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Imazethapyr is an herbicide that is used in a variety of crops worldwide, including soybean and corn. The aim of the present study was to evaluate the biomarkers responses of adult Leptodactylus latinasus exposed to the formulation Pivot® H (10.59% imazethapyr) in the laboratory at concentrations and under conditions that simulate two potential field exposure scenarios: an immersion in field runoff (Scenario 1: 10 mg/L) and a direct exposure to the droplets emitted by spray noozles (Scenario 2: 1000 mg/L). In both scenarios, the experimental procedure involved completely immersing the frogs over a period of 15 s. Different endpoints were evaluated at several ecotoxicological levels 48 and 96 h after the herbicide exposure. These included individual (biometric indices and behavior alterations), histological (liver pigments and lesions), biochemical (catalase, glutathione system and cholinesterase activities) and genotoxic effects (micronuclei induction and nuclear abnormalities). Forty-eight hours after imazethapyr exposure, frogs submitted to Scenario 1 presented an inhibition of liver glutathione-S-transferase activity, whereas histological alterations and increased hepatic cholinesterase levels were observed in frogs exposed under Scenario 2. Ninety-six hours after exposure to the imazethapyr formulation, frogs from the Scenario 1 treatment presented a decrease in liver melanin and hemosiderin, increased hepatic catalase activity and micronuclei induction. For their part, frogs exposed to Scenario 2 presented a decrease in the hepatosomatic index, an increase in liver alterations, melanin reduction and micronuclei induction. The multivariate analysis enables correlations to be made between biomarkers of different organizational level in exposed anurans. Our result indicates that real exposure to imazethapyr formulations under field conditions may pose a risk to Leptodactylus latinasus populations living in the agroecosystems., Facultad de Ciencias Naturales y Museo

Acute genotoxicity of commercial glyphosate (GLY) (Credit®)-, 2,4-D-acid (2,4-D) (Dedalo Elite)-, 2,4-D-amine (2,4-D DMA) (Weedar Full®)- and 2,4-D-ester (2,4-D BE) (Herbifen Super®)-based herbicide formulations alone and their combinations were analysed in Cnesterodon decemmaculatus. Mortality was evaluated as a lethal end-point and the single cell gel electrophoresis (SCGE) bioassay was used as a sublethal end-point. LC50₉₆ₕ values for Dedalo Elite was 0.46 mg/L and Herbifen Super® was 2.67 mg/L based on 2,4-D and 2,4-D BE, respectively. Results reveal a higher toxicity exerted on C. decemmaculatus after exposure to 2,4-D- rather than 2,4-D BE-based herbicide formulations. Overall, results demonstrated an enhancement in the genetic damage index committed to an enhancement of damaged erythrocytes of C. decemmaculatus when exposed to Credit®, Dedalo Elite, Weedar Full® and Herbifen Super® at 5% and 10% of LC50₉₆ₕ values alone as well as in their combinations. Overall, the combination of GLY plus 2,4-D or GLY plus 2,4-D DMA showed a synergistic pattern whereas the combination of GLY plus 2,4-D BE was antagonic. Furthermore, this research is pioneer in the assessment of lethality and genotoxicity induced by 2,4-D-, 2,4-D DMA- and 2,4-D BE-based formulations when combined with GLY-based formulated herbicides in fish after they are acutely exposed., Facultad de Ciencias Naturales y Museo

We analyzed the acute toxicity of the 48% glyphosate (GLY)-based Credit®, the 57.71% dicamba (DIC)-based Kamba®, and the 83.5% 2,4-dichlorophenoxyacetic acid (2,4-D)-based Weedar® Full, alone and as mixtures on the fish Cnesterodon decemmaculatus. Mortality revealed the LC50 96h values of 91.73 mg L−1 (range: 86.80-98.00 mg L−1), 1401.57 mg L−1 (range: 1243.78-1527.35) and 678.04 mg L−1 (range: 639.35-718.04 mg L−1) for GLY, DIC and 2,4-D, respectively. Mean values for the toxic unit (TU) that induced 50% mortality (TU50 96h) of fish exposed to equitoxic mixtures were 1.67 (range: 1.65-1.69) for Credit® and Kamba® and 1.28 (range: 1.20-1.36) for Credit® and Weedar® Full suggesting that both mixtures are antagonic. Non-equitoxic combinations demonstrated an antagonistic interaction of herbicides Credit® and Kamba®, whereas a synergistic effect was observed for Credit® and Weedar® Full formulations. GLY and DIC as a mixture demonstrated lower toxicity on non-target species compared to GLY and 2,4-D in combination, at least for C. decemmaculatus, leading to the conclusion that the former combination could be strongly recommended in further agricultural practices. Equitoxic and non-equitoxic mixtures of glyphosate with auxinic herbicides evidence opposite effects in C. decemmaculatus. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) are two of the most applied auxinic herbicides worldwide, both individually and as part of a mixture. However, the toxicity and interactions achieved when applied as a mixture have not yet been characterised. The equitoxic and non-equitoxic acute toxicity exerted by binary mixtures of Banvel® (57.71% DIC) and DMA® (58.4% 2,4-D) on the Neotropical fish Cnesterodon decemmaculatus were evaluated. Results revealed mean values of 1.02 (range, 0.96–1.08) for the toxic unit (TU) that induced 50% mortality (TU50 96 h) to the fish exposed to binary equitoxic mixtures of the commercial formulations Banvel®–DMA®. These results suggest that the mixture is nearly concentration additive. Furthermore, results demonstrated the occurrence of synergistic interaction when non-equitoxic combinations of Banvel®-or DMA®-formulated herbicides were assayed. In this context and regardless of their concentrations, either Banvel®- or DMA®-induced toxicity were synergised by the presence of the counterpart within mixtures. The present study represents the first evidence of the lethality exerted by mixtures of two auxinic herbicides—namely, DIC and 2,4-D—reported to date for fish and other biotic matrices. When C. decemmaculatus is used as the target organism, a synergistic pattern is observed following exposure to a mixture of both herbicides. Synergic effects of equitoxic and non-equitoxic mixtures of auxinic herbicides in C. decemmaculatus. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Glyphosate (GLY)-dicamba (DIC) and GLY-flurochloridone (FLC) are herbicide mixtures which are widely used for treating fallow containing glyphosate resistant weeds. The aim of this study was to evaluate the acute toxic effects and the prevailing interactions on stage 36 tadpoles of the anuran species Rhinella arenarum when exposed to equitoxic and non-equitoxic combinations of these herbicide combinations. Experiments were realized using the following combinations of commercial formulations: 48% GLY-based Credit® + 57.71% DIC-based Banvel® and 48% GLY-based Credit® + 25% FLC-based Twin Pack Gold®. GLY-DIC and GLY-FLC equitoxic mixtures were assayed mixing each constituent with an equivalent individual toxicity able to induce the same lethality effect. After 96 h of exposure, GLY-DIC and GLY-FLC equitoxic mixtures presented toxic unit 50 values (TU50 96h) of 1.74 (confidence interval: 1.58–1.92) and 1.54 (confidence interval: 1.46–1.62) respectively, indicating the presence of a weak antagonistic interaction as TU values were greater than 1. For their part, most non-equitoxic combinations of GLY-DIC and GLY-FLC tested did not significantly differ from additivity, the only exception being when DIC and FLC were fixed at 0.33 TUs, where a weak antagonism was observed. Overall, results indicate that the toxicity of both GLY-DIC and GLY-FLC mixtures to R. arenarum tadpoles vary from additive to slightly antagonistic, depending on the proportion of constituting herbicide formulations present in the mixture.

Pesticides might increase the production of reactive oxygen species (ROS). Dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) are auxinic herbicides commonly applied in agroecosystems to control unwanted weeds. We analysed the oxidative damage exerted on the fish Cnesterodon decemmaculatus by an acute exposure to DIC- and 2,4-D-based herbicides formulations Banvel® and DMA®, respectively. The Endo III- and Fpg-modified alkaline comet assay was employed for detecting DNA damage caused by oxidative stress, whereas enzymatic and non-enzymatic biomarkers such as the activities of catalase (CAT), glutathione-S-transferase (GST), acetylcholinesterase (AChE), and glutathione content (GSH) were used to assess antioxidant response to these two herbicides. At the DNA level, results demonstrate that both auxinic herbicides induce oxidative damage at purines level. An increase on CAT and GST activities were detected in 48 h- and 96 h-treated specimens with both auxinics. GSH content decreased in fish exposed to DIC during 48 h and to 2,4-D after 96 h of exposure. Additionally, a diminished AChE activity in specimens treated with DIC and 2,4-D was observed only after 96 h. Total protein content decreased in fish exposed to both auxinics during 96 h. These results represent the first evaluation of oxidative damage related to DIC and 2,4-D exposure on a fish species as the Neotropical freshwater teleost C. decemmaculatus., Facultad de Ciencias Naturales y Museo

Glyphosate (GLY) and imazethapyr (IMZT) are two herbicides commonly used worldwide, either alone or in mixtures. They represent key pesticides in modern agricultural management. The toxicity that results when employed as mixtures has not been characterized so far. Acute toxicity of the 48% GLY-based herbicide (GBH) Credit® and the 10.59% IMZT-based herbicide (IBH) Pivot® H alone and their binary combinations was analyzed in Rhinella arenarum tadpoles exposed in a semi-static renewal test. Lethal effects were determined using mortality as the end-point, whereas sublethal effects were determined employing the single-cell gel electrophoresis (SCGE) bioassay. Based on mortality experiments, results revealed LC5096 h values of 78.18 mg/L GBH and 0.99 mg/L IBH for Credit® and Pivot® H, respectively. An increase in the genetic damage index (GDI) was found after exposure to Credit® or Pivot® H at 5 and 10% of LC5096 h values. The combinations of 5% Credit®-5% Pivot® H LC5096 h and 10% Credit®-10% Pivot® H LC5096 h concentrations significantly enhanced the GDI in comparison with tadpoles exposed only to Credit® or Pivot® H. Thus, the effect of interaction between GBH and IBH inducing DNA damage in R. arenarum blood cells can be considered to be synergistic., Facultad de Ciencias Naturales y Museo

Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold® and Rainbow® were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold® or Rainbow® induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold®-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold®-, or Rainbow®-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold® exposure are due to the excipients contained within the technical formulation rather than FLC itself. Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

The organophosphorus herbicide glyphosate (GLY) and the organophosphate insecticide chlorpyriphos (CPF) are key pesticides in modern management cultures worldwide. Sublethal toxicity of the commercial herbicide formulation Roundup® and the insecticide formulation Terfos® were evaluated on Eisenia fetida coelomocytes exposed under in vivo and ex vivo laboratory conditions. Induction of DNA single-strand breaks evaluated by the single cell gel electrophoresis assay and coelomocyte viability as well as alterations in coelomocyte trophic indexes were employed as endpoints for genotoxicity and cytotoxicity, respectively. Specimens were exposed at concentrations corresponding to recommended pesticide field application rate, and endpoints were evaluated after 7 and 14 days of treatment (in vivo exposure). In addition, coelomocytes were exposed to aqueous leachate of pesticide-contaminated soils during 1 h (ex vivo exposure). Earthworms exposed to Roundup® and Terfos® showed an increased frequency of DNA damage. Also, a decrease of coelomocyte viability and decrease of trophic indexes were observed in all treatments. The results demonstrate that either GLY- and CPF-based formulations exerted genotoxic as well as cytotoxic effects in coelomocytes of E. fetida exposed in vivo and ex vivo. Fil: Curieses, Silvana Patricia. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Programa de Investigación en Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sáenz, María Elena. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Programa de Investigación en Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alberdi, José Luis. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Programa de Investigación en Ecotoxicología; Argentina Fil: Martinez, Ricardo Santiago. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Programa de Investigación en Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Di Marzio, Walter Dario. Universidad Nacional de Luján. Departamento de Ciencias Básicas. Programa de Investigación en Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Long-term genotoxic effects of two auxinic herbicide formulations, namely, the 58.4% 2,4-dichlorophenoxyacetic acid (2,4-D)-based DMA® and the 57.7% dicamba (DIC)-based Banvel® were evaluated on Cnesterodon decemmaculatus. Primary DNA lesions were analyzed by the single-cell gel electrophoresis methodology. Two sublethal concentrations were tested for each herbicide corresponding to 2.5% and 5% of the LC5096h values. Accordingly, fish were exposed to 25.2 and 50.4 mg/L or 41 and 82 mg/L for 2,4-D and DIC, respectively. Fish were continuously exposed for 28 days with replacement of test solutions every 3 days. Genotoxicity was evaluated in ten individuals from each experimental point at the beginning of the exposure period (0 day) and at 7, 14, 21 and 28 days thereafter. Results demonstrated for first time that 2,4-D-based formulation DMA® induced primary DNA strand breaks after 7–28 days exposure on C. decemmaculatus regardless its concentration. On the other hand, DIC-based formulation Banvel® exerted its genotoxic effect after exposure during 7–14 days and 7 days of 2.5 and 5% LC5096h, respectively. The present study represents the first evidence of primary DNA lesions induced by two widely employed auxinic herbicides on C. decemmaculatus, namely 2,4-D and DIC, following long-term exposure. 2,4-D and dicamba exert primary DNA lesions in blood cells of C. decemmaculatus after long-term exposure. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Facultad de Ciencias Naturales y Museo, Universidad Nacional de la Plata; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Facultad de Ciencias Naturales y Museo, Universidad Nacional de la Plata; Argentina Fil: Soloneski, Sonia Maria Elsa. Facultad de Ciencias Naturales y Museo, Universidad Nacional de la Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

In the present study, the damage recovery capabilities of Boana pulchella tadpoles after acute exposure (96 h) to 0.39 mg/L concentration of the imazethapyr (IMZT)-based herbicide formulation Pivot® H (25% IMZT LC50 value) were assessed during a period of 7 to −21 days. To appraise the recovery capabilities, frequency of micronuclei (MNs), other nuclear abnormalities and DNA single-strand breaks evaluated by single cell gel electrophoresis assay on circulating blood cells were employed as endpoints for genotoxicity. Growth, development, body mass, and morphological abnormalities were also employed as individual endpoints in the recovery assay. Results demonstrated that IMZT induced sublethal effects at both the individual (i.e., loss of keratodonts) and cytogenetic levels (e.g., increase of MN frequency, other nuclear abnormalities and DNA single-strand breaks). At 11 days of the exposure phase, tadpoles recovered their basal levels of frequency of MNs, other nuclear abnormalities, and comets. However, loss of keratodonts, observed at the end of the exposure period, was present up to 21 days thereafter. Finally, axial abnormalities and delay in development stage were observed only during the postexposure phase in IMZT-exposed tadpoles at 18 and 25 days, respectively and were observed until the end of the experiment. This is the first evidence of use the comet assay as cytogenetic biomarker of genotoxicity in evaluating the recovery capabilities of amphibians in general and also those of B. pulchella after exposure to IMZT., Facultad de Ciencias Naturales y Museo, Facultad de Ciencias Exactas, Centro de Investigaciones del Medioambiente

Acute lethal and sublethal toxicity of the pirimicarb-based commercial formulation Aficida® were evaluated on Boana pulchella tadpoles. Whereas mortality was used as end point for lethality, frequency of micronuclei and other nuclear abnormalities as well as alterations in the frequency of erythroblasts in circulating blood as biomarkers for genotoxicity and cytotoxicity, respectively. Swimming, growth, developmental and morphological abnormalities were also employed as sublethal end points. Results show that the species is within the 13th percentile of the distribution of acute sensitivity of species to pirimicarb for aquatic vertebrates. Results revealed values of 23.78 and 101.45 mg/L pirimicarb as LC5096 h for GS25 and GS36 tadpoles, respectively. The most evident effects were related with the swimming activity with NOEC and LOEC values within the 0.005–0.39 mg/L pirimicarb concentration range. Aficida® induced DNA damage at the chromosomal level by increasing micronuclei frequency and other nuclear abnormalities, i.e., lobbed and notched nuclei and binucleated cells. Cellular cytotoxicity was found after Aficida® treatment. The presence of abdominal oedemas in exposed organisms and thus flotation response of organisms could be proposed as a new sensitive exposure parameter. The multiple end point assessment approach used allowed a complete understanding the multi level of effects occurring by exposure to pirimicarb, at least in B. pulchella. Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina Fil: Vera Candioti, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Ronco, Alicia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina

We evaluated the role of oxidative stress in the genotoxic damage induced by imazethapyr (IMZT) and its formulation Pivot® in mammalian CHO-K1 cell line. Using the alkaline comet assay, we observed that a concentration of 0.1 μg/mL of IMZT or Pivot® was able to induce DNA damage by increasing the frequency of damaged nucleoids. To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which convert base damage to strand breaks, were used. Our results demonstrate that after treatment of CHO-K1 cells with the pure active ingredient as well as the commercial formulation Pivot®, an increase in DNA strand breaks was observed after incubation of both Endo III and Fpg enzymes, indicating that both compounds induce DNA damage involving both pyrimidine and purine-based oxidations, at least in CHO-K1 cells. Our findings confirm the genotoxic potential of IMZT and suggest that this herbicide formulation must be employed with great caution, especially not only for exposed occupational workers but also for other living species., Facultad de Ciencias Naturales y Museo

Acute toxicity and genotoxicity of the dicamba-based commercial herbicide formulation Banvel® were evaluated on Cnesterodon decemmaculatus (Pisces, Poeciliidae) exposed under laboratory conditions. A lethal effect was used as the end point for mortality, whereas frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Mortality studies revealed an LC50 96 h value of 1639 mg/L (range, 1471–1808) of dicamba. Furthermore, behavioral changes, e.g., gathering at the bottom of the aquarium, slowness in motion, abnormal swimming, and slow reaction, were observed. Whereas increased frequency of MNs was observed when 1229 mg/L dicamba was assayed for 48 h, no induction of MNs was observed in fish exposed to the herbicide for 96 h, regardless of the concentration of dicamba. Furthermore, other nuclear abnormalities, i.e., binucleated cells and lobed and notched nuclei, were induced in fish exposed for 48 h but not 96 h. Increase in the genetic damage index was observed in those treatments (lasting for both 48 and 96 h) within the 410–1229 mg/L dicamba concentration-range. This study represents the first evidence of acute lethal and sublethal effects exerted by dicamba on a piscine species native to Argentina. The results could indicate that dicamba-based formulation Banvel® is the less toxic emerging pollutant reported so far for C. decemmaculatus. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to this agrochemical. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

The ability of two 48 percent chlorpyrifos-based insecticides (Lorsban* 48E® and CPF Zamba®), two 50 percent pirimicarb-based insecticides (Aficida® and Patton Flow®), and two 48 percent glyphosate-based herbicides (Panzer® and Credit®) to induce DNA single-strand breaks in peripheral blood erythrocytes of Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) exposed under laboratory conditions was evaluated by the single-cell gel electrophoresis (SCGE) assay. In those fish exposed to Lorsban* 48E®, CPF Zamba®, Aficida®, Patton Flow®, Credit®, and Panzer®, a significant increase of the genetic damage was observed for all formulations regardless of the harvesting time. This genotoxic effect was achieved by an enhancement of Type II-IV comets and a concomitant decrease of Type 0-I comets over control values. A regression analysis revealed that the damage varied as a negative function of the exposure time in those Lorsban* 48E®- and Aficida®-treated fish. On the other hand, a positive correlation between damage increase and exposure time was achieved after Patton Flow® and Credit® treatment. Finally, no correlation was observed between increase in the genetic damage and exposure time after treatment with CPF Zamba® or Panzer®. These results highlight that all agrochemicals inflict primary genotoxic damage at the DNA level at sublethal concentrations, regardless of the exposure time of the aquatic organisms under study, at least within a period of 96 h of treatment. Fil: Vera Candioti, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico la Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico la Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina

Fil: Vera Candioti, Josefina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

Mortality, genotoxicity, and cytotoxicity of the 48% glyphosate-based formulations Panzer and Credit ® were evaluated on Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) under laboratory conditions. Induction of micronuclei (MN) and alterations in the erythrocytes:erythroblasts ratio were employed as end points for genotoxicity and cytotoxicity, respectively. For Panzer ® , mean values of 16.70 and 15.68 mg/L were determined for LC 50 at 24 and 96 h, respectively, and these concentrations reached mean values of 98.50 and 91.73 mg/L for Credit ® . LC 50 values decreased as a negative linear function of Panzer ® exposure time within the 0–96 h period, but not for Credit ® . LC 50 values indicated that the fish were more sensitive to Panzer ® than to Credit ® . Both 3.9 and 7.8 mg/L of Panzer ® increased MN frequency at 48 and 96 h of treatment. When fish were exposed to Credit ® , an increased frequency of MN over control values was found after 96 h for all concentrations assayed, but not after 48 h. No cellular cytotoxicity was found after Panzer ® and Credit ® treatment, regardless of both the concentration and the sampling time. Furthermore, our results demonstrated that Panzer ® and Credit ® should be considered as glyphosate-based commercial formulations with genotoxic but not cytotoxic effect properties.

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC50 (96 h) value (0.39 mg/L IMZT) during 48 and 96 h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay., Centro de Investigaciones del Medioambiente, Facultad de Ciencias Naturales y Museo

Amphibians as animal models have long been employed as indicators of environmental quality and to detect toxicity risks of environmental pollutants. The increased attention to the use of non-traditional species of amphibians in ecotoxicology and genotoxicology research lies in their ability not only to reveal the toxic and genotoxic effects of many potential environmental xenobiotics, but also to help researchers understand the behaviour of real ecosystems. This chapter presents an overview of selected research that has led to the use of the common tree frog Hypsiboas pulchellus as a reliable and valid model in in vivo and in situ studies of aquatic pollution.

The acute toxicity of two herbicide formulations, namely, the 57.71 % dicamba (DIC)-based Banvel® and the 48 % glyphosate (GLY)-based Credit®, alone as well as the binary mixture of these herbicides was evaluated on late-stage Rhinella arenarum larvae (stage 36) exposed under laboratory conditions. Mortality was used as an endpoint for determining acute lethal effects, whereas the single-cell gel electrophoresis (SCGE) assay was employed as genotoxic endpoint to study sublethal effects. Lethality studies revealed LC50₉₆ ₕ values of 358.44 and 78.18 mg L⁻¹ DIC and GLY for Banvel® and Credit®, respectively. SCGE assay revealed, after exposure for 96 h to either 5 and 10 % of the Banvel® LC50₉₆ ₕ concentration or 5 and 10 % of the Credit® LC50₉₆ ₕ concentration, an equal significant increase of the genetic damage index (GDI) regardless of the concentration of the herbicide assayed. The binary mixtures of 5 % Banvel® plus 5 % Credit® LC50₉₆ ₕ concentrations and 10 % Banvel® plus 10 % Credit® LC50₉₆ ₕ concentrations induced equivalent significant increases in the GDI in regard to GDI values from late-stage larvae exposed only to Banvel® or Credit®. This study represents the first experimental evidence of acute lethal and sublethal effects exerted by DIC on the species, as well as the induction of primary DNA breaks by this herbicide in amphibians. Finally, a synergistic effect of the mixture of GLY and DIC on the induction of primary DNA breaks on circulating blood cells of R. arenarum late-stage larvae could be demonstrated., Facultad de Ciencias Naturales y Museo

The management and final disposal of industrial wastes are a matter of considerable human concern. The present study evaluates the cyto/genotoxic effects and changes of the coelomic cell formulas exerted by aqueous leachates and solid waste (SW) of two industrial residues using coelomocytes extruded from Eisenia fetida. The assayed wastes corresponded to industrial foundry and cosmetic activities. After 14 days of exposure, we obtained a group of endpoints that reflect the toxicity/genotoxicity, coelomocyte formula and indexes; and the mortality classical value (LC50-14d). Among the variables measured, total coelomocytes formula (eleocytes + amebocytes + granulocytes) appears as a single and easy parameter to assess the toxicity of eluates at short exposure times. We applied a set of assays using earthworms as test organism that would allow evaluating SW as well as its aqueous leachates. It is easy to run trials combining exposures of 1 h to 14 days, which can be integrated into the implementation of the traditional test for evaluating acute toxicity., Facultad de Ciencias Naturales y Museo

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.

The intensification of modern agriculture may impact amphibian populations through habitat loss and the direct and indirect effects of the pesticides upon which it relies. The increased homogeneity of modern agricultural landscapes may be detrimental in times of extreme low and high temperatures associated with climate change, as refuge abundance and habitat connectivity decrease. In this study, we evaluated the impacts of the herbicide glyphosate and subsequent intense drought on the Common Tree Frog, Hypsiboas pulchellus, inhabiting an agricultural landscape. We examined a series of organismic indices (stomach content index, hepatosomatic index, body fat index, gonadosomatic index, condition factor) as well as biomarkers of oxidative stress (hepatic catalase activity and reduced glutathione [GSH] content), exposure to contaminants (hepatic gluthatione-S-transferase activity), and genotoxicity (frequency of micronuclei). No significant differences were observed in the parameters measured when co...

The in vitro effects of flurochloridone (FLC) and its formulations Twin Gold Pack® (25% a.i.) and Rainbow® (25% a.i.) were evaluated on Chinese hamster ovary (CHO-K1) cells by genotoxicity [sister chromatid exchange (SCE)] and cytotoxicity [cell-cycle progression, proliferative rate index (PRI), mitotic index (MI), MTT, and neutral red] end points. Cells were treated for 24. h within the 0.25-15 μg/ml concentration range. FLC and Twin Pack Gold® induced a significant and equivalent increase in SCEs regardless of the concentration. Rainbow®-induced SCEs at concentrations higher than 2.5 μg/ml; however, the increases were always lower than those induced by FLC and Twin Pack Gold®. For all compounds, the PRI decreased as a function of the concentration titrated into cultures. Whereas only the highest FLC and Twin Pack Gold® concentrations induced a significant reduction of the MI, all tested Rainbow® concentrations induced MI inhibition. Overall, the results demonstrated that although all compounds were not able to reduce the lysosomal activity, the mitochondrial activity was diminished when the highest concentrations were employed. These observations represent the first study analyzing the genotoxic and cytotoxic effects exerted by FLC and two formulated products on mammalian cells in vitro, at least on CHO-K1 cells. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Zineb [ethylene bis(dithiocarbamate) zinc] is a widely employed foliar fungicide for agricultural and industrial applications. Allium cepa L. is a reliable model for the assessment of xenobiotic genotoxicity and cytotoxicity. We evaluated the effects of the zineb-containing commercial formulation Azzurro(®) (70% zineb) in cell cycle stages of the meristem root cells of A. cepa. The mitotic index (MI), chromosomal aberrations at anaphase/telophase (CAs), micronuclei (MN), and abnormalities in immunodetected microtubule structures, e.g., preprophasic band (PPB), mitotic spindle (MS), and phragmoplast (Phrag), were used as end-points. Azzurro(®) (1 and 10μg/ml) induced a significant increase in the frequency of CAs (P

The purpose of this study was to further investigate the cytotoxic and genotoxic effects of dicamba and Banvel® employing the cytokinesis-block micronucleus cytome (CBMN-cyt) assay estimated by the analysis of the nuclear division index (NDI), the frequency of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs). Besides, for mechanism of MN induction CREST anti-kinetochore antibody analysis was performed. The activities of both compounds were tested within the range of 50–500 μg/ml on Chinese hamster ovary (CHO-K1) cells. Overall, dicamba and Banvel® produced a NDI dose-dependent decrease but the response was statistically significant only in cultures treated with Banvel® at a 100–500 μg/ml concentration range. A dose-dependent induction of MN was observed after dicamba- and Banvel®-treatments within the 50–400 μg/ml and 50–500 μg/ml concentration-ranges, respectively. Induction of NPBs and NBUDs was significantly enhanced by both test compounds. The NPBs/MN ratio values found for dicamba and Banvel® were 0.04–0.11 and 0.05–0.18, respectively. Results clearly demonstrated that dicamba and Banvel® exerted both cyto- and genotoxic damage on CHO-K1 cells. Furthermore, the CBMN-cyt assay employed confirmed our previous investigations concerning the cellular and DNA damaging capabilities of dicamba and highlights that both clastogenic and aneugenic mechanisms are implicated in the MN induction. Fil: González, N.V.. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Baccharis articulata (Lam.) Persoon, known in South America as “carqueja or carquejilla”, is used as folk medicine infusion for gastrointestinal and liver disease treatments in Southern Brazil, Uruguay, and Argentina. The antimutagenic effect of aqueous extracts from aerial parts of B. articulata against the mutagens 2-aminofluorene (2-AF), 2-nitrofluorene, and sodium azide was evaluated by the Ames test, with both the classic plate and pre-incubation assays. Results demonstrated that the antimutagenic effect of the extract depends not only on the mutagen tested but also on the incubation method used. The antimutagenicity curves for the pro-mutagen 2-AF requiring metabolic activation showed clear differences between TA98 and TA100 bacterial test strains. The presence of chlorogenic acid was detected by high performance liquid chromatography. This finding indicates that the antimutagenic capability of B. articulata could be related to its antioxidative properties.

The acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida ® (50% pirimicarb) were evaluated on Cnesterodon decemmaculatus (Pisces, Poeciliidae) exposed under laboratory conditions. Micronucleus (MN) induction as well as alterations in the erythrocytes:erythroblasts ratios were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) and cyclophosphamide were used as positive controls for the toxicity and geno-cytotoxicity assays, respectively. Mean values of 344.3 and 225.5 mg Aficida ® /L were determined for LC-50 24 h and LC-50 96 h , respectively. In 48 h-exposed fish, a MN increase was found in Aficida ® -treated fish in the 50–157 mg/L concentration range. When fish were exposed to Aficida ® for 96 h, only those animals treated at 50–100 mg/L showed an increase in MN frequency. Cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts, was found after 48 h of exposure in 50–157 mg Aficida ® /L-treated fish, while, after 96 h exposure, only 100–157 mg Aficida ® /L induced the same effect. This species provides a useful experimental model for the biomonitoring of aquatic ecosystems.

Genotoxic effects of ivermectin (IVM) and its commercial formulation ivomec® (IVM 1.0%) were studied on Aedes albopictus larvae (CCL-126™) cells by sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE) while cytotoxicity was determined by cell-cycle progression (CCP), proliferative rate index (PRI), mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR) endpoints within a 1–250 µg mL−1 concentration range. While IVM and ivomec® did not markedly affect SCE frequencies, these agents induced DNA-strand breaks enhancing both slightly damaged and damaged cells at 25–50 and 5–50 µg mL−1 IVM and ivomec®, respectively. Both compounds exerted a delay in CCP and reduction of PRI at 10 µg mL−1. Cytotoxicity was observed at concentrations higher than 25 µg mL−1. A marked reduction of about 98% and 94% of MI compared to controls was noted with 25 µg mL−1 of IVM and ivomec®, respectively. NR and MTT assays revealed that both compounds induced a c...

Acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida® (50% pirimicarb) were evaluated on Rhinella arenarum (Anura, Bufonidae) tadpoles exposed under laboratory conditions. Lethal and sublethal effects were employed as bioassays for acute toxicity, whereas micronuclei (MNi) induction and alterations in the ratio erythrocytes:erythroblasts were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) (23 mg L-1) and cyclophosphamide (40 mg L-1) were employed as positive controls for toxicity and geno-cytotoxicity assays, respectively. In Gosner stage 25 (STD25), the results revealed mean values of 402.0 and 223.6 mg Aficida® L-1 for LC-5024 h and LC-5096 h, respectively. When STD37-39 tadpoles were exposed, the LC-5024 h and LC-5096 h reached values of 239.4 and 181.7 mg Aficida® L-1, respectively. Sublethal effects revealed a mean EC-5096 h of 133.85 and 104.2 mg Aficida® in those STD25 and STD37-39 treated tadpoles, respectively. The results demonstrated that in 48-h-exposed tadpoles, a MNi increase was found only in those 80.0 mg L-1 Aficida®-treated individuals. When tadpoles were exposed to Aficida® for 96 h, only the 160 mg L-1-treated individuals showed a significant increase in MNi frequency. Concentrations ranging from 80.0 to 250.0 mg Aficida® L-1 resulted in cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts. Accordingly, this species could provide a suitable and useful experimental model for biomonitoring aquatic ecosystems. © 2009 Elsevier Ltd. All rights reserved. Fil: Vera Candioti, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Laboratorio de Citogenetica; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Laboratorio de Citogenetica; Argentina Fil: Ronco, Alicia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Laboratorio de Citogenetica; Argentina

The effects of ivermectin (IVM) and its commercial formulation ivomec (IVM 1.0%) were studied on Chinese hamster ovary (CHO(K1)) cells by several genotoxicity [sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE)] and cytotoxicity [cell-cycle progression (CCP), mitotic index (MI), proliferative replication index (PRI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)] bioassays within the 1.0-250 microg/ml concentration-range. While IVM and ivomec did not modified SCE frequencies, they induced DNA-strand breaks revealed by SCGE. An enhancement of slightly damaged cells and a decrease in undamaged cells were observed in IVM-treated cultures with 5.0-50.0 microg/ml. In ivomec((R))-treated cells, while an increase in slightly damaged cells was induced with 5.0-50.0 microg/ml, the damaged and undamaged cells increased and decreased only with 50.0 microg/ml. Both compounds exerted a delay in CCP and a reduction in PRI when 25.0 microg/ml was employed whereas cytotoxicity was observed at higher concentration than 50.0 microg/ml. No MI alteration was observed with 1.0-10.0 and 1.0-5.0 microg/ml of IVM and ivomec, respectively. A concentration-related trend to an increase in MI was achieved within 1.0-10.0 microg/ml. An increase in the MI was induced in 10.0 microg/ml ivomec-treated cultures. A marked reduction of about 89% and 62% in regard to controls was observed with 25.0 microg/ml of IVM and ivomec, respectively. NR and MTT assays revealed a cell growth inhibition when 0.25-250.0 microg/ml of both compounds was employed. The results highlighted that IVM and ivomec exert both genotoxicity and cytotoxicity in mammalian cells in vitro, at least in CHO(K1) cells.

Leiomyosarcoma (LMS) is the third most common type of soft tissue sarcoma after malignant fibrous histiocytoma (MFH) and liposarcoma. Comparative genomic hybridization (CGH) has shown similar DNA copy number imbalances in LMS and MFH. It has been suggested that both tumors may correspond to different differentiation states of a single tumor entity and that a large proportion of MFHs could correspond to undifferentiated LMS. We report CGH results from 102 MFH and 82 LMS cases, as well as a subsequent clustering analysis. The distribution pattern of DNA copy number changes could not differentiate LMS from MFH, suggesting that most MFHs could represent an ultimate state of tumor progression of LMS. Even if an oncogenic pattern common to LMS and MFH is valid, the genes relevant to smooth muscle cell differentiation may reside in one or more chromosomal imbalances that are not shared by both tumor types. Further explorative analysis identified a small cluster of tumors (9% of the samples: 2 LMS and 10 MFH) characterized by the presence of high-level amplifications at 1p33 approximately p34.3, 17q22 approximately q23, 17q25 approximately qter, 19p, 22p, and 22q, and associated with a higher proportion of tumors located in the thigh (P=0.003) and with male sex (P=0.079).

The in vitro geno- and cytotoxicity exerted by the N-methylcarbamate pesticide carbofuran (CF) and its commercial formulation furadan (F) were studied in Chinese hamster ovary (CHO(K1)) cells by several bioassays for both genotoxicity (e.g., the sister chromatid exchange (SCE) and micronuclei (MNi) frequencies), and cytotoxicity (e.g., cell-cycle progression, mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)). Both CF and F activities were tested within the range of 5-100 microg/ml. CF within a 10-100 microg/ml concentration-range induced a significant dependent increase of SCE frequency and MNi over control values. At the same concentration-range, F increased significantly the SCE frequencies over control values although in a non-dependent manner while only an enhanced frequency of MNi was found in those 50 microg/ml-treated cultures. No binucleated cytokinesis-block cells were found in 100 microg/ml F-treated cultures. The NDI index revealed a delay in the onset of cell-division with 50 and 100 microg/ml of CF and F, respectively. The delayed rate of nuclear division induced by 100 microg/ml of F was higher than that induced by an equal concentration of CF. CF and F induced both a significant concentration-dependent delay in cell-cycle progression and a decrease in the proliferative replication index within 5-100 microg/ml and 50-100 microg/ml concentration-range, respectively. Decreased cell viability was found in up to 26% and 47% in 100 microg/ml CF- and F-treated cultures, respectively. The NR and MTT assays revealed a clear cell growth inhibition when concentrations of 50 and 100 microg/ml of either CF or F were employed. Accordingly, the results highlight that CF by itself and F, even in a greater extend exerts both genotoxicity and cytotoxicity in mammalian cells in culture, at least in CHOK1 cells.

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0-500.0 microg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r=0.48; P>0.05) nor for the commercial formulation (r=0.58, P>0.05). For the 200.0 microg/ml and 500.0 microg/ml dicamba doses and the 500.0 microg/ml banvel dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r=-0.98, P

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.

The amplification or gain of the p-arm of chromosome 17 is common in sarcomas, suggesting its role in carcinogenesis. Here, we report the architectural structure and targets of 17p aberrations commonly shared by osteosarcoma (OS), leiomyosarcoma (LMS) and malignant fibrous histiocytoma (MFH) of soft tissue. Two low-grade and two high-grade soft tissue LMS, three OS, and two MFH samples were studied using fine-resolution oligonucleotide-based microarray comparative genomic hybridization. Eight of the nine samples showed a loss of 17pter→p13, the locus of tumor suppressor TP53 preceding the amplified area 17p12→p11.2. The size and detailed architecture of the amplified region of 17p differed between the studied sarcoma entities. OS and high-grade LMS showed similar complex patterns of discontinuous amplifications with regions of gain in between. MFH and low-grade LMS showed continuous regions of gains and amplifications. Precise boundaries of the lost or gained regions were determined, and in addition to the previously suggested targets of the region, ELAC and FLCN were amplified in all the sarcoma entities.

Acute toxicity and genotoxicity of the 54.8% 2,4-D-based commercial herbicide DMA® were assayed on Cnesterodon decemmaculatus (Pisces, Poeciliidae). Whereas lethal effect was used as the end point for mortality, frequency of micronuclei (MNs), other nuclear abnormalities and primary DNA damage evaluated by the single cell gel electrophoresis (SCGE) assay were employed as end points for genotoxicity. Mortality studies demonstrated an LC50 96 h value of 1008 mg/L (range, 929-1070) of 2,4-D. Behavioral changes, e.g., gathering at the bottom of the aquarium, slowness in motion, slow reaction and abnormal swimming were observed. Exposure to 2,4-D within the 252-756 mg/L range increased the frequency of MNs in fish exposed for both 48 and 96 h. Whereas blebbed nuclei were induced in treatments lasting for 48 and 96 h, notched nuclei were only induced in fish exposed for 96 h. Regardless of both concentration and exposure time, 2,4-D did not induce lobed nuclei and binucleated erythrocytes. In addition, we found that exposure to 2,4-D within the 252-756 mg/L range increased the genetic damage index in treatments lasting for either 48 and 96 h. The results represent the first experimental evidence of the lethal and several sublethal effects, including behavioral alterations and two genotoxic properties namely the induction of MNs and primary DNA strand breaks, exerted by 2,4-D on an endemic organism as C. decemmaculatus. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Acute lethal and sublethal toxicity of the imidazolinone imazethapyr (IMZT)-based commercial formulation herbicide Pivot H® (10.59% IMZT) was evaluated on Hypsiboas pulchellus tadpoles. Whereas mortality was used as the end point for lethality, frequency of micronuclei (MNs) and other nuclear abnormalities as well as DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed to test genotoxicity. Behavioral, growth, developmental, and morphological abnormalities were also employed as sublethal end points. Mortality studies revealed equivalent LC50 (96h) values of 1.49mg/L (confidence limit, 1.09-1.63) and 1.55mg/L (confidence limit, 1.51-1.60) IMZT for Gosner stage (GS) 25 and GS36, respectively. Behavioral changes, i.e., irregular swimming and immobility, as well as a decreased frequency of keratodonts were observed. The herbicide increased the frequency of MNs in circulating erythrocytes of tadpoles exposed for 48h to the highest concentration assayed (1.17mg/L). However, regardless of the concentration of the herbicide assayed, an enhanced frequency of MNs was observed in tadpoles exposed for 96h. The herbicide was able to induce other nuclear abnormalities, i.e., blebbed and notched nuclei, only when tadpoles were exposed for 96h. In addition, we observed that exposure to IMZT within the 0.39-1.17mg/L range increased the genetic damage index in treatments lasting for both 48 and 96h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMZT on amphibians. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to IMZT. Fil: Pérez Iglesias, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

We analyzed the aspects of lethality, genotoxicity, and cytotoxicity in the ten spotted live-bearer exposed under laboratory conditions to the pirimicarb-based formulation Patton Flow1 (50% active ingredient (a.i.)). Acute effects were evaluated using different end points for lethality, genotoxicity, and cytotoxicity. Median lethal concentration (LC50) estimation was employed as a bioassay for lethality, whereas micronucleus (MN) induction and alterations in erythrocyte/erythroblast frequency were used as end points for genotoxicity and cytotoxicity, respectively. Results demonstrated an LC5096h value of 88 mg/L. Patton Flow1 increased the MN frequency in fish erythrocytes after 48 h of exposure at a concentration of 66 mg/L, whereas a concentration range of 22?66 mg/L was able to exert the same genotoxic effect at 96 h of treatment. Furthermore, cytotoxicity was also observed by alterations in erythrocyte/erythroblast frequencies within the concentration range of 22? 66 mg/L, regardless of the exposure time. Our current observations provide evidence that Patton Flow1 (50% a.i.) should be considered a clear lethal, cytotoxic, and genotoxic agent on Cnesterodon decemmaculatus. Thus, repeated applications of this carbamic insecticide can enter the aquatic environment and exert deleterious effects on aquatic organisms other than the evaluated species C. decemmaculatus. Fil: Vera Candioti, Josefina. Universidad Nacional de la Plata. Facultad de Cs.naturales y Museo. Laboratorio de Citogenetica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de la Plata. Facultad de Cs.naturales y Museo. Laboratorio de Citogenetica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de la Plata. Facultad de Cs.naturales y Museo. Laboratorio de Citogenetica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

The cytogenetic effects exerted by the phenoxy herbicide dicamba and one of its commercial formulations banvel (57.71% dicamba) were studied in in vitro whole blood human lymphocyte cultures. The genotoxicity of herbicides was measured by analysis of the frequency of sister chromatid exchanges (SCEs) and cell-cycle progression assays. Both dicamba and banvel activities were tested within 10.0-500.0 microg/ml doses range. Only concentrations of 200.0 microg/ml of dicamba and 500.0 microg/ml of banvel induced a significant increase in SCE frequency over control values. The highest dose of dicamba tested (500.0 microg/ml) resulted in cell culture cytotoxicity. The cell-cycle kinetics was affected by both test compounds since a significant delay in cell-cycle progression and a significant reduction of the proliferative rate index were observed after the treatment with 100.0 and 200.0 microg/ml of dicamba and 200.0 and 500.0 microg/ml of banvel. For both chemicals, a progressive dose-related inhibition of the mitotic activity of cultures was observed. Moreover, only the mitotic activity statistically differed from control values when doses of both chemicals higher than 100.0 microg/ml were employed. On the basis of our results, the herbicide dicamba is a DNA damage agent and should be considered as a potentially hazardous compound to humans.

DNA copy number changes were investigated in 51 (19 uterine and 32 nonuterine) primary leiomyosarcomas by comparative genomic hybridization. The aim was to evaluate whether true biological differences exist between uterine and nonuterine leiomyosarcoma and whether changes revealed by comparative genomic hybridization have prognostic value. Genomic imbalances were found in 48 (94%) cases. The most frequent DNA copy number changes were losses in 10q (35%), 13q (57%), and 16q (41%), gains in 1q (41%), and gains and high-level amplifications in 17p (39%). Gains were nearly as frequent as losses in both uterine and nonuterine leiomyosarcoma. Correlation-based tree modeling revealed two clusters that segregated significantly a group of uterine (gains at 1q11-q24) and a group of nonuterine (losses at 13q14-q34, 16q11.1-q24, and 10q21-q26) cases. The nonuterine cluster was associated with subcutaneous origin and a trend toward increased metastasis-free survival. Further explorative analyses identified aberrations associated with shorter metastasis-free survival time, including losses at 2q32.1-q37 and gains at 8q24.1-q24.3, whereas the cases with losses at 6cen-p25 showed longer metastasis-free survival time., Facultad de Ciencias Naturales y Museo

To contribute to a more accurate characterization of the mutagenic and aneugenic effects of thiabendazole (TBZ), a widely used antiparasitic and food preservative drug, the induction of sister chromatid exchanges (SCEs) and mitotic spindle anomalies as cytogenetic end-points were investigated. Studies were carried out in Chinese hamster ovary (CHO) cells and human peripheral blood lymphocytes. A significant dose-dependent increase in SCE frequency was observed in CHO cells with S9-Mix (P < 0.01) in the 50-100 microg ml(-1) dose-range, while in the absence of S9-Mix, an enhancement of the SCE frequency was exhibited at the highest dose (P < 0.01). In CHO-K1 cells a significant increase in mitotic spindle anomalies (P < 0.01) was observed with the highest concentration assayed reflecting the specific effect of TBZ formulation at the microtubule level. Cell proliferation kinetics (CPK) were not modified by the addition of this pharmaceutical product. In human lymphocyte cultures, exposure to 100 microg ml(-1) TBZ formulation resulted in a significant decrease of the mitotic index (MI) (P < 0.003) and changes in the replication index (RI) (P < 0.05).

The amount, composition and location of heterochromatin in Athaumastus haematicus (Stål, 1859), Leptoglossus impictus (Stål, 1859), Phthia picta (Drury, 1770) (Coreidae), Largus rufipennis Laporte, 1832 (Largidae) and Jadera sanguinolenta (Fabricius, 1775) (Rhopalidae) are analyzed by C-banding and DAPI/ CMA fluorescent banding. As the rule for Heteroptera the possession of holokinetic chromosomes and a pre-reductional type of meiosis cytogenetically characterize these five species. Besides, all of them (except L. rufipennis) present a pair of m chromosomes. C-banding technique reveals the absence of constitutive heterochromatin in A. haematicus, scarce C-positive blocks in L. impictus and J. sanguinolenta, and C-positive heterochromatin terminally located in P. picta and L. rufipennis. All C-bands are DAPI bright, except for a DAPI dull/CMA bright band at one telomeric end of the X chromosome in L. rufipennis, which probably corresponds to a nucleolar organizing region. The results of the banding techniques are analyzed in relation to the chiasma frequency and distribution in the five species, and it is concluded that there should exist some constraints to the acquisition and/ or accumulation of heterochromatin in their karyotypes.

Genotoxicity of the 2,4-dichlorophenoxyacetic acid (2,4-D) and a commercially-used derivative, 2,4-D dimethylamine salt (2,4-D DMA), was evaluated in CHO cells using SCE and single cell gel electrophoresis (SCGE) assays. Log-phase cells were treated with 2.0-10.0 microg/ml of herbicides and harvested 24 and 36 h later for SCE analysis. Both agents induced significant dose-dependent increases in SCE, regardless of the harvesting time (2,4-D: r=0.98 and r=0.88, P0.01, for 24 and 36 h harvesting times; 2,4-D DMA: r=0.97 and r=0.88, P0.01, for 24 and 36 h harvesting times). Neither test compound altered cell-cycle progression or proliferative replication index (P0.05), but the higher doses of both compounds reduced the mitotic index of cultures harvested at 24 and 36 h (P0.05). A 90-min treatment with 2.0-10.0 microg/ml 2,4-D and 2,4-D DMA produced dose-dependent increases in the frequency of DNA-strand breaks detected in the SCGE assay, both in cultures harvested immediately after treatment and in cultures harvested 36 h later. The doses of 2,4-D and 2,4-D DMA were equally genotoxic in all of the assays. The results indicate that 2,4-D induces SCE and DNA damage in mammalian cells, and should be considered as potentially hazardous to humans.

A method for simultaneous detection of fluorescence in situ hybridization of DNA probes and high resolution fluorescent R banding is described. Human lymphocytes were stimulated with phytohemagglutinin and synchronized using a fluorouracil block followed by exposure to bromodeoxyuridine and Hoechst 33258 prior to harvest. Metaphase preparations were treated with Hoechst 33258 and exposed to UV light. Thereafter they were incubated in sodium phosphate buffer and dried prior to in situ hybridization with a biotin-labelled centromere-specific alpha-satellite DNA probe for chromosome 1 (pUCl.77) and two digoxigenin-labelled probes, i.e., a PCR-generated chromosome 8-specific alphoid probe (#8) and a cosmid probe for FLT4 gene on 5q33-qter (class III receptor tyrosine kinase). Hybridization signals were detected by an indirect immunofluorescence method using fluorescein isothiocyanate. The chromosomes were counterstained with propidium iodide and 4',6-diamidino-2-phenylindole dihydrochloride. This simple method allows unambiguous chromosome band identification simultaneously with detection of the hybridized probes.

Comparative genomic hybridization (CGH) was used to search for gains, high-level amplifications and losses of DNA sequences along all chromosome arms in 19 primary Merkel cell carcinomas (MCC). Extensive genetic aberrations, with a mean value of 5.5+/-1.1 changes per tumor were detected in 13 out of the 19 samples analyzed. Our CGH results reveal several new and other previously known chromosomal regions that are involved in the pathogenesis of MCC. The majority of the alterations were gains of whole chromosomes or whole chromosome arms. Compared to losses, the frequency of DNA copy number gains was two-fold. DNA sequence copy number gains were most common in chromosomes 6 (42%), 1 (37%), and 5 (32%). The most frequent minimal common regions of gains were 6pterqter (42%), 1q11q31 (32%), and 5p (32%). No recurrent high-level amplifications were observed. High-level amplifications of small chromosomal regions were found in four samples out of the 19 tumors analyzed (21%). Amplifications affected 1q22q24 (5%), 4p (5%), and 5p (5%). Losses most frequently affected chromosomes 13 (21%) and 4 (16%). Minimal common regions with the most frequent losses were 13q13q31 (21%), 4q (16%), and 16q (11%). No significant statistical correlation between genomic aberrations and clinicopathological factors was revealed, despite the fact that there was an obvious tendency towards it. Primary MCC expressing DNA alterations were predominantly distinguished in large tumors, and risk of metastatic dissemination was three-fold compared to tumors with no DNA alterations.