Increase In Micronucleus Frequency Research Articles

Evaluation of DNA damage in Wistar rat tissues with hyperlipidemia induced by tyloxapol

Hyperlipidemia is characterized by high levels of plasma triglycerides and LDL-cholesterol, accompanied by reduced HDL-cholesterol levels, and is often associated with an increased risk of cardiovascular diseases. However, few studies have shown the effects of hyperlipidemia on genomic stability. The aim of this study was to evaluate DNA damage provided by tyloxapol induced hyperlipidemia. Tyloxapol, a non-ionic surfactant, which increases the activity of the enzyme HMG-CoA reductase and decreases clearance of lipoproteins, was used to induce hyperlipidemia in Wistar rats. Genomic instability was assessed using the comet assay which evaluates DNA strand breaks in several tissues, and the micronucleus assay in bone marrow to detect chromosomal mutagenicity for clastogenic and/or aneugenic effects. Biochemical analyses confirmed hyperlipidemia in tyloxapol-treated rats, accompanied by hyperglycemia. Higher creatinine and urea levels were observed, suggesting kidney injury. The comet assay indicated increased DNA damage in blood, liver, and kidney, but not in brain tissue. However, no increase in micronucleus frequency was observed, indicating lack of mutagenic effects. Simvastatin, used as lipid lowering drug, decreased cholesterol and triglycerides in rats treated with tyloxapol. Those findings indicate that tyloxapol-induced hyperlipidemia is able to increase genomic instability, which is associated with higher cancer risk. Therefore, this surfactant might be used in models to evaluate new hypolipidemic drugs with associated chemopreventive properties.

Buccal mucosa micronuclei counts in relation to exposure to low dose-rate radiation from the Chornobyl nuclear accident and other medical and occupational radiation exposures

BackgroundIonizing radiation is a well-known carcinogen. Chromosome aberrations, and in particular micronuclei represent an early biological predictor of cancer risk. There are well-documented associations of micronuclei with ionizing radiation dose in some radiation-exposed groups, although not all. That associations are not seen in all radiation-exposed groups may be because cells with micronuclei will not generally pass through mitosis, so that radiation-induced micronuclei decay, generally within a few years after exposure.MethodsBuccal samples from a group of 111 male workers in Ukraine exposed to ionizing radiation during the cleanup activities at the Chornobyl nuclear power plant were studied. Samples were taken between 12 and 18 years after their last radiation exposure from the Chornobyl cleanup. The frequency of binucleated micronuclei was analyzed in relation to estimated bone marrow dose from the cleanup activities along with a number of environmental/occupational risk factors using Poisson regression adjusted for overdispersion.ResultsAmong the 105 persons without a previous cancer diagnosis, the mean Chornobyl-related dose was 59.5 mSv (range 0–748.4 mSv). There was a borderline significant increase in micronuclei frequency among those reporting work as an industrial radiographer compared with all others, with a relative risk of 6.19 (95% CI 0.90, 31.08, 2-sided p = 0.0729), although this was based on a single person. There was a borderline significant positive radiation dose response for micronuclei frequency with increase in micronuclei per 1000 scored cells per Gy of 3.03 (95% CI -0.78, 7.65, 2-sided p = 0.1170), and a borderline significant reduction of excess relative MN prevalence with increasing time since last exposure (p = 0.0949). There was a significant (p = 0.0388) reduction in MN prevalence associated with bone X-ray exposure, but no significant trend (p = 0.3845) of MN prevalence with numbers of bone X-ray procedures.ConclusionsThere are indications of increasing trends of micronuclei prevalence with Chornobyl-cleanup-associated dose, and indications of reduction in radiation-associated excess prevalence of micronuclei with time after exposure. There are also indications of substantially increased micronuclei associated with work as an industrial radiographer. This analysis adds to the understanding of the long-term effects of low-dose radiation exposures on relevant cellular structures and methods appropriate for long-term radiation biodosimetry.

Apoptotic gene expression profiles and DNA damage levels in rat liver treated with perfluorooctane sulfonate and protective role of curcumin

Perfluorinated compounds (PFCs) such as PFOS and PFOA, are xenobiotics that can be detected worldwide in the environment and humans. PFOS (C8F17SO3−) is a fluorinated organic compound has been used for decades in industrial and commercial products. We investigated the genotoxic and apoptotic impact of PFOS in rat liver using comet assay, micronucleus test and apoptotic gene expression methods for caspase 3, caspase 8 and the protective role of curcumin on the PFOS- induced damage under chronic exposure. In this study, rats were treated either with three different PFOS doses only (0.6, 1.25 and 2.5mg/kg) or one dose of curcumin (80mg/kg) or three different doses of PFOS combined with 80mg/kg dose of curcumin by gavage for 30days at 48h intervals. We evaluated the DNA damage via comet assay and micronucleus test. Doses of PFOS increased micronucleus frequency (p<0.05) and strongly induced DNA damage in liver in two different parameters; i: the damaged cell percentage and ii: genetic damage index. Curcumin prevented the formation of DNA damage induced by PFOS and curcumin substance applied with PFOS caused a decrease in the micronucleus frequency. PFOS increased apoptotic gene expression but curcumin decreased the expression levels of caspase 3 and 8.

The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus)

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.

Buccal Cell Micronucleus Frequency Is Significantly Elevated in Patients with Spinocerebellar Ataxia Type 2

Spinocerebellar ataxia type 2 (SCA2) is part of a group of at least nine dominantly inherited disorders characterized by progressive degeneration of specific neuronal populations and a shared mutational mechanism involving the expansion of a CAG repeat tract in coding regions of novel genes. Efforts have been made to identify biomarkers of disease progression, which would allow timely preventive therapeutic interventions. In the present study was assessed the influence of several genome instability biomarkers on SCA2 clinical severity. A case-control design was applied on exfoliated epithelial buccal cells to determine micronuclei frequency and others nuclear anomalies, using 5% Giemsa stains. The slides were analyzed under 1000X magnification and nuclei morphological anomalies were identified according to Tolbert PE, etal. (1992) and Bolognesi C, etal. (2013) criteria. It was found a highly significant increase in micronuclei frequency in cases related to age and sex-matched healthy controls (p <0.001). There was a trend for karyolytic, pyknotic and condensed chromatin cells to be increased in SCA2 cases, and a significant association was found between binucleated cells and disease duration (r=0.46; p=0.027). Nor the CAG repeat length neither the age at onset correlated significantly with any of the studied markers (p >0.05). Our results are consistent with report previous in similar neurodegenerative diseases, and suggest that micronuclei and binucleated cells constitute potential peripheral biomarkers for SCA2. These results should be validated by other studies.

Evaluation of cytotoxicity, genotoxicity, and apoptosis of wastewater before and after disinfection with performic acid

Disinfection with performic acid (PFA) represents an emerging technology in wastewater treatment. Many recent studies indicate its effectiveness and suitability as a disinfectant for different applications; several have demonstrated its reliability as an alternative to chlorine for disinfecting secondary effluents from urban wastewater treatment plants (WWTPs). Some disinfection technologies, in relation to their oxidative power, lead to the formation of disinfection by-products (DBPs), some of which are of concern for their toxic and carcinogenic potential. The aim of this study was to investigate potential genotoxic, cytotoxic, and mutagenic effects of this disinfection agent on treated secondary effluent coming from a municipal WWTP. A strategy with multiple short-term tests and different target cells (bacterial, plant, and mammalian) was adopted to explore a relatively wide range of potential genotoxic events. The Ames test (point mutation in Salmonella), the micronucleus (chromosomal damage) and Comet tests (primary DNA damage) on human hepatic cells (HepG2) were conducted to detect mutagenicity and chromosomal DNA alterations. DNA fragmentation and mitochondrial potential assays were conducted to evaluate apoptosis in the same kinds of cells. Mutagenic and clastogenic effect potentials were evaluated by examining micronucleus formation in Allium cepa root cells. In all the in vitro tests, carried out on both disinfected and non-disinfected effluents, negative results were always obtained for mutagenic and genotoxic effects. In the Allium cepa tests, however, some non-concentrated wastewater samples after PFA treatment induced a slight increase in micronucleus frequencies in root cells, but not in a dose-related manner. In conclusion, PFA applied for disinfection to a secondary effluent from a municipal wastewater treatment plant did not contribute to the release of genotoxic or mutagenic compounds. Further studies are required to establish to which extent these findings can be generalized to support PFA for other disinfection applications.

Micronucleus, cell-free DNA, and plasma glycan composition in the newborns of healthy and diabetic mothers

Diabetes is associated with certain environmental exposures, heritable factors, and metabolic conditions of intrauterine development due to diabetes in the mother. We evaluated genomic damage, cell-free DNA, N-glycosylation of umbilical cord plasma proteins (PG), and nuclear division index (NDI) as possible prognostic biomarkers of health risk in the newborns of mothers with treated pregestational diabetes (NBDM; 22 mothers), compared these parameters with those from newborns of healthy mothers (NBHM; 89 mothers), and associated the results with the mothers’ lifestyle in both groups, based on a detailed questionnaire. Genomic damage was estimated by the in vitro micronucleus (MN) assay. NDI was detected on MN slides. Glycans were analyzed by ultra-performance liquid chromatography that separates the plasma N-glycome into 46 glycan peaks. Cell-free DNA was analyzed by real-time PCR. For the association between biomarkers and individual characteristics, generalized linear/nonlinear analysis was performed. No significant difference was found between NBHM and NBDM for cell-free DNA levels. There was no association between cell-free DNA levels and lifestyle. MN frequency was significantly higher in NBDM than in NBHM (median, 0.6 vs. 0.3%, p<0.001). MN frequency and NDI were significantly associated with residence (urban vs. rural). PG differed significantly between NBHM and NBDM (p<0.001). A significant association was found between PG and increase of MN frequency (p<0.001). As both MN frequency and altered N-glycosylation are associated with cancer risk, our study indicates need for further investigations.

Association of Genomic Instability with HbA1c levels and Medication in Diabetic Patients

Diabetes Mellitus type 2 (DM2) is associated with increased cancer risk. Instability of the genetic material plays a key role in the aetiology of human cancer. This study aimed to analyse genomic instability with the micronucleus cytome assay in exfoliated buccal cells depending on glycated haemoglobin (HbA1c) levels and medication in 146 female DM2 patients. The occurrence of micronuclei was significantly increased in DM2 patients compared to healthy controls. Furthermore, it was doubled in DM2 patients with HbA1c > 7.5% compared to subjects with HbA1c ≤ 7.5%. Positive correlations were found between micronuclei frequencies and HbA1c as well as fasting plasma glucose. Patients under insulin treatment showed a two-fold increase in micronuclei frequencies compared to subjects under first-line medication (no drugs or monotherapy with non-insulin medication). However, after separation of HbA1c (cut-off 7.5%) only patients with severe DM2 characterised by high HbA1c and insulin treatment showed higher micronuclei frequencies but not patients with insulin treatment and low HbA1c. We demonstrated that the severity of DM2 accompanied by elevated micronuclei frequencies predict a possible enhanced cancer risk among female DM2 patients. Therapy, therefore, should focus on a strict HbA1c control and personalised medical treatments.

In vitro and in vivo investigation of the genotoxic potential of waters from rivers under the influence of a petroleum refinery (São Paulo State – Brazil)

In recent years concern about the chemical composition of wastewater generated by the oil refining industry has increased, even after its treatment. These wastewaters contain substances that can harm both the entire aquatic ecosystem and the health of any exposed organisms. The aim of this study was to evaluate the genotoxic and mutagenic potentials of the effluent generated by the largest Brazilian petroleum refinery, the effectiveness of the treatments used by the refinery, and whether its effluent can compromise the water quality of the river where it is discarded. Chromosomal aberration and micronucleus assays were performed in Allium cepa and micronucleus test in mammalian cell culture (CHO-K1). The samples were collected in three sites at the refinery: one site on the Jaguari River and two sites on the Atibaia Rivers (upstream and downstream of the discharged effluent), under three different climatic conditions. Tests with A. cepa showed increased frequencies of chromosomal aberrations and micronuclei in meristematic cells for the effluent after physico-chemical treatment, but the samples after treatment biological and stabilization pond presented none of these abnormalities. It was observed that the induced damage in the meristematic cells was not observed in the F1 cells of A. cepa roots. The micronucleus test performed with mammalian cell culture also indicated that the effluent, after physico-chemical treatment, induced a significant increase in micronucleus frequencies. Plant and hamster cells exposed to the other samples collected inside the refinery and in the Jaguari and Atibaia Rivers did not present evidence of genotoxicity and mutagenicity in the tests performed. This study showed that the effluent treated carried out by the refinery (biological treatment followed by a stabilization pond) proved to be efficient for the removal of the toxic load still present after the physico-chemical treatment, since no change in the quality of the Atibaia River was observed. However, because this is an industry with a high production of effluent with toxic potential, its effluents must be constantly monitored, so that there is no compromise of the water quality of the receiving river.

Adverse and beneficial effects in Chinese hamster lung fibroblast cells following radiofrequency exposure.

In this study, the effect of radiofrequency (RF) exposure to 1950 MHz, Universal Mobile Telecommunication System signal, was investigated in Chinese hamster lung fibroblast cell line (V79). Genotoxic and cytotoxic effects of 20-h exposure at specific absorption rate (SAR) values from 0.15 W/kg to 1.25 W/kg were measured by means of cytokinesis-block micronucleus (MN) assay. Exposure was carried out blinded under strictly controlled conditions of dosimetry and temperature. The effect of RF exposure alone at four SAR values was tested, that is, 0.15, 0.3, 0.6, and 1.25 W/kg. A statistically significant increase in MN frequency was found in cultures exposed to 0.15 and 0.3 W/kg (P < 0.05) compared to sham-exposed ones, in the absence of cytotoxicity. SAR values of 0.6 and 1.25 W/kg did not exert any effect. Moreover, to evaluate the ability of RF to exert protective effects with respect to a chemical mutagen, cell cultures were also pre-exposed for 20 h at 0.3 or 1.25 W/kg, and then treated with 500 ng/ml of mitomycin-C (MMC). A significant reduction in the frequency of MN was detected in cultures pre-exposed to 1.25 W/kg compared to cultures treated with MMC alone (P < 0.05), indicating induction of adaptive response. Such a decrease was not induced by pre-exposure at 0.3 W/kg SAR. Taken together, our results indicated that V79 is a sensitive cell model to evidence either adverse or beneficial effects of RF exposure, depending on experimental conditions applied. Bioelectromagnetics. 38:245-254, 2017. © 2017 Wiley Periodicals, Inc.

DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells.

Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 μg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 μM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 μM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 μM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 μM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system.

Increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of bladder cancer

Background:Bladder cancer (BC) is among the most common malignancies worldwide. The identification of new biomarkers for early BC detection, recurrence/progression is urgently needed. The cytokinesis-block micronucleus assay (CBMN) evaluates chromosome damage in cultured human lymphocytes and micronuclei (MN) provide a convenient and reliable index of both chromosome breakage and loss.Methods:Chromosomal damage (expressed as frequencies of MN, nucleoplasmic bridges and nuclear buds (NBUD)) was evaluated by CBMN assay in cryopreserved lymphocytes from 158 age/smoking-matched pairs of cases and controls in relation to BC risk, recurrence or progression. Moreover, non-muscle invasive BC (NMIBC) patients were characterised for 783 DNA repair gene polymorphisms for their possible association with the investigated cytogenetic end points.Results:MN and NBUD frequencies were significantly higher in cases than in controls (P=0.001 and P=0.006, respectively), with the associations being stronger in NMIBC. In a logistic regression model, for each increase of one unit in the MN frequency, a 1.12 increased risk of developing NMIBC was observed. In NMIBC cases, 10 polymorphisms were associated with different MN frequencies after genotype stratification.Conclusions:A model including traditional BC risk factors, MN frequency and the selected polymorphisms differentially distributed in cases and controls improved BC patient identification. Understanding the meaning of systemic chromosomal damage in BC patients with respect to the general population may help to adopt specific prevention strategies and therapeutic intervention.

Evaluation of the automated MicroFlow\xae and Metafer\u2122 platforms for high-throughput micronucleus scoring and dose response analysis in human lymphoblastoid TK6 cells

The use of manual microscopy for the scoring of chromosome damage in the in vitro micronucleus assay is often associated with user subjectivity. This level of subjectivity can be reduced by using automated platforms, which have added value of faster with high-throughput and multi-endpoint capabilities. However, there is a need to assess the reproducibility and sensitivity of these automated platforms compared with the gold standard of the manual scoring. The automated flow cytometry-based MicroFlow® and image analysis-based Metafer™ were used for dose response analyses in human lymphoblastoid TK6 cells exposed to the model clastogen, methyl methanesulfonate (MMS), aneugen, carbendazim, and the weak genotoxic carcinogen, ochratoxin A (OTA). Cells were treated for 4 or 30 h, with a 26- or 0-h recovery. Flow cytometry scoring parameters and the Metafer™ image classifier were investigated, to assess any potential differences in the micronucleus (MN) dose responses. Dose response data were assessed using the benchmark dose approach with chemical and scoring system set as covariate to assess reproducibility between endpoints. A clear increase in MN frequency was observed using the MicroFlow® approach on TK6 cells treated for 30 h with MMS, carbendazim and OTA. The MicroFlow®-based MN frequencies were comparable to those derived by using the Metafer™ and manual scoring platforms. However, there was a potential overscoring of MN with the MicroFlow® due to the cell lysis step and an underscoring with the Metafer™ system based on current image classifier settings. The findings clearly demonstrate that the MicroFlow® and Metafer™ MN scoring platforms are powerful tools for automated high-throughput MN scoring and dose response analysis.

Water-soluble and organic extracts of airborne particulate matter induce micronuclei in human lung epithelial A549 cells

The in vitro genotoxic effects of organic and water-soluble fractions of airborne particulate matter (PM10) with the cytokinesis blocked micronucleus (MN) test in human alveolar carcinoma cells A549 were investigated. Samples were collected in three different sites of São Paulo State, Brazil, and fifteen soluble metals and the sixteen EPÁs priority polycyclic aromatic hydrocarbons (PAH) were chemically determined. PAHs prevailing were fluoranthene and benzo(ghi)perylene. In the water-soluble extracts, highest concentration of metals was found for zinc, iron, and copper in all places of collection. Although PM10 concentration in all samples was in the range of 33.5–110.1μg/m3, lower than 120μg/m3 (limit established by São Paulo State’s legislation for PM10 in 24h), MN results showed that of the 24 samples analyzed, five organic and seven water-soluble extracts presented a significant increase in MN frequency. The frequency of MN correlates with the total PAH concentration of the three sites investigated, and the concentration of PM10 is correlated with the biological effect in two of them. For the water-soluble fraction, all the sites presented a relation between the PM10 concentration and the MN frequency. Again, the genotoxic response showed a correlation with the total concentration of water-soluble metals in two of the three sites. Our results confirm the importance of the soluble fraction of PM10 to the genotoxic effect of airborne PM even at low concentration of water-soluble compounds. Thus, together with chemical analysis, the implementation of the MN protocol for both organic and water-soluble fraction biological monitoring could be used as a strategy in a routine air-quality monitoring program, complementing other usual analyses for air pollution control and protection of populations.

Chemical and toxicological characterisation of anticancer drugs in hospital and municipal wastewaters from Slovenia and Spain

Anticancer drugs are continuously released into hospital and urban wastewaters, where they, most commonly, undergo conventional treatment in wastewater treatment plants (WWTPs). Wastewaters contain complex mixtures of substances including parent compounds, their metabolites and transformation products (TPs). In this study, samples of hospital effluents and WWTP influents and effluents from Slovenia and Spain were analyzed for twenty-two selected anticancer drugs, their metabolites and transformation products. Acute and chronic toxicity tests were performed on the crustacean Ceriodaphnia dubia, genotoxicity was determined with Tradescantia and Allium cepa micronucleus (MN) assays and in vitro comet assay in zebrafish (Danio rerio) liver cell line (ZFL cells). Sixty of the two hundred-twenty determinations revealed detectable levels of anticancer drug residues. Among the targeted compounds, platinum based were most frequently detected (90%). Furthermore, erlotinib was detected in 80%, cyclophosphamide and tamoxifen in 70% and methotrexate in 60% of the samples. Seven of ten samples were toxic to C. dubia after acute exposure, whereas after chronic exposure all samples reduced reproduction of C. dubia at high sample dilutions. Allium cepa proved insensitive to the potential genotoxicity of the tested samples, while in Tradescantia increased MN frequencies were induced by a hospital effluent and WWTP influents. In ZFL comet assay all but one sample induced a significant increase of DNA strand breaks. Correlations of chemotherapeutics or their TPs were detected for all bioassays except for Allium cepa genotoxicity test, however for each test the highest correlations were found for different substances indicating differential sensitivities of the test organisms.

Detection of genotoxic effects of drinking water disinfection by-products using Vicia faba bioassay

Plant-based bioassays have gained wide use among the toxicological and/or ecotoxicological assessment procedures because of their simplicity, sensitivity, low cost, and reliability. The present study describes the use of Vicia faba (V. faba) micronucleus (MN) test and V. faba comet assay in the evaluation of the genotoxic potential of disinfection by-products (DBPs) commonly found in chlorine-disinfected drinking water. Five haloacetic acids and three halogenated acetonitriles were chosen as representatives of DBPs in this study because they are of potentially great public health risk. Results of the MN test indicated that monochloroacetic acid (MCA), monobromoacetic acid (MBA), dichloroacetic acid (DCA), dibromoacetic acid (DBA), trichloroacetic acid (TCA), and trichloroacetonitrile (TCAN) caused a statistically significant increase in MN frequency in V. faba root tip cells. However, no genotoxic response was observed for dichloroacetonitrile (DCAN) and dibromoacetonitrile (DBAN). Results of the comet assay showed that all tested DBPs induced a statistically significant increase in genomic DNA damage to V. faba root tip cells. On considering the capacity to detect genomic damage of a different nature, we suggest that a combination of V. faba MN test and V. faba comet assay is a useful tool for the detection of genotoxic effects of DBPs. It is worthy of assessing the feasibility of using V. faba comet assay combined with V. faba MN test to screen for the genotoxic activity of chlorinated drinking water in future work.

Long-term genotoxic effects in the hematopoietic system of prenatally X-irradiated mice

Purpose: To investigate the genotoxic effects of prenatal X-irradiation in mice and the possible presence of late genomic instability.Materials and methods: Pregnant mice were exposed to 0, 1 or 2 Gy at embryonic day 11.5. Blood smears were obtained from pups at birth and on post-natal day 11, 21, 42 and 140. Hematological data (diameter of erythrocytes, percentage of reticulocytes and Granulocyte-to-Lymphocyte ratio [GLR]) and genotoxicity (micronucleated erythrocytes, micronucleated reticulocytes, CREST-positive and negative micronuclei) were assessed.Results: Prenatal irradiation caused perinatal reticulocytosis (which ended on postnatal day 11) and a dose-dependent increase of GLR (indicative of myeloid skewing) on postnatal days 42 and 140. Two temporally distinct genotoxic effects were observed: an early, acute damage (still detectable at birth and soon after) and a late, long-term damage.Conclusions: Increases in micronuclei frequencies and GLR observed from day 42 on are both ascribable to DNA damage. Time of appearance of this late effect may be linked to the shift of hematopoiesis from spleen to bone marrow and to cell-extrinsic factor such as the microenvironment. This study confirms that ionizing radiation can induce long-term genotoxic effects in the hematopoietic system and shows that prenatal irradiation determines genomic instability in blood-forming tissues of adult mice.

Tradescantia as a biomonitor for pesticide genotoxicity evaluation of iprodione, carbaryl, dimethoate and 4,4′-DDE

There is a current tendency to develop and apply environmentally friendly techniques that meet the requirements of green analytical chemistry as an alternative to conventional analytical methods. For toxicity evaluation, these alternatives may be found in bioassays such as Tradescantia. This technique, developed in the 1980s, is highly sensitive to evaluate environmental mutagens, simple and cheap. In this paper, the sensibility of both the Tradescantia micronucleus bioassay (Trad-MCN) and the Tradescantia stamen hair bioassay (Trad-SH) were studied for carbaryl, dimethoate and iprodione, common agricultural and domestic pesticides that are currently used in Chile, which have never been tested with such bioassays. Biomonitor exposures were performed by capillary absorption for each individual pesticide over a wide range of concentrations, from maximum residue limits (trace levels) up to the application dose in agricultural fields. In addition, the organochloride 4,4′-DDE was included but only in the concentration range from 0.01mgL−1 to 1mgL−1, mimicking residue concentrations since it is not a commercial product but, rather, the main breakdown product of the persistent organochloride pesticide 4,4-DDT, whose use was discontinued in Chile in the 1980s.The Trad-MCN bioassay revealed a significant increase in micronucleus frequency at the early tetrads of meiotic pollen mother cells of the biomonitor Tradescantia pallida var. purpurea, induced by 4,4′-DDE (for 1mgL−1), dimethoate (for 40mgL−1, 200mgL−1, 400mg/L−1) and carbaryl (for 889mgL−1). Iprodione did not generate any significant change at the tested concentration. Meanwhile, the Trad-SH bioassay was carried out by analysis of the phenotype variations of the stamen hair cells of the Tradescantia clone KU-20 for the same pesticides and doses. This bioassay was not sufficiently sensitive for toxicity evaluation of most of the pesticides tested, with exception of dimethoate in low doses (2 and 5mg/L−1).

Association and interaction of heat shock proteins B1 gene and tumor-suppressor protein p53 gene with chromosome damage levels among coke oven workers

Objective: To investigate the association and interaction of heat shock proteins B1(HSPB1)gene rs2868371 and tumor-suppressor protein p53(TP53)gene rs1042522 polymorphisms with chromosome damage levels among coke oven workers. Methods: We recruited 1 333 male workers from a state-run coke oven plant in Wuhan in September-October 2010. Among them, 949 who had worked in coke oven workplaces, including auxiliary facilities and bottom, side, and top ovens, were nominated as coke oven workers(i.e., exposed), and 384 administrative or medical staff whose workplaces were offices were used as controls. General characteristics and 5 ml of venous blood were collected from each participant. The plasma concentrations of benzo[a]pyrene-diolepoxide(BPDE)-albumin adducts and the lymphocytic micronucleus(MN)frequencies for each individual were detected by ELISA and cytokinesis-block micronucleus assay, respectively. Gene polymorphisms were genotyped using TaqMan assays via quantitative PCR(ABI Prism 7900HT), and the corresponding frequency ratios(FR)with 95% confidence intervals(CI)were computed for all assays. Results: In the exposed group, the MN frequencies were higher in HSPB1 rs2868371 GC, CC, and GC+ CC genotype carriers((3.88 ± 2.88)‰,(4.00 ± 2.66)‰, and(3.91 ± 2.83)‰, respectively)than in rs2868371 GG genotype carriers((3.52±2.67)‰; FR=1.10, 1.13, and 1.11; 95% CI: 1.02-1.19, 1.02-1.25, and 1.03-1.19, respectively), and the HSPB1 rs2868371C allele was associated with increased MN frequency(Ptrend=0.006). Further, in the exposed group, the MN frequencies were lower in TP53 rs1042522 CG and CG+GG genotype carriers((3.63±2.61)‰ and(3.66±2.61)‰, respectively)than in TP53 rs1042522 CC genotype carriers(3.95±3.06)‰(FR=0.87 and 0.90; 95% CI: 0.83-0.96 and 0.84-0.97, respectively). The effect of gene-gene interaction between HSPB1, rs2868371, and TP53 rs1042522 on MN frequency was significant among coke oven workers(P=0.001). Further stratified analyses showed that the effects of the HSPB1 rs2868371C allele in increasing MN frequencies were robust in subjects aged >40 years(FR=1.07, 95% CI: 1.01-1.12), those working >20 years(FR=1.08, 95% CI: 1.02-1.14), those with BMI ≤24 kg/m2(FR=1.07, 95% CI: 1.01-1.13), drinkers(FR=1.09, 95% CI: 1.02-1.16), and workers with higher BPDE-albumin adduct levels(FR=1.07, 95% CI: 1.01-1.13)(Ptrend=0.023, 0.013, 0.029, and 0.020, respectively). The decreasing effect of the TP53 rs1042522 G allele on MN frequencies was robust in subjects aged >40 years(FR=0.94, 95% CI: 0.89-0.99), those with BMI ≤24 kg/m2(FR=0.94, 95% CI: 0.88-0.99), and drinkers(FR=0.94, 95% CI: 0.88-1.00)(Ptrend=0.031, 0.023, and 0.038, respectively). In addition, there were interactions between HSPB1 rs2868371 and age and between HSPB1 rs2868371 and working years in terms of MN frequency(P=0.030 and 0.013, respectively). Conclusion: In coke oven workers, the HSPB1 rs2868371 C and TP53 rs1042522 G alleles were associated with increased and decreased chromosome damage levels, respectively, and their interaction effect on chromosome damage levels was significant.

Maternal diet during pregnancy and micronuclei frequency in peripheral blood T lymphocytes in mothers and newborns (Rhea cohort, Crete).

The study assessed whether diet and adherence to cancer prevention guidelines during pregnancy were associated with micronucleus (MN) frequency in mothers and newborns. MN is biomarkers of early genetic effects that have been associated with cancer risk in adults. A total of 188 mothers and 200 newborns from the Rhea cohort (Greece) were included in the study. At early-mid pregnancy, we conducted personal interviews and a validated food frequency questionnaire was completed. With this information, we constructed a score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention guidelines on diet, physical activity and body fatness. At delivery, maternal and/or cord blood was collected to measure DNA and hemoglobin adducts of dietary origin and frequencies of MN in binucleated and mononucleated T lymphocytes (MNBN and MNMONO). In mothers, higher levels of red meat consumption were associated with increased MNBN frequency [2nd tertile IRR=1.34 (1.00, 1.80), 3rd tertile IRR=1.33 (0.96, 1.85)] and MNMONO frequency [2nd tertile IRR=1.53 (0.84, 2.77), 3rd tertile IRR=2.69 (1.44, 5.05)]. The opposite trend was observed for MNBN in newborns [2nd tertile IRR=0.64 (0.44, 0.94), 3rd tertile IRR=0.68 (0.46, 1.01)], and no association was observed with MNMONO. Increased MN frequency in pregnant women with high red meat consumption is consistent with previous knowledge. Our results also suggest exposure to genotoxics during pregnancy might affect differently mothers and newborns. The predictive value of MN as biomarker for childhood cancer, rather than adulthood, remains unclear. With few exceptions, the association between maternal carcinogenic exposures during pregnancy and childhood cancer or early biologic effect biomarkers remains poorly understood.