Chemical composition, toxicity and larvicidal and antifungal activities of Persea americana (avocado) seed extracts

The present study had the aim of testing the hexane and methanol extracts of avocado seeds, in order to determine their toxicity towards Artemia salina, evaluate their larvicidal activity towards Aedes aegypti and investigate their in vitro antifungal potential against strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis through the microdilution technique. In toxicity tests on Artemia salina, the hexane and methanol extracts from avocado seeds showed LC50 values of 2.37 and 24.13mg mL-1 respectively. Against Aedes aegypti larvae, the LC50 results obtained were 16.7mg mL-1 for hexane extract and 8.87mg mL-1 for methanol extract from avocado seeds. The extracts tested were also active against all the yeast strains tested in vitro, with differing results such that the minimum inhibitory concentration of the hexane extract ranged from 0.625 to 1.25mg L-¹, from 0.312 to 0.625mg mL-1 and from 0.031 to 0.625mg mL-1, for the strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis, respectively. The minimal inhibitory concentration for the methanol extract ranged from 0.125 to 0.625mg mL-1, from 0.08 to 0.156mg mL-1 and from 0.312 to 0.625mg mL-1, for the strains of Candida spp., Cryptococcus neoformans and Malassezia pachydermatis, respectively.

Toxicity of carcinogenic and non-carcinogenic Polycyclic Aromatic Hydrocarbons and their mixtures to aquatic organisms under ecologically-relevant scenarios

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous priority pollutants that tend to be trapped in aquatic sediments due to their high hydrophobicity. Nonetheless, the differential toxicological effects and mechanisms between the various classes of PAHs and their mixtures, as they invariably occur in the environment, are scarcely known, especially under ecologically-relevant scenarios. This thesis aimed at establishing a bridge between the study of mechanistic pathways and environmental monitoring of carcinogenic and non-carcinogenic PAHs, by introducing ecological-relevance in the research with model PAHs. A first bioassay conducted in situ with the mussel Mytilus edulis demonstrated that, dredging operations in harbours increase PAH bioavailability, eliciting genotoxicity, and showed that established environmental guidelines underestimate risk. Subsequent ex situ bioassays were performed with the carcinogenic benzo[b]fluoranthene (B[b]F) and non-carcinogenic phenantrene (Phe), selected following preceding results, and revealed that low-moderate concentrations of these PAHs in spiked sediments induce genotoxic effects to the clam Ruditapes decussatus, therefore contradicting the general notion that bivalves are less sensitive to PAHs than vertebrates due to inefficient bioactivation. Also, it was demonstrated that passive samplers permit inferring on PAH bioavailability but not on bioaccumulation or toxic effects. On the other hand, sea basses (Dicentrarchus labrax), yielded a complex pattern of effects and responses, relatively to genotoxicity, oxidative stress and production of specific metabolites, especially when exposed to mixtures of the PAHs which led to additive, if not synergistic, effects. It was shown that Phe may elicit significant genotoxicity especially in presence of B[b]F, even though the low, albeit realistic, exposure concentrations diluted dose- and time-independent relationships. The present work demonstrated that environmental quality guidelines underestimate the effects of PAHs in realistic scenarios and showed that the significant genotoxic and histopathological effects caused by mixed PAHs may not be reflected by oxidative stress- or CYP-related biomarkers. Besides important findings on the metabolism of PAH mixtures, the work calls for the need to re-evaluate the criteria for assessing risk and for the disclosure of more efficient indicators of toxicological hazard.

Evaluation of larvicidal activity and brine shrimp toxicity of rhizome extracts of Zingiber zerumbet (L.) Smith

Introduction In this study, we used dichloromethane (DCM) and methanol (MeOH) extracts of the Zingiber zerumbet rhizome to evaluate brine shrimp lethality and larvicidal activity on Aedes aegypti and Anopheles nuneztovari mosquitoes. Methods Bioassays were performed by exposing third-instar larvae of each mosquito species to the DCM or MeOH extracts. Results Probit analysis with DCM and MeOH extracts demonstrated efficient larvicidal activity against A. aegypti and A. nuneztovari larvae. Conclusions The DCM and MeOH extracts showed higher activity against A. nuneztovari larvae than against A. aegypti larvae, suggesting that the extracts have species-specific activity.

Effects of piperonyl butoxide on the toxicity of the organophosphate temephos and the role of esterases in the insecticide resistance of Aedes aegypti

IntroductionThe effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated.MethodsA. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined.ResultsAt concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only.ConclusionsThe results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae.

Cholestasis in a murine experimental model: lesions include hepatocyte ischemic necrosis

OBJECTIVE: To establish a murine experimental model of bile duct obstruction that would enable controlled observations of the acute and subacute phases of cholestasis. METHODOLOGY: Adult male isogenic BALB/c mice underwent a bile duct ligation (22 animals) or a sham operation (10 animals). Fifteen days after surgery, or immediately after the animal's death, macroscopic findings were noted and histological study of the liver, biliary tree, and pancreas was performed (hematoxylin-eosin and Masson trichromic staining). RESULTS: Beginning 24 hours after surgery, all animals from the bile duct ligation group presented progressive generalized malaise. All animals presented jaundice in the parietal and visceral peritoneum, turgid and enlarged liver, and accentuated dilatation of gallbladder and common bile duct. Microscopic findings included marked dilatation and proliferation of bile ducts with accentuated collagen deposits, frequent areas of ischemic necrosis, hepatic microabscesses, and purulent cholangitis. Animals from the sham operation group presented no alterations. CONCLUSION: We established a murine experimental model of induced cholestasis, which made it possible to study acute and subacute tissue lesions. Our data suggests that in cholestasis, hepatic functional ischemia plays an important role in inducing hepatic lesions, and it also suggests that the infectious process is an important factor in morbidity and mortality.

Toxicity measurement techniques for building materials with wastes

The innovative Horizon 2020 program sponsored by the European Union (EU) aims to promote and develop processes of waste integration in construction materials. However, several potential health hazards caused by building materials have been identified and, there-fore, there is an ongoing need to develop new recycling methods for hazardous wastes and effi-cient barriers in order to prevent toxic releases from the new construction solutions with wastes. This paper presents an overview that focus on two main aspects: the identification of the health risks related to radioactivity and heavy metals present in building materials and identification of these toxic substances in new construction solutions that contain recycled wastes. Different waste materials were selected and distinct methodologies of toxicity evaluation are presented to analyse the potential hazardous, the feasibility of using those wastes and the achievement of op-timal construction solutions involving wastes.

Analysis of toxicity on Bacillus sphaericus from amazonian soils to Anopheles darlingi and Culex quinquefasciatus larvae

Bioassays under laboratory conditions aiming to determine the larvicidal activity of Bacillus sphaericus were carried out on Anopheles darlingi and Culex quinquefasciatus. In order to estimate the toxicity through median lethal concentration (LC50) and the relative potency of the strains to B. sphaericus standard strain 2362, probit analysis was performed utilizing the POLO-PC program. The findings of LC50 pointed out high effectiveness on strains IB15 (0.040 ppm), IB19 and S1116 (0.048 ppm), IB16 (0.052 ppm) and S265 (0.057 ppm). Strain IB15 presented nearly 50% more potency than strain 2362 in bioassays conducted on A. darlingi. It was observed that IB16 and S1116 strains were the most powerful against C. quinquefasciatus, showing to be about 300-400% stronger than 2362 strain. The results show that laboratory conditioned evaluation can be an important way to select promising bacteria with entomopathogenic action on biolarvicides production for use on mosquitoes breeding sites.

Toxicity and repellency of essential oils to Zabrotes subfasciatus (Boheman) (Coleoptera, Chrysomelidae, Bruchinae) in Phaseolus vulgaris L

The effects of tangerine (Phaseolus vulgaris Blanco), lemon (Citrus medica limonum Lush), pear orange (Citrus sinensis L. Osbeck), red copaiba (Copaifera langsdorffii Desf.), rosemary (Baccharis dracunculifolia De Candole), Eucalyptus (Eucalyptus globulus Labillardière and E. citriodora Hook), lemongrass (Cymbopogon citratus Stapf.) and citronella (Cimbopogon nardus Linnaeus) oils at several concentrations on Zabrotes subfasciatus (Boheman) were studied. In toxicity tests, grains of Phaseolus vulgaris L. cv. Rajadinho were impregnated with oils and infested with adults of Z. subfasciatus up to 24 hours old. All tested oils were effective in reducing the viable egg-laying and adult emergence of this pest, in function of the concentrations used, highlighting E. citriodora and E. globulus oils which caused 100% effectiveness from 0.5 mL Kg-1 concentration. In repellency tests, two arenas consisting of plastic containers, connected symmetrically to a central box by two plastic tubes were used. In one of the boxes, untreated beans were placed and on the other ones beans treated with each oil concentration were used. In the central box, five couples of Z. subfasciatus were released. Grains of P. vulgaris treated with oils of E. citriodora, C. citratus and C. oleifera reduced the attraction percentage of Z. subfasciatus adults, while the E. globulus increased this percentage. The percentages of reduced viable eggs ranged from 17.9% (C. medica limonum) to 93.3% (C. nardus), while the reduction on the number of emerged insects was 23.9% and 95.9%, respectively for these same oils.

Copper toxicity for Scinax ruber and Rhinella granulosa (Amphibia: Anura) of the Amazon: Potential of Biotic Ligand Model to predict toxicity in urban streams

In the last years many populations of anurans have declined and extinctions have been recorded. They were related to environmental pollution, changes of land use and emerging diseases. The main objective of this study was to determine copper sensitivity of the anuran of the Amazon Rhinella granulosa and Scinax ruber tadpoles at stage 25 and Scinax ruber eggs exposed for 96 h to copper concentrations ranging from 15 µg Cu L-1 to 94 µg Cu L-1. LC50 at 96 h of Rhinella granulosa Gosner 25, Scinax ruber Gosner 25 and Scinax ruber eggs in black water of the Amazon were 23.48, 36.37 and 50.02 µg Cu L-1, respectively. The Biotic Ligand Model was used to predict the LC50 values for these species and it can be considered a promising tool for these tropical species and water conditions. Copper toxicity depends on water physical-chemical composition and on the larval stage of the tadpoles. The Gosner stage 19-21 (related to the appearance of external gills) is the most vulnerable and the egg stage is the most resistant. In case of contamination by copper, the natural streams must have special attention, since copper is more bioavailable.

Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Toxicity induced by a metal mixture (Cd, Pb and Zn) in the yeast Pichia kudriavzevii: The role of oxidative stress

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2,7-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.

Predicting hematologic toxicity in patients undergoing radioimmunotherapy with 90Y-ibritumomab tiuxetan or 131I-tositumomab.

This study aimed at identifying clinical factors for predicting hematologic toxicity after radioimmunotherapy with (90)Y-ibritumomab tiuxetan or (131)I-tositumomab in clinical practice. Hematologic data were available from 14 non-Hodgkin lymphoma patients treated with (90)Y-ibritumomab tiuxetan and 18 who received (131)I-tositumomab. The percentage baseline at nadir and 4 wk post nadir and the time to nadir were selected as the toxicity indicators for both platelets and neutrophils. Multiple linear regression analysis was performed to identify significant predictors (P < 0.05) of each indicator. For both platelets and neutrophils, pooled and separate analyses of (90)Y-ibritumomab tiuxetan and (131)I-tositumomab data yielded the time elapsed since the last chemotherapy as the only significant predictor of the percentage baseline at nadir. The extent of bone marrow involvement was not a significant factor in this study, possibly because of the short time elapsed since the last chemotherapy of the 7 patients with bone marrow involvement. Because both treatments were designed to deliver a comparable bone marrow dose, this factor also was not significant. None of the 14 factors considered was predictive of the time to nadir. The R(2) value for the model predicting percentage baseline at nadir was 0.60 for platelets and 0.40 for neutrophils. This model predicted the platelet and neutrophil toxicity grade to within ±1 for 28 and 30 of the 32 patients, respectively. For the 7 patients predicted with grade I thrombocytopenia, 6 of whom had actual grade I-II, dosing might be increased to improve treatment efficacy. The elapsed time since the last chemotherapy can be used to predict hematologic toxicity and customize the current dosing method in radioimmunotherapy.

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by &gt;1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.