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In vitro testing for genotoxicity of the herbicide terbutryn: Cytogenetic and primary DNA damage
Abstract
Terbutryn is a widely used preemergence and postemergence s-triazine herbicide. This pesticide is used in agriculture as a control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Unexpectedly, this compound was found to persist in the environment (240 and 180 days in pond and river sediment, respectively) and to have the tendency to move from treated soils to water compartments through water runoff and leaching. However, only scant information is available about the genotoxic properties of terbutryn. In the present in vitro study, we investigated the relationship between cytogenetic damage, as evaluated in the sister-chromatid exchange (SCE) assay and the micronucleus (MN) test, and primary DNA damage (as evaluated by the “comet” assay). Cytogenetic and primary DNA damage were recorded in vitro in freshly isolated human peripheral blood leukocytes. Our results showed that the tested compound failed to produce any significant increases in SCE or MN, neither in the absence nor in the presence of S9-mix. However, terbutryn was found to induce primary DNA damage, more pronounced without S9 mix, even though in the absence of a clear trend for dose-dependence and in the presence of a concomitant mild cytotoxic effect.
... Er eignet sich daher sehr gut als Screeningtest, um das Ausmaß des biologischen Schädigungspotentials von Sedimenten und Schwebstoffen abzuschätzen. Gentoxizitätstest: Die Kontamination von Gewässern mit gentoxischen Substanzen anthropogener Herkunft, ist sowohl aus human-als auch aus ökotoxikologischen Gesichtspunkten bedenklich (Deventer 1996, Helma et al. 1994, Moretti et al. 2002. Gentoxizität kann über Absterben von Gameten und erhöhte Mortalität von Entwicklungsstadien negative Folgen auf die Reproduktion haben, oder durch die Induktion von Mutationen einen starken Einfluss auf die Populationsdynamik und damit auch auf die Stabilität von Ökosystem ausüben (Anderson und Wild 1994, Calmano et al. 1992, Helma et al. 1994. ...
... Die Anzahl der durch Frameshift-oder durch Basenaustauschmutationen entstanden histidinunabhängigen (his + ) Kolonien (Revertanten) dient als Maß für die mutagene Aktivität (Ames et al. 1975, Maron und Ames 1983. Mit dem Comet-Assay lassen sich in nahezu allen eukaryontischen Zelltypen DNA-Strangbrüche auf dem Niveau der Einzelzelle detektieren, die z.B. durch Umweltproben verursacht werden (McKalvey-Martin et al. 1993, Moretti et al. 2002, Schurstein und Braunbeck 1998. Nachweisbare Effekte sind Einzel-und Doppelstrangbrüche, AP (apurinische/apyrimidinische) Stellen, Cross-Links und die Intensität der zellulären Reparatur, ein indirektes Maß für die Bildung von Addukten (Moretti et al. 2002, Schnurstein und Braunbeck 1998, Schnurstein 2000. ...
... Mit dem Comet-Assay lassen sich in nahezu allen eukaryontischen Zelltypen DNA-Strangbrüche auf dem Niveau der Einzelzelle detektieren, die z.B. durch Umweltproben verursacht werden (McKalvey-Martin et al. 1993, Moretti et al. 2002, Schurstein und Braunbeck 1998. Nachweisbare Effekte sind Einzel-und Doppelstrangbrüche, AP (apurinische/apyrimidinische) Stellen, Cross-Links und die Intensität der zellulären Reparatur, ein indirektes Maß für die Bildung von Addukten (Moretti et al. 2002, Schnurstein und Braunbeck 1998, Schnurstein 2000. Bakterienkontakttest: Der Bakterienkontakttest ist besonders gut zur Untersuchung von Sedimenten, Schwebstoffen und Bodenproben geeignet, da der Testorganismus Arthrobacter globiformis als einer der häufigsten Bodenorganismen auch in der Natur direkt mit dem zu untersuchenden Medium in Kontakt steht (Ulrich 2002). ...
... Years ago, Moretti et al. (2002) tested in vitro the genotoxicity of the s-triazine herbicide terbutryn on stimulated human peripheral blood lymphocytes. They found that while the herbicide induced primary DNA damage revealed by the comet assay, it failed to produce a significant increase in MN frequency, either in the absence or presence of S9-mix. ...
... They found that while the herbicide induced primary DNA damage revealed by the comet assay, it failed to produce a significant increase in MN frequency, either in the absence or presence of S9-mix. Furthermore, these authors suggested that the cells were able to induce either the DNA lesions evaluated by the comet assay or a selective elimination of heavily damaged cells that could not survive long enough to contribute to MN formation (Moretti et al., 2002). Our results accord well with this concept. ...
... Finally, in agreement with our previous observations (Nikoloff et al., 2012), the current findings are in accord with the genotoxic profile shown by s-triazine herbicides like terbutryn (Moretti et al., 2002) and atrazine (Kligerman et al., 2000;Malik et al., 2004;Zeljezic et al., 2006). Several studies have reported that these herbicides show a suspected endocrine activity (Simić et al., 1991;Stoker et al., 2000). ...
The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 μg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 μg/mL. Furthermore, 10 and 15 μg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 μg/mL of both flurochloridone and Rainbow® but only when 15 μg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
... https://www.mdpi.com/journal/separations Separations 2023, 10, 363 2 of 18 miniaturized techniques in monitoring human health and environment exposure [39][40][41][42][43][44][45][46] and in continuation of our investigations on occupational exposure of herbicides in soil and water bodies. The residual nature of herbicide atrazine and its metabolites was determined simultaneously using the LCMS/MS method in soil and water [43] from agricultural production, leading to environmental contamination. ...
... The crop safety and soil degradation of triazine compared with chloro sulfuron and its derivatives were evaluated by the proton and carbon-13 NMR techniques [45]. The newly synthesized triazine herbicides in the 4,6 di-substitution accelerated the high degradation rate on acidic and alkaline soil. ...
A new multi-residue method using gas chromatography–mass spectrometry electron ionization selective ion monitoring mode (SIM) has been developed for the simultaneous determination of eight 1,3,5-triazine herbicides such as 1,3,5-triazine-2,4-diamine (atrazine), ametryn, prometryn, propazine, terbuthylazine, terbutryn, and simazine simetryn in water and soil samples. Quantification is done using lindane (gamma benzene hexachloride) as an internal standard. A specific Capillary DB-Wax column of 30 m length, 0.32 mm internal diameter, and 0.25 µm film thickness is used for the separation of eight 1,3,5-triazine-2,4-diamine. The method was applied for the determination of residues in groundwater and soil samples. The lowest detection limit by GC-MS-SIM (selective ion monitoring mode) is 0.1 pg/mL. Recovery in water samples is in the range of 93–103%, and in soil samples, 91–102% for different individual compounds. Forty-five groundwater and soil samples were collected in and around Kancheepuram district in Tamilnadu (India), and they were analyzed for the respective residues. A detailed discussion of the GC-MS analysis results has been presented.
... Pesticides have widely been used worldwide since the 1940s in agriculture applications to control pests and weed (Moretti et al. 2002;Benedetti et al. 2013). Pesticide applications have increased crop yields and reduced post-harvest losses. ...
... Pesticide applications have increased crop yields and reduced post-harvest losses. On the other hand, many pesticides are a potential danger for environment and non-target organisms like human beings because of the contamination of air, water, food, and household use (Moretti et al. 2002;Grover et al. 2003;Bolognesi and Holland 2016). ...
Since many different pesticides have been used occupationally, there have been inconsistent results regarding DNA damages among greenhouse workers. Thus, the aim of the study is to evaluate DNA damages, cell death, and chromosomal instability by using the buccal micronucleus cytome (BMcyt) assay in greenhouse workers and to compare those with a non-exposed group. The BMcyt assay was applied to the exfoliated buccal cell samples collected from 66 pesticide-exposed and 50 non-exposed individuals. We evaluated the frequency of micronucleus (MN), nuclear bud (NBUD), binucleated (BN) cells, and karyolitic (KL), pyknotic (PY), and karyorrhectic (KH) cells. The results showed that the MN, BN, PY, and KH frequencies of the pesticide-exposed group were significantly higher than those of the controls (P ˂ 0.05, P ˂ 0.05, P ˂ 0.01, and P ˂ 0.05, respectively). We observed that the MN, BN, PY, and KH frequencies in the autumn were statistically different compared with those in the control group (P = 0.037 for MN, P = 0.001 for BN, P = 0.016 for PY, and P = 0.033 for KH). The same comparison was done in the spring for the control, and there was a statistically significant difference for MN (P = 0.046) and PY (P = 0.014). We can conclude that pesticide exposure in greenhouse workers was one of the factors that altered DNA damages, cell death, and chromosomal instability in oral mucosa cells.
... The herbicide terbutryn is an s-triazine herbicide used pre-and post-emergence and widely used worldwide as an agent to control grass, sedge, and broadleaf weeds in vegetables, cereals and fruit trees. It is an herbicide persistent in the environment, which tends to dislocate by the flow of water and leachate [108]. ...
... An in vitro study performed by Moretti et al. [108] investigated the genotoxicity of the herbicide terbutryn, by analyzing the relationship between the cytogenetic damage, evaluated by the assays of SCE (sister chromatid exchanges) and MN (micronucleus), and the primary damage in the DNA, assessed by the comet assay, in leukocytes newly-isolated from peripheral human blood. The results showed that terbutryn did not produce significant increases of SCE or MN, both in the absence and in the presence of the metabolic activation system from rat liver (S9 fraction), although terbutryn has induced primary damages in the DNA in a more pronounced form in the absence of S9. ...
... The demand for increased crop yields and reduced post-harvest loss encourages agricultural producers to make extensive use of pesticides (Moretti et al. 2002). For similar reasons, the use of pesticides in combating pests is indispensable in comparison with other methods. ...
... Primary DNA damage was evaluated in the in vitro culture of isolated human peripheral blood lymphocytes with the comet assay after a 1-h treatment period. To date, several studies have been performed to evaluate the genotoxic and/or cytotoxic potential of different commercial pesticide formulations by means of various test systems (Koller et al. 2012;Moretti et al. 2002;Ribas et al. 1997;Santovito et al. 2011;Undeger and Basaran 2005;Villarini et al. 1998). As far as is known, there are no reports related to DNA damage induced by the commercial formulation CP. ...
Cabrio Plus, a commercial fungicide, is used in agriculture as the control agent for a broad spectrum of diseases including black dot, early blight, late blight and powdery mildew. This study aimed to evaluate the genotoxicity of commercial formulation of Cabrio Plus which has been inadequately evaluated. The genotoxic potential of Cabrio Plus in isolated human peripheral blood lymphocytes was measured by means of an alkaline version of the comet assay (pH > 13) and in whole blood by use of the in vitro micronucleus test. Cabrio Plus induced a statistically significant increase in DNA damage assessed with the in vitro micronucleus assay and the comet assay. Cabrio Plus also induced cytotoxicity in a dose-dependent manner with the in vitro micronucleus assay. It can be concluded that a commercially available pesticide formulation, Cabrio Plus, has the ability to cause DNA damage and cytotoxicity.
... The herbicide terbutryn is an s-triazine herbicide used pre-and post-emergence and widely used worldwide as an agent to control grass, sedge, and broadleaf weeds in vegetables, cereals and fruit trees. It is an herbicide persistent in the environment, which tends to dislocate by the flow of water and leachate [108]. ...
... An in vitro study performed by Moretti et al. [108] investigated the genotoxicity of the herbicide terbutryn, by analyzing the relationship between the cytogenetic damage, evaluated by the assays of SCE (sister chromatid exchanges) and MN (micronucleus), and the primary damage in the DNA, assessed by the comet assay, in leukocytes newly-isolated from peripheral human blood. The results showed that terbutryn did not produce significant increases of SCE or MN, both in the absence and in the presence of the metabolic activation system from rat liver (S9 fraction), although terbutryn has induced primary damages in the DNA in a more pronounced form in the absence of S9. ...
... The formulation for pesticides is performed in two ways, as liquids including aqueous and dispersions and solids including wettable powders and water-dispersible granules or as controlled-release systems [61]. Factors that are considered in formulations include the mode of application, the crop and agricultural practices [62]. The application of nanotechnology for formulation of new products, using polymer-based nanocapsules, or encapsulation with metallic nanoparticles could result in increased stability and efficacy of EOs and therefore reduce the required dosage for application. ...
Crop diseases due to fungal pathogens cause significant resulting economic losses in agriculture. For management of crop diseases, farmers use synthetic pesticides. However, the frequent application of these chemicals leads to accumulation in soil and therefore presenting pollution problems. Essential oils (EOs) sourced from aromatic plants are safer alternatives and are effective against a variety of crops pathogens. In addition to their role as the sources of EOs, aromatic plants are gaining much attention in rehabilitation strategies. In phytoremediation processes, suitable plants species are used to clean-up polluted sites. Mining activities and electricity generation processes have resulted in significant amounts of tailings and coal fly ash. Mine tailings and coal fly ash are disposed in dumpsites, converting productive lands to unusable waste sites. These solid waste materials contain toxic metals and therefore posing serious risks to the health of the environment. Aromatic plants can be cultivated in contaminated sites and therefore be used for restoration of polluted lands. The EOs can be sourced from these aromatic plants as they are free from metal-toxicity and can therefore be used to generate revenues. This review highlights the role of aromatic plants in the control of crops pathogens and also their application in phytoremediation processes.
... With the gradual rise of chemical consumption in diaspora, there arises a need to check for adversity that follows. The exponential rise in the consumption of OCs has compelled scientists to explore the probable toxicity caused to the flora and fauna at the genetic level starting from the building block of life, i.e., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (Moretti et al., 2002;Tsuboy et al., 2007). OCs have long been identified as carcinogenic or mutagenic materials in several ecologically important species including humans along with various bacterial cell lines such as Salmonella typhimurium (Chung and Cerniglia, 1992;Chung et al., 1997;Rashid et al., 1987;Sabbioni, 1994;Tomita et al., 1982). ...
In an attempt to describe the underlying causes of mutagenicity mainly due to organic chemicals, quantitative structure-activity relationship (QSAR) models have been developed using two different Salmonella typhimurium mutagenicity endpoints with or without presence of liver metabolic microsomal enzymes (S9) namely TA98-S9 and TA98 + S9. The models were developed using simple 2D variables having definite physicochemical meaning calculated from Dragon, SiRMS, and PaDEL-descriptor software tools. Stepwise regression followed by partial least squares (PLS) regression was used in model development following the strict OECD guidelines for QSAR model development and validation. The models were validated using coefficient of determination R2, cross-validation coefficient Q2LOO (leave one out) while the test set predictions were analyzed using Q2F1 (coefficient of determination for the test set). Several other internationally accepted validation metrics like MAE95%train, rm(LOO)2 and Δrm(LOO)2 were used to check model robustness while predictive efficiency was evaluated using MAE95%test, rm(LOO)2 and Δrm(LOO)2. The scope of predictions was defined by applicability domain analysis using the DModX approach, a recommended tool for PLS models. The major contributing features related to mutagenicity include lipophilicity, electronegativity, branching and unsaturation, etc. The present manuscript is the first attempt to undertake modeling of two different endpoints (TA98-S9 and TA98 + S9) in order to explore major contributing molecular features linked directly or indirectly to mutagenicity.
... Thus, it is crucial to evaluate the genotoxicity and cytotoxicity of active substances, as well as their PPPs. Many studies have investigated the genotoxicity and cytotoxicity of pesticides by using different methods (Koller et al., 2012;Moretti et al., 2002;Ribas et al., 1997;Santovito et al., 2011;Undeger & Basaran, 2005;Villarini et al., 1998). The genotoxicity of Signum®, evaluated by the cytokinesisblock micronucleus (CBMN) assay on human peripheral blood lymphocytes, showed that some concentrations of Signum® increased micronucleus frequencies when compared with control (Çayır et al., 2014). ...
The genotoxic potential of the plant protection product Signum? and its two active substances (pyraclostrobin and boscalid) was investigated by the comet assay (pH>13). Leukocytes isolated from whole blood were treated with different concentrations (0.1-25 ?g/ml) of the fungicide for 2 h and 20 h. The Trypan Blue exclusion test showed higher cell viability (>84%) under all three concentrations and in the two treatment periods. The obtained results revealed that both Signum? and pyraclostrobin induced statistically significant DNA damage. In contrast, boscalid did not cause statistically significant DNA damage after 2 h exposure although it caused DNA damage at higher concentrations after a longer time exposure (20 h). It is deducible that pyraclostrobin and Signum? might be genotoxic. However, within the studied concentration ranges, none of the fungicides was found to be cytotoxic in the two treatment periods.
... Analysis of blind samples was carried out at 200× magnification by using an Olympus BΧ41 (Japan) fluorescence microscope (excitation filter 515-560 nm, emission filter 590 nm) equipped with a high-sensitivity black and white CCD camera (Mod. 4912, Cohu; USA) and connected with a computerized system for the analysis of images (BComet Assay III^, Perspective Instruments Ltd., Suffolk, UK) (Moretti et al. 2002). One hundred cells were analyzed for each experimental point (50 cells/ slide); the median of the scored comets for each slide was used to calculate the group means. ...
Road tunnel construction involves the use of explosives and diesel-powered machines in a very limited indoor space. Tunnel construction workers are thus exposed to a variety of toxic substances, including dust, asbestos, silica, concrete, diesel fumes, and oil mist. In this study, the low noise miniaturized Sioutas Cascade Impactor (SKC) has been used to collect PM samples as a function of particle size (i.e., aerodynamic diameter). Airborne particulates were sampled into a road tunnel (final length 2391 m) under construction near the village of Pale, Municipality of Foligno, Umbrian Apennines (State Highway 77 “Val di Chienti”). Three sampling sessions were performed: (i) in the West yard, during dislodging of rocks; (ii) in the East yard during drilling of rocks, and (iii) during scaling and grouting with quick-drying shotcrete. The aim of this study was to characterize size-fractionated PM samples for their cytotoxic/genotoxic potentials. Toxicological evaluation was carried out in vitro on A549 lung carcinoma cells: cytotoxicity was assessed by Trypan blue dye exclusion assay, whereas genotoxicity was assessed by comet assay (primary DNA damage) and cytokinesis-block micronucleus (CBMN) Cytome assay (cytogenetic effects). Primary DNA damage (i.e., strand breakage) was mainly caused by coarse fractions A (Ø > 2.5 μm) sampled in the West yard (sample i) and in the East yard (sample iii). Cytogenetic effects were mainly caused by the fine fractions AF (aerodynamic Ø < 0.25 μm) sampled in the East yard (sample ii). Results reported in this article show that particulate matter (PM) samples collected underground (i.e., during road tunnel construction) may trigger various cytotoxic/genotoxic effects. The recent classification of air pollution and PM itself as carcinogenic to humans (group 1) by the IARC makes indoor/outdoor air pollution a growing matter of concern. Our results highlight the need in this occupational setting for a reduction, if applicable, of PM emission (prevention), or an implementation of work practices by stressing the importance of proper use of protective equipment (protection).
... For each experimental point, 100 randomly chosen cells were examined. DNA integrity in individual cell was scored with the "comet assay III" imaging analysis software (21,22). Tail intensity, that is the percentage of DNA in comet tails, was used as the parameter for DNA damage (23). ...
... For each experimental point, 100 randomly chosen cells were examined. DNA integrity in individual cell was scored with the "comet assay III" imaging analysis software (21,22). Tail intensity, that is the percentage of DNA in comet tails, was used as the parameter for DNA damage (23). ...
Epilobium angustifolium L. is a medicinal plant belonging to the Onagraceae family, which includes more than 200 different species from all over the world. Traditional medicinal applications include treatment of prostate, gastrointestinal, menstrual disorders and recently it has been used for its analgesic and anti-inflammatory activity. In this investigation E. angustifolium was collected in Ternopil region of Ukraine. The obtained data demonstrated that E. angustifolium herb extract, rich in polyphenolic compounds such as flavonoids and tannins, display high antioxidant properties. In addition the potential anticancer activity has been investigated in vitro on human hepatocellular carcinoma cells (HepG2). Furthermore the cytotoxic and genotoxic effects of E. angustifolium have been investigated respectively by MTT and Comet assay. Results showed that at low concentration, up to 25 μg/mL, the cytotoxic effect was not observed. Increasing concentration from 50 to 75 μg/mL reduced significantly cell viability and induced an important DNA damage in hepatocellular carcinoma. These promising data were also confirmed with mitochondrial potential test. It is possible to conclude that E. angustifolium has beneficial properties in low concentration, in term of antioxidant activity, and it could be a potential antitumoral natural product if it will be used at high concentration
... The extent of induced DNA strand breakage was measured by a computer-based semi-automated image analysis system (Comet Assay III, Perceptive Instruments, Haverhill, UK). Computerized imaging was described in detail elsewhere (Moretti et al., 2002) and was performed (blind) on coded slides. Percent of fluorescence migrated in the comet tail (i.e., tail intensity; Collins, 2004) was used to measure the level of DNA damage produced by FW incubation. ...
This study analyzes the composition of viable fecal bacteria and gut toxicology biomarkers of 29 healthy volunteers, who followed omnivorous, lacto-ovo-vegetarian, or vegan diets. In particular, the research was focused on the prevalence of some representative viable bacteria from the four dominant phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria) commonly present in human feces, in order to evaluate the relationship between microorganisms selected by the habitual dietary patterns and the potential risk due to fecal water (FW) genotoxicity and cytotoxicity, considered as biomarkers for cancer risk and protective food activity. The relative differences of viable bacteria among dietary groups were generally not statistically significant. However, compared to omnivores, lacto-ovo-vegetarians showed low levels of total anaerobes. Otherwise, vegans showed total anaerobes counts similar to those of omnivores, but with lower number of bifidobacteria and the highest levels of bacteria from the Bacteroides–Prevotella genera. FW genotoxicity of lacto-ovo-vegetarians resulted significantly lower either in relation to that of omnivores and vegans. Lacto-ovo-vegetarians also showed the lowest levels of cytotoxicity, while the highest were found for vegans. These results highlighted that lacto-ovo-vegetarian diet was particularly effective in a favorable modulation of microbial activity, thus contributing to a significant reduction of the genotoxic and cytotoxic risk in the gut.
... Recent in vitro laboratory studies have shown that pesticides, such as organophosphates, carbamates, and pyrethoids, can induce increased DNA damage in human peripheral lymphocytes. 39,40 Certain pesticides, such as DDT, pyrethroids, and chlorinated pesticides, can also dysregulate the immune system, a key pathway in the development of childhood ALL. 41,42 Most toxicologic studies of pesticides have focused on active ingredients; however, the so-called inert ingredients added to enhance pesticide activity may have significant toxicological properties, as shown for glyphosate. ...
In contrast to most pediatric cancers, there is a growing body of literature, nationally and internationally, that has implicated the role of several environmental indoor and outdoor hazards in the etiology of childhood leukemia. For example, exposures to solvents, traffic, pesticides, and tobacco smoke have consistently demonstrated positive associations with the risk of developing childhood leukemia. Intake of vitamins and folate supplementation during the preconception period or pregnancy has been demonstrated to have a protective effect. Despite the strength of these findings, the dissemination of this knowledge to clinicians has been limited. Some children may be more vulnerable than others as documented by the high and increasing incidence of childhood leukemia in Hispanics. To protect children's health, it is prudent to establish programs to alter exposure to those factors with well-established associations with leukemia risk rather than to suspend judgment until no uncertainty remains. This is particularly true because other serious health outcomes (both negative and positive) have been associated with the same exposures. We draw from historical examples to put in perspective the arguments of association versus causation, as well as to discuss benefits versus risks of immediate and long-Term preventive actions.
... Respecto a los resultados, los agricultores mostraron una longitud media de la cola de los cometas de ± D.E 62,32 ± 5,86µm; la población floricultores una media de ± D.E 25,61 ± 3,1µm y el grupo control presentó una de ± D.E 23,46 ± 4,15µm; estos resultados se presentan resumidos en la tabla 1. Se evidenció un incremento significativo en los valores del largo de la cola de los cometas analizados en la población de agricultores, respecto a las poblaciones de floricultores y el grupo control (p < 0.001), resultados también observados en trabajos anteriores de Garaj-Vrhovac & Zeljezic. (1999, 2000y 2002, Moller et al. (2000), Elhajouji (2001) y Moretti et al. (2002). Estos resultados se deben a que en la población de agricultores, la exposición ocupacional a plaguicidas en el cultivo es alta, ya que fue evidente la falta de implementos adecuados para su protección y el poco acatamiento de las medidas básicas de seguridad para el manejo de plaguicidas. ...
RESUMEN Algunas investigaciones demuestran el potencial genotóxico de ciertos plaguicidas sobre los seres humanos, lo cual, hace necesario el desarrollo de metodologías que permitan evaluar el impacto de dichas sustancias, para alterar el ADN. El ensayo del cometa alcalino es un método económico, rápido y sensible que detecta, principalmente, rompimientos de cadena sencilla y sitios lábiles al-álcali en células individuales. El objetivo del presente estudio fue evaluar el daño en el ADN, en una población de floricultores y agricultores ocupacionalmente en contacto con plaguicidas y compararlo con un grupo control. Se obtuvieron muestras de sangre periférica de 101 personas, entre trabajadores expuestos e individuos del grupo control. El ensayo del cometa, se realizó utilizando sangre total y linfocitos aislados, con muestras embebidas en agarosa y puestas sobre una lámina portaobjetos; luego expuestas a una solución de lisis con detergente y, posteriormente, sometidas a una corriente eléctrica, con buffer alcalino. El daño en el ADN fue evaluado por medición del largo de la cola (migración de ADN) y por la morfología que presentaban los cometas. En los agricultores, el ensayo mostró un incremento significativo (p < 0,001) del daño en el ADN, comparado con los floricultores y el grupo control; entre estos últimos, no se hallaron diferencias. Los resultados sugieren que el ensayo de cometa puede ser un buen biomarcador de exposición ocupacional a plaguicidas y que puede ser utilizado para este tipo de estudios. Palabras clave: Genotoxicidad, ensayo del Cometa, plaguicidas, exposición ocupacional.
... Respecto a los resultados, los agricultores mostraron una longitud media de la cola de los cometas de ± D.E 62,32 ± 5,86µm; la población floricultores una media de ± D.E 25,61 ± 3,1µm y el grupo control presentó una de ± D.E 23,46 ± 4,15µm; estos resultados se presentan resumidos en la tabla 1. Se evidenció un incremento significativo en los valores del largo de la cola de los cometas analizados en la población de agricultores, respecto a las poblaciones de floricultores y el grupo control (p < 0.001), resultados también observados en trabajos anteriores de Garaj-Vrhovac & Zeljezic. (1999, 2000y 2002, Moller et al. (2000), Elhajouji (2001) y Moretti et al. (2002). Estos resultados se deben a que en la población de agricultores, la exposición ocupacional a plaguicidas en el cultivo es alta, ya que fue evidente la falta de implementos adecuados para su protección y el poco acatamiento de las medidas básicas de seguridad para el manejo de plaguicidas. ...
RESUMEN Algunas investigaciones demuestran el potencial genotóxico de ciertos plaguicidas sobre los seres humanos, lo cual, hace necesario el desarrollo de metodologías que permitan evaluar el impacto de dichas sustancias, para alterar el ADN. El ensayo del cometa alcalino es un método económico, rápido y sensible que detecta, principalmente, rompimientos de cadena sencilla y sitios lábiles al-álcali en células individuales. El objetivo del presente estudio fue evaluar el daño en el ADN, en una población de floricultores y agricultores ocupacionalmente en contacto con plaguicidas y compararlo con un grupo control. Se obtuvieron muestras de sangre periférica de 101 personas, entre trabajadores expuestos e individuos del grupo control. El ensayo del cometa, se realizó utilizando sangre total y linfocitos aislados, con muestras embebidas en agarosa y puestas sobre una lámina portaobjetos; luego expuestas a una solución de lisis con detergente y, posteriormente, sometidas a una corriente eléctrica, con buffer alcalino. El daño en el ADN fue evaluado por medición del largo de la cola (migración de ADN) y por la morfología que presentaban los cometas. En los agricultores, el ensayo mostró un incremento significativo (p < 0,001) del daño en el ADN, comparado con los floricultores y el grupo control; entre estos últimos, no se hallaron diferencias. Los resultados sugieren que el ensayo de cometa puede ser un buen biomarcador de exposición ocupacional a plaguicidas y que puede ser utilizado para este tipo de estudios. Palabras clave: Genotoxicidad, ensayo del Cometa, plaguicidas, exposición ocupacional.
... The alkaline comet assay was used since this method allows for the sensitive detection and quantification of a variety of lesions responsible for primary DNA damage in individual cells (Tice, 1995). It has also been previously established as a valuable endpoint in research on pesticide toxicology (Ribas et al., 1995; Villarini et al., 2000; Tennant et al., 2001; Moretti et al., 2002; Zelje zi c et al., 2006; Sharma and Sharma, 2012; Nikoloff et al., 2012 Nikoloff et al., , 2014b). The CBMN Cyt assay, on the other hand, detects cytogenetic damage that persists for at least one mitotic cycle. ...
Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL) on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.
... The micronucleus test and the comet assay conducted on various fish species exposed to a number of genotoxicants indicated that the micronucleus test was much less sensitive than the comet assay (Petras et al., 1995;Lee & Steinert, 2003), which explains the damage indicated by the comet assay and the lack of damage indicated by the micronucleous test in the present work. Moretti et al. (2002) studied Terbutryn (up to 100 or 150 μg mL -1 ) in vitro using freshly isolated human peripheral blood leukocytes; they did not observe any significant increase in the number of micronuclei, but the comet assay evidenced DNA damage. The comet assay also identified a significant genotoxic effect of diuron on oyster spermatozoa exposed to the herbicide at concentrations higher than 0.05 μg L -1 (Akcha et al., 2012). ...
Pesticides have made possible a safer and plentiful supply of food; however, the ultimate sink for many of these contaminants is the aquatic environment. We analyzed the commercial mixture Velpar K® WG, which is composed of the pesticides diuron (46.8% m/m) and hexazinone (13.2% m/m), as well as inert ingredients (40.0% m/m). The present study aimed to evaluate the effects of the herbicide mixture on Oreochromis niloticus of different sizes. To this end, we analyzed biomarkers in small and large O. niloticus exposed to a mixture of herbicides at 125, 250 and 500 ug L-1 for 72h. EROD increased activity in small fish exposed to the herbicide mixture at 250 and 500 ug L-1. The GST activity and levels of the antioxidant enzymes GPx and CAT remained the same in the treated fish, compared with the control. The level of the antioxidant enzyme SOD measured in the fish gills was changed in animals exposed to the herbicide mixture at 250 ug L-1. MDA analysis did not show lipid peroxidation. The comet assay evidenced widespread DNA damage, but the micronucleus test did not show mutagenicity. Hepatosomatic (HSI) analysis did not indicate any alterations in liver morphology. The biomarkers response in the fish depended on the size of the individuals.
... Many results showed that the pesticides had genotoxic effects on human and animals. For example, in vitro studies with human and Chinese hamster cell lines showed that positive evidence was existed between the genotoxicity and pesticides application [15]. It is important to note that genotoxicty is related with the generation of cancers. ...
Although it is known that the advised or lower than advised doses of eco-friendly pesticides cause a genotoxic effect on non targeted higher cells as well as on targeted organisms, their very low or ultra low doses, however, have not been tested with an efficient method. The single cell gel electrophoresis (SCGE) or comet assay was used to detect the DNA damages on higher cells in vitro exposed to insecticides such as dimethoate (100-, 50-, 10 μg ml -1), methyl parathion (90-, 45-, 5 μg ml-1), alphacypermethrin (25-, 15, 5 μg ml-1) and dichlorvos (100-, 50-, 10 μg ml-1) in which the concentration of each insecticide was designated as "very low volume" (vlv), "very very low volume" (vvlv) and "ultra low volume" (ulv) doses, respectively. The induction of DNA damage was evident on human peripheral lymphocytes (PBL) after 1 h treatment in vitro with the above insecticides. The results showed that the "vlv" and "vvlv" doses of methyl parathion (LD 50=3 μg ml-1), alphacypermethrin (LD50=57 μg ml-1) and dichlorvos (LD50=50 μg ml-1) caused significantly higher DNA damages in treated groups than those of their controls; on the other hand, dimethoate (LD50=387 μg ml -1) caused a significant damage in blood cells only in "vlv" dose. DNA damage was not detected in "ulv" doses of all insecticides. It is probable that the "vlv" or "vvlv" doses of highly toxic chemicals expressing DNA damages on blood cells might be related with their very low LD50 values, which may play an important role on the impact of environmental pollution and human health even with their very low doses. It would be also beneficial to determine the genotoxic effect of "ulv" doses of all insecticides along with the non-harmful doses of dimethoate with extended incubation period if time has potential to create extensive DNA damages.
... Their toxicity was assayed with the lethality test of Artemia salina (Finney, 1971) in order to use sublethal quantities to study cell damage in the experiments of genotoxicity. The comet assay (Moretti et al., 2002) was applied for the analysis of DNA damage because it provides a direct determination of DNA singleand double-strand breaks in individual cells. This test was selected because it was applied for the in vivo and in vitro assays with several cell lines (CHO, V79, HepG2, among others) (Valentin-Severin et al., 2003). ...
... This assay appeared not to be as sensitive as the comet assay in detecting genotoxicity, as we found no significant changes in micronucleus formation with any of the particle treatments or when dispersed in different media. Moretti et al. (2002) suggested that a lack of sensitivity in the micronucleus assay compared with the comet assay may be due to differences in the actual type of damage to the cell. The authors hypothesized in their study that there may be differences in 'cytogenetic damage' as measured in the micronucleus assay compared with 'primary DNA damage' as measured using the comet assay. ...
Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks’ balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle’s activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.
... Die WHO hat Chloridazon als "bei normalem Gebrauch nicht akut gefährlich" klassifiziert (WHO 2005). Es konnten jedoch Schäden an der DNA menschlicher Leukozyten nachgewiesen werden (Moretti et al. 2002). Die WHO hat den Wirkstoff in die Gefährdungsklasse III ("class III, slightly hazardous", WHO 2005) eingestuft. ...
Bei der heterotrophen Denitrifikation mit biologisch abbaubaren Kunststoffen dienen diese der im Reaktor wachsenden Biozönose als organisches Substrat und gleichzeitig als Aufwuchskörper. Da die bioabbaubaren Kunststoffe in Form eines festen Granulats vorliegen, das wasserunlöslich ist, müssen diese hydrolysiert werden, bevor sie für die Denitrifikation genutzt werden können. Es wurde angenommen, dass die Menge an freigesetztem "gelöstem Kohlenstoff" durch die Biozönose selbst reguliert wird, was die Gefahr einer Überdosierung deutlich begrenzen sollte.
Um die Vorteile, die bioabbaubare Kunststoffe bei der Denitrifikation bieten - gleichzeitig organisches Substrat und Aufwuchskörper - mit denen eines kontinuierlich betriebenen Systems zu verbinden, wurde als Reaktor ein Roto-Bioreaktor (RBR) ausgewählt. Dieser war mit einer ca. 60 L großen, in drei Kammern aufgeteilten Reaktortrommel ausgestattet.
Betrieben wurde der RBR hauptsächlich bei einer Drehzahl von 5 h-1 und einem Volumenstrom von 75 L/h. Bei dem im RBR kontinuierlich denitrifizierten Wasser wurde die entstehende überschüssige Biomasse selbstständig ausgetragen. Unterbrechungen des Betriebs, wie z. B. bei Festbettreaktoren zum Spülen der Schüttung notwendig, waren beim RBR nicht erforderlich.
Um den RBR an die kontinuierliche Denitrifikation mit einem in der Größe veränderlichen Granulat anzupassen und Verluste davon zu minimieren, wurde die ursprüngliche Spaltweite der Reaktorsiebe von 3,0 mm auf 1,5 mm verringert. Damit wurde erreicht, dass statt 42 % nur etwa 5 % des eingesetzten Polycaprolactons (PCL) als Partikel verloren ging.
Die für den Verbrauch von PCL erstellte Massenbilanz zeigte, dass über diesen Anteil an partikulärem Substrat hinaus auch bereits gelöstes Substrat ungenutzt aus dem Reaktor ausgeschleust wurde. Dieser unerwünschte Austrag schränkt den Einsatz von PCL in der Trinkwasseraufbereitung ein, da in diesem Fall ein weiterer Behandlungsschritt des denitrifizierten Wassers zur Entfernung dieser Stoffe notwendig wäre.
Anhand von simulierten Störungsszenarien, die beim Betrieb auftreten können, wurde festgestellt, dass sich die Denitrifikation am besten bei einer Drehzahl von 5 h-1 und 75 L/h einstellt. Die zu Anfang in falscher Reihenfolge angeordnete Kammersiebe führten, durch die im Verlauf der Reaktion kleiner werdenden Granulatkörner, vermehrt zu Verstopfungen und dadurch zu einem erhöhten Wartungsaufwand. Nach dem Vertauschen der Siebe verlängerten sich die Intervalle zwischen den Wartungen wieder. Es konnte so gezeigt werden, dass ein Betrieb des RBR mit einem größenveränderlichen Granulat prinzipiell möglich ist.
Ein Mehrnutzen von PCL gegenüber flüssigen organischen Substraten ergibt sich aus der Möglichkeit der Sorption von PBSM, die bereits für das Insektizid Endosulfan nachgewiesen wurde.
Um das Sorptionsvermögen für weitere PBSM zu erkunden, wurden elf PBSM ausgewählt und in den RBR dosiert. Für einige der ausgewählten PBSM schienen die ermittelten Kontaktzeiten zwischen PCL und PBSM im Reaktor zu gering, als dass eine deutliche Sorption an PBSM hätte erwartet werden können. Die für die untersuchten PBSM berechneten Wiederfindungsraten (z. B. ca. 74 % für Metolachlor) zeigten allerdings, dass für einzelne PBSM durchaus eine deutliche Konzentrationsminderung über Sorption möglich war.
Im Rahmen der PBSM-Analytik wurde im Wasser des Reaktorablaufs die unerwünschte Substanz Diisopropylanilin (DIPA) gefunden, für die die Europäische Behörde für Lebensmittelsicherheit (EFSA) eine Begrenzung des Migrationswertes aus Lebensmittelverpackungen empfohlen hat (< 0,05 mg DIPA/kg Lebensmittel). Das in dieser Arbeit verwendete PCL (Capa 6500, Fa. Solvay), aus dem dieser Stoff freigesetzt wird, sollte in der Trinkwasseraufbereitung nicht zum Einsatz kommen.
Als Alternativen zu dem verwendeten PCL wurden vom Kooperationspartner MLU (TP V) verschiedene Biocompounds (Mischungen von PCL mit anderen bioabbaubaren Kunststoffen, EP 1 068 152 B1) hergestellt und mit dem analytischen Verfahren der Thermoextraktion auf potenziell mobilisierbare Stoffe untersucht.
Die Ergebnisse der Analysen zeigten, dass im Falle der Biocompounds die Verarbeitung während des Herstellungsprozesses einen wesentlichen Einfluss auf die im Kunststoff enthaltene Menge an DIPA hat. Die Fa. Solvay stellte - nachdem sie auf das Problem der DIPA-Kontamination hingewiesen worden war - eine DIPA-freie PCL-Charge zur Verfügung. Sowohl Biocompounds als auch PCL selbst lassen sich demnach DIPA-frei herstellen.
Sollen biologisch abbaubare Kunststoffe in der Trinkwasseraufbereitung verwendet werden, so ergibt sich die Notwendigkeit, dass entsprechende Mindestqualitätsforderungen an die Kunststoffe gestellt werden müssen. Insbesondere müssen Vorgaben gemacht werden, die die Gefahr von unerwünschten, ggf. sogar gesundheitsschädlichen Zusätzen, die potenziell aus abbaubaren Kunststoffen während des Denitrifikationsprozesses freigesetzt werden, minimieren.
... The genotoxic effects of pesticides on higher cells are reported in many in vivo and in vitro studies. For example, in vitro studies on human and Chinese hamster cell lines showed positive evidence for genotoxicity when treated with different pesticides [34]. Toxicity resulting from pesticides is capable of inducing choromosomal aberrations [35] and increase in the number of miconuclei in lymphocytes [36]. ...
In this study, the genotoxic effects of commonly used eco-friendly pesticides selected from herbicide (tribenuron methyl), fungicide (triadimenol) and insecticide (lambdacyhalothrin) were evaluated on mononuclear leukocytes isolated from higher cells using the single cell gel electrophoresis (SCGE) assay. Mononuclear cells were exposed for a period of 30 min to 50-, 25-, 15-, 10- and 5 mu g ml(-1) doses of lambda-cyhalothrin and triadimenol while tribenuron methyl was applied at 10-, 7.5-, 5-, 2.5- and 1 mu g ml(-1) doses on those cells. DNA damages characterized with Olive tail moment (OTM), comet length and % Tail DNA (% T-DNA) increased while % Head DNA (% H-DNA) values significantly decreased with the increase of concentrations of all pesticides used. The results showed that the pesticide lambda-cyhalothrin was found more genotoxic than triadimenol and tribenuron methyl at all doses. However, the lowest doses of triadimenol and tribenuron methyl (last two doses) had only marginal DNA damaging effects on the leukocytes while lambda-cyhalothrin had a remarkable effect at 10 mu g ml(-1). We conclude that the pesticide lambda-cyhalothrin had a toxic effect on human and could have significant impact on environmental health even if they are applied at low doses. It would be crucially important to determine the effects of genotoxicity and oxidative stress of those pesticides with descending doses on higher cells as well as on plants and animals with extended incubation period if time creates extensive DNA damages.
... The concentration of PM extract that decreases cell viability by more than 30% was not used for the Comet assay. The Comet assay was performed under alkaline conditions (pH > 13), according to the method of Tice et al. (2000) after slight modifications (Moretti et al., 2002). A549 cells (approximately 6 × 10 5 cells) were mixed with 300 L low melting point agarose (LMA), placed on the slides coated with 1% of normal melting agarose (NMA), with LMA added as the top layer. ...
... The microscope, equipped with a high sensitivity black and white CCD camera (PE2020, Pulnix Europe Ltd., UK), was connected to a computer-based semiautomated image analysis system (Comet Assay III, Perceptive Instruments Ltd., Haverhill, Suffolk, UK). Computerized imaging, described in detail elsewhere (Moretti et al., 2002;Villarini et al., 2000), was performed on coded slides and the percent of DNA in the comet tail (i.e. tail intensity) was used to measure the level of DNA damage (Figure 1). ...
The aim of the present study was to investigate the impact of a probiotic product commercially distributed, consisting of a mix of four different species of Bifidobacterium [i.e. (B. bifidum, B. breve, B. longum, B. infantis) (Bifiselle)], on the DNA-damaging effects of 2-amino-l-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) or acrylamide. Male CD1 mice were treated orally with PhIP or acrylamide. Suspensions of bifidobacteria were given by gavage to the animals 3 h before administration of model genotoxins. Subsequently, the extent of DNA migration was measured in colon and liver cells by the single-cell microgelelectrophoresis (comet) assay. The Bifidobacterium strains mix suspension caused a significant inhibition of DNA damage induced by PhIP in the colon and by acrylamide in the liver.
... As a consequence of this massive use of pesticides in agriculture, they have become a significant ecological burden especially in aquatic ecosystems (Cerejeira et al. 2003;Flynn & Spellman 2009). An important group of herbicides are triazines, which have been used extensively or selectively primarily to control broadleaf and some grassy weeds in agriculture and in non-agricultural areas worldwide for more than 50 years (Moretti et al. 2002;Arufe et al. 2004;Breckenridge et al. 2008). The application of pesticides such as herbicides or insecticides, and their mixtures, may often result in negative impacts on nontarget organisms in the aquatic environment, including fish (Di Giulio & Hinton 2008;Slaninová et al. 2009;Zhang et al. 2010). ...
Triazines are an important group of herbicides, which have been used extensively or selectively both in agricultural and non-agricultural areas worldwide for more than fifty years to control broadleaf and some grassy weeds. As a consequence of their massive application, they have become and remain significant environmental pollutants, especially in aquatic ecosystems. Fish are an integral part of the aquatic environment and are, therefore, suitable models for the study of the behavioral, biological, and biochemical effects of triazine exposure. We have summarized and evaluated the effects of triazine herbicides on fish in order to provide an overview of current information on triazines. The overall effects of triazine herbicide exposure on the physiology of fish were evaluated by considering a variety of parameters in a number of reports. Haematological and biochemical profiles of blood provide important information about the internal environment of the organism and the general physiology and health status of triazine exposed fish. According to studies using biotransformation and bioaccumulation indices to estimate the effects of triazines on fish, changes in fish metabolism reflect the pollution of the environment by these herbicides. The responses of antioxidant defence systems in fish to triazine exposure could be an adaptive mechanism to protect the fish from triazine-induced oxidative stress. Acute exposure to triazines affects reproduction or reproductive development in fish; however, some triazines did not affect fish behavior during long-term exposure to low concentrations. Nevertheless, an impact on the overall behavioral response of fish cannot be excluded.
... The comets in each microgel were analyzed using a computerbased semi-automated image analysis system (Comet Assay III, Perceptive Instruments Ltd., Suffolk, UK). Computerized imaging, described in detail elsewhere [31], was performed on coded slides and the percent of DNA in the comet tail (i.e., tail intensity; TI) was used to measure the level of DNA damage. ...
... Terbutryn (2-(tert-butylamino)-4-(ethylamino)-6-(methylthio)-s-triazine) belongs to substituted symmetrical triazines. Triazines form a group of similar herbicides used extensively in agriculture and non-agricultural sites, primarily to control broadleaf and some grassy weeds that have become ubiquitous contaminants of the environment (Muir 1980;Moretti et al. 2002;Arufe et al. 2004). Triazine herbicides are divided into two groups, asymmetrical triazines or triazinones, such as metribuzin, and symmetrical triazines. ...
The aim of the present study was to determine and compare acute toxicity of terbutryn in Danio rerio and Poecilia reticulata, and in two different developmental stages of D. rerio - embryonic and juvenile. Acute toxicity tests were performed according to OECD methodology. The LC50 values were assessed by probit analysis using EKO-TOX 5.2 programme. The 96hLC50 terbutryn mean value of 5 tests was 2.85 +/- 0.75 mg.l(-1) for the juvenile stage of P. reticulata and 5.71 +/- 0.46 mg.l(-1) for the juvenile stage of D. rerio. For the embryonic stage of D. rerio the 144hLC50 terbutryn mean value of 6 tests was estimated as 8.04 +/- 1.05 mg.l(-1). The study proved significantly higher (p < 0.01) sensitivity of the juvenile stage of D. rerio to terbutryn compared to the embryonic stage of D. rerio and significantly higher (p < 0.01) sensitivity of the juvenile stage of P. reticulata to terbutryn compared to the juvenile stage of D. rerio. herbicides. This study documented differences in sensitivity of several fish species and different developmental stages of fish to one of triazine.
... The in vivo micronucleus assay in amphibians detects fixed DNA damages persisting after at least one mitotic cycle, i.e. chromosomal and/or genomic mutations (Mouchet et al., 2005). Alkaline Comet assay detects primary DNA damages which represent reversible damages (Moretti et al., 2002;Van Goethem et al., 1997). Unlike the micronucleus assay, which highlights the cumulative effect of chronic exposure, the Comet assay provides instantaneous information about the current exposure (Maluf and Erdtmann, 2000). ...
The potential impact of Multiwalled Carbon NanoTubes (MWCNTs) was investigated on Xenopus laevis tadpoles exposed to 0.1, 1 and 10mg/L. Oxidative stress was measured in entire larvae exposed and DNA damage (Comet assay) was carried out in erythrocytes of circulating blood from 2h to 24h according to standardized recommendations. Results showed significant H2O2 production when larvae were exposed to 1mg/L and 10mg/L of MWCNTs after 4h and 2h of exposure, respectively. Antioxidant enzyme activities showed significant induction of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) from only 2h of exposure to 10mg/L of MWCNTs. In presence of 1mg/L of MWCNTs, only GR and CAT activities were significantly induced at 4h. Enzyme activities do not follow a simple dose-effect relation, but the time of induction is shortened in relation with the tested concentration. The Comet assay results showed significant DNA damages with a dose dependent response. The profiles of DNA damages show fluctuations, in course of time, which are characteristics of oxidative stress response in relation with the continuous balance between damage and compensation process.
... In our work, triazole formulation did not induce the SCE frequency increase either during 24 h nor 48 h treatment period suggesting that the fungicide probably doesn't belong to genotoxic agents describing as capable of inducing DNA damage that interferes with DNA replication. [32] It is known that G1 lymphocytes are supposed to exert a high capacity for repair or selective elimination of heavily damaged cells; thus Moretti et al., [33] who tested in vitro genotoxic potential of herbicide terbutryn by SCE assay, concluded that probably only a limited fraction of the induced primary DNA damage lead to fixed mutations, such as those quantified by determining SCE in metaphases after 24 h assay. On the other hand, the decreased PI values indicated that the fungicide tested in our experiment induced differences in cell proliferation kinetics: cytotoxicity and/or cell cycle delay was seen following the 24 h treatment. ...
To date, most data about the possible genotoxic effect of triazole pesticides are focused on laboratory animals resulting in limited information on further non-target organisms such as cattle. The objective of the present study was to investigate the effect of triazole (tebuconazole/prothioconazole) fungicide formulation on the induction of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and DNA fragmentation in bovine cultured lymphocytes. Our results showed that the fungicide formulation did not induce significant number of CAs in bovine cells after 24 h treatment. Nevertheless, the dose-dependent reduction of mitotic division was observed, with the strongest effect at 30.0 μg mL(-1) in both donors (P < 0.01 and P < 0.001, respectively). Prolonged 48 h exposure caused the increased level of breaks in treated cultures (3.0-15.0 μg mL(-1); P < 0.05) and significant decrease in mitotic index (MI). The tested fungicide failed to produce any statistical changes in the SCE frequency neither after 24 h nor 48 h treatment. However, the significant decline of the proliferation index (PI) was observed after 24 h indicating the fungicide influence on cell cycle kinetics. Prolonged 48 h exposure caused cytotoxicity reflecting in lower PI value relative to control mainly at the highest fungicide concentrations (30.0 μg mL(-1), P < 0.001). Using painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) only low levels of aneuploidies were detected. Significant increase of polyploidy cells (P < 0.05) was induced by a 3.0 μg mL(-1) dose of the fungicide after 48 h. DNA fragmentation assay didn't reveal the presence of DNA nucleosome ladder in cell cultures at any time (24 h and 48 h) and fungicide concentration.
... The mean lethal toxicity of terbutryn (96hLC50) to be 4 mg l À1 for common carp [19]. Little information is available on the mutagenic and/or genotoxic potential of terbutryn [20,21], and no effect was observed in several reviews [19,22]. ...
... Exposure to insecticides can lead to numerous and diverse side effects throughout the kingdom Animalia: from a decreased lipid and glycogen content [8] and changes in DNA structure [22] through carcinogenic and genotoxic activity [18,24] and ultrastructural abnormalities [21,20,2,3] to reproductive malfunctions and malformations [10]. Moreover, pesticides can cause hereditary malformations both in vertebrates [7] and in invertebrates [4]. ...
We present data on the effects of various concentrations of the organophosphorus insecticide, fenitrothion, on development, reproduction and eggshell structure of Spodoptera exigua. The insecticide did not change the male/female ratio in tested populations. The pesticide-exposed larvae were significantly smaller than the controls and their pupation was delayed. These changes were not proportionally concentration-dependent. After imaginal molting experimental females laid significantly fewer eggs than controls. This effect was the same whether one or both members of a mating pair were exposed to fenitrothion during the larval stage. In the egg stage the insecticide caused serious malformations of the eggshell. The exposure caused other types of clearly abnormal development of eggs: two micropylar regions were noticed. Hence, even a low concentration of fenitrothion in the diet can lead to serious disturbances in reproduction and thus possibly at the population level.
... Their side effects are observed among evolutionarily distinct groups within the animal kingdom. These xenobiotics can change DNA structure (VRHOVAC & ZELJEZIC 2002, MORETTI et al. 2002), generate reactive oxygen species (BAGCHI et al. 1995, WRIGHT et al. 2000) and act as inducers of heat stress proteins (SACHANA et al. 2001, YOSHIMI et al. 2002). The carcinogenic and genotoxic activity of some pesticides has been proven (KEVEKORDES et al. 1996, BLASIAK et al. 1999, SAFI 2002). ...
According to our previous observations, larvae of the moth Spodoptera exigua can develop resistance to the phenyl organothiophosphate insecticide fenitrothion, and survive long-term exposure to it. Therefore we examined the effect of similar fenitrothion doses on cell structure in S. exigua cell line UCR-Se-1. The treatment resulted in several kinds of ultrastructural changes in these cells. The most prominent were malformations in mitochondrial and nuclear structure: swelling of the nuclear envelope and extension of perinuclear space, improper organization of genetic material (enhanced ratio of condensed chromatin), destruction of mitochondria (loss of cristae), and presence of electron-lucent spaces within the matrix. The cytoplasm was heterogeneous, with electron-lucent vesicles. The observed malformations became more evident with prolonged exposure to fenitrothion. Finally, disintegration of several cells was noted. We suggest that fenitrothion may act directly on cell organelles, apart from its known blocking effect on synaptic transmission.
... It is becoming an important tool for evaluating the genotoxic potential of compounds in vitro and in vivo. 4,[9][10][11][12][13] Under alkaline conditions (pH > 13), the assay is able to evaluate DNA damage and other lesions, such as alkali-labile sites, DNA-protein and DNA-DNA cross-links 14,15 and incomplete excision repair events. 16 The genotoxic effect caused by pesticides could be evaluated using a very small sample of cells. ...
The present study investigated the genotoxic effects of flumorph in various organs (brain, liver, spleen, kidney and sperm) of mice. The DNA damage, measured as comet tail length (µm), was determined using the alkaline comet assay. The comet assay is a sensitive assay for the detection of genotoxicity caused by flumorph using mice as a model. Statistically significant increases in comet assay for both dose-dependent and duration-dependent DNA damage were observed in all the organs assessed. The organs exhibited the maximum DNA damage in 96 h at 54 mg/kg body weight. Brain showed maximum DNA damage followed by spleen > kidney > liver > sperm. Our data demonstrated that flumorph had induced systemic genotoxicity in mammals as it caused DNA damage in all tested vital organs, especially in brain and spleen.
... Imaging was performed by using a specialized analysis system ( ' Comet Assay III ' , Perceptive Instruments, Ltd, Haverhill, Suff olk, UK). Computerized imaging, described in detail elsewhere (Villarini et al. 2000, Moretti et al. 2002, was performed on coded slides and the percent of DNA in the comet tail (i.e., tail intensity) was used to measure the level of DNA damage. ...
Purpose:
To determine whether a dose-response relationship exists among exposure to extremely low frequency magnetic fields (ELF-MF) at different densities and 70-kDa heat shock protein (hsp70) expression and DNA damage in mouse brain.
Materials and methods:
Male CD1 mice were exposed to ELF-MF (50 Hz; 0.1, 0.2, 1 or 2 mT) for 7 days (15 h/day) and sacrificed either at the end of exposure or after 24 h. Hsp70 expression was determined in cerebral cortex-striatum, hippocampus and cerebellum by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Primary DNA damage was evaluated in the same tissues by comet assay. Sham-exposed mice were used as controls.
Results:
No changes in both hsp70 mRNA and corresponding protein occurred following exposure to ELF-MF, except for a weak increase in the mRNA in hippocampus of exposed mice to 0.1 mT ELF-MF. Only mice exposed to 1 or 2 mT and sacrificed immediately after exposure presented DNA strand breaks higher than controls in all the cerebral areas; such DNA breakage reverted to baseline in the mice sacrificed 24 h after exposure.
Conclusions:
These data show that high density ELF-MF only induce reversible brain DNA damage while they do not affect hsp70 expression.
... luble, it can easily accumulate in biological membranes, and change their structure and fluidity (Domenech et. al. 1977;Antunes-Madeira & Madeira 1979). Within the exposed groups DNA was more con-densed and the heterochromatin patches were less regular and larger. Organophosphate insecticides can change DNA structure (Garaj Vrhovac & Zeljezic 2002;Moretti et. al. 2002). The nuclei of cells exposed to the highest concentrations show symptoms of advanced disintegration: deep invaginations and large heterochromatin patches. Consequently, the malfunctioning of follicle cells must have led to crackings within the lamellar layer, as well as under-development of the columnar layer. Secondly, we also observed ...
The effect of four sublethal concentrations (from 0.01165 to 0.3025 µg per insect, responding to LC10/10, LC10, LC30 and LC50) of the organophosphorus insecticide, fenitrothion, on Spodoptera exigua (Lepidoptera, Noctuidae) larval development and ultrastructure of follicular cells of adult female moths was tested. The
most prominent malformations were: breaks in the cuticle, increased mortality during larval/pupal molting, alterations of
nuclear envelope, changed heterochromatin pattern and abnormalities in mitochondrial ultrastructure. Some of the alterations
are dose-dependent, but the other ones, like organization of rough endoplasmic reticulum (RER), show non-linear response:
cells exposed to lower concentrations (LC10/10 and LC10) of the insecticide possessed clusters of accumulated RER, which were absent within the cells exposed to higher ones (LC30 and LC50). Formation of the eggshell was also markedly postponed within the groups exposed to lower doses. These findings prove that
even minute amounts of xenobiotics may cause significant changes within exposed populations.
Terbutryn (2-(ethylamino)-4-(tert-butylamino)-6-(methylthio)-1,3,5-triazine) is a substituted symmetrical triazine herbicide used in agricultural fields to prevent undesired vegetation growth by inhibiting photosynthesis in target weeds. Although terbutryn has various benefits, long-term exposure, misuse, or abuse of terbutryn may cause non-target toxicity and severe ecosystem pollution. To provide a detailed description of the embryonic developmental toxicity of terbutryn, zebrafish (Danio rerio) were exposed to 2, 4, and 6 mg/L of terbutryn and the morphological changes, pathological abnormalities, and developmental endpoints were assessed relative to that of a solvent control. The results showed that terbutryn induces a loss of survivability, reduction in body and eye size, and edema in the yolk sac. Through fluorescence microscopy, blood vessels, motor neurons, and liver development were investigated using transgenic zebrafish models based on fluorescently tagged genes (fllk1:eGFP, olig2:dsRed, and L-fabp:dsRed). Furthermore, cell death by apoptosis in zebrafish caused by terbutryn exposure was evaluated via acridine orange staining, which is a selective fluorescent staining agent. To support the preceding results, gene expression alterations caused by terbutryn exposure in zebrafish larvae were assessed. The overall results indicate that exposure to terbutryn induces apoptosis and disrupts organ development. These embryonic developmental toxicity results suggest that terbutryn should be applied in the right areas at the appropriate rates, concentrations, and quantities.
Amorphous silica nanoparticles (SNPs) are widely used in biomedical applications and consumer products. Little is known, however, about their genotoxicity and potential to induce gene expression regulation. Despite recent efforts to study the underlying mechanisms of genotoxicity of SNPs, inconsistent results create a challenge. A variety of factors determine particle–cell interactions and underlying mechanisms. Further, high-throughput studies are required to carefully assess the impact of silica nanoparticle physicochemical properties on induction of genotoxic response in different cell lines and animal models. In this article, we review the strategies available for evaluation of genotoxicity of nanoparticles (NPs), survey current status of silica nanoparticle gene alteration and genotoxicity, discuss particle-mediated inflammation as a contributing factor to genotoxicity, identify existing gaps and suggest future directions for this research.
Terbuthylazine is a selective pre- and post-emergency chloro-triazine herbicide used for a broad spectrum of weed control. We evaluated the potential of low doses of terbuthylazine to induce oxidative stress and cytogenetic damage in peripheral blood samples of adult male Wistar rats. Following 28-day repeated oral exposure at 0.004 mg/kg b.w./day, 0.4 mg/kg b.w./day and 2.29 mg/kg b.w./day, parameters of lipid peroxidation, total antioxidant capacity, and activities of antioxidant enzymes were measured in blood samples. Alkaline comet assay on leukocytes and erythrocyte micronucleus assay were used to measure DNA damage. In addition, the concentration of terbuthylazine and its metabolite in urine and plasma were determined using high performance liquid chromatography with UV diode-array detector (HPLC-UV-DAD). The fraction of terbuthylazine excreted in urine was negligible and was not found in plasma. Deethylterbuthylazine was only compound detected in plasma samples. Exposure to terbuthylazine did not induce significant lipid peroxidation products. The significant changes in antioxidant enzyme activities and the elevated total antioxidant capacity indicated that terbuthylazine at experimental conditions applied has potential to disturb oxidative/antioxidant balance. Results regarding the alkaline comet assay as well as micronucleated reticulocyte frequency indicated that treatment led to low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.
Punjab being the major state of India that utilizes pesticides comprises different populations, which are directly or indirectly exposed to the pesticides, mainly the pesticides manufacturers, formulators and distributors. Amongst these pesticide distributors are least affected considered despite their continuous exposure to them. The present study was undertaken to investigate the occurrence of genotoxic effects in pesticide distributors from three cities in Punjab. The methods used included Comet Assay to assess DNA damage. The results showed that a significant increase in the frequency of DNA damage was found. An increasing trend in genetic damage was observed between the workers with increasing years of exposure as revealed by an ANOVA test. Similarly, the effect of other confounding factors such as age, diet and alcohol drinking habits were also studied. Conclusively, the use of protective measures and other safety regulations is emphasized among the pesticide distributors to prevent further exposure to this group.
Organophosphate (OP) pesticides are the most widely used synthetic chemicals purposefully added in the environment for agricultural and household pest control. Chlorpyrifos (CPF), methyl parathion (MPT) and malathion (MLT) are among the most widely used OP pesticides. The aim of present study is to determine the genotoxicity of these commonly used OP pesticides individually and in mixture, in in vitro system, using isolated lymphocytes from peripheral blood of healthy Wistar rats. Genotoxicity was estimated by measuring DNA single strand breaks and double strand breaks by single cell gel electrophoresis (SCGE). To find out whether DNA lesions were caused due to oxidative stress, combination of bacterial DNA repair enzymes namely formamidoaminopyrimidine glycosylase (Fpg) and Endonuclese (Endo III) which convert base damage to breaks are used. Significant increase in DNA strand breaks measured by increase in tail length, % tail DNA and tail moment, were observed in rat lymphocytes treated with pesticides in modified comet assay. The levels of reactive oxygen species (ROS) namely, superoxide anion and hydrogen peroxide, were also increased in rat lymphocytes on treatment with CPF, MPT and MLT individually and in combination for different period of time, which further shows the involvement of oxidative stress in pesticide induced DNA damage. MPT exposure caused maximum DNA damage followed by CPF and MLT which has also correlated with ROS production. The results of the study show genotoxic potential of these selected OP pesticides.
The aim of the present study was to investigate the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC501; DNA damage was induced by one representative food mutagen, 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP). Mice were treated orally with suspensions of lactobacilli for 10 days before administration of food mutagen. During the treatment, the abundance of lactobacilli in feces, as assessed by qPCR analysis, increased, while β-glucuronidase and N-acetyl-β-glucosaminidase activities decreased. The extent of DNA damage was measured in colon and liver cells by comet assay. In colonocytes, diet supplementation with IMC501 resulted in a significant inhibition of DNA damage induced by PhIP. The results obtained in this in vitro study suggest that Lactobacillus rhamnosus IMC501 used as a dietary supplement can provide a useful integration of antimutagen food components of the normal diet, which are generally lower than the protective level.
The impact of pesticide movement via overland flow or tile drainage water on the quality of receiving water bodies has been a serious concern in the last decades; thus, for remediation of water contaminated with herbicides, bioreaction systems designed to retain biomass have been proposed. In this context, the aim of this study was to evaluate the atrazine and terbutryn biodegradation capacity of a microbial consortium, immobilized in a biofilm reactor (PBR), packed with fragments of porous volcanic stone. The microbial consortium, constituted by four predominant bacterial strains, was used to degrade a commercial formulation of atrazine and terbutryn in the biofilm reactor, intermittently or continuously operated at volumetric loading rates ranging from 44 to 306 mg L(-1) d(-1). The complete removal of both herbicides was achieved in both systems; however, higher volumetric removal rates were obtained in the continuous system. It was demonstrated that the adjuvants of the commercial formulation of the herbicide significantly enhanced the removal of atrazine and terbutryn.
Henna is extracted from the crushed leaves of Lawsonia inermis Linn. (Lythraceae) and the principal natural dye contained at 1.0-1.4% is 2-hydroxy-1,4-naphthoquinone (lawsone), a red-orange pigment insoluble in water. Henna is also used in the traditional medicine as an antirheumatic and antineuralgic agent. Antimicrobial, antifungine as well as antiviral properties have been reported for Henna. Henna is largely considered to be safe, however it has been reported to cause a wide spectrum of adverse effects, such as allergic contact dermatitis from temporary Henna tattoos; Henna has also been reported to cause urticarial rash, severe angioneurotic edema and hemolytic anemia. The aim of this study was to evaluate the possible genotoxic activity of extracts of L. inermis and a commercial Henna product in an in vitro model. Induced primary DNA damage was evaluated in human liver cells (HepG2 cell line) by comet assay. In order to assess if some processing stages affect genotoxicity, both extracts of leaves of L. inermis and a commercial Henna product were analyzed. L. inermis did not show any genotoxic activity after 4 or 24 h exposure in HepG2 cells in the evaluated dose range. Similarly, Henna did not show any genotoxic activity as well. The results of this study are largely negative in terms of genotoxicity, however further study in animal model is needed to rule out in vivo genotoxicity.
Semipermeable membrane device (SPMD) is a passive sampler that sequesters lipophilic contaminants, mimicking the bioconcentration in the fatty tissue of organisms. This study was designed to assess the use of SPMD and biological tests (Comet assay and Ames test) for air monitoring. For this purpose an occupational environment with expected polycyclic aromatic hydrocarbons (PAHs) contamination (coke plant) was selected for a case study. The SPMDs were deployed in five occupational contaminated sites and in a control site. The SPMD dialysates were chemically analysed and examined for in vitro DNA-damaging activity in human cells (Jurkat) by Comet assay and for mutagenicity with the Ames test (TA98 strain, w/o S9). Total suspended particulates were also collected and analysed (GC–MS). No biological effect of SPMD extract was revealed in the control site. On the other hand, air samples collected with SPMDs within the coke plant showed variable degrees of genotoxic and mutagenic activity. The highest effects were associated with the highest PAH level recovered in the SPMDs extracts and in particulate samples.Results obtained support the sensitivity of biological tests associated to SPMD sampling for evaluating the health risk of potentially contaminated work environments highlighting the usefulness of SPMDs for environmental air quality monitoring.
Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 μM) at 4 °C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.
Sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test in human blood cultures in vitro. Both metals induced DNA damage in white blood cells that was expressed and detected as DNA migration in the SCG assay. Dose dependent effects were seen for cadmium in concentrations from 5 × 10−4-5 × 10−3 M and for arsenic in concentrations from 2 × 10−4-1.5 × 10−3 M. The distribution of DNA migration among cells, a function of dose, revealed that the majority of exposed cells expressed more DNA damage than cells from control cultures and that with increasing length of DNA migration the variability in migration among cells increased as well.
Treatment of cells for 2 hr or 24 hr beginning 48 hr after the start of the blood cultures did not increase the SCE frequency in the case of cadmium but caused a small but significant SCE induction with arsenic at the highest concentration. The metal concentrations which could be investigated in the SCE test were much lower due to a strong toxic effect. Metal concentrations which were toxic in the SCE test were without visible effect in the SCG assay. Thus the two endpoints for the determination of genotoxic effects in vitro differed markedly with respect to the detection of genotoxicity induced by metals. These differences and the biological significance of the findings are discussed.
Occupational exposure to styrene was studied in nine workers of a hand lamination plant in Bohemia, Personal dosimeters were used to monitor the styrene workplace exposure, and the levels of styrene in blood and mandelic acid in urine were measured, Blood samples were taken at four occasions during a 7 month period to determine styrene-specific O-6-guanine DNA adducts in lymphocytes and granulocytes, DNA strand breaks and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutant frequency in T-lymphocytes, Seven administrative employees in the same factory (factory controls) and eight persons in a research laboratory (laboratory controls) were used as referents, DNA adduct levels determined by the P-32-postlabelling method in lymphocytes of laminators were remarkably constant and significantly higher (P < 0.0001) than in factory controls at all four sampling times, HPRT mutant frequencies (MF) measured by the T-cell cloning assay were higher in the laminators (17.5x10(-6), group mean) than in the factory controls (15.7x10(-6), group mean) at three of the four sampling times, but the differences were not statistically significant. However, a statistically significant (P = 0.021) difference between MF in the laminators (18.0X10(-6), group mean) and laboratory controls (11.8X10(-6), group mean) was observed at sampling time 4 (the only sampling time when this latter group was studied), This result indicates that styrene exposure may induce gene mutation in T-cells in vivo. DNA strand breaks were studied by the 'Comet assay' at the fourth sampling time, The laminators were found to have significantly higher levels of DNA strand breaks than the factory controls (P = 0.032 for tail length, TL; P = 0.007 for percentage of DNA in tail, T%; and P = 0.020 for tail moment, TM), A statistically significant correlation was also found between the levels of lymphocyte DNA adducts and all three DNA strand break parameters (TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary, no significant correlations were found between DNA adduct levels and the HPRT mutant frequencies or between the mutant frequencies and DNA strand breaks, Taken together, these results add further support to the genotoxic and possibly mutagenic effects of styrene exposure in vivo. However, no simple quantitative relationship seems to exist between the levels of styrene-induced DNA damage and frequency of HPRT mutation in T-lymphocytes.
A combination of cytological and leukocyte culture techniques is described which constitutes a convenient, reliable approach for chromosome studies of humans and yields the following advantages: 1.1. Relative ease of obtaining blood and small volume required.2.2. Adequate mitotic yield from the short-term culture of leukocytes.3.3. High numbers of “exact count” quality metaphase spreads, permitting a critical analysis of chromosome morphology.
The alkaline single-cell microgel-electrophoresis (comet) assay, allowing the evaluation of damage occurring at the DNA level in individual cells associated with exposure to genotoxic compounds, appears to be a promising tool for human biomonitoring studies. In a molecular epidemiology approach aimed at evaluating the eventual genetic damage induced by occupational exposure to pesticides, peripheral blood leukocytes of 17 male farm workers and of 17 age-matched controls living in the same area were analysed for the presence of DNA damage using the comet assay. In this paper we report results obtained after a re-analysis by a computerized system of photomicrographs previously used for the direct manual measurement of comet length. Exposed subjects (farm workers) showed a statistically significant increase in the number of damaged cells (cells with an abnormal size tail, AST) for the tail length parameter, as compared with controls. We did not observe any statistically significant effect of cigarette smoking on the extent of DNA damage (both exposed and controls) and trends similar to those obtained for the groups in toto were observed when smoking habits was considered. Our results support data reported in the literature indicating that occupational exposure to pesticides may evoke some genotoxic effect and, consequently, confirm the necessity to improve preventive measures and to perform accurate health surveillance of individuals exposed to pesticides.
A recent biochemical technique, the alkaline single‐cell microgel‐electrophoresis ("comet") assay, allows the evaluation of damage occurred at the DNA level in individual cells associated with exposure to genotoxic compounds. The extent of DNA damage may be directly evaluated by the use of simple visual techniques. However, the development of computerized image analysis systems specifically devoted to this assay has made possible an easier and more detailed measure. In a biological monitoring approach aimed to assess the genotoxic hazard following rubber processing, peripheral blood leukocytes of 19 male workers from the rubber industry and of age‐matched controls living in the same area were analyzed for the presence of DNA damage using the “comet”; assay. In this paper we report results obtained after a re‐analysis by a computerized system of photomicrographs previously used for the direct manual measurement of comet length. Considering tail parameters such as tail intensity and tail moment, a statistically significant higher extent of DNA damage was demonstrated in exposed subjects, as compared to controls. On the contrary, visual analysis gave negative results.
A rapid assay for chromosomal damage would greatly speed studies of the mechanism by which chromosomal aberrations are formed. The characteristics of such an assay — micronuclei produced in cultured human lymphocytes — are given here, together with the evidence that the assay accurately measures X-ray-induced chromosomal damage. Micronuclei arise from chromosomal fragments that are not incorporated into daughter nuclei at mitosis because they lack a centromere. In our experiments the response of lymphocytes from different donors was very uniform and agreed well with what was expected from metaphase analysis of aberrations: (1) the increase in micronucleus frequency begins at the time of the first mitoses, 48 hours after the cultures are started, (2) the exponent of the dose response equation (y = kDn) was 1.2 for micronuclei. For one-hit aberrations n = 1 whereas for two-hit aberrations n = 2. Since two-hit aberrations predominate in these cultures, a value of n = ∼1.8 was expected if no increase in mitotic delay or cell death occurred at higher doses, and n < 1.8 if an increase occurred, (3) the frequency of micronuclei was decreased by a factor of about two when the dose was fractionated, as expected when most of the aberrations are two-hit. The rejoining time for four of five donors was between 30 and 60 minutes, (4) the X-ray-induced micronucleus frequency in cells from people with Down's syndrome (trisomy-21) was twice that of control donors as expected from metaphase analysis [22,23]. Since the micronucleus assay reflects the aberration frequencies so well and is so fast, it is suitable for a rapid assessment of chromosomal damage.
An advantage of using freshly isolated intact cells of different organs in toxicology is that they reflect more closely the in vivo situation than do long-term cultures. In vitro, primary cells provide the possibility of determining cell-specific xenobiotic metabolism, in the absence of artificial extracellular activation systems, which may result in cytotoxic and genotoxic effects. After in vivo exposure of animals to xenobiotics, isolated primary cells can be studied to elucidate toxicokinetic effects. In the review presented here, selected methods are described for isolating cells with high viability from pig liver and avian embryonic liver, and from the nasal cavity, lungs, kidneys, gastro-intestinal tract, urinary bladder, testes and thymus of the rat. Two techniques for preparing rat lymphocytes are also described. Cell isolation may be initiated with an in situ perfusion to clear the organ of blood. Steps to loosen cell-to-cell contacts and to digest the intercellular connective material may then follow. Also, in situ digestion may be performed, as described for the epithelial cells from different mucosal tissues. Following initial digestion, a single-cell suspension is prepared by tissue mincing and a second digestive step with proteolytic enzymes. Frequently used digestive enzymes are collagenase (types I, IV and P; from Clostridium histolyticum), trypsin and proteinase K. Follow-up filtration is usually required to remove undigested material. The quantities and viabilities of the harvested cells vary with the organ of choice and the procedure used; the values obtained are stated.
Forty substances were tested for antialgal activity against Chlorella pyrenoidosa (Wis. 2005) and Phormidium inundatum (Wis. 1093). C. pyrenoidosa exhibited greater resistance to adverse effects of test compounds than did P. inundatum. Although several structurally unrelated compounds were inhibitory to both alga species, even at an initial concentration of 1.0 mg/liter, methylthio-s-triazines, ametryne, prometryne, and terbutryne, at 0.1 mg/liter, restricted growth to less than 25% of control (untreated) cultures. The methylthio-s-triazines were virtually chemically unreactive with free iodine. Inhibition of Staphylococcus aureus by free iodine dosages of 0.25, 0.5, 0.75, and 1.0 mg/liter was unaffected by the presence of 2.0 mg of terbutryne per liter.
Sister-chromatid exchanges (SCE), both spontaneous and chemically-induced [bleomycin (BLM), mitomycin-C (MMC), streptonigrin (SN), and 4-nitroquinoline-1-oxide (4NQO)], were studied in the lymphocytes of 24 normal individuals on 2 or 3 different occasions, separated by periods of up to 2 years. For all BLM-induced SCEs, the variation in SCE frequency among the samples from a single individual was significantly greater than the variation between replicate cultures on a given day. These results raise questions concerning the validity of conclusions based on a single observation of chemically-induced SCEs.
The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S-9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.
The induction of micronuclei (MN) by vincristine, mitomycin C and cyclophosphamide was compared in purified lymphocytes and in whole-blood cultures. With both assays, cytokinesis was blocked by cytochalasin B and MN were only scored in binucleate cells. The data suggest that whole-blood cultures may be considered a better experimental condition for the detection of MN induced by chemicals in vitro.
A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the G0 (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sister-chromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in G0 cells and that G1 cells can repair both DEB and MNU induced lesions.
The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.
IF human lymphocytes1 or Chinese hamster2 cells are treated with the base analogue 5-bromodeoxyuridine (BrdU) in the latter part of the S period, Giemsa stained chromosomes exhibit a pattern of condensed and extended segments along their length. This phenomenon has been attributed to a delay in the spiralisation pattern of the late replicating regions along the chromosomes. Other experiments3 with Chinese hamster ovary (CHO) cells have shown that after two rounds of replication in the presence of BrdU or IdU, sister chromatids stain differentially with Giemsa, allowing the identification of the two chromatids, and the observation of sister chromatid exchanges (SCEs) without recourse to autoradiography. The chromatid with the bifilarly substituted DNA (BrdU substituted in both strands of DNA) is less condensed and stains more weakly with Giemsa than the unifilarly substituted sister chromatid. The yield of SCEs is approximately that observed by autoradiography.
The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.
The effects of the synthetic dibromo-pyrethroid insecticide deltamethrin on some hepatic phase I and II enzyme activities were studied in rat liver. The animals were treated with daily doses of 5 and 10 mg/kg of both pure insecticide or its commercial formulation (Decis), administered i.p. in corn oil for 7 days. The following enzyme activities were studied: NADPH-cytochrome-P450 reductase, aryl-hydrocarbon hydroxylase, aminopyrine N-demethylase, glutamyl cysteine synthetase, glutathione S-transferase, glutathione peroxidase, peroxisomal acyl-CoA oxidase, catalase, and urate oxidase. Both deltamethrin and its commercial formulation were effective in modifying the activities of several of these hepatic xenobiotic-metabolizing enzymes. However, some differences in enzyme modifications were found between treatment with pure or commercial deltamethrin, the latter being more active. This effect could be ascribed to additives, solvents, and chemical intermediates present in the Decis formulation. These results suggest that exposure to this deltamethrin commercial formulation could be more dangerous than exposure to deltamethrin alone, both in terms of its hepatotoxicity and/or alterations in the hepatic biotransformation of other occupational/environmental xenobiotics.
The genotoxicity of the herbicides, alachlor, atrazine, maleic hydrazide, paraquat and trifluralin has been evaluated in the single-cell gel electrophoresis (SCGE) assay by using human peripheral blood lymphocytes. All treatments were conducted with and without the presence of an external bioactivation source (S9 mix). The results indicate that all the herbicides tested are able to give positive results by increasing the comet tail length, which would confirm both the genotoxicity of the herbicides and the sensitivity of the assay in front of these chemicals. Alachlor and atrazine give similar results in treatments with and without S9, while when the S9 mix was not used paraquat and trifluralin genotoxicity was higher. On the other hand, although maleic hydrazide genotoxicity was higher when S9 mix was used at normal pH (7.4), our data show that its genotoxicity depends largely on the pH solution, increasing as the pH decreased.
Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.
The alkaline single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to assess and compare the level of DNA damage in peripheral blood leukocytes/lymphocytes from well-nourished children with infection, well-nourished children with infection and under drug treatment, and from malnourished infected children with and without previous drug treatment. The present study shows that severe infection is associated with a significant increase in DNA damage. The average migration length was five times greater in severely infected well-nourished children compared to that found in healthy, well-nourished children. The results obtained in this study indicate that malnutrition is another factor associated with an increase in DNA migration. The average tail length for malnourished, severely infected children was twice as great as that obtained for cells from well-nourished, severely infected children. We also detected a variable increase in DNA migration associated with treatment for severe infection. Nevertheless, the excessive heterogeneity, the concurrent number of drugs used and the limited size of the treated population precludes an accurate assessment of which drugs were involved in the increase in DNA damage. Further studies will be necessary involving a large number of patients to address the relation between levels of DNA damage and specific kinds of infection, various drug treatments, and the type and severity of malnutrition. The increased level of DNA damage in severely infected and malnourished children could be related to negative effects such as a deficient immune response resulting in an increased susceptibility to malignant transformation.
Lymphocytes and granulocytes were separated from human peripheral blood and irradiated with low doses of gamma-rays (0.05-0.5 Gy) from a 137Cs source. The magnitude and intercellular distribution of DNA damage, i.e., single-strand breaks and alkali-labile lesions, were compared with those obtained in unfractionated leukocytes irradiated in whole blood, using the alkaline single-cell gel-electrophoresis technique. Based on the extent of DNA migration, irradiation resulted in a linear and dose-dependent increase in DNA damage in all 3 cell populations, with a significant increase being detected at 0.05 Gy. The dose-dependent increase for DNA migration was not significantly different between separated lymphocytes and granulocytes, but their responses were significantly elevated over that obtained for leukocytes irradiated in whole blood. Based on an analysis of the ratio of the range to the standard deviation for each cell population at each dose of radiation, the distribution of damage among cells was relatively homogeneous and independent of dose and cell population. These results are consistent with a hypothesis that irradiation of leukocytes in whole blood partially protects against radical-induced DNA damage.
The use of a wide range of chemicals to destroy pests and weeds is an important aspect of agricultural practice in both developed and developing countries. Undoubtedly, this has increased crop yield and reduced postharvest losses. However, the expanded use of such pesticides expectedly results in residues in foods, which has led to widespread concern over the potential adverse effects of these chemicals on human health. It is clear that the possibility for exposure to pesticides is greatest among farm workers. Also, it is exceedingly plausible that less controlled and regulated uses of pesticides may offer the greatest opportunity for exposure to toxicologically significant quantities. Very limited epidemiological data are available for evaluation of the health effects of pesticides on humans. Only a small proportion of a population is likely to receive a pesticide dose high enough to cause acute severe effects; however, many more may be at risk of developing chronic effects (such as cancer, adverse reproductive outcome, and immunological effects) depending on the type of pesticide they are exposed to. The pesticides currently in use include a wide variety of chemicals with great differences in their mode of action, uptake by the body, metabolism, elimination from the body, and toxicity to humans. With pesticides that have a highly acute toxicity but are readily metabolized and eliminated, the main hazard lies in acute, short-term exposures. With others that have a lower acute toxicity but show a strong tendency to accumulate in the body, the main hazard is connected with long-term exposure, even to comparatively small doses. Other pesticides that are rapidly eliminated but induce persistent biological effects also present a hazard connected with long-term, low-dose exposures. Adverse effects may be caused not only by the active ingredients and the associated impurities, but also by solvents, carriers, emulsifiers, and other constituents of the formulated product. This review attempts to describe several aspects of the problem.
The herbicide linuron and a mixture of 15 pesticides commonly found in the Italian diet have been assayed for promoting activity in rat liver carcinogenesis. Composition of the pesticide mixture was: benomyl (19.55%); dithiocarbamates (20.67%); thiabendazole (14.94%); diphenylamine (14.25%); chlorthalonil (13.13%); procymidone (7.96%); fenarimol (1.95%); chlorpropham (0.70%); vinchlozolin (0.28%); methidathion (2.37%); chlorpyriphos-ethyl (2.09%); parathionmethyl (1.00%); chlorfenvinphos (0.27%); parathion (0.70%); pyrimiphos-ethyl (0.14%). To determine promoting activity we evaluated induction of preneoplastic foci in diethylnitrosamine-initiated hepatocytes, by positive gammaglutamyl-transpeptidase (GGTase) staining in liver slides, and peroxisome proliferation by peroxisomal-dependent catalase and palmitoyl-CoA-oxidase dosage. For the assay, groups of male Sprague-Dawley rats were initiated with 100 mg/kg diethylnitrosamine intraperitoneally and, one week later, given 150 mg/kg/day linuron or 10 mg/kg/day pesticide mixture, administered by gavage three days a week. All rats were 2/3 hepatectomized at the beginning of the 3rd week. All treatments were terminated at the end of the 8th week, and the rats were sacrificed one week later. No significant increases in number and area (mm2) per slide unit area (cm2) of GGTase-positive foci could be observed in linuron-treated rats (5.84 +/- 1.62/cm2; 0.139 +/- 0.041 mm2/cm2) with respect to controls only initiated with diethylnitrosamine (4.47 +/- 1.30/cm2; 0.182 +/- 0.078 mm2/cm2). After treatment with the pesticide mixture, the number of preneoplastic foci was instead significantly increased (6.91 +/- 2.05/cm2) although the area was not (0.188 +/- 0.128 mm2/cm2). Moreover, no increases in the peroxisome proliferation enzymatic markers were observed in either treated groups. The results imply a possible carcinogenic risk for the population stemming from promoting activities of pesticide mixtures.
The effect of the ureic herbicide Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] on the levels of some hepatic xenobiotic metabolizing enzymes was studied in rats. The cytochrome P450-dependent monooxigenase activities of aryl hydrocarbon hydroxylase (AHH) and of aminopyrine N-demethylase (APD) were measured in rat livers after a 14-d treatment by gavage with Linuron. AHH was employed as a marker of the catalytic activity of P450IA1 and APD as a marker of the catalytic activity of P450IIB1/2. Furthermore, the enzymatic activities of the cytosolic via glutathione detoxifying enzymes glutathione peroxidase and glutathione S-transferase were assessed. Three doses of Linuron (both as pure compound and as commercial preparation) were tested. The doses tested were 150, 300, and 450 mg/kg body weight for the pure compound and 315.8, 631.6, and 947.4 mg/kg for the commercial preparation. Differences were found in the relative liver weight only in rats treated with the commercial formulation. The aryl hydrocarbon hydroxylase activity was increased with all the tested doses of pure and commercial Linuron. A reduction in the aminopyrine N-demethylase activity was noted for the highest dose of pure Linuron, whereas an increment in this activity was observed for all the doses of the commercial preparation tested. The activity of glutathione peroxidase was not affected by treatment with the pure product; however, an increment in activity was observed at all the tested doses of the commercial preparation. The glutathione S-transferase activity was reduced in both cases.
The DNA damaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from C57BL/6 female mice using a gel electrophoresis assay for DNA from single cells ('the single cell gel electrophoresis assay') under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of 750 mg/kg body weight. The cytostatic agent cyclophosphamide (150 mg/kg, i.p.) was used as a reference substance, and vehicle-treated mice as controls. DNA damage was recorded 16 h after the (last) injection, using an automated computerized image analysis system specifically designed for the single cell gel electrophoresis assay. There was evidence of chlorobenzene-induced DNA damage after 3 days of repeated exposure in peripheral lymphocytes, but no indications of such an effect in bone marrow cells. Cyclophosphamide induced significant damage to DNA both in bone marrow cells and lymphocytes, the effect being most pronounced in the latter cells. It is concluded that high-dose exposure to chlorobenzene is associated with genotoxicity to peripheral lymphocytes. However, this solvent is apparently not a major hazard to bone marrow cells, even after repeated high-dose exposure.
Single cell gel electrophoresis under alkaline conditions is a technique used to detect primary DNA damage in individual mammalian cells. Cells embedded in agarose on microscope slides are subjected to lysis, unwinding of DNA and electrophoresis at high pH. After staining with a fluorescent dye, cells with DNA damage display increased migration of genetic material from the cell nucleus. The damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. The torsional moment of the tail ('tail moment') has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. In the present paper it will be shown that the moment of inertia ('tail inertia'), a not previously described tail parameter, provides a more precise description of the distribution of individual DNA fragments within the tails. The tail inertia was also found to be the most sensitive indicator of the DNA damage induced in peripheral lymphocytes from mice given a single intraperitoneal injection of cyclophosphamide (150 mg/kg b.w.). It is concluded that the tail inertia is an important complement to other tail parameters when looking for damage of DNA with the single cell gel electrophoresis assay.
In order to study the metabolic differences between whole blood and isolated lymphocyte cultures, two indirectly acting mutagens cyclophosphamide (CP) and benzo[a]pyrene (B[a]P) were assessed for their potential to induce micronuclei (MN) in the presence and absence of S9 microsomal fractions. In isolated lymphocyte cultures supplemented with S9, CP and B[a]P induced a statistically significant increase in MN which was not observed in whole blood cultures. However, the direct-acting agent methyl methanesulphonate (which was used as a positive control) showed an increase in MN frequency in a dose-dependent manner in both culture methods. The effect of erythrocytes was then investigated by treating isolated lymphocyte cultures simultaneously with CP and S9 mix in the presence of purified erythrocyte concentrate (PEC). A clear reduction in the MN frequency was observed compared to the frequencies of MN induced in isolated lymphocyte cultures treated with CP and S9 mix in the absence of PEC. Thus, isolated lymphocyte cultures may represent a more sensitive test system for the evaluation of potential indirect-acting mutagens. However, whole blood cultures may reflect the 'real life' situation more accurately as a consequence of the presence of erythrocytes.
Sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test in human blood cultures in vitro. Both metals induced DNA damage in white blood cells that was expressed and detected as DNA migration in the SCG assay. Dose dependent effects were seen for cadmium in concentrations from 5 x 10(-4)-5 x 10(-3) M and for arsenic in concentrations from 2 x 10(-4)-1.5 x 10(-3) M. The distribution of DNA migration among cells, a function of dose, revealed that the majority of exposed cells expressed more DNA damage than cells from control cultures and that with increasing length of DNA migration the variability in migration among cells increased as well. Treatment of cells for 2 hr or 24 hr beginning 48 hr after the start of the blood cultures did not increase the SCE frequency in the case of cadmium but caused a small but significant SCE induction with arsenic at the highest concentration. The metal concentrations which could be investigated in the SCE test were much lower due to a strong toxic effect. Metal concentrations which were toxic in the SCE test were without visible effect in the SCG assay. Thus the two endpoints for the determination of genotoxic effects in vitro differed markedly with respect to the detection of genotoxicity induced by metals. These differences and the biological significance of the findings are discussed.
The frequency of sister chromatid exchanges (SCE) and micronuclei in peripheral blood lymphocytes was evaluated in 48 agricultural workers and 50 control subjects living in central Italy. No difference in SCE frequency was found between the control and the exposed populations with respect to age, smoking habits, and duration of exposure, although smokers, both farmers and controls, had a higher SCE frequency than nonsmokers. However, the comparison of proliferative rate index values found in the two groups revealed a significant decrease in the activation capability of lymphocytes in the pesticide-exposed workers, probably related to the toxic properties of chemicals to which the farmers were exposed. On the contrary, the analysis of micronuclei frequency indicated that there were differences between the exposed and control subjects with respect to smoking habits, age, and duration of exposure. Our results indicate that, in the study population occupationally exposed to a complex mixture, including insecticides, fungicides, and herbicides, there is clear, although slight, evidence of clastogenic activity in peripheral blood lymphocytes but no corresponding effects on SCE induction. Moreover, our data show clear evidence of cell proliferation delay relatable to chemical compounds used in agriculture.
The ureic herbicide linuron [3-(3, 4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and D-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: 'comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and D-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel-electrophoresis technique ('comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.
Deltamethrin (CAS registry No. 52918-63-5), a synthetic dibromo-pyrethroid insecticide is highly effective against a broad spectrum of insects, and is widely used on crops and in public health programs. Data on the genotoxicity and carcinogenicity of deltamethrin are rather controversial, depending on the genetic system or the assay used. The aim of the present study was to analyze previously demonstrated metabolic changes using aspecific noninvasive methods in rats which are potentially applicable for monitoring occupational exposure. Since human exposure to pesticides occurs not only to active principles but to all chemicals present in a commercial formulation, we tested both the pure compound and a deltamethrin-based commercial formulation. Groups of rats were treated, i.p., consecutively for 7 days. The daily doses tested were 5 and 10 mg/kg body weight for pure deltamethrin, corresponding to volumes of 178.57 and 377.14 microliter/kg body weight for the commercial formulation (containing 2.8% deltamethrin). Urine was analyzed for mutagenic metabolites, thioethers, and D-glucaric acid content. Faeces extracts were tested for mutagenicity. Results show that DGA urinary excretion values did not mirror the phase I enzyme induction capability of the insecticide. Results obtained for urinary thioethers do not agree completely with those obtained on the influence of deltamethrin on glutathione S-transferase activity in rat liver. In fact, after administration of the deltamethrin commercial formulation, highest thioether excretion values were obtained during the treatment time for treated animals, as compared to controls. The mean values (+/-SEM) of thioether excretion were 0. 033 +/- 0.002 micromole -SH/24 h for control animals, 0.122 +/- 0. 004 and 0.185 +/- 0.025 for the two treatment groups. Thence, thioether determination in urine samples seems to be a suitable aspecific noninvasive method for assessing exposure to deltamethrin-based formulations, particularly those containing xylene and mesitylene as solvents, as in the tested formulation. Negative or toxic results obtained in the urinary and faecal mutagenicity test seem to exclude the formation and excretion of mutagenic metabolites following treatment with deltamethrin.
Deltamethrin, a synthetic dibromo-pyrethroid insecticide, is extensively used in agriculture, forestry and in household products because of its high activity against a broad spectrum of insect pests (both adults and larvae), its low animal toxicity and its lack of persistence in the environment. Data on the genotoxicity and carcinogenicity of deltamethrin are rather controversial, depending on the genetic system or the assay used. The aim of this study was to further evaluate the potential genotoxic activity of deltamethrin. The in vitro genotoxicity of deltamethrin has been evaluated by assessing the ability of the insecticide to damage DNA (as evaluated using the single-cell microgel-electrophoresis or 'comet' assay) or induce sister-chromatid exchanges (SCE) and micronuclei (MN) in human peripheral blood leukocytes. All treatments were conducted with and without the presence of an external bioactivation source (+/- S9mix). The results indicate that deltamethrin, in the presence of metabolic activation (+ S9mix), is able to induce DNA damage (double- and single-strand breaks, alkali-labile sites and open excision repair sites) as revealed by the increasing tail moment values observed with increasing doses. The frequency of SCE and MN were not statistically increased in deltamethrin-treated cells as compared to controls, both with and without S9mix. However, lower deltamethrin doses were tested, as compared to 'comet' assay, because of cytotoxicity.
Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
The genotoxic effects of X-ray radiation on human lymphocytes were measured using the single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (CBMN) test; both were carried out in vitro on isolated human lymphocytes in order to compare the relationship and sensitivity of these two detecting methods. The radiation-doses were 0.00, 0.02, 0.05, 0.10, 0.25, 0.50, 1.00 and 2.00 Gy. In the comet assay, the average comet length (38.6+/-0.8 microm) of 0.05 Gy was significantly longer than that (29.4+/-1.1 microm) of 0 Gy (P<0.01), moreover, the average comet length increased with the dose of X-ray radiation. In the CBMN, both the average micronucleus rate (MN) and micronucleated cell rate (MNC) of 0.05 Gy were 11.5+/-4.5 per thousand, which showed no difference with that (7.5+/-0.5 per thousand) of 0 Gy (P>0.05). The lowest dose, which induced significant increase of average MN and MNC, was 0.25 Gy. The average MN and MNC rates increased with radiation-dose. The results showed that there was correlation between SCGE and CBMN, and the sensitivity of SCGE was significantly higher than that of CBMN.
Terbutryn, a s-triazine herbicide, is extensively used in agriculture as a selective pre- and postemergence control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Terbutryn was reported to degrade slowly, with half-lives of 240 and 180 days in pond and river sediment, respectively. The tendency of this herbicide to move from treated soils to water compartments through water runoff and leaching was demonstrated and residual amounts of terbutryn and its metabolites have been found in drinking water, and industrial food products, long after application. Although this herbicide may be regarded as a contaminant of our environment, only limited and inconsistent data exist concerning its genotoxic properties. In this study, the DNA-damaging ability of the herbicide was evaluated in the alkaline single-cell microgel-electrophoresis ("comet") assay by testing terbutryn in the presence of S9mix (rat liver homogenate containing microsomal enzymes plus cofactors) prepared with liver homogenate from both uninduced (basal) and aroclor 1254-induced rats. DNA damage was recorded in freshly isolated human peripheral blood leukocytes. A statistically significant increase in the extent of primary DNA damage, more pronounced in the absence of S9mix, took place only when terbutryn concentrations were high (100 and 150 microg/ml), in the presence of a concomitant mild cytotoxic effect.
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