Showing papers in "Mutation Research-genetic Toxicology and Environmental Mutagenesis in 2002"
TL;DR: Of all the additives, dyes were the most genotoxic and induced DNA damage in the colon at close to the acceptable daily intakes (ADIs), and more extensive assessment of food additives in current use is warranted.
Abstract: We determined the genotoxicity of 39 chemicals currently in use as food additives. They fell into six categories—dyes, color fixatives and preservatives, preservatives, antioxidants, fungicides, and sweeteners. We tested groups of four male ddY mice once orally with each additive at up to 0.5×LD50 or the limit dose (2000 mg/kg) and performed the comet assay on the glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24 h after treatment. Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal organs at a low dose (10 or 100 mg/kg). Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs). Two antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs. Based on these results, we believe that more extensive assessment of food additives in current use is warranted.
619 citations
TL;DR: The mutagenic risk posed by simple, well-characterized mixtures of priority PAHs can reasonably be estimated as the sum of the risks posed by the mixture components.
Abstract: Risk assessment of complex environmental samples suffers from difficulty in identifying toxic components, inadequacy of available toxicity data, and a paucity of knowledge about the behavior of geno(toxic) substances in complex mixtures. Lack of information about the behavior of toxic substances in complex mixtures is often avoided by assuming that the toxicity of a mixture is simply the sum of the expected effects from each mixture component, i.e. no synergistic or antagonistic interactions. Although this assumption is supported by research investigating non-genotoxic end-points, the literature describing the behavior of genotoxic substances in complex mixtures is sparse and, occasionally, contradictory. In this study, the results of polycyclic aromatic hydrocarbon (PAH) analyses on freshwater bivalves were used to prepare realistic mixtures containing up to 16 PAHs. The SOS genotoxicity of the mixtures and each component were then assessed in an effort to evaluate the additivity of PAH genotoxicity. At nominal PAH concentrations above 1 microg/ml, observed genotoxic responses were far lower than those predicted under the assumption of additivity. At nominal concentrations below 0.75 microg/ml, differences are smaller and occasionally negligible, indicating that the genotoxicity of unsubstituted homocyclic PAHs is additive or slightly less than additive. Other researchers who have investigated the mutagenicity, carcinogenicity, and DNA binding activity of mixtures containing unsubstituted homocyclic PAHs have also reported additive effects. Therefore, the mutagenic risk posed by simple, well-characterized mixtures of priority PAHs can reasonably be estimated as the sum of the risks posed by the mixture components. Current data indicate that less-than-additive effects likely result from saturation of metabolic pathways needed to activate mutagenic PAHs.
196 citations
TL;DR: The data strongly indicate a genotoxic potential of intermittent ELF-EMF, which points to the need of further studies in vivo and consideration about environmental threshold values for ELF exposure.
Abstract: Results of epidemiological research show low association of electromagnetic field (EMF) with increased risk of cancerous diseases and missing dose–effect relations. An important component in assessing potential cancer risk is knowledge concerning any genotoxic effects of extremely-low-frequency-EMF (ELF-EMF). Human diploid fibroblasts were exposed to continuous or intermittent ELF-EMF (50 Hz, sinusoidal, 24 h, 1000 μT). For evaluation of genotoxic effects in form of DNA single- (SSB) and double-strand breaks (DSB), the alkaline and the neutral comet assay were used. In contrast to continuous ELF-EMF exposure, the application of intermittent fields reproducibly resulted in a significant increase of DNA strand break levels, mainly DSBs, as compared to non-exposed controls. The conditions of intermittence showed an impact on the induction of DNA strand breaks, producing the highest levels at 5 min field-on/10 min field-off. We also found individual differences in response to ELF-EMF as well as an evident exposure–response relationship between magnetic flux density and DNA migration in the comet assay. Our data strongly indicate a genotoxic potential of intermittent EMF. This points to the need of further studies in vivo and consideration about environmental threshold values for ELF exposure.
194 citations
TL;DR: It is revealed that fish MN assay can be used as a genotoxicological test-system since some methodological particularities were observed.
Abstract: A comparative analysis between mouse and fish erythrocyte micronuclei (MN) assays was carried out using cyclophosphamide, mitomycin C and various pesticides such as alliete, brestanid, decis 25 CE (deltamethrin), kelthane 480 CE (dicofol), roundup (glyphosate), imazapyr and thiram. The aim of this study was to evaluate the fish species Tilapia rendalli as a suitable organism for the detection of genotoxicants in water. The clastogens cyclophosphamide and mitomycin C induced MN in both test-systems. Insecticides: decis 25 CE increased T. rendalli MN frequencies at doses of 1.0 and 5.0mg/kg, but not at the highest dose, and in mice there was no MN induction. Kelthane 480 CE also induced a significant MN frequency in T. rendalli, but not in mice. Fungicides: alliete and brestanid induced MN only in T. rendalli, while thiram was negative in both assays. Herbicides: imazapyr induced MN in T. rendalli at the maximum tolerated dose only, while roundup induced MN at three dosed levels. In mice both herbicides presented negative results. This study revealed that fish MN assay can be used as a genotoxicological test-system since some methodological particularities were observed.
186 citations
TL;DR: The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations but further research is required to better understand the potential and limitations of the RAPD assay.
Abstract: The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.
184 citations
TL;DR: Results show that ribavirin is mutagenic to rat germ cells in a transient fashion.
Abstract: Ribavirin (1-β- d -ribofuranosyl-1,2,4, triazole-3 carboxamide) is a broad-spectrum antiviral drug. This study was aimed to investigate the mutagenicity of ribavirin on germ cells by employing sperm morphology assay. Male Wistar rats were treated with water, cyclophosphamide (CP) 40 mg/kg, and ribavirin 20, 100 and 200 mg/kg (i.p.) for 5 consecutive days at intervals of 24 h. Following the last exposure, at 14, 28, 35, 42 and 70 days, the epididymal sperm smears were obtained and stained according to the standard procedure. One thousand sperms per animal were classified into normal and different abnormal types. Both CP and ribavirin-induced anomalies of head and tail of sperm except at 70 days. In CP groups, maximum incidence was observed at 28, 35 and 42 days. Ribavirin 20 mg/kg induced maximum incidence at 14 and 42 days, 100 mg/kg at 28 and 42 days and 200 mg/kg at 28-42 days. These results show that ribavirin is mutagenic to rat germ cells in a transient fashion.
169 citations
TL;DR: Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.
Abstract: The meristematic mitotic cells of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. For assessing genotoxicity of pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor), 50% effective concentration (EC(50)), c-mitosis, stickiness, chromosome breaks and mitotic index (MI) were used as endpoints of genotoxicity. EC(50) values for PCP and butachlor are 0.73 and 5.13 ppm, respectively. 2,4-D evidently induced morphological changes at higher concentrations. Some changes like crochet hooks, c-tumours and broken roots were unique to 2,4-D at 5-20 ppm. No such abnormalities were found in PCP and butachlor treated groups, however, root deteriorated and degenerated at higher concentrations (<3 ppm) in PCP. MI in 2,4-D showed a low average of 14.32% followed by PCP (19.53%), while in butachlor it was recorded 71.6%, which is near to the control value. All chemicals induced chromosome aberrations at statistically significant level. The highest chromosome aberration frequency (11.90%) was recorded in PCP at 3 ppm. Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.
162 citations
TL;DR: The micronucleus test in fish erythrocytes has increasingly been used to detect the genotoxic effects of environmental mutagens and its frequency is considered to reflect thegenotoxic damage to cells, mainly the chromosomes.
Abstract: The micronucleus test (MNT) in fish erythrocytes has increasingly been used to detect the genotoxic effects of environmental mutagens and its frequency is considered to reflect the genotoxic damage to cells, mainly the chromosomes. Besides, morphologically altered erythrocyte is taken as an index of cytotoxicity. Both parameters were used in the present study by two herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D, in 25, 50 and 75 ppm concentrations) and 2-chloro-2,6-diethyl- N -(butoxymethyl) acetanilide (butachlor, in 1, 2 and 2.5 ppm concentrations) for genotoxic and cytotoxic endpoints. The study was carried out by an in vivo method on peripheral erythrocytes of catfish Clarias batrachus using multiple sampling times (48, 72 and 96 h). Cytogenetic preparations were made by haematoxylin-eosin staining technique. Pycnotic and granular micronuclei (MN) were consistently observed irrespective of chemical tested. A wide range of altered cells was also observed. Echinocytes accompanied by altered nuclei and vacuoles were prominent feature of 2,4-D, whereas, anisochromasia and anisocytosis of erythrocytes were characteristic of butachlor. Increase in MN as well as altered cells frequencies were significant. A positive dose-response relationship in all exposures and sampling times was observed. Herbicides used were found to be genotoxic as well as cytotoxic in this fish. The suitability of the adopted parameters for the screening of the aquatic genotoxicants is discussed.
159 citations
TL;DR: Results suggest that oxidative stress in testicular milieu is associated with DNA damage and produces higher frequency of abnormal sperms with significant effect on male fertility.
Abstract: Our previous work has shown that prooxidant treatment has the propensity to induce male-mediated dominant lethal (DL) type mutations in mice. The present investigation is aimed to understand the effect of oxidative stress (OS) on DNA damage in testis, epididymal sperms and its propensity to induce sperm head abnormalities as well as its implications on male fertility in mice. Initially, employing two organic hydroperoxides, (t-butyl hydroperoxide, t-bHP and cumene hydroperoxide, cHP) as model prooxidants, induction of oxidative stress was ascertained following single/multiple sublethal doses. Further, the multiple exposure model was utilized to characterize effects on testicular weights, histoarchitecture, caudal sperm counts, lipid peroxidation, DNA damage and frequency of abnormal sperms. Single sublethal doses (1/20, 1/10 and 1/5 LD50) of t-bHP and cHP administered (i.p.) to adult mice resulted in only a marginal increase (20% at the highest dosage) in testicular MDA levels. However, multiple doses (1/10 and 1/5 LD50 per day for 5 days) induced marked OS in testis and epididymal sperms as evidenced by a marked increase in lipid peroxidation at 24 h after the last dose. This was associated with significant increase in the DNA damage (FADU assay) in the testicular tissue. While caudal sperm counts determined at all sampling weeks showed no treatment related alterations, analysis for head abnormalities revealed nearly 2–3-fold increase in the percent abnormal sperms among the hydroperoxide treated mice during the first 3 weeks. Furthermore, mating of prooxidant treated males sequentially for a period of 5 weeks with untreated females resulted in a significant reduction in average pup number per litter during the first 3 weeks. These results suggest that oxidative stress in testicular milieu is associated with DNA damage and produces higher frequency of abnormal sperms with significant effect on male fertility.
150 citations
TL;DR: Overall the data clearly indicate that Cr(VI) compounds are cytotoxic and genotoxic to human lung cells.
Abstract: Hexavalent chromium (Cr(VI)) is a human lung carcinogen. Cr(VI) is a particularly important and dangerous carcinogen, because there is widespread exposure to it both occupationally and to the general public. However, despite the potential for widespread exposure and the fact that the lung is its target organ, there are few reports of the genotoxicity of Cr(VI) in human lung cells. Clearly, in order to better understand this carcinogen, its effects in its target cells need to be evaluated. Accordingly, we determined the cytotoxicity and clastogenicity of both particulate (water-insoluble) and soluble Cr(VI) in primary human bronchial fibroblasts (PHBFs). We used lead chromate (PbCrO 4 ) and sodium chromate (Na 2 CrO 4 ) as prototypical particulate and soluble Cr(VI) salts, respectively. Both compounds induced concentration-dependent cytotoxicity after a 24 h exposure in PHBFs. The relative survival was 87, 46, 26 and 2% after exposure to 0.1, 0.5, 1 and 5 μg/cm 2 PbCrO 4 , respectively, and 74, 57, 13 and 0% after exposure to 1, 2.5, 5 and 10 μM Na 2 CrO 4 , respectively. Similarly, the amount of chromosome damage increased with concentration after 24 h exposure to both compounds. Specifically, 0.1, 0.5 and 1 μg/cm 2 PbCrO 4 damaged 15, 34 and 42% of metaphase cells with the total amount of damage reaching 18, 40 and 66 aberrations per 100 metaphases, respectively. PbCrO 4 (5 μg/cm 2 ) induced such profound cell cycle delay that no metaphases were found. Na 2 CrO 4 (1 and 2.5 μM) damaged 18 and 33% of metaphase cells with the total amount of damage reaching 19 and 43 aberrations per 100 metaphases, respectively. Na 2 CrO 4 (5 and 10 μM) induced such profound cell cycle delay that no metaphases were found. Overall the data clearly indicate that Cr(VI) compounds are cytotoxic and genotoxic to human lung cells.
149 citations
TL;DR: From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.
Abstract: Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.
TL;DR: Modifications of the neutral comet method are described, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture, and point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB.
Abstract: Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion. In this report, we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture. The analysis carried out according to this protocol takes less time than those most often applied. Also, the use of lysis at 50 degrees C is avoided; this is important in view of the presence of heat-labile sites in the chromatin of irradiated cells, recently reported by Rydberg [Radiation-induced heat-labile sites that convert into DNA double-strand breaks, Radiation Research 153 (2000) 805-812]. The comets have well-defined, sharp limits, suitable for image analysis. The chromatin of the hydrogen peroxide-treated or UV-C-irradiated cell remains condensed similarly to that of the control cells. We checked the neutral comets for the presence of single-stranded DNA by means of a specific antibody. The results point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB. The minimum detection level of the modified neutral comet assay is about 5 Gy.
TL;DR: The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.
Abstract: The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10 mg/kg) of malathion tested in the present study, induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations, two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.
TL;DR: It is demonstrated that NIN is able to protect mouse bone marrow cells against the radiation-induced DNA damage and decline in the cell proliferation as observed by a reduction in the micronucleus frequency and an increase in PCE/NCE ratio, respectively, in the NIN-pretreated irradiated group.
Abstract: The effect of various doses, viz. 0, 0.5, 1, 2, 4, 6 and 8 mg/kg body weight of naringin (NIN) (a citrus flavanone) was studied on the alteration in the radiation-induced micronucleated polychromatic (MPCE) and normochromatic (MNCE) erythrocytes in mouse bone marrow exposed to 2 Gy of 60 Co γ-radiation. The treatment of mice with various doses of NIN before exposure to 2 Gy resulted in a significant decline in the frequency of MPCE when compared to the non-drug-treated irradiated control. However, the greatest reduction in MPCE was observed for 2 mg/kg body weight NIN, accompanied by a highest PCE/NCE ratio when compared with the non-drug-treated irradiated control. Therefore, further studies were carried out using this dose of NIN, where the animals were administered with 2 mg/kg body weight of NIN before exposure to 0, 0.5, 1, 2, 3 and 4 Gy of γ-radiation. The frequency of MPCE and MNCE increased in a dose-dependent manner in both the non-drug-treated irradiated control and NIN-pretreated irradiated groups up to a dose of 2 Gy, while a further increase in the irradiation dose resulted in a significant decline in MPCE and MNCE frequencies in both groups. Pretreatment of mice with 2 mg/kg body weight of NIN resulted in a significant decline in the frequencies of MPCE and MNCE. NIN treatment not only reduced the frequency of MPCE with one micronucleus, but also of MPCE with multiple micronuclei (MN), indicating its ability to reduce complex chromosome aberrations. Conversely, the PCE/NCE ratio declined in a dose-dependent manner in both groups. The treatment of mice with NIN before exposure to different doses of γ-radiation resulted in the inhibition in this decline in the PCE/NCE ratio. Our study demonstrates that NIN is able to protect mouse bone marrow cells against the radiation-induced DNA damage and decline in the cell proliferation as observed by a reduction in the micronucleus frequency and an increase in PCE/NCE ratio, respectively, in the NIN-pretreated irradiated group.
TL;DR: The results of the present study show that the Trad-MN assay is suitable for the detection of genotoxic effects of metal contamination in soils and furthermore, that the DNA-damaging potential of soils from different origin cannot be predicted on the basis of chemical analyses of their metal concentrations.
Abstract: The aim of this study was to investigate correlation between genotoxic effects and changes of microbial parameters caused by metal contamination in soils. In total, 20 soils from nine locations were examined; metal contents and physicochemical soil parameters were measured with standard methods. In general, a pronounced induction of the frequency of micronuclei (MN) in the Tradescantia micronucleus (Trad-MN) assay was seen with increasing metal concentration in soils from identical locations. However, no correlations were found between metal contents and genotoxicity of soils from different locations. These discrepancies are probably due to differences of the physicochemical characteristics of the samples. Also, the microbial parameters depended on the metal content in soils from identical sampling locations. Inconsistent responses of the individual enzymes were seen in soils from different locations, indicating that it is not possible to define a specific marker enzyme for metal contamination. The most sensitive microbial parameters were dehydrogenase and arylsulfatase activity, biomass C, and biomass N. Statistical analyses showed an overall correlation between genotoxicity in Tradescantia on the one hand and dehydrogenase activity, biomass C, and the metabolic quotient on the other hand. In conclusion, the results of the present study show that the Trad-MN assay is suitable for the detection of genotoxic effects of metal contamination in soils and furthermore, that the DNA-damaging potential of soils from different origin cannot be predicted on the basis of chemical analyses of their metal concentrations.
TL;DR: The results suggest that lead-exposure induces an increase of DNA breakage with an alternate cellular redox state and a significant down-regulation of PKC alpha, suggesting that this metal may act as a tumor promoter.
Abstract: Lead and lead compounds play a significant role in modern industry; a wide variety of population is at risk of occupational exposure and lead is suspected to be a human carcinogen. The biochemical and molecular mechanisms of lead toxicity are poorly understood, but emerging data suggest that some of the effects of lead may be due to its interference with calcium in the activation of protein kinase C (PKC) and/or through production of reactive oxygen species (ROS). Many of these results are conducted in vitro on cell lines or ex vivo on human lymphocytes treated in vitro. We, therefore, performed a study on the induction of DNA damage, using the alkaline comet assay, in lymphocytes of battery plant workers. To elucidate in vivo the mechanism(s) responsible for this effect, we determined ROS production, and glutathione (GSH) levels in living cells using the fluorescent probe (2′,7′-dichlorofluorescein and monochlorobimane, respectively). Subcellular fractions were obtained from sonicated lymphocytes; cytosolic and membrane expression of PKC isoforms (α, and ζ) was evaluated after electrophoresis by immunoblot analysis. The results indicate that lead-exposed workers have significantly elevated levels of DNA breaks compared to the unexposed group. A multivariate analysis of variance (ANOVA) shows that the most common confounding factors (smoking, drinking and age) have no synergistic effects with lead-exposure on the comet parameters or on GSH levels and ROS production. The logistic regression analysis distinguishing the exposed and non-exposed indicates that only GSH with tail moment are selected as significant risk factors. There is a significant positive correlation with ROS production and negative correlation with GSH levels. The content of PKC α in cytosol and membranes is decreased 40% (indicating a down-regulation of protein), whereas PKC ζ isoform is not modified in an evident manner. Our results suggest that lead-exposure induces an increase of DNA breakage with an alternate cellular redox state and a significant down-regulation of PKC α, suggesting that this metal may act as a tumor promoter.
TL;DR: The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk Assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process.
Abstract: Recent advances in genetic toxicity (mutagenicity) testing methods and in approaches to performing risk assessment are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. The US Environmental Protection Agency (EPA) first published Guidelines for Mutagenicity Risk Assessment in 1986 that focused mainly on transmissible germ cell genetic risk. Somatic cell genetic risk has also been a risk consideration, usually in support of carcinogenicity assessments. EPA and other international regulatory bodies have published mutagenicity testing requirements for agents (pesticides, pharmaceuticals, etc.) to generate data for use in genotoxicity risk assessments. The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process. A classification strategy for agents based on inherent genotoxicity, dose-responses observed in the data, and an exposure analysis is proposed. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.
TL;DR: It is suggested that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.
Abstract: Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro-o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and D-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p'-DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p,p'-DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.
TL;DR: Examination of BaP-arsenic mixtures provides strong support for the positive interaction between arsenic and PAH-induced cancer observed in epidemiology studies, and helps to identify additional mechanistic steps likely to be involved in arsenic comutagenesis.
Abstract: Co-exposures to complex mixtures of arsenic and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are common in the environment. These two environmental pollutants are carcinogenic, but the nature of their molecular interactions in the induction of cancer is not well understood. Additive or synergistic interactions have been proposed to explain why arsenic, which is not a potent mutagen itself, is comutagenic with a variety of DNA-damaging agents. We have examined the genotoxicity of BaP-arsenic mixtures. We find that exposure of mouse hepatoma Hepa-1 cells to low concentrations of arsenite increases BaP-DNA adduct levels by as much as 18-fold. This effect requires the activation of BaP by cytochrome p450 1A1 (CYP1A1), although arsenite does not alter BaP-inducible CYP1A1 enzymatic activity, suggesting that arsenite acts downstream of metabolic BaP activation. Glutathione homeostasis was important in modulating the potency of arsenite. In cells depleted of reduced glutathione, arsenite increased BaP-DNA adduct formation by an even greater degree than in cells co-treated with BaP and arsenite in control medium. Although arsenic comutagenicity has been attributed to inhibition of DNA repair, arsenite treatment did not alter adduct removal kinetics in BaP-treated cells, suggesting that mechanisms upstream of DNA repair are responsible for increased adduct levels. Concentrations of arsenite and BaP that had no measurable mutagenic effect alone, increased mutation frequency at the Hprt locus by eight-fold when given in combination, demonstrating a comutagenic response between BaP and arsenite. These results provide strong support for the positive interaction between arsenic and PAH-induced cancer observed in epidemiology studies, and help to identify additional mechanistic steps likely to be involved in arsenic comutagenesis.
TL;DR: Iron is taken up by human colon cells and participates in the induction of oxidative DNA damage, which means that iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor.
Abstract: Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.
TL;DR: In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr (III) involvement in Cr(VI)-induced cancers, it is cautioned against ingestion of large doses of CrPic for extended periods.
Abstract: Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48 h exposure. The highest dose tested was 80 μg/cm2 CrPic, which, if fully soluble, would be equivalent to 1 mM or 0.44 mg/ml CrPic, and would correspond to 1 mM Cr(III) or 52 μg/ml Cr(III). This exposure resulted in 68±16% cell survival based on 48 h cell counts, and 24±11% survival by 7-day colony formation. Exposure of CHO cells to CrPic produced a statistically significant increase in 6-thioguanine (6-TG)-resistant cells over the dose range tested. The 80 μg/cm2 CrPic exposure resulted in an average induced mutation frequency (MF) of 58 per 106 surviving cells, or an average 40-fold increase in hprt mutants relative to untreated cells. An equivalent dose of 3 mM Pic was highly cytotoxic and did not yield hprt mutants. The dose range of 0.375–1.5 mM Pic produced a slight increase in hprt mutants, but the increase was not statistically significant. An equivalent dose of 1 mM chromic chloride yielded an induced MF of 9 per 106 surviving cells, or a 10-fold increase in mutants with cell survivals of >100%. The coordination of Cr(III) with picolinic acid may make the metal more genotoxic than other forms of Cr(III). In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr(III) involvement in Cr(VI)-induced cancers, we caution against ingestion of large doses of CrPic for extended periods.
TL;DR: Observations indicate that the curcumin-induced G2/M arrest previously described for MCF-7 cells is due to the assembly of aberrant, monopolar mitotic spindles that are impaired in their ability to segregate chromosomes.
Abstract: The dietary phytochemical curcumin possesses anti-inflammatory, -oxidant, and cytostatic properties, and exhibits significant potential as a chemopreventative agent in humans. Although many cell types are arrested in the G2/M-phase of the cell cycle after curcumin treatment, the mechanisms by which this occurs are not well understood. The purpose of this study was to examine the effects of curcumin on the cell cycle of MCF-7 breast cancer cells to determine whether growth arrest is associated with structural changes in cellular organization during mitosis. For this purpose, MCF-7 breast cancer cells were treated with 10-20 microM curcumin, and the effects on cell proliferation and mitosis studied. Structural changes were monitored by immunolabeling cells with antibodies to a number of cytoplasmic and nuclear proteins, including beta-tubulin, NuMA, lamins A/C and B1, lamin B receptor, and centromere antigens. At the concentrations used, a single dose of curcumin does not induce significant apoptosis, but is highly effective in inhibiting cell proliferation for over 6 days. During the first 24-48 h of treatment, many cells are arrested in M-phase, and DNA synthesis is almost completely inhibited. Remarkably, arrested mitotic cells exhibit monopolar spindles, and chromosomes do not undergo normal anaphase movements. After 48 h, most cells eventually leave M-phase, and many form multiple micronuclei instead of individual daughter nuclei. These observations indicate that the curcumin-induced G2/M arrest previously described for MCF-7 cells is due to the assembly of aberrant, monopolar mitotic spindles that are impaired in their ability to segregate chromosomes. The production of cells with extensive micronucleation after curcumin treatment suggests that at least some of the cytostatic effects of this phytochemical are due to its ability to disrupt normal mitosis, and raises the possibility that curcumin may promote genetic instability under some circumstances.
TL;DR: First evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation is reported, with TUNEL-positive phenotype observed in both cases.
Abstract: An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H(2)O(2) or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G(1) population was pointed out by flow cytometry. Sub-G(1) cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.
TL;DR: The results support previous epidemiological studies that XRCC1 may play a role in cigarette smoking-induced lung cancer and support the hypothesis that DNA repair modulate smoking-related DNA damage.
Abstract: The association between metabolic polymorphisms and cigarette smoking-induced cancers has been documented. However, the role of DNA repair polymorphism in carcinogenesis is less clear. To investigate if the polymorphisms of metabolic traits and DNA repair modulate smoking-related DNA damage, we used sister chromatid exchange (SCE) as a marker of genetic damage to explore the relationship of microsomal epoxide hydrolase (mEH), glutathione S-transferase M1 (GSTM1), and X-ray cross-complementing group 1 (XRCC1) and cigarette smoking-induced SCE. Sixty-one workers without significant exposure to mutagens were recruited. Questionnaires were completed to obtain detailed occupational, smoking, and medical histories. SCE frequency in peripheral lymphocytes was determined using a standard cytogenetic assay and GSTM1, mEH (exons 3 and 4), XRCC1 (codon 399) genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP). Smokers had higher SCE frequency than non-smokers (8.4 versus 7.1, P<0.05). Among workers who had smoked equal to or greater than 10 cigarettes each day, those with XRCC1 Arg/Gln+Gln/Gln had higher SCE frequency than those with XRCC1 Arg/Arg after adjusting for potential confounders (9.0 versus 7.9, P<0.05). The interaction of XRCC1 and cigarettes smoked per day on SCE frequency was also observed (P=0.02). There was no significant interaction between cigarettes smoked per day with GSTM1 and mEH on SCE frequency. Our results support previous epidemiological studies that XRCC1 may play a role in cigarette smoking-induced lung cancer.
TL;DR: Evaluation of the micronuclei formation in oral mucosa cells, urothelial cells and peripheral blood lymphocytes was carried out in symptomatic individuals exposed to arsenic through drinking water in West Bengal, India and results indicate that the symptomatic Individuals exposed to arsenicism through drinkingWater in this region have significant cytogenetic damage.
Abstract: In West Bengal, India arsenic in ground water has been found to be above the maximum permissible limit in seven districts covering an area of 37,493km2. In the present study, evaluation of the micronuclei (MN) formation in oral mucosa cells, urothelial cells and peripheral blood lymphocytes was carried out in the symptomatic individuals exposed to arsenic through drinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11 microg/l of As in drinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning and residing in two unaffected districts (5.49 microg/l of As) were considered as controls. The exposed and control groups had similar age distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conducted to ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in the study participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair and urine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45 microg/l, 12.58 and 6.97 microg/g, respectively which were significantly high in comparison to corresponding control group values of 4.88 microg/l, 0.51 and 0.34 microg/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oral mucosa, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with the controls (0.77, 0.56 and 0.53/1000 cells, respectively). Thus, the above results indicate that the symptomatic individuals exposed to arsenic through drinking water in this region have significant cytogenetic damage.
TL;DR: The study revealed the antigenotoxic potential of curcumin against CP induced chromosomal mutations and the incidence of aberrant cells was found to be reduced by both the doses ofCurcumin when compared to CP treated group.
Abstract: Curcumin, a yellow pigment commonly used as a spice and food coloring agent is obtained from rhizomes of Curcuma longa and is a major chemopreventive component of turmeric. In the present set of investigations the antimutagenic potential of curcumin has been evaluated using in vivo chromosomal aberration assay in Wistar rats. Cyclophosphamide (CP), a well-known mutagen was given by intraperitoneal (i.p.) injection at the dose of 40 mg/kg body weight (b.w.). Curcumin was given at the dose of 100 and 200 mg/kg b.w. through gastric intubation for seven consecutive days prior to CP treatment. The animals were sacrificed at the sampling time of 24 h after treatment and their bone marrow tissue was analyzed for chromosomal damage and mitotic index. In CP treated animals a significant induction of chromosomal aberration was recorded with decrease in mitotic index. However, in curcumin-supplemented animals, no significant induction in chromosomal damage or change in mitotic index was recorded. In different curcumin-supplemented groups, a dose dependent significant decrease in CP induced clastogenicity was recorded. The incidence of aberrant cells was found to be reduced by both the doses of curcumin when compared to CP treated group. The anticytotoxic potential of curcumin towards CP was also evident as the status of mitotic index was found to show increment. The study revealed the antigenotoxic potential of curcumin against CP induced chromosomal mutations.
TL;DR: The results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported, indicating that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells.
Abstract: In order to elucidate the health effects of occupational exposure to traffic fumes, a few biomarkers of early genetic effect were investigated in Rome traffic policemen. One hundred and ninety healthy subjects engaged in traffic control (133 subjects) or in office work (57 subjects) participated the study. For all subjects, detailed information on smoking habits and other potential confounders were recorded by questionnaires. Average exposure of the study groups to benzene and other aromatic hydrocarbons was evaluated in a parallel exposure survey. All workers were genotyped for the following metabolic polymorphisms: CYP1A1 (m1, m2, and m4 variants), CYP2E1 (PstI and RsaI), NQO1 (Hinf1), GSTM1 and GSTT1 (null variants). In this paper, the results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported. No statistically significant difference in the frequency of SCE or high frequency cells (HFC) was observed between traffic wardens and office workers (controls), despite the significantly higher exposure to benzene of the former (average group exposure 9.5 versus 3.8microg/m(3), 7h TWA). Conversely, both SCE per cell and HFC were highly significantly (P<0.001) increased in smokers compared to nonsmokers, showing a significant correlation (P<0.001) with the number of cigarettes per day. Multiple regression analyses of data, with metabolic polymorphisms, smoking habits, alcohol consumption, age, gender, and family history of cancer as independent variables, showed that smoking habits, and possibly the CYP2E1 variant genotypes, were the main factors explaining the variance of both SCE and HFC. Within smokers, an association of borderline significance between the CYP1A1 variant genotypes and increased SCE (P=0.050) and HFC (P=0.090) was found. This effect was mainly observed in light smokers (<15 cigarettes per day). The analysis of DNA damage by comet assay did not highlight any statistically significant difference between the exposed and control workers. Moreover, no significant model explaining tail moment variance was obtained by multiple regression analysis using the independent variables shown above. On the whole, these results indicate that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells. This evidence does not rule out any possibility of adverse effects, but strongly suggests that in urban residents life-style related factors, such as tobacco smoking, give the prevailing contribution to individual genotoxic burden.
TL;DR: The genotoxicity experiments with the ground water collected from an area under the influence of textile dyeing and bleaching industries in Tirupur, Tamilnadu, India demonstrate the application of the comet assay in environmental monitoring studies.
Abstract: This paper presents the genotoxicity experiments with the ground water collected from an area under the influence of textile dyeing and bleaching industries in Tirupur, Tamilnadu, India. The alkaline single cell gel electrophoresis (SCGE) assay was performed in vitro with human peripheral blood lymphocytes. The cells were exposed to two doses of non-volatile organic agents extracted from ground water samples. Ground water samples were collected from 12 locations distributed in and around Tirupur and extracts were taken at different pHs (without pH adjustment and acidic pH 2.0). The persistence of the DNA damage after exposure to the organic extracts was also studied. All the samples were found to contain substances capable of inducing DNA damage in human lymphocytes. Extracts from acidified waters (pH=2.0) were found to induce more DNA damage than extracts from without pH adjustment (natural pH). The DNA damage was not fully repaired after incubation for 2h at 37 degrees C. The chemical characterization of the sub-fractions revealed the existence of aromatic amines in the extracts, which may be responsible for the DNA damaging activity of the water samples. The results of this investigation demonstrate the application of the comet assay in environmental monitoring studies.
TL;DR: To the authors' knowledge, this report has, for the first time, demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.
Abstract: The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.
TL;DR: It is demonstrated that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay.
Abstract: Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC50=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC50=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[β- d -arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos, black-currants, and black tea strongly reduced the genotoxicity of BaP-7,8-OH, onions, rooibos tea, and red wine were less potent while red beets and spinach were inactive. On the other hand, red beets and spinach strongly inhibited the genotoxicity of N-OH-PhIP, rooibos tea was weakly active while all other items were inactive. These results are suggestive for enzyme inhibition as mechanism of protection by complex mixtures of plant origin. Taken together, our results demonstrate that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay [Food Chem. Toxicol. 32 (1994) 443; Mutat. Res. 341 (1995) 303].