Dysregulation of the Hippo pathway signaling in aging and cancer.

Human beings are facing emerging degenerative and cancer diseases, in large part, as a consequence of increased life expectancy. In the near future, researchers will have to put even more effort into fighting these new challenges, one of which will be prevention of cancer while continuing to improve the aging process through this increased life expectancy. In the last few decades, relevance of the Hippo pathway on cancer has become an important study since it is a major regulator of organ size control and proliferation. However, its deregulation can induce tumors throughout the body by regulating cell proliferation, disrupting cell polarity, releasing YAP and TAZ from the Scribble complexes and facilitating survival gene expression via activation of TEAD transcription factors. This pathway is also involved in some of the most important mechanisms that control the aging processes, such as the AMP-activated protein kinase and sirtuin pathways, along with autophagy and oxidative stress response/antioxidant defense. This could be the link between two tightly connected processes that could open a broader range of targeted molecular therapies to fight aging and cancer. Therefore, available knowledge of the processes involved in the Hippo pathway during aging and cancer must necessarily be well understood.

Half-Wave Potentials and In Vitro Cytotoxic Evaluation of 3-Acylated 2,5-Bis(phenylamino)-1,4-benzoquinones on Cancer Cells.

A broad range of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones were synthesized and their voltammetric values, as well as in vitro cancer cell cytotoxicities, were assessed. The members of this series were prepared from acylbenzoquinones and phenylamines, in moderate to good yields (47-74%), through a procedure involving a sequence of two in situ regioselective oxidative amination reactions. The cyclic voltammograms of the aminoquinones exhibit two one-electron reduction waves to the corresponding radical-anion and dianion, and two quasi-reversible oxidation peaks. The first and second half-wave potential values (E1/2) of the members of the series were sensitive to the push-pull electronic effects of the substituents around the benzoquinone nucleus. The in vitro cytotoxic activities of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones against human cancer cells (bladder and prostate) and non-tumor human embryonic kidney cells were measured using the MTT colorimetric method. The substitution of both aniline groups, by either methoxy (electron donating effect) or fluorine (electron withdrawal effect), decreased the cytotoxicity in the aminoquinones. Among the members of the unsubstituted phenylamino series, two of the 18 compounds showed interesting anti-cancer activities. A preliminary assay, looking for changes in the expression of selected genes, was performed. In this context, the two compounds increased TNF gene expression, suggesting an association with an inflammatory-like response.

Heavy metals in wild house mice from coal-mining areas of Colombia and expression of genes related to oxidative stress, DNA damage and exposure to metals.

Coal mining is a source of pollutants that impact on environmental and human health. This study examined the metal content and the transcriptional status of gene markers associated with oxidative stress, metal transport and DNA damage in livers of feral mice collected near coal-mining operations, in comparison with mice obtained from a reference site. Mus musculus specimens were caught from La Loma and La Jagua, two coal-mining sites in the north of Colombia, as well as from Valledupar (Cesar Department), a city located 100km north of the mines. Concentrations in liver tissue of Hg, Zn, Pb, Cd, Cu and As were determined by differential stripping voltammetry, and real-time PCR was used to measure gene expression. Compared with the reference group (Valledupar), hepatic concentrations of Cd, Cu and Zn were significantly higher in animals living near mining areas. In exposed animals, the mRNA expression of NQ01, MT1, SOD1, MT2, and DDIT3 was 4.2-, 7.3-, 2.5-, 4.6- and 3.4-fold greater in coal mining sites, respectively, than in animals from the reference site (p<0.05). These results suggest that activities related to coal mining may generate pollutants that could affect the biota, inducing the transcription of biochemical markers related to oxidative stress, metal exposure, and DNA damage. These changes may be in part linked to metal toxicity, and could have implications for the development of chronic disease. Therefore, it is essential to implement preventive measures to minimize the effects of coal mining on its nearby environment, in order to protect human health.

Biochemical effects induced by the hexachlorocyclohexanes.

The hexachlorocyclohexanes (HCHs) are synthetic compounds that have been widely used for the control of pests. The most common HCH isomers are the α-, ß-, δ-, and γ-HCH. Although the have the same chlorine substitution pattern, the spatial orientation of chlorine atoms is different on each one of them, resulting in unique structures that have distinct molecular properties. Humans are exposed to individual HCH isomers through various routes, including ingestion of contaminated water or food, absorbed through the skin or by inhalation, and because of their liposolubility, these chemicals are mostly stored in fat-containing tissues. The isomer-specific spectrum of biochemical actions for these compounds has been wee characterized for different endpoints such as enzyme activation, calcium homeostasis, gap junctional intercellular communication, endocrine disruption, and cancer, among others. The interaction with the GABA reception has been one of the most extensively studied properties of the HCHs. For instance, γ-HCH acts as a GABAA channel blocker, whereas α- and δ-HCH potentiate currents , all working as allosteric modulators of the receptor. The changes in calcium homeostasis elicited by HCHs are both isomer and cell type specific. For example, in neurons, both the δ- and γ-isomers of HCH stimulate Ca²+ influx through different voltage-gated Ca²+ channels. In human neutrophils, α-,δ-, and γ-HCH, but not ß-HCH, increase intracellular Ca²+ concentrations. This isomer-dependent behavior is also similar to that observed for phospholipase A2 activation and also correlates with oxidative stress generation. On the other hand, there are several lines of evidence suggesting that HCHs alter genomic integrity, and, therefore, these compounds have been classified as possibly carcinogenic to humans . Finally, HCHs have been reported to be endocrine disrupters. In fact, γ- and ß-HCH have been shown to have weak estrogenic activity, and together with the α- and the δ-isomer, also interfere with steroidogenesis. In short, the HCH isomers are good examples of structurally related chemicals, for which the geometrical patterns present in each one of the different conformers create structures that possess specific mechanisms of action and toxicological properties.

New 2-Acetyl-3-aminophenyl-1,4-naphthoquinones: Synthesis and In Vitro Antiproliferative Activities on Breast and Prostate Human Cancer Cells.

The reaction of 2-acyl-1,4-naphthoquinones with N,N-dimethylaniline and 2,5-dimethoxyaniline, promoted by catalytic amounts of CeCl3·7H2O under "open-flask" conditions, produced a variety of 2-acyl-3-aminophenyl-1,4-naphthoquinones structurally related to the cytotoxic 2-acetyl-3-phenyl-1,4-naphthoquinone, an inhibitor of the heat shock chaperone protein Hsp90. The members of the 2-acyl-3-aminophenyl-1,4-naphthoquinone series were isolated in good yields (63-98%). The cyclic voltammograms of the 2-acyl-3-aminophenyl-1,4-naphthoquinone exhibit two one-electron reduction waves to the corresponding radical-anion and dianion and two quasireversible oxidation peaks. The first and second half-wave potential values (E 1/2) of the members of the series are sensitive to the push-pull electronic effects of the substituents in the naphthoquinone scaffold. Furthermore, the in vitro antiproliferative properties of these new quinones were evaluated on two human cancer cells DU-145 (prostate) and MCF-7 (mammary) and a nontumorigenic HEK-293 (kidney) cell line, using the MTT colorimetric method. Two members, within the series, exhibited interesting cytotoxic activities on human prostate and mammary cancer cells.

Targeting pro-senescence mitogen activated protein kinase (Mapk) enzymes with bioactive natural compounds.

Aging is a multifactorial universal process characterized by a gradual decrease in physiological and biochemical functions. Given that life expectancy is on the rise, a better understanding of molecular mechanisms of the aging process is necessary in order to develop anti-aging interventions. Uncontrolled cellular senescence promotes persistent inflammation and accelerates the aging process by decreasing tissue renewal, repair and regeneration. Senescence of immune cells, immunesenescence, is another hallmark of aging. Targeting pro-senescent enzymes increases survival and therefore the lifespan. Although the upregulation of Mitogen Activated Protein Kinases (MAPK) enzymes in aging is still controversial, increasing evidence shows that dysregulation of those enzymes are associated with biological processes that contribute to aging such as irreversible senescence. In this manuscript components of the MAPK pathway will be summarized, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38, as well as natural flavonoids, phenolic and diterpenoids with anti-senescence activity that shows positive effects on longevity and MAPK inhibition. Although more studies using additional aging models are needed, we suggest that these selected natural bioactive compounds that regulate MAPK enzymes and reduce senescent cells can be potentially used to improve longevity and prevent/treat age-related diseases.

Advantages and disadvantages of apoptosis in the aging process.

Researchers cannot predict as yet how long a human being can live. Life expectancy has been steadily increasing in the last century, but perhaps not always the quality of life in parallel with it. Future generations will be faced with the problems of an increased life expectancy along with the emergence of new age-related diseases. A deeper understanding of the aging process is crucial to ameliorate, if not to prevent, these projected new old-age diseases. One of the mechanisms responsible for healthy aging is through the effective maintenance of physiological, biochemical, and immunological functions. To carry this out, the organism needs to create new cells to replace old ones and to induce the disappearance of old and damaged cells. Apoptosis is involved in all these processes. However, if apoptosis is dysregulated, premature senescence-associated diseases are likely to appear. In our review, the focus will be on a better understanding of the role of apoptosis in the aging process. These signaling pathways will most assuredly be pharmacologically targeted in antiaging medicine therapies.

Toxic effects of a methanolic coal dust extract on fish early life stage.

Coal dust is a contaminant that impacts the terrestrial and aquatic environment with a complex mixture of chemicals, including PAHs and metals. This study aims to evaluate the toxic effect of a methanolic coal dust extract on a fish early life stage by analyzing phenotypic alterations, transcriptome changes, and mortality in zebrafish (ZF) embryos. ZF embryos were exposed to methanolic coal dust extract at 1-5000 mg·L-1 and monitored using bright field microscopy 24 and 48 hpf to determine malformations and mortality. In situ hybridization, RNA sequencing, and qRT-PCR were employed to identify transcriptome changes in malformed embryos. Three malformed phenotypes were generated in a dose-dependent manner. In situ hybridization analysis revealed brain, somite, dorsal cord, and heart tube development biomarker alterations. Gene expression profile analysis identified changes in genes related to structural constituent of muscle, calcium ion binding, actin binding, melanin metabolic process, muscle contraction, sarcomere organization, cardiac myofibril assembly, oxidation-reduction process, pore complex, supramolecular fiber, striated muscle thin filament, Z disc, and intermediate filament. This study shows, for the first time, the malformations generated by a mixture of pollutants from a methanolic coal dust extract on a fish early life stage, constituting a potential risk for normal embryonic development of other aquatic vertebrate organisms. Furthermore, we establish that phenotypes and changes in gene expression induced by the extract constitute a target for future studies about mechanical toxicity and their utility as sensitive tools in environmental risk assessments for biota and humans exposed to coal mining activities.

Signaling Pathways in Inflammation and Anti-inflammatory Therapies.

During the past decade, an abundance of new evidence highlighted the importance of inflammation in the development of chronic pathologies such as neurodegeneration, cancer, diabetes, cardiovascular disease and inflammatory bowel disease. However, most of the current therapies do not address the underlying problem and better therapies are urgently needed. A growing number of researchers have discovered various signaling pathways that are associated with the initiation and progression of inflammation. Among different pathways, we will focus on three classical inflammatory pathways: p38 MAPK, IL-6/JAK/STAT3 and PI3K; and a non-classical inflammatory pathway, the Hippo. Recently, the Hippo pathway has been linked to various inflammatory modulators such as FoxO1/3, TNFα, IL-6, COX2, HIF-1α, AP-1, JAK and STAT. In this review, the molecular mechanisms, associated pathologies and selected drugs (both preclinical and clinical) of these signaling pathways will be summarized. Finally, limitations and potential risks of anti-inflammatory drugs will also be discussed.

Chemical and toxicological characterization of sediments along a Colombian shoreline impacted by coal export terminals.

Extraction, transport and utilization of coal spread out coal dust. Nowadays, Colombia is an important producer of this mineral in South America, being the Santa Marta area one of the largest coal exporting ports in the country. The aim of this work was to assess the pollutants levels and toxicity of shoreline sediments from this place. 16 PAHs and 46 elements were measured in nine locations during dry and rainy seasons. HepG2 cells were exposed to 1% sediment extracts and mRNA expression evaluated for selected genes. PAHs levels were greater during the rainy season. The highest ∑PAHs (89.9 ng g(-1)) appeared at a site located around 300 m far from the coast line at close proximity to the area where coal is loaded into cargo vessels for international shipments, being naphthalene the most abundant PAH. At Santa Marta Bay port, ∑PAHs were 62.8 ng g(-1) and 72.8 ng g(-1) for dry and rainy seasons, respectively, with greatest levels for fluoranthene. Based on sediment standards, most stations have poor condition regarding Cr, but moderate contamination on Cu, Pb and Zn. Sediments from the port and coal transport sites, the most polluted by PAHs and metals, induced CYP1A1 and NQO1 during the dry season. Data showed the sediments from this shoreline have bioactive chemicals that determine their toxicological profile.

Altered gene expression in HepG2 cells exposed to a methanolic coal dust extract.

Exposure to coal dust has been associated with different chronic diseases and mortality risk. This airborne pollutant is produced during coal mining and transport activities, generating environmental and human toxicity. The aim of this study was to determine the effects of a coal dust methanolic extract on HepG2, a human liver hepatocellular carcinoma cell line. Cells were exposed to 5-100ppm methanolic coal extract for 12h, using DMSO as control. MTT and comet assays were used for the evaluation of cytotoxicity and genotoxicity, respectively. Real time PCR was utilized to quantify relative expression of genes related to oxidative stress, xenobiotic metabolism and DNA damage. Coal extract concentrations did not induce significant changes in HepG2 cell viability after 12h exposure; however, 50 and 100ppm of the coal extract produced a significant increase in genetic damage index with respect to negative control. Compared to vehicle control, mRNA CYP1A1 (up to 163-fold), NQO1 (up to 4.7-fold), and GADD45B (up to 4.7-fold) were up regulated, whereas PRDX1, SOD, CAT, GPX1, XPA, ERCC1 and APEX1 remained unaltered. This expression profile suggests that cells exposed to coal dust extract shows aryl hydrocarbon receptor-mediated alterations, changes in cellular oxidative status, and genotoxic effects. These findings share some similarities with those observed in liver of mice captured near coal mining areas, and add evidence that living around these industrial operations may be negatively impacting the biota and human health.

Chemical composition, in vitro cytotoxic and antioxidant activities of the essential oil of Peruvian Minthostachys mollis Griseb / Composición química, actividades citotóxicas y antioxidantes in vitro del aceite esencial de Minthostachys mollis Griseb peruano

The composition of the essential oil obtained by hydrodistillation from Minthostachys mollis Griseb (Lamiaceae) aerial parts was determined by GC and GC/MS. Menthone (13.2%), pulegone (12.4%), cis-dihydrocarvone (9.8%) and carvacrol acetate (8.8%) were the main essential oil components. The cytotoxic activity of the essential oil was in vitro measured using the MTT colorimetric assay. IC50 values were calculated on healthy non-tumor cells (HEK-293) and three human cancer cell lines (T24, DU-145 and MCF-7). In such latter cells, the estimated values were around 0.2 mg/mL. In addition, the antioxidant activity was determined by interaction with the stable free radical 2,2"-diphenyl-1-picrylhydrazyl. The essential oil was almost devoid of antioxidant activity indicating that its anti-proliferative action relies on other unknown mechanism.

La composición del aceite esencial obtenido por hidrodestilación a partir de partes aéreas de Minthostachys mollis Griseb (Lamiaceae) se determinó mediante GC y GC/MS. Mentona (13.2%), pulegona (12.4%), junto con cis-dihidrocarvona (9.8%) y acetato de carvacrol (8.8%) fueron los principales componentes del aceite esencial. La actividad citotóxica del aceite esencial se midió in vitro utilizando el ensayo colorimétrico MTT tanto en células sanas no tumorales (HEK-293) como en tres líneas celulares de cáncer humano (T24, DU-145 y MCF-7). Los valores de IC50 calculados fueron de alrededor de 0.2 mg/mL. Además, se determinó la actividad antioxidante por su interacción con el radical libre 2,2"-difenil-1-picrilhidrazilo. El aceite esencial tiene baja actividad antioxidante, lo que indica que su acción antiproliferativa depende de otro mecanismo desconocido.

Direct effect of p, p'-DDT on mice liver

ABSTRACT Contact with the pesticide dichlorodiphenyltrichloroethane (p,p′-DDT) can be the cause of various harmful effects in humans, wildlife, and the environment. This pesticide is known to be persistent, lipophilic, resistant to degradation, and bioaccumulive in the environment and to be slowly released into bloodstream. Growing evidence shows that exposure to DDT is linked to type 2 diabetes mellitus. Individuals exposed to elevated levels of DDT and its metabolite have an increased prevalence of diabetes and insulin resistance. To evaluate these possible relationships, experiments were performed on eight-week-old female mice, divided into three groups (n = 10 per group): Group 1 received a vehicle-control intraperitoneal (i.p.) injection of sesame oil; Groups 2 and 3 received an i.p. dose of 50 and 100 µg/g p,p′-DDT respectively, dissolved in sesame oil. All groups were treated once daily for four days. Real-time PCR analysis of several genes was undertaken. Additionally, biochemical parameters and histopathological changes were measured. NQO1, HMOX1, NR1I3 and NR3C1 were up-regulated in DDT-exposed animals compared to the vehicle control group, while only SREBP1 was down-regulated in the 100 µg/g group. MTTP and FABP5, not previously reported for DDT exposure, but involved in regulation of fatty acid fluxes, could also function as biomarkers cross-talking between these signaling pathways. These results suggest that beyond epidemiological data, there is increasing molecular evidence that DDT may mimic different processes involved in diabetes and insulin resistance pathways.

RESUMO O contato com o praguicida diclorodifeniltricloroetano (p, p'-DDT) pode ser a causa de vários efeitos nocivos sobre os seres humanos, animais silvestres e o meio ambiente. Sabe-se de sua característica de bioacumulação, ser altamente persistente no meio ambiente, lipofílico, resistente à degradação e lentamente liberado na corrente sanguínea. Existe uma evidência crescente de que a exposição ao DDT pode ser ligada a Diabetes mellitus tipo 2. Os indivíduos expostos a níveis elevados de DDT e seu metabólito apresentam maior prevalência de diabetes e resistência à insulina. A fim de obter informações sobre essas possíveis relações, camundongos fêmeas de oito semanas de idade foram divididos em três grupos (n = 10 por grupo): Grupo 1 recebeu um veículo de óleo de gergelim via i.p.; os Grupos 2 e 3 receberam, via i.p., 50 e 100 µg/g de p, p'-DDT, respectivamente, dissolvidos em óleo de gergelim. Todos os grupos foram tratados uma vez ao dia durante quatro dias. Além da análise de PCR em Tempo Real de vários genes, os parâmetros bioquímicos e alterações histopatológicas também foram medidos. A expressão gênica do mRNA dos genes NQO1, HMOX1, NR1I3 e NR3C1 foi maior nos animais expostos ao DDT, em comparação ao grupo controle, enquanto a expressão gênica do SREBP1 diminuiu na concentração de 100 µg/g. Os genes MTTP e FABP5 envolvidos na regulação do fluxo de ácidos graxos, embora não estudados quanto à exposição ao DDT, também podem funcionar como biomarcadores de resposta cruzada entre essas vias de sinalização. Esses resultados sugerem que, além de dados epidemiológicos, há cada vez mais evidências moleculares de que o DDT poderia, de fato, imitar diferentes processos que envolvem as rotas de diabetes e de resistência à insulina.

Perfil de la expresión génica en células sanguíneas aisladas de carpinteros expuestos a solventes orgánicos en Sucre (Colombia) / Gene expression profile in blood cells isolated from carpenters exposed to organic solvents in Sucre (Colombia)

Resumen Objetivo: El objetivo de este estudio fue evaluar la expresión de genes asociados con estrés oxidativo, inflamación y daño al ácido desoxirribonucleico (ADN) en trabajadores de carpinterías en Sucre (Colombia). Materiales y métodos: Aleatoriamente fueron seleccionados 41 individuos de sexo masculino: 28 expuestos y 13 controles, con edades entre 32.3±7.9 y 33.2±8.4 años, respectivamente. Se colectaron muestras de sangre periférica y se realizaron análisis hematológicos y de marcadores de daño hepático. En 24 individuos expuestos y 10 controles se realizó análisis de expresión génica para marcadores de estrés oxidativo, inflamación y daño al ADN usando reacción en cadena de la polimerasa en tiempo real. Resultados: Los parámetros hematológicos y de daño hepático estuvieron dentro de los valores de referencia. La expresión génica de la P53 y BCL-2, genes asociados con el daño al ADN, fue significativamente mayor para el grupo expuesto en comparación con el grupo control. Conclusión: En ausencia de cambios en marcadores hematológicos o de daño hepático, personas expuestas a solventes en Sucre tienen niveles de expresión elevados para los genes P53 y BCL-2. Estos genes podrían ser candidatos útiles como biomarcadores moleculares relacionados con la exposición a solventes.

Abstract Objective: The aim of this study was to evaluate the expression of genes related to oxidative stress, inflammation and deoxyribonucleic acid (DNA) damage in carpentry workers from Sucre, Colombia. Materials and methods: 41 male individuals were randomly selected, 28 exposed and 13 controls, with ages 32.3 ± 7.9 and 33.2 ± 8.4 years old, respectively. Peripheral blood samples were collected and used for hematological and liver damage markers analysis. Gene expression analysis for oxidative stress, inflammation and DNA markers was performed using Real-Time Polymerase Reaction on 24 exposed and 10 controls. Results: Hematological parameters and liver damage markers were found within the reference values. Gene expression of P53 and BCL-2, genes related to DNA damage, was significantly greater for the exposed group when compared with the control group. Conclusion: In the absence of hematological or hepatic damage markers, individuals exposed to solvents in Sucre have increased gene expression for P53 and BCL2. These genes may be useful candidates as molecular biomarkers related to solvent exposure.

Toxicity of the essential oil of the cytral chemotype of Lippia alba (Mill.) N. E. Brown

The essential oil (EO) of Lippiaalba (Mill.) N. E. Brown (Verbenaceae) has been traditionally used to treat several diseases. In this study, the acute toxic effects of the citral chemotype of L. alba EO were evaluated in mice. Animals were treated via intraperitoneal receiving the L. alba essential oil at doses between 50 and 2500 mg/kg, and the control group received sesame oil (vehicle). The EO induced dose-dependent neurotoxic effects at doses greater than 1000 mg/kg, including decreased locomotion, motor skills and muscle strength, hypotonia, dyspnea, kyphosis and convulsions. The EO was lethal at a dose of 2500 mg/kg. Animals receiving 1000 mg/kg were euthanized at the end of the treatment period and their blood and livers were collected for analysis. Mice exposed to L. alba EOpresented significantly greater plasma alanine aminotransferase (ALT) activities than the control group. Liver histological changes included mild inflammation, in particular, an increase in nuclear size. Compared to vehicle control group, changes in expressionfor selected genes were significant for FABP5, a fatty acid transport related gene. In summary, the intraperitoneal administration of L. alba EO (citral chemotype) causes neurological damage in mice at doses equal or greater than 1500 mg/kg, whereas at 1000 mg/kg, it generates mild liver damage. Therefore, the systemic use of this EO raises concerns about its safety.

El aceite esencial (AE) de Lippia alba(Mill.) NE Brown (Verbenaceae) ha sido utilizado tradicionalmente para tratar varias enfermedades. En este estudio, los efectos tóxicos agudos del AE de Lippia alba quimiotipo citral fueron evaluados en ratones. Los animales fueron tratados por vía intraperitonealrecibiendo el AE en dosis entre 50 y 2500 mg/kg de peso, y el grupo control aceite de sésamo (vehículo). Dosis superiores a 1000 mg/kg del AE mostraron efectos neurotóxicos incluyendo disminución de la locomoción e hipotonía, disnea, cifosis y convulsiones. El AE fue letal a la dosis de 2500 mg/kg. Veinticuatro horas después de que los animales fueron tratados con 1000 mg/kg del AE se les realizó eutanasia y su sangre e hígado fueron recolectados para análisis. Los ratones expuestos al AE de L. alba, presentaron actividad alanina aminotransferasa (ALT) en plasma significativamente mayor que el grupo control. Dentro de los cambios histológicos hepáticos se incluyen inflamación leve, en particular, un aumento del tamaño nuclear. En comparación con el grupo control, la expresión de genes seleccionados tuvo diferencias significativas para FABP5, un gen relacionado con el transporte de ácidos grasos. En conclusión, la administración intraperitoneal del AE de L. alba (quimiotipo citral) causa dañosneurológicos en ratones a una dosis igual o superior a 1500 mg/kg, mientras que a 1000 mg/kg, genera daño hepático leve. Por lo tanto, el uso sistémico de este AE plantea preocupaciones en cuanto a su seguridad.