Genotoxicity and Cytotoxicity Exerted by Pesticides in Different Biotic Matrices-An Overview of More Than a Decade of Experimental Evaluation

Agrochemicals represent one of the most important sources of environmental pollution. Although attempts to reduce agrochemical use through organic agricultural practices and the use of other technologies to control pests continue, the problem is still unsolved. Recent technological advances in molecular biology and analytical science have allowed the development of rapid, robust, and sensitive diagnostic tests (biomarkers) that can be used to monitor exposure to, and the effects of pollution. One of the major goals of our research laboratory is to evaluate comparatively the genotoxic and cytotoxic effects exerted by several pure agrochemicals and their technical formulations commonly used in Argentina on vertebrate cells in vitro and in vivo employing several end-points for geno and cytotoxicity. Among them are listed the herbicides dicamba and flurochloridone, the fungicide zineb, the insecticides pirimicarb and imidacloprid. Overall, the results clearly demonstrated that the damage induced by the commercial formulations is in general greater than that produced by the pure pesticides, suggesting the presence of deleterious components in the excipients with either a putative intrinsic toxic effect Larramendy et al. 4 or with the capacity of exacerbating 52 the toxicity of the pure agrochemicals, or both. Accordingly, the results highlight that: 1) A complete knowledge of the toxic effect/s of the active ingredient is not enough in biomonitoring studies; 2) Pesticide/s toxic effect/s should be evaluated assaying to the commercial formulation available in market; 3) The deleterious effect/s of the excipient/s present within the commercial formulation should not be either discarded nor underestimated, and 4) A single bioassay is not enough to characterize the toxicity of a agrochemical under study.Facultad de Ciencias Naturales y Muse

Vitamin E prevents ethylene bis(dithiocarbamate) pesticide zineb-induced sister chromatid exchange in Chinese hamster ovary cells

The in vitro effect of the antioxidant α-tocopherol, vitamin E, on deleterious effects induced by the dithiocarbamate fungicide zineb and its commercial formulation azzurro on Chinese hamster ovary (CHO) cells was studied by using frequency of sister chromatid exchanges (SCEs), cell cycle progression and mitotic index (MI) as genetic end points. Both zineb and azzurro activities were tested within the range 0.1-100.0 μg/ml on exponentially growing CHO cells preincubated for 24 h in the presence or absence of 50.0 μg/ml vitamin E. SCE frequencies increased significantly over control values in a concentration-dependent manner in zineb- and azzurro-treated cultures at concentrations of 0.1-10.0 and 0.1-25.0 μg/ml, respectively. When target cells were preincubated with vitamin E, the number of SCEs was significantly lower than that observed in cells exposed only to 1.0-10.0 μg/ml zineb or 1.0-25.0 μg/ml azzurro, but higher than control values. Cytotoxicity was observed at concentrations higher than 25.0 and 50.0 μg/ml zineb and azzurro, respectively, regardless of the absence or presence of vitamin E. Regression analysis showed that the proliferative rate index decreased as a function of the concentration of zineb (0.1-10.0 μg/ml concentration range) and azzurro (0.1-25.0 μg/ml concentration range) titrated into cultures. For both chemicals, progressive concentration-related inhibition of the mitotic activity from cultures was observed when 10.0 μg/ml zineb or 1.0-25.0 μg/ml azzurro was employed. However, no significant alteration in cell cycle progression or MI was observed between vitamin E-preincubated cultures and those treated only with zineb and azzurro.Facultad de Ciencias Naturales y Muse

Cytotoxic and genotoxic assessments of 2,4-dichlorophenoxyacetic acid (2,4-D) in in vitro mammalian cells

A combined approach employing alkaline single cell gel electrophoresis (SCGE) and cytokinesis-blocked micronucleus (MNs) cytome bioassays was adopted to assess the deleterious properties of the auxinic 2,4-dichlorophenoxyacetic acid (2,4-D) and its microparticulated low volatility product Dedalo Elite (30% a.i.) on Chinese hamster ovary (CHO-K1) cells. Cytotoxicity was estimated by neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptosis assessment. Both compounds were assayed at 0.1–10 μg/ml concentration range. Whereas exposed CHO-K1 cells revealed a statistically significant enhancement of MNs when 10 μg 2,4-D/ml was assayed, MNs were only achieved in cells treated with 2 μg Dedalo Elite/ml. A diminution in the nuclear division index was only achieved after exposure to Dedalo Elite within the 1–10 μg/ml concentration range. Whereas increased genetic damage index was achieved when 6 and 10 μg 2,4-D/ml were assayed, GDI induction was observed in treatments employing 4 μg Dedalo Elite/ml. Both compounds induced cytotoxicity by inhibition of both lysosomal and MTT activities by enhancing the frequencies of early and late apoptotic cells. Our results not only indicate the genotoxic and cytotoxic potential of 2,4-D and its microparticulated marketplace formulation, but also highlight the risk of these agrochemicals present towards the biota and human health.Facultad de Ciencias Naturales y MuseoConsejo Nacional de Investigaciones Científicas y Técnica

Genotoxicidad inducida por el herbicida fitohormonal ácido 2,4- diclorofenoxiacético contenido en la formulación comercial DMA® en <i>Cnesterodon decemmaculatus</i> (Pisces: Poeciliidae)

El 2,4- diclorofenoxiacético (2,4-D) es un herbicida sistémico ampliamente usado en Argentina que imita la acción de las fitohormonas auxinas. Estas actúan alterando el desarrollo y crecimiento de las plantas. El presente trabajo tiene como objetivo evaluar los efectos letales y genotóxicos inducidos por el formulado DMA® (58,4% 2,4-D como sal de dimetilamina) en ejemplares adultos de C. decemmaculatus expuestos en condiciones de laboratorio. Se determinó la CL5096h, y a partir de ésta se expusieron individuos a concentraciones subletales de 252, 504 y 756 mg 2,4-D mg/l (correspondientes al 25, 50 y 75% de CL5096h) durante 48 y 96 h. Se utilizó ciclofosfamida (10 mg/l) y agua de red declorinada como control positivo y negativo, respectivamente. Como método de estudio se emplearon el ensayo de micronúcleos (MN) y de anormalidades nucleares (AN) en células circulantes sanguíneas. Los datos fueron analizados estadísticamente por ANOVA simple y test a posteriori de Dunnett. La CL5096h obtenida fue 1008,16 mg 2,4-D/l (LC95% 928,72-1070,31 mg 2,4-D/l). Los resultados demuestran que el 2,4-D incrementó la frecuencia de micronúcleos con todas las concentraciones ensayadas tanto a las 48 como a las 96 h de exposición (pC. decemmaculatus.Universidad Nacional de La Plat

Genotoxicidad inducida por el herbicida fitohormonal ácido 2,4- diclorofenoxiacético contenido en la formulación comercial DMA® en <i>Cnesterodon decemmaculatus</i> (Pisces: Poeciliidae)

El 2,4- diclorofenoxiacético (2,4-D) es un herbicida sistémico ampliamente usado en Argentina que imita la acción de las fitohormonas auxinas. Estas actúan alterando el desarrollo y crecimiento de las plantas. El presente trabajo tiene como objetivo evaluar los efectos letales y genotóxicos inducidos por el formulado DMA® (58,4% 2,4-D como sal de dimetilamina) en ejemplares adultos de C. decemmaculatus expuestos en condiciones de laboratorio. Se determinó la CL5096h, y a partir de ésta se expusieron individuos a concentraciones subletales de 252, 504 y 756 mg 2,4-D mg/l (correspondientes al 25, 50 y 75% de CL5096h) durante 48 y 96 h. Se utilizó ciclofosfamida (10 mg/l) y agua de red declorinada como control positivo y negativo, respectivamente. Como método de estudio se emplearon el ensayo de micronúcleos (MN) y de anormalidades nucleares (AN) en células circulantes sanguíneas. Los datos fueron analizados estadísticamente por ANOVA simple y test a posteriori de Dunnett. La CL5096h obtenida fue 1008,16 mg 2,4-D/l (LC95% 928,72-1070,31 mg 2,4-D/l). Los resultados demuestran que el 2,4-D incrementó la frecuencia de micronúcleos con todas las concentraciones ensayadas tanto a las 48 como a las 96 h de exposición (pC. decemmaculatus.Universidad Nacional de La Plat

Erythrocytes modulate cell cycle progression but not the baseline frequency of sister chromatid exchanges in pig lymphocytes

The effect of co-culturing varying concentrations of pig and human red blood cells (RBCs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression in pig plasma (PLCs) and whole blood leukocyte cultures (WBCs) was studied. No variation in SCE frequency was observed between pig control WBC and PLC. Addition of pig and human RBCs to pig PLCs did not modify the baseline frequency of SCEs. On the other hand, cell proliferation was slower in PLCs than in WBCs. The addition of pig or human RBCs to PLCs accelerated the cell-cycle progression of pig lymphocytes. When RBCs were added to PLCs the concentration and time sequence of RBC incorporation affected the cell-cycle progression of swine lymphocytes. When doses of pig or human RBCs equivalent to those present in WBCs were added immediately after PLC stimulation, the cell-cycle kinetics were similar to those of WBCs. Shorter co-incubation periods or a reduction in the dose of RBCs made cell-cycle progression intermediate between PLC and WBC values. Thus, pig and human RBCs modulated the in vitro cell-cycle progression of pig lymphocytes in a time- and dose-dependent manner, and the low baseline frequency of SCEs of pig lymphocytes is independent of the presence or absence of erythrocytes in culture.Facultad de Ciencias Naturales y Muse

Are the damaging effects induced by the imazethapyr formulation Pivot® H in <i>Boana pulchella</i> (Anura) reversible upon ceasing exposure?

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 MuseoFacultad de Ciencias ExactasCentro de Investigaciones del Medioambient

Genotoxicidad y carcinogénesis

La Genotoxicología comprende el estudio de la acción nociva de xenobióticos sobre los componentes hereditarios de los seres vivos y las consecuencias que su exposición genera sobre los ecosistemas y la biota. Por lo tanto, los xenobióticos pueden ser agentes físicos tales como la temperatura, luz ultravioleta, radiaciones ionizantes, radiaciones electromagnéticas; agentes químicos, tales como metales, halógenos, ácidos orgánicos e inorgánicos, entre otros y agentes biológicos tales como algunos parásitos, bacterias, hongos y virus (Repetto Jiménez y Reppeto Kuhn, 2009). Esta disciplina estudia las modificaciones de la estructura genética y sus manifestaciones en la reproducción de la célula, tejido o del individuo, en procesos conocidos como mutagénesis, carcinogénesis y teratogénesis. Todo agente xenobiótico capaz de interactuar de manera negativa, tanto física como químicamente, con las bases del ADN y alterar su estructura es considerado un mutágeno. Por otro lado, el término “genotóxico” es más amplio, ya que incluye los agentes que inducen no sólo mutaciones sino cualquier otro tipo de daño acontecido en el ADN celular (Mudry y Carballo, 2006).Facultad de Ciencias Naturales y MuseoFacultad de Ciencias Exacta

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae)

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 MedioambienteFacultad de Ciencias Naturales y Muse

Auxinic herbicides induce oxidative stress on Cnesterodon decemmaculatus (Pisces: Poeciliidae)

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 Muse