Genotoxic and antiproliferative properties of Endopleura uchi bark aqueous extract.

The bark extract from Endopleura uchi has been widely used in traditional medicine to treat gynecological-related disorders, diabetes, and dyslipidemias albeit without scientific proof. In addition, E. uchi bark extract safety, especially regarding mutagenic activities, is not known. The aim of this study was to determine the chemical composition, antitumor, and toxicological parameters attributed to an E. uchi bark aqueous extract. The phytochemical constitution was assessed by colorimetric and chromatographic analyzes. The antiproliferative effect was determined using sulforhodamine B (SRB) assay using 4 cancer cell lines. Cytotoxic and genotoxic activities were assessed utilizing MTT and comet assays, respectively, while mutagenicity was determined through micronucleus and Salmonella/microsome assays. The chromatographic analysis detected predominantly the presence of gallic acid and isoquercitrin. The antiproliferative effect was more pronounced in human colon adenocarcinoma (HT-29) and human breast cancer (MCF-7) cell lines. In the MTT assay, the extract presented an IC50 = 39.1 µg/ml and exhibited genotoxic (comet assay) and mutagenic (micronucleus test) activities at 20 and 40 µg/ml in mouse fibroblast cell line (L929) and mutagenicity in the TA102 and TA97a strains in the absence of S9 mix. Data demonstrated that E. uchi bark possesses bioactive compounds which exert cytotoxic and genotoxic effects that might be associated with its antitumor potential. Therefore, E. uchi bark aqueous extract consumption needs to be approached with caution in therapeutic applications.

Gamma-Decanolactone Alters the Expression of GluN2B, A1 Receptors, and COX-2 and Reduces DNA Damage in the PTZ-Induced Seizure Model After Subchronic Treatment in Mice.

Gamma-decanolactone (GD) has been shown to reduce epileptic behavior in different models, inflammatory decreasing, oxidative stress, and genotoxic parameters. This study assessed the GD effect on the pentylenetetrazole (PTZ) model after acute and subchronic treatment. We evaluated the expression of the inflammatory marker cyclooxygenase-2 (COX-2), GluN2B, a subunit of the NMDA glutamate receptor, adenosine A1 receptor, and GD genotoxicity and mutagenicity. Male and female mice were treated with GD (300 mg/kg) for 12 days. On the tenth day, they were tested in the Hot Plate test. On the thirteenth day, all animals received PTZ (90 mg/kg), and epileptic behavior PTZ-induced was observed for 30 min. Pregabalin (PGB) (30 mg/kg) was used as a positive control. Samples of the hippocampus and blood were collected for Western Blotting analyses and Comet Assay and bone marrow to the Micronucleus test. Only the acute treatment of GD reduced the seizure occurrence and increased the latency to the first stage 3 seizures. Males treated with GD for 12 days demonstrated a significant increase in the expression of the GluN2B receptor and a decrease in the COX-2 expression. Acute and subchronic treatment with GD and PGB reduced the DNA damage produced by PTZ in males and females. There is no increase in the micronucleus frequency in bone marrow after subchronic treatment. This study suggests that GD, after 12 days, could not reduce PTZ-induced seizures, but it has been shown to protect against DNA damage, reduce COX-2 and increase GluN2B expression.

Anti-hyperlipidemic effects of Campomanesia xanthocarpa aqueous extract and its modulation on oxidative stress and genomic instability in Wistar rats.

The use of natural products from herbs may be a therapeutic option in dyslipidemia treatment. Campomanesia xanthocarpa (Mart.) O. Berg (Myrtaceae) leaves have been used to decrease cholesterol levels. However, studies to determine activities of this plant on triglycerides metabolism have received little attention. The aim of this study was to examine anti-hyperlipidemic effects of a C. xanthocarpa aqueous leaf extract (CxAE) and assess protective actions against oxidative stress and DNA damage. The tyloxapol-induced hyperlipidemia model was used in Wistar rats. Rats were treated orally with CxAE either 250 or 500 mg/kg/day for 7 days prior to tyloxapol administration. Biochemical parameters, oxidative stress levels, and genomic instability were assessed in several tissues. CxAE decreased cholesterol and triglyceride levels in serum and hepatic and renal DNA damage in tyloxapol-treated rats. There was no marked effect on the micronucleus frequency in bone marrow. The extract increased catalase activity and decreased glutathione S-transferase activity in kidney tissue. CxAE showed anti-hyperlipidemic effects, improved oxidative parameters, and protected DNA against damage induced by tyloxapol-induced hyperlipidemia, suggesting C. xanthocarpa leaves may be useful in preventing dyslipidemias.Abbreviations: ALP: Alkaline phosphatase; ALT: Aspartate aminotransferase; ANOVA: Analysis of variance; AST: Aspartate aminotransferase; Ator: Atorvastatin; CAT: Catalase; Chol: Cholesterol; CxAE: Campomanesia xanthocarpa aqueous extract; GST: Glutathione S-transferase; HDL: High density cholesterol; i.p.: Intraperitoneal; NCE: Normochromatic erythrocyte; PBS: Phosphate buffer solution; PCE: Polychromatic erythrocyte; ROS: Reactive oxygen species; SD: Standard deviation; SOD: Superoxide dismutase; T: Tyloxapol; TBARS: Thiobarbituric acid reacting substances; TG: Triglyceride.

Toxicological aspects of Campomanesia xanthocarpa Berg. associated with its phytochemical profile.

Campomanesia xanthocarpa leaves are used as tea to treat diarrhea, inflammation, and hypercholesterolemia. Some pharmacological studies noted its beneficial uses of C. xanthocarpa; however, few investigations examined the toxicological profile of this plant. The aim of this study was to determine the chemical composition, genotoxic, and mutagenic potential of an aqueous extract of C. xanthocarpa leaves (CxAE), and potential protective effects against oxidative damage. Phytochemical constituents were determined using HPLC, and antioxidant effect in vitro was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay. Genotoxic effects and chromosomic mutations were assessed using comet assay and micronucleus (MN) test in Wistar rats treated with CxAE at 250, 500 or 1000 mg/kg for 7 consecutive days. Lipid peroxidation and antioxidant enzyme activities were measured in several tissues. CxAE induced mutations in TA98, TA97a, and TA102 strains. However, in the presence of metabolic activation, data were negative for all strains tested. Lack of mutagenicity was also observed in the MN test. This extract did not induce DNA damage, except when the highest concentration was used. DNA oxidative damage induced by hydrogen peroxide (H2O2) decreased in blood after treatment with CxAE. Lipid peroxidation levels were reduced while superoxide dismutase (SOD) activity increased in kidneys. The inhibitory concentration of CxAE required to lower DPPH levels to 50% was 38.47 ± 2.06 µg/ml. In conclusion, frameshift and oxidative mutations were observed only in the absence of metabolic activation which may be attributed to the presence of flavonoids such as quercetin. It is of interest that CxAE also showed protective effects against DNA oxidative damage associated with presence of ellagic acid, a phenolic acid with antioxidant activities. CxAE did not induce in vivo mutagenicity, suggesting that this extract poses a low toxic hazard over the short term.

DNA damage and oxidative stress induced by seizures are decreased by anticonvulsant and neuroprotective effects of lobeline, a candidate to treat alcoholism.

The alkaloid lobeline (Lob) has been studied due to its potential use in treatment of drug abuse. This study evaluates the possible anticonvulsant and neuroprotective activities of Lob to obtain new information on its properties that could confirm it as a candidate in the treatment of alcohol addiction. The anticonvulsant effect of Lob was evaluated using a pilocarpine-induced seizure model. In addition, possible neuroprotective effects were investigated measuring DNA damage using the comet assay, assessing free radical levels by dichlorofluorescein diacetate (DCF) oxidation, and measuring the antioxidant potential using the α, α-diphenyl-ß-picrylhydrazyl (DPPH) scavenging assay, besides measuring superoxide dismutase (SOD) and catalase (CAT) enzyme activities in brain tissues. Lobeline increased the latency to the first seizure and decreased the percentage of seizures in a similar way as diazepam, used as control. DNA damage induced by Pil and hydrogen peroxide were decreased in hippocampus and cerebral cortex from mice treated with Lob. The levels of free radicals and CAT activity increased in cortex and hippocampus, respectively, in mice treated with Pil. Lobeline decreased CAT in hippocampus, leading to similar values as in the saline negative control. In conclusion, Lob has anticonvulsant and neuroprotective actions that may be mediated by antioxidant-like mechanisms, indicating its potential as candidate drug in alcoholism therapy.

Amantadine mitigates the cytotoxic and genotoxic effects of doxorubicin in SH-SY5Y cells and reduces its mutagenicity.

Amantadine (AMA) is a useful drug in neuronal disorders, but few studies have been performed to access its toxicological profile. Conversely, doxorubicin (Dox) is a well-known antineoplastic drug that has shown neurotoxic effects leading to cognitive impairment. The aims of this study are to evaluate the cytotoxic, genotoxic, and mutagenic effects of AMA, as well as its possible protective actions against deleterious effects of Dox. The Salmonella/microsome assay was performed to assess mutagenicity while cytotoxicity and genotoxicity were evaluated in SH-SY5Y cells using MTT and comet assays. Possible modulating effects of AMA on the cytotoxicity, genotoxicity, and mutagenicity induced by Dox were evaluated through cotreatment procedures. Amantadine did not induce mutations in the Salmonella/microsome assay and decreased Dox-induced mutagenicity in the TA98 strain. AMA reduced cell viability and induced DNA damage in SH-SY5Y cells. In cotreatment with Dox, AMA attenuated the cytotoxicity of Dox and showed an antigenotoxic effect. In conclusion, AMA does not induce gene mutations, although it has shown a genotoxic effect. Furthermore, AMA decreases frameshift mutations induced by Dox as well as the cytotoxic and genotoxic effects of Dox in SH-SY5Y cells, suggesting that AMA can interfere with Dox mutagenic activity and attenuate its neurotoxic effects.

Caenorhabditis elegans as a suitable model to evaluate the toxicity of water from Rolante River, southern Brazil.

Urbanization and agricultural activities increased environmental contaminants. Integrated analysis of water parameters and bioassays represents an essential approach to evaluating aquatic resource quality. This study aimed to assess water quality by microbiological and physicochemical parameters as well as the toxicological effects of water samples on the Ames test and Caenorhabditis elegans model. Samples were collected during (collection 1) and after (collection 2) pesticide application in the upper (S1), middle (S2), and lower (S3) sections of the Rolante River, southern Brazil. Metals were determined by GFAAS and pesticides by UPLC-MS/MS. Bioassays using the Ames test and the nematode C. elegans were performed. Levels of microbiological parameters, as well as Mn and Cu were higher than the maximum allowed limits established by legislation in collection 2 compared to collection 1. The presence of pesticide was observed in both collections; higher levels were found in collection 1. No mutagenic effect was detected. Significant inhibition of body length of C. elegans was found in collection 1 at S2 (P < 0.001) and S3 (P < 0.001) and in collection 2 at S2 (P = 0.004). Comparing the same sampling site between collections, a significant difference was found between the site of collection (F(3,6)=8.75, P = 0.01) and the time of collection (F(1,2)=28.61, P = 0.03), for the S2 and S3 samples. C. elegans model was useful for assessing surface water quality/toxicity. Results suggest that an integrated analysis for the surface water status could be beneficial for future approaches.

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. AIMS OF THE STUDY: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. MATERIALS AND METHODS: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. RESULTS: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. CONCLUSION: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

Evaluation of DNA Damage in HepG2 Cells and Mutagenicity of Garcinielliptone FC, A Bioactive Benzophenone.

Garcinielliptone FC (GFC) is a polyprenylated benzophenone isolated from the hexanic extract of Platonia insignis seeds with potential pharmacological effects on the central nervous system. In a pre-clinical study, this compound showed anticonvulsant action, becoming a candidate to treat epilepsy disorders. However, genotoxicological aspects of GFC should be known to ensure its safe use. This study investigated the cytotoxic, genotoxic, and mutagenic effects of GFC. Cytotoxicity was evaluated using the colorimetric assay of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in human hepatoma cells (HepG2) (2-100 µg/mL) for 3, 6 and 24 hr. The genotoxic and mutagenic potentials were analysed using the alkaline version of the comet assay, the cytokinesis-block micronucleus cytome assay in HepG2 cells, and the Salmonella/microsome assay with the strains TA98, TA97a, TA100, TA102 and TA1535, with and without metabolic activation. GFC concentrations above 50 µg/mL were cytotoxic at all experimental times. Viability of HepG2 cells was higher than 70% after exposure to GFC 2-30 µg/mL for 3 hr in the MTT test. No GFC concentration was mutagenic or genotoxic in the Salmonella/microsome and comet assays. Nuclear division index decreased, indicating the cytotoxic effect of the compound, while micronucleus and nuclear bud frequencies rose after treatment with the highest GFC concentration tested (30 µg/mL). Nucleoplasmatic bridges were not observed. The results indicate that GFC is cytotoxic and mutagenic to mammalian cells, pointing to the need for further studies to clarify the toxicological potentials of this benzophenone before proceeding to clinical studies.