ABSTRACT
Today, a great interest in Jatropha-based products exists worldwide, mainly for the production of biofuel.However, the oil obtained from this plant is known to be toxic due to contained curcins andphorbol esters. Bioassays, including plant cytogenetic assays based on cell cycle observation, are useful for determining the toxicity of J. curcas oil. Hence, the aim of this study was to describe the mechanism of action of J. curcas oil by cell cycle analysis using Lactuca sativa as plant testing model. A decrease in root growth was observed, closely related to the reduction in mitotic index, along with an increase in condensed nuclei. J. curcas chemicals act both as aneugenic agents, leading to the formation of lagged, sticky chromosomes and c-metaphase cells, as well as clastogenic agents, inducing the formation of chromosome bridges and fragments. The cytotoxicity and genotoxicity of phorbol esters and other chemical components of J. curcas oil was determined and discussed.
Um grande interesse mundial existe em produtos à base de pinhão manso, principalmente para a produção de biocombustíveis. No entanto, o óleo obtido a partir desta planta é conhecidamente tóxico por conter curcina e ésteres de forbol. Bioensaios, incluindo ensaios citogenéticos em plantas-modelo com base na observação do ciclo celular, são úteis para determinar a toxicidade do óleo de J. curcas. Assim, o objetivo deste estudo foi descrever o mecanismo de ação do óleo de J. curcas por análise do ciclo celular usando Lactuca sativa como modelo de teste em plantas. Foi observada uma redução no crescimento das raízes, intimamente relacionada com a redução do índice mitótico e com um aumento de núcleos condensados. Os constituintes químicos de J. curcas atuam simultaneamente como agentes aneugênicos, levando à formação de cromossomos perdidos e pegajosos e células em c-metáfase, bem como agentes clastogênicos, induzindo a formação de pontes e fragmentos cromossômicos. A citotoxicidade e genotoxicidade do éster de forbol e outros componentes químicos do óleo de J. curcas foram determinados e discutidos.
Subject(s)
Cell Cycle , Aneugens , Jatropha , Toxicity , Mitotic IndexABSTRACT
Abstract The tropical and subtropical naturalized physic nut (Jatropha curcas L.), has been explored for biodiesel production in recent times. The oil is extracted from the seeds and, for the production to be feasible, utilization of the residual seed cake is crucial. Although the cake could be employed as a protein source in animal feed, it is rich in phorbol ester, which is toxic for animals. Therefore, breeding programs have been working to reduce or eliminate the phorbol ester content in physic nut. In this context, the present work aimed to evaluate the physic nut oil of toxic and non-toxic varieties (containing known or undetectable amounts of phorbol ester, respectively) with regards to phytotoxicity in a model experiment with Lactuca sativa L. For this, the percentage of germinated seeds was evaluated after 8, 16, 24, 36 and 48 hours of exposure to the treatments with toxic and non-toxic oil at concentrations of 22.5 %, 45 % and 67.5 % of emulsion (physic nut oil energetically mixed with distilled water). Root growth was determined after 48 hours of exposure and the germination speed index was obtained. The different stages of mitotic division as well as possible chromosomal and nuclear alterations were also recorded. The mitotic index was calculated as the number of dividing cells, as a fraction of the total number of cells, and the frequency of chromosome and nuclear alterations, expressed as the percentage of number of alterations divided by the total number of cells. Both varieties exhibited phytotoxicity, inducing significant reductions in percentage of germinated seeds (reduction of 98 %), germination speed index (reduction of 24.44) and root growth (reduction of 8.54 mm). In microscopic analysis, a mitodepressive effect was observed for both oils at the three concentrations used when compared to the negative control; however, it was possible to distinguish between the toxic and the non-toxic varieties based on the more expressive reduction of division promoted by the first, 2.19 %. Significant increments in the frequency of mitotic cells showing chromosome alterations as well, as the presence of condensed nuclei, were observed in the treated cells. However, these parameters were not significantly different from the control in the cells treated with both physic nut oils. In conclusion, the evaluation of root growth and cell division in the plant model L. sativa, can be proposed as an alternative to animal tests to distinguish the varieties with high and low phorbol ester concentration, thus contributing to the detection of toxicity in varieties used in breeding programs.
Resumen Jatropha curcas L., naturalizado tropical y subtropical, ha sido explorado para la producción de biodiesel. El aceite se extrae de las semillas y, para que la producción sea factible, la utilización de la torta de semillas residual es crucial. Aunque la torta se puede emplear como una fuente de proteína en la alimentación animal, es rica en éster de forbol, que es tóxico para los animales. Por lo tanto, los programas de mejoramiento han procurado reducir o eliminar el contenido de éster de forbol de J. curcas. En este contexto, el presente trabajo tuvo como objetivo evaluar el aceite de J. curcas de las variedades tóxicas y no tóxicas (con cantidades conocidas o indetectables de éster de forbol, respectivamente) con respecto a la fitotoxicidad en el modelo Lactuca sativa L. El porcentaje de semillas germinadas se evaluó después de 8, 16, 24, 36 y 48 horas de tratamiento. El crecimiento de la raíz se determinó después de 48 horas de exposición y se obtuvo el índice de velocidad de germinación. Se registraron las diferentes etapas de la división mitótica así como posibles alteraciones cromosómicas y nucleares. El índice mitótico se calculó como el número de células en división como una fracción del número total de células y la frecuencia de las alteraciones cromosómicas y nucleares, expresada como el porcentaje del número de alteraciones dividido entre el número total de células. Ambas variedades exhibieron fitotoxicidad, induciendo reducciones significativas en el porcentaje de semillas germinadas (Reducción del 98 %), índice de velocidad de germinación (Reducción de 24.44) y crecimiento de raíces (Reducción de 8.54 mm). En el análisis microscópico, se observó un efecto mitodepresivo para ambos aceites. Sin embargo, fue posible distinguir entre las variedades tóxicas y las no tóxicas basándose en la reducción más expresiva de la división promovida por la primera, 2.19 %. Se observaron incrementos significativos en la frecuencia de células mitóticas que mostraban alteraciones cromosómicas, así como la presencia de núcleos condensados en las células tratadas. Sin embargo, estos parámetros no fueron significativamente diferentes del control en las células tratadas con ambos aceites de J. curcas. En conclusión, la evaluación del crecimiento de las raíces y la división celular en el modelo L. sativa se puede proponer como una alternativa a las pruebas en animales para distinguir las variedades con concentraciones altas y bajas de éster de forbol, contribuyendo así a la detección de toxicidad en variedades utilizadas en programas de mejoramiento genético.