposed to 6 and 10 μg/ml, and a significant increase in CEN-positive MNi at all concentrations of CYN tested (1, 3, 6, and 10 μg/ml). However, despite this apparently greater sensitivity of WIL2-NS cells to induction of CEN-positive MNi at low CYN concentrations, at the higher concentrations the magnitude of the increase in CEN-positive MNi did not account for the greater increase in MNi in BNCs, indicating that both CEN-positive and CEN-negative MNi were induced. This suggests that CYN acts to induce cytogenetic damage via two mechanisms, one at the level of the DNA to induce strand breaks, the other at the level of kinetochore/spindle function to induce loss of whole chromosomes (aneuploidy). C. raciborskii occurs in a number of human drinking water sources worldwide and so these findings may have important public health implications.

Genetic polymorphisms in human xenobiotica metabolizing enzymes as susceptibility factors in toxic response

Abstract: Biotransformation plays an important role in the carcinogenic activity and organ specificity of environmental carcinogens. Large interindividual variation in the biotransformation has been reported, and genetic polymorphisms in some xenobiotica metabolizing enzymes can in part explain some of these differences. The concentration of the ultimate carcinogen, that will react with DNA, is determined by the rate of activation and detoxification. Individuals with a decreased rate of detoxification, i.e., lacking the glutathione S-transferase M1 gene, have a slightly higher level of bulky carcinogen-DNA adduct in some tissues, and do also have an increased level of chromosomal aberrations. In addition, the genotype may also influence the type of mutations, e.g., in tumor suppressor gene, transversion being predominant in the GSTM1 null group. People with slow N-acetyltransferase activity do generally have a higher adduct level of aromatic amines in bladder tissues. Genetic polymorphism in either CYP1A1 or glutathione S-transferase is linked to an increased risk of smoking related cancers, while N-acetyltransferase activity is related to cancers in which aromatic amines are the main risk factor. Combination of the high risk genotypes for activating and detoxification enzymes, e.g., CYP1A MspI/GSTM1 null is not only associated with an increased risk of cancer development, but also an increased level of markers of the biological active dose and early markers of effect. Additional studies on the role of genetic variants of xenobiotica metabolizing enzymes and combinations thereof at relevant low levels of exposure are important in order to establish guidance values for toxic compounds.

A comparison of the in vitro genotoxicity of tri- and hexavalent chromium.

Abstract: Chromium can be found in the environment in two main valence states: hexavalent (Cr(VI)) and trivalent (Cr(III)). Cr(VI) salts are well known human carcinogens, but the results from in vitro studies are often conflicting. Cr(VI) primarily enters the cells and undergoes metabolic reduction; however, the ultimate product of this reduction, Cr(III) predominates within the cell. In the present work, we compared the effects of tri- and hexavalent chromium on the DNA damage and repair in human lymphocytes using the alkaline single cell gel electrophoresis (comet assay). Potassium dichromate induced DNA damage in the lymphocytes, measured as the increase in comet tail moment. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Cr(III) caused greater DNA migration than Cr(VI). The lymphocytes did not show measurable DNA repair. Vitamin C at 50 μM reduced the extent of DNA migration. This was either due to a decrease in DNA strand breaks and/or alkali labile sites induced by Cr(VI) or to the formation of DNA crosslinks by Cr(VI) in the presence of vitamin C. Vitamin C, however, did not modify the effects of Cr(III). Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by Cr(VI) but not the one induced by Cr(III). Lymphocytes exposed to Cr(VI) and treated with endonuclease III, which recognizes oxidized pyrimidines, displayed greater extent of DNA damage than those not treated with the enzyme. Such an effect was not observed when Cr(III) was tested. The results obtained suggest that reactive oxygen species and hydrogen peroxide may be involved in the formation of DNA lesions by hexavalent chromium. The comet assay did not indicate the involvement of oxidative mechanisms in the DNA-damaging activity of trivalent chromium and we speculate that its binding to cellular ligands may play a role in its genotoxicity.

A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay

Abstract: The single-cell gel electrophoresis (or comet) assay has gained widespread acceptance as a cheap and simple genotoxicity test, but it requires a computer-assisted image-analysis system. As commercial programs are expensive and inflexible, we decided to develop an image-analysis system based on public domain programs and make it publicly available for the scientific community. Our system is based on the scientific image-processing program NIH Image, and was written in its Pascal-like macro language. User interaction was kept as simple as possible, to enable the measurement of a large number of cells with a few keystrokes. Therefore, the time for image analysis is very low, even on slow computers. The comet macro can be obtained from http://mailbox.univie.ac.at/christoph.helma++ +/comet/, NIH Image is available at http://rsb.info.nih.gov/nih-image/. Both programs are free of charge.

An investigation on the antimutagenic properties of South African herbal teas

Abstract: The antimutagenic properties of South African herbal teas were investigated using the Salmonella typhimurium mutagenicity assay. Aqueous extracts of fermented and unfermented rooibos tea (Aspalathus linearis) and honeybush tea (Cyclopia intermedia) both possess antimutagenic activity against 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1)-induced mutagenesis using tester strains TA98 and TA100 in the presence of metabolic activation. A far less inhibitory effect was noticed against the direct acting mutagens, methyl methanesulfonate (MMS), cumolhydroperoxide (CHP), and hydrogen peroxide (H2O2) using TA102, a strain designed to detect oxidative mutagens and carcinogens. Depending on the mutagen used, the unfermented tea exhibited the highest protective effect. A similar response regarding the protection against mutagenesis was obtained when utilising different variations of the double layer Salmonella assay. The double layer technique proved to be more effective to detect the protective effect of the different tea preparations against the direct acting mutagens. With respect to indirect mutagens, the highest protection was noticed when the carcinogen was metabolically activated in the presence of the tea extract as compared with when the tea extract was incubated in a separate layer with the bacteria. The current data suggest that two mechanisms seem to be involved in the antimutagenicity of the tea extracts towards carcinogens that require metabolic activation: (i) the tea components may interfere with cytochrome P450-mediated metabolism of these mutagens and (ii) the direct interaction between the tea constituents, presumably the polyphenolic compounds, with the promutagens and/or the active mutagenic metabolites. However, the mild and/or lack of protection and in some cases even enhancement of mutagenesis induced by direct acting or oxidative mutagens, provide new perspectives regarding the role of the polyphenolic compounds known to exhibit antioxidant properties, in the protection against mutagenesis in the Salmonella assay. The present study provides the first evidence on the antimutagenic activity of honeybush tea and further evidence on the antimutagenicity of rooibos tea.

Cytogenetic biomonitoring in a Mexican floriculture worker group exposed to pesticides.

Abstract: The cytogenetic damage in floriculturists of Morelos State, Mexico, exposed to pesticides, was evaluated by mean of biological tests based on sister chromatid exchanges (SCE) in lymphocytes of peripheral blood and micronuclei (MN) in exfoliated cells of the buccal mucosa. Besides the cytogenetic analysis, the effects of pesticides exposure on the cell proliferation kinetics (CPK) by the replication index (RI) were also studied. The mitotic index (MI) to detect cytotoxic effects was also determined. Greenhouses of the towns of Santa Catarina, Jiutepec and Yecapixtla were selected for the study, because the application of chemicals to the flowers is uncontrolled. As non-exposed group, people of the town of Temisco were chosen; their activity was not related to pesticides. The SCE were analyzed in the peripheral blood of 30 persons, 22 women and 8 men, with 10 and 1.5 years of exposure to pesticides, respectively, and of 30 persons, 28 women and 2 men, that were considered as the non-exposed group. Samples of buccal mucosa were also taken from each person. Significant differences between exposed and non-exposed groups were found in SCE, CKP and MI. Besides, the MN frequencies in the exposed group were three times higher than in the non-exposed group.

Follow-up study of the genetic damage in lymphocytes of pharmacists and nurses handling antineoplastic drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay.

Abstract: A follow-up study was carried out 4 years after an initial evaluation of the micronucleus frequency in 10 healthy individuals who had been occupationally exposed to antineoplastic drugs in a Brazilian hospital Upon the first evaluation, these 10 exposed individuals were compared with 10 non-exposed individuals matched for age, sex and smoking habits; the results revealed that the frequency of micronucleated lymphocytes in individuals exposed to antineoplastic drugs was significantly higher (P=0038) than in controls The frequency of dicentric bridges was also increased, although not significantly (P=00545) After the first analysis, the workers handling antineoplastic drugs were advised to modify their work schedule to limit exposure, and the number of workers in the group was increased from 10 to 12 individuals In the follow-up study, 12 individuals from the same work area were assessed In addition to micronucleus frequency, alkaline single cell gel electrophoresis was also used to monitor genetic hazard This exposed group was compared to 12 non-exposed workers from the same hospital, matched for age, sex and smoking habits In the follow-up study, no statistical difference was found between exposed workers and controls in terms of micronucleus and dicentric bridge frequency with the Mann–Whitney U-test (P=0129 and 0373, respectively) However, the mean value of SCGE analysis was significantly higher in the exposed group than in the controls (P=00006) Although the micronucleus analysis seems to be less sensitive to assess DNA damage, it detects chromosome aberrations and not just repairable DNA breakage and alkali-labile sites Combination of the alkaline single cell gel electrophoresis and cytokinesis blocked micronucleus assay appears to be commendable to monitor populations chronically exposed to genotoxic agents

Evaluation of the single cell gel electrophoresis assay with human hepatoma (Hep G2) cells.

Abstract: Human Hep G2 cells have retained the activities of phase I and phase II enzymes which are involved in the metabolism of environmental genotoxins. The present study describes the results of single cell gel electrophoresis (SCGE) assays with a panel of different model compounds with these cells. With genotoxic carcinogens such as aflatoxin B(1) (AFB(1)), benzo(a)pyrene (B(a)P), nitrosodimethylamine (NDMA) and cyclophosphamide (CP), statistically significant dose dependent induction of DNA migration was measured. With the two heterocyclic amines, 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and also with rodent carcinogens such as safrole, hexamethylphosphoramide (HMPA) and the pyrrolizidine alkaloid isatidine, which give negative results in other in vitro genotoxicity tests, positive results were obtained in Hep G2/SCGE assays. Nitrosomethylurea (NMU) was the only directly acting compound tested in the study and was by far (ca. 10(3)-fold) more active than the corresponding nitrosamine. The exposure concentrations required to cause significant effects varied over a broad range. The most pronounced effect was seen with AFB(1) (0.008 microM) followed by HMPA (15 microM), B(a)P (25 microM), NMU (100 microM), isatidin (500 microM), CP (900 microM), IQ (1200 microM), safrol (4000 microM), and NDMA (90x10(3) microM). Numbers in parenthesis give the lowest concentrations, which caused a significant increase of DNA migration. With two compounds, namely, the non-carcinogen pyrene and the synthetic hormone tamoxifen (TF), negative results were obtained under all test conditions. These findings are in agreement with the results of recent investigations which indicated that human hepatocytes are unable to convert TF to DNA reactive metabolites, whereas it is activated by rat liver cells and causes DNA adducts in these cells. Comparisons of the present results with data from earlier experiments indicate that the Hep G2/SCGE assay enables the detection of genotoxins including compounds which give misleading results in other in vitro genotoxicity tests and appears to be an alternative to tests with primary liver cells from laboratory animals.

Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay.

Abstract: The comet assay, also called the single-cell gel electrophoresis (SCGE) assay, is a rapid and sensitive method for the detection of DNA damage (strand breaks and alkali-labile sites) in individual cells. The assay is based on the embedding of cells in agarose, their lysis in alkaline buffer and finally subjection to an electric current. In the present study, alkaline SCGE was used to evaluate the extent of primary DNA damage and DNA repair in peripheral blood lymphocytes of workers employed in pesticide production. After the period of high pesticide exposure, lymphocytes of the occupationally exposed workers manifested increased tail length and tail moment compared to the control group. After the workers spent 6 months out of the pesticide exposure zone, both endpoints were still above that of the control but significantly decreased as compared to the results of the first analysis.

Cytogenetic biomonitoring of Spanish greenhouse workers exposed to pesticides: micronuclei analysis in peripheral blood lymphocytes and buccal epithelial cells.

Abstract: In the present study, we evaluate whether or not occupational exposure to a complex mixture of pesticides results in a significant increase of micronuclei (MN) in both peripheral blood lymphocytes and buccal cells. Sixty four greenhouse workers from Almeria (Southeastern Spain), together with 50 men from the same area, without indication of exposure to pesticides, that served as controls were used in this investigation. The results obtained indicate that there are no statistically significant differences in the MN frequencies between the two groups. Each donor was assessed for the presence or absence of glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1), to look for relationships between the genotypes and the cytogenetic reponses. According to the GSTT1 genotype, there is a difference between both groups only for the cytokinesis-block proliferation index (CBPI). Neither GSTM1 nor smoking habit and age showed any effect in the overall analysis.

Comet assay: rapid processing of multiple samples.

Abstract: The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds.

Abstract: Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.

Sesamol exhibits antimutagenic activity against oxygen species mediated mutagenicity.

Abstract: The effects of sesamol, a phenolic compound responsible for the high resistance of sesame oil to oxidative deterioration as compared with other vegetable oils, have been investigated after mutagen treatment in various strains of Salmonella typhimurium. Sesamol was shown to exhibit strong antimutagenic effects in the Ames tester strains TA100 and TA102. The TA102 strain has been shown to be highly sensitive to reactive oxygen species. Mutagenicity was induced by the generation of oxygen radicals by tert-butylhydroperoxide (t-BOOH) or hydrogen peroxide (H2O2); therefore, the antimutagenic property of sesamol was attributed to its antioxidant properties. The superoxide and hydroxyl radical scavenging capabilities have further been elucidated using in vitro test systems. It was further shown to have a desmutagenic effect on t-BOOH-induced mutagenesis in TA102 strain. Sesamol also inhibited the mutagenicity of sodium azide (Na-azide) in TA100 tester strain while it had no effect on nitroquinoline-N-oxide (NQNO)-induced mutagenesis in TA98 strain of Salmonella typhimurium. Since active oxygen species are involved in multiple stage processes of carcinogenicity, this compound may also exhibit anticarcinogenic properties.

Validation and implementation of an internal standard in comet assay analysis

Abstract: The comet assay is widely used to detect DNA damage in single cells. However, only moderate attention has been paid to the experimental variability of this assay, especially during electrophoresis. To take into account this variation and to be able to compare measurements from different electrophoretic runs, as would be necessary when large numbers of samples need to be analysed, it is important to integrate an internal standard into the assay. This study presents a first step in the validation and implementation of an internal standard in the alkaline comet assay. Untreated and ethyl methanesulfonate treated cells (K562 human erythroleukemia cell line) were used as negative and positive internal standards, respectively, in each electrophoresis run. Three steps were followed: (1) assessment of the different levels of variability which may influence the damage levels of the internal standards, (2) evaluation of the variability across separate electrophoresis runs on the quantification of DNA damage in the internal standards by three experimenters involved in different studies and (3) proposal of an adequate calculation system to integrate the internal standards into test sample data. The application of the two proposed models to samples from a human biomonitoring study is presented. The model which calibrates the measurements against the negative internal standard is the most useful since this negative standard was the most stable across experiments and among the three experimenters. The percentage of DNA in the tail is the most appropriate parameter to analyse induced DNA damage, because its interelectrophoresis and interexperimenter variation is less pronounced than that of tail length.

Detection of micronuclei in haemocytes of zebra mussel and great ramshorn snail exposed to pentachlorophenol

Abstract: The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 μg/l, and for ramshorn snail from 10 to 450 μg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50‰, and between 2.07 and 13.80‰ in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75‰, and in snails from 1.56 to 2.00‰. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.

The effect of dibenzo[a,l]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21WAF1 proteins and cell cycle distribution

Abstract: Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture DNA binding was detectable even at the lowest concentration used (24h exposure, 001 microM for both PAHs) The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 01 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively) The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts The DNA adduct levels correlated with both p53 (R=0832, P or = 15-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 01 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells Cell treatment with 10 microM B[a]P resulted in more subtle alterations We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established

Improved Bacterial SOS Promoter: Lux Fusions For Genotoxicity Detection

Abstract: Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

The modifying effect of β-carotene on gamma radiation-induced elevation of oxidative reactions and genotoxicity in male rats

Abstract: The aim of the present work was to evaluate the modulatory role of β-carotene on the radiation-induced changes in certain biochemical and cytogenetic parameters. β-Carotene was given by gavage at a dose of 5 mg/kg body weight for 7 consecutive days before whole body gamma irradiation with 7 Gy (single dose). The levels of β-carotene in plasma, thiobarbituric acid-reactive substances (TBARS) in plasma and liver, the activities of superoxide dismutase (SOD) and catalase in blood and liver were the selected parameters. Furthermore, the frequency of micronuclei (MN) of polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), the ratio of PCEs/NCEs and the mitotic index (MI) of bone marrow cells were also evaluated. The biochemical and cytogenetic determinations were carried out 1, 24, and 72 h after radiation exposure. The results obtained revealed that administration of β-carotene pre-irradiation significantly inhibited the decrease in plasma β-carotene, significantly reduced the levels of TBARS in plasma and liver. Significant protection of the radiation-induced changes in the activities of SOD and catalase was also recorded in the blood and liver of β-carotene-treated and -irradiated rats. β-Carotene resulted in significant inhibition in the frequency of radiation-induced MN, as well as in the ratio of PCEs/NCEs and the MI of bone marrow cells. These results suggest that β-carotene as a natural product with its antioxidant capacity and capability of quenching singlet oxygen, could play a modulatory role against the cellular damage affected by free radicals induced by whole body irradiation.

Concepts of threshold in mutagenesis and carcinogenesis.

Abstract: Although the existence of a threshold in the dose effect relationship is well documented for many, if not most, types of toxicological effects the existence of a threshold for the mutagenic effects of ionising radiation and of certain chemicals has been questioned since the middle of the century and only recently the question of thresholds for radiation and chemical carcinogenesis has been addressed. The essential facts for the interpretation of threshold dose-response curves are common to all type of effects and are: (i) the number and the identity of the target; (ii) the type and sensitivity of the endpoint used to quantify the effect. We therefore will first try to model the type of interactions which may be expected between a mutagen and its target and define from this whether a threshold dose-effect can be expected; in a second step the concept will be extended to heritable mutations and carcinogenesis.

Comparative evaluation of the in vitro micronucleus test and the comet assay for the detection of genotoxic effects of X-ray radiation.

Abstract: The genotoxic effects of X-ray radiation on human lymphocytes were measured using the single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (CBMN) test; both were carried out in vitro on isolated human lymphocytes in order to compare the relationship and sensitivity of these two detecting methods. The radiation-doses were 0.00, 0.02, 0.05, 0.10, 0.25, 0.50, 1.00 and 2.00 Gy. In the comet assay, the average comet length (38.6±0.8 μm) of 0.05 Gy was significantly longer than that (29.4±1.1 μm) of 0 Gy (P 0.05). The lowest dose, which induced significant increase of average MN and MNC, was 0.25 Gy. The average MN and MNC rates increased with radiation-dose. The results showed that there was correlation between SCGE and CBMN, and the sensitivity of SCGE was significantly higher than that of CBMN.

Induction of chromosome aberrations, micronucleus formation and sperm abnormalities in mouse following carbofuran exposure.

Abstract: Carbofuran was tested to study in vivo cytogenetic effects in mouse bone marrow cells and morphological alterations in sperms. The acute oral and intraperitoneal (i.p.) LD(50) of carbofuran was determined to be 9.5 or 2.0 mg/kg b.w. in mice, respectively. The animals were orally administered 1.9, 3.8 or 5.7 mg/kg b.w. (20, 40 and 60% of LD(50)) of carbofuran for 24 h or 1.9 mg/kg b.w. for 4 consecutive days (cumulative 7.6 mg/kg or 80% of LD(50)) to analyse chromosome aberrations (CAs). For micronucleus test (MT) animals were orally exposed to 5.7 mg/kg b.w. for 24 and 48 h or 1.9 mg/kg b.w. for 4 consecutive days. For reference mice were exposed to peanut oil (negative control) and cyclophosphamide (20 mg/kg) or ethyl methanesulfonate (EMS: 100 mg/kg) positive control for CAs and MT respectively. To analyse the effect on sperm morphology mice were exposed to single i.p. dose of 1 and 2 mg/kg b.w. of carbofuran and repeatedly to 0.5 mg/kg for 5 consecutive days. Cytogenetic analysis revealed that all the test doses induced mitotic inhibition, CAs, micronucleus (MN) formation and sperm abnormalities in a dose dependent manner. Present observations concurrent with earlier reports substantiate the genotoxic potential of carbofuran and possible risk to human beings.

Effects of the antioxidants curcumin and vitamin C on cisplatin-induced clastogenesis in Wistar rat bone marrow cells

Abstract: The use of dietary antioxidants to prevent antitumor agent-induced chromosomal damage in nontumor cells is currently eliciting considerable interest. Curcumin (CMN) is a dietary antioxidant that has been reported to protect against clastogenesis in in vivo and in vitro assays. This study was undertaken to investigate the modulatory effects of CMN on cisplatin-induced chromosomal aberrations in Wistar rat bone marrow cells and whether there is any potentiation of these effects with the combination between CMN and vitamin C (VC), which has been reported to reduce the clastogenic effect of many antitumor agents in in vivo assays. Animals treated with CMN plus a single dose of cisplatin, at 18, 24 or 72 h following treatment, presented a statistically significant reduction in the total amount of chromosomal damage and in the number of abnormal metaphases. The results also indicate that the combination between antioxidants would not be effective in protecting against cisplatin-induced chromosomal damage in animals sacrificed 24 h after cisplatin treatment. Under the present experimental conditions, CMN could prevent cisplatin-induced clastogenesis by acting as a free radical scavenger.

Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals.

Abstract: Issues of biological relevance and thresholds for genotoxicity are discussed here based upon the background of experience with the submissions for the approval of new pharmaceuticals to the German regulatory authority over the period between 1990 and 1997. This experience shows that out of the genotoxicity test systems which are required according to existing guidelines in the European Union (EU), the in vitro tests for chromosomal aberrations (CA) and the mouse lymphoma tk assays (MLA) yield a rate of positives that is about four-fold higher than that of other genotoxicity tests. A detailed analysis of chemical and pharmacological classes of compounds and their effects in these systems reveals that in addition to direct DNA reactivity several mechanisms of indirect genotoxicity such as nucleoside analogue incorporation into DNA, interaction with microtubule assembly, topoisomerase inhibition and high levels of cytotoxicity are relevant. New pharmaceuticals, for which the latter mechanisms apply, often display threshold-like characteristics in their genotoxic effects in vitro or even in vivo in experimental animals. This casts doubt upon the relevance of positive in vitro test results for such compounds. However, the discussion of examples shows that it may not be easy to demonstrate the exact thresholded mechanism of genotoxicity in a given case. In particular, the demonstration of a coincidence of genotoxicity and high levels of cytotoxicity, which seems to be a major factor for biologically non-relevant in vitro positive new pharmaceuticals, usually requires quite extensive testing. Hence, for new pharmaceuticals it is practice to provide in addition to in vitro results that may be thresholded a wealth of information from in vivo studies on genotoxicity, carcinogenicity, metabolism, pharmacokinetics, etc. the results of which help in assessing the biological relevance of in vitro positives. The regulatory acknowledgement of biologically non-relevant, thresholded mechanisms of (in vitro) genotoxicity in addition to those that are considered relevant for human risk ensures a better understanding of test results and is needed for the credibility of genotoxicity testing practice in general.

Increased cytogenetic damage in outdoor painters.

Abstract: Painters are exposed to an extensive variety of hazardous substances such as organic solvents, lead-containing pigments and residual plastic monomers. In this particular case, workers used commercially available exterior paints and occasionally gasoline or thinner as solvents. The application or removal of paints was performed without protection (masks or gloves). To determine occupational exposure risk, a monitoring study was designed. Group selection was made after a questionnaire administration, which included questions about lifestyle and medical history to exclude exposure to other potential sources of genotoxics. Smoking and drinking habits were also considered. Blood and buccal cell samples were obtained from 25 public building male painters and from a similar number of age- and gender-matched controls. Lead levels were measured in paint samples and in individuals' blood. Organic solvents and/or its metabolites were also determined in blood. Chromosomal aberrations (CA) and sister chromatid exchanges (SCE) were determined in peripheral blood lymphocyte cultures. Also, the frequency of micronuclei (MN) in buccal cells was investigated. Painters had higher lead levels in blood (p<0.05); CA and SCE in lymphocytes and MN in epithelial cells were also elevated (p<0.05). Cytogenetic damage was significantly associated with occupational exposure time but not with the levels of lead found in blood.

Cytotoxicity of PM2.5 and PM2.5–10 ambient air pollutants assessed by the MTT and the Comet assays

Abstract: Ambient air particulate matters are classified into two distinct modes in size distribution, namely the coarse and fine particles. Correlation between high particulate concentration and adverse effects on human populations has long been recognized, however, the toxicology of these adverse effects has not been clarified. In the current report, the cytotoxic effects of the solvent-extractable organic compounds (SEOC) from fine particles smaller than 2.5 microm (PM(2.5)) and from coarse particles between 2.5-10 microm (PM(2.5-10)) were studied. Nine 24h consecutive monthly samples were tested to determine the correlation between cytotoxicity and total SEOC in two size fractions of particulate air pollution. Cytotoxicity of SEOC was measured by two micro-scale mammalian cells-based bioassays: the MTT cell proliferation assay, and the Comet assay for the detection of DNA damage. A well-defined mammalian cell line - Rat 6 rodent fibroblast was employed in the study. The SEOC extracts of air particulate matters were sub divided into two equal parts. One part was dissolved in DMSO, the other in KOH/hexane and then conjugated with bovine serum albumin to produce a lipid-soluble fraction for testing. The DMSO fraction would contain mainly the polycyclic aromatic hydrocarbons (PAH), alkanes and alkanols, while the lipid-soluble fraction would be enriched with fatty acids. The results from MTT assay showed that cytotoxicity of the PM(2.5) was much more severe than the PM(2.5-10), suggesting that toxic SEOC were confined to the fine particles. By and large, the DMSO solubles were much more toxic than the lipid solubles. The degree of cytotoxicity of the DMSO soluble samples is positively correlated to the amount of particulates present in the ambient air. For the PM(2.5), the winter samples were significantly more toxic than the summer samples in terms of cell killing, which seemed to be a direct reflection of the total loading of organic matter in the samples. Results from Comet assays showed that SEOC samples of PM(2.5) derived from winter months induced DNA damage at dosages resulting in no obvious cell killing in the MTT assay. Thus, long-term exposure to non-killing dosage of air pollutants may lead to the accumulation of DNA lesions, which may be one of the mechanisms responsible for the chronic adverse health effects of particulate air pollution.

Spontaneous micronuclei in peripheral blood erythrocytes from 54 animal species (mammals, reptiles and birds): part two.

Abstract: The normal numbers of micronucleated erythrocytes (MNE) observed in peripheral blood samples differ among species. This depends on the effectiveness of the spleen (or the rest of the reticuloendothelial system) to withdraw them from circulation. In our previous report, we assessed the number of MNE in the peripheral blood of 35 mammalian species. Here we show the results observed in 54 species including mammals, reptiles and birds. We obtained 212 peripheral blood samples from different species. In 14 species, only one individual was studied. Slides were stained with acridine orange. The total number of MNE (normo and polychromatic) in 10,000 erythrocytes per animal are shown. The species that display the higher MNE were: ocelote, lynx, owl, gray squirrel, hedgehog, lion, orange fronted parakeet and common barn owl. For this reason, these species could be tested as monitors for genotoxic events. Another interesting observation was that in the gray squirrel, we found the highest values of MNE in the smaller (younger) animals when compared with the larger (older) of the same species.

DNA damage and repair: consequences on dose-responses.

Abstract: Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances.

Abstract: A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.

Detection of genotoxins in the marine environment: adoption and evaluation of an integrated approach using the embryo–larval stages of the marine mussel, Mytilus edulis

Abstract: In genetic ecotoxicology or eco-genotoxicology, there is lack of well-validated systems which could demonstrate the utility of multiple endpoints in environmental quality assessment. For an evaluation of genotoxic potential of heterogeneous marine sediment samples collected from a small fishing harbour in the UK, an in vivo test system using embryo–larval stages of the common mussel, Mytilus edulis was validated against direct and indirect acting reference mutagens. The system appeared to be sensitive and reproducible for cytogenetic endpoints analysed (sister chromatid exchanges (SCEs) and chromosomal aberrations (CAbs)). Following validation and chemical characterisation of the environmental samples, multiple endpoints were measured. Determination of the maximum tolerated dose (MTD) was carried out as a measure to determine cytotoxic effects as a confounding factor for genotoxicity, based on developmental and cytotoxic (in terms of proliferative rate index or PRI) effects. Evaluation of the genotoxic potential of the samples gave a positive response for all the endpoints tested, linking different levels of biological organisation (i.e., chromosomal, cellular and organismal) for the observed effects. The study also emphasises the need for the assessment of the short and long-term impacts of dredge disposal on marine biota by including laboratory-based bioassays and incorporating an integrated approach which could yield as much useful information as possible in overall hazard and risk assessment for aquatic genotoxicity.