Brosimum gaudichaudii Trecul (Moraceae): Da planta ao medicamento

The chemical, biological, pharmacological and toxicological study of plants used in medical treatments by different societies around the world has been intensifying over the years, primarily in the search for new herbal, homeopathic or even allopathic medicines. Motivated by this interest, we are investigating some Brazilian plant species, among them a tree, Brosimum gaudichaudii Trecul (Moraceae), found in Brazil's savannah-like prairies and scrub-forest (cerrado) and mesophytic forest. Several extracts have been made and separated into fractions and some compounds have been isolated - principally coumarins - and their respective structures analyzed by spectrophotometric and other methods. By collaborating with researchers in other fields, we have been able to carry out various bioassays and even basic clinical tests, namely: determination of anti-helminthic, anti-microbial, photosensitizing, anticancer and mutagenic activities, toxicological and pathogenic studies, with the aim of discovering a genuinely Brazilian herbal or homeopathic medicine.

Effects of diuron on toxicological and hematological parameters and lymphohematopoietic organs in a rat chemical hepatocarcinogenesis model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of insecticides used in corn on immature stages of trichogramma atopovirilia (hymenoptera: trichogrammatidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of the Atibaia River water quality using Lecane bulla as a test organism

In view of the diversity of environments found in the Brazilian territory, it is understandable that the use of native species can provide more relevant information for ecotoxicological studies. The purpose of this work was to evaluate the quality of water samples from the Atibaia River in an area that is under the influence of petroleum refinery using a native test-organism and submitting the data to PCA statistical analysis. Therefore, acute toxicity assays with Lecane bulla (Rotifera) were performed in four locations of the river, as well as physical-chemical analyses. Sampling was drawn in the dry and rainy seasons. The bioassays were static and lasted 48 hours; dead organisms were quantified at the end of the tests. Toxicological differences among the samples/per location and control were compared by means of the Analysis of Variance. Physical-chemical and mortality variables were simultaneously analyzed by multivariate analysis of the principal components and the Pearson correlation coefficient. Water samples from the exit of the refinery stabilization pond (location S.1) were toxic to L. bulla in both seasons, with significant differences in relation to the control and between the seasons. The statistical treatment of data showed that mortality was strong and positively correlated with total hardness, chlorides and EC, which together with pH presented higher values in location S.1, in the dry and in the rainy seasons. Due to its sensibility to the quality of the Atibaia river water samples, the potential use of L. bulla for ecotoxicological studies as an alternative test organism could be demonstrated.

Atrazine levels in the Jaboticabal water stream (Sao Paulo State, Brazil) and its toxicological effects on the pacu fish Piaractus mesopotamicus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hematological changes and cytogenotoxicity in the tilapia Oreochromis niloticus caused by sub-chronic exposures to mercury and selenium

Fish bioassays are valuable tools that can be used to elucidate the toxicological potential of numerous substances that are present in the aquatic environment. In this study, we assessed the antagonistic action of selenium (Se) against the toxicity of mercury (Hg) in fish (Oreochromis niloticus). Six experimental groups with six fish each were defined as follows: (1) control, (2) mercury (HgCl2), (3) sodium selenite (Na2Se4O3), (4) sodium selenate (Na2Se6O4), (5) mercury + sodium selenite (HgCl2 + Na2Se4O3), and (6) mercury + sodium selenate (HgCl2 + Na2Se6O4). Hematological parameters [red blood cells (RBC), white blood cells (WBC), and erythroblasts (ERB)] in combination with cytogenotoxicity biomarkers [nuclear abnormalities (NAs) and micronuclei (MN)] were examined after three, seven, ten, and fourteen days. After 7 days of exposure, cytogenotoxic effects and increased erythroblasts caused by mercury, leukocytosis triggered by mercury + sodium selenite, leukopenia associated with sodium selenate, and anemia triggered by mercury + sodium selenate were observed. Positive correlations that were independent of time were observed between WBC and RBC, ERB and MN, and NA and MN. The results suggest that short-term exposure to chemical contaminants elicited changes in blood parameters and produced cytogenotoxic effects. Moreover, NAs are the primary manifestations of MN formation and should be included in a class characterized as NA only. Lastly, the staining techniques used can be applied to both hematological characterization and the measurement of cytogenotoxicity biomarkers.

Allium cepa and Tradescantia pallida bioassays to evaluate effects of the insecticide imidacloprid

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analytical method for determination of nitric oxide in zebrafish larvae: Toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coil lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae. (C) 2011 Elsevier Inc. All rights reserved.

Applicability of in vitro bioassays for the diagnosis of ivermectin resistance in Rhipicephalus microplus (Acari: Ixodidae)

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of Sao Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R microplus. (C) 2011 Elsevier B.V. All rights reserved.

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.

Ultrasensitive chemiluminescence bioassays based on microfluidics in miniaturized analytical devices

The activity carried out during my PhD was principally addressed to the development of portable microfluidic analytical devices based on biospecific molecular recognition reactions and CL detection. In particular, the development of biosensors required the study of different materials and procedures for their construction, with particular attention to the development of suitable immobilization procedures, fluidic systems and the selection of the suitable detectors. Different methods were exploited, such as gene probe hybridization assay or immunoassay, based on different platform (functionalized glass slide or nitrocellulose membrane) trying to improve the simplicity of the assay procedure. Different CL detectors were also employed and compared with each other in the search for the best compromise between portability and sensitivity. The work was therefore aimed at miniaturization and simplification of analytical devices and the study involved all aspects of the system, from the analytical methodology to the type of detector, in order to combine high sensitivity with easiness-of-use and rapidity. The latest development involving the use of smartphone as chemiluminescent detector paves the way for a new generation of analytical devices in the clinical diagnostic field thanks to the ideal combination of sensibility a simplicity of the CL with the day-by-day increase in the performance of the new generation smartphone camera. Moreover, the connectivity and data processing offered by smartphones can be exploited to perform analysis directly at home with simple procedures. The system could eventually be used to monitor patient health and directly notify the physician of the analysis results allowing a decrease in costs and an increase in the healthcare availability and accessibility.

Stereoselective determination of drugs and metabolites in body fluids, tissues and microsomal preparations by capillary electrophoresis (2000-2010)

During the past two decades, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs and their metabolites in body fluids, tissues and in vitro preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed during the past 10 years and presents an overview of the key achievements encountered in that time period. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and bioanalysis of drug enantiomers of toxicological, forensic and doping interest. Chiral CE was extensively employed for research purposes to investigate the stereoselectivity associated with hydroxylation, dealkylation, carboxylation, sulfoxidation, N-oxidation and ketoreduction of drugs and metabolites. Enantioselective CE played a pivotal role in many biomedical studies, thereby providing new insights into the stereoselective metabolism of drugs in different species which might eventually lead to new strategies for optimization of pharmacotherapy in clinical practice.

Zebrafish (Danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies

Aquatic toxicology is facing the challenge to assess the impact of complex mixtures of compounds on diverse biological endpoints. So far, ecotoxicology focuses mainly on apical endpoints such as growth, lethality and reproduction, but does not consider sublethal toxic effects that may indirectly cause ecological effects. One such sublethal effect is toxicant-induced impairment of neurosensory functions which will affect important behavioural traits of exposed organisms. Here, we critically review the mechanosensory lateral line (LL) system of zebrafish as a model to screen for chemical effects on neurosensory function of fish in particular and vertebrates in general. The LL system consists of so-called neuromasts, composed of centrally located sensory hair cells, and surrounding supporting cells. The function of neuromasts is the detection of water movements that is essential for the fish's ability to detect prey, to escape predator, to socially interact or to show rheotactic behaviour. Recent advances in the study of these organs provided researchers with a broad area of molecular tools for easy and rapid detection of neuromasts dysfunction and/or disturbed development. Further, genes involved in neuromasts differentiation have been identified using auditory/mechanosensory mutants and morphants. A number of environmental toxicants including metals and pharmaceuticals have been shown to affect neuromasts development and/or function. The use of the LL organ for toxicological studies offers the advantage to integrate the available profound knowledge on developmental biology of the neuromasts with the study of chemical toxicity. This combination may provide a powerful tool in environmental risk assessment.

Expert opinion on toxicity profiling--report from a NORMAN expert group meeting.

This article describes the outcome and follow-up discussions of an expert group meeting (Amsterdam, October 9, 2009) on the applicability of toxicity profiling for diagnostic environmental risk assessment. A toxicity profile was defined as a toxicological "fingerprint" of a sample, ranging from a pure compound to a complex mixture, obtained by testing the sample or its extract for its activity toward a battery of biological endpoints. The expert group concluded that toxicity profiling is an effective first tier tool for screening the integrated hazard of complex environmental mixtures with known and unknown toxicologically active constituents. In addition, toxicity profiles can be used for prioritization of sampling locations, for identification of hot spots, and--in combination with effect-directed analysis (EDA) or toxicity identification and evaluation (TIE) approaches--for establishing cause-effect relationships by identifying emerging pollutants responsible for the observed toxic potency. Small volume in vitro bioassays are especially applicable for these purposes, as they are relatively cheap and fast with costs comparable to chemical analyses, and the results are toxicologically more relevant and more suitable for realistic risk assessment. For regulatory acceptance in the European Union, toxicity profiling terminology should keep as close as possible to the European Water Framework Directive (WFD) terminology, and validation, standardization, statistical analyses, and other quality aspects of toxicity profiling should be further elaborated.

[Sense and nonsense of toxicological analyses in the daily clinical routine]

Screening tests for drugs of abuse are regularly used in the clinical routine. These tests identify the targeted substances very differently if tests from different manufacturers are used and sometimes also react positive after the intake of drugs which are not intended to be detected. Therefore, implausible results have to be questioned. A test result can be falsely negative, if a patient has taken a compound which is not detected by the antibody used in the test system. Chromatographic confirmation and screening assays are more laborious to perform and more demanding for the interpretation and are therefore only offered by several specialized clinical laboratories. However, their specificity is excellent and many different compounds can be detected depending on the number of compounds which are part of the mass spectra library used. If the clinical evaluation results in the differential diagnosis of an acute intoxication, screening tests for drugs of abuse can help to identify a single compound or a group of substances. The clinical picture, however, can usually not been explained by a qualitative test result. In addition, there are no published data demonstrating that these tests meaningfully influence triage, treatment, diagnosis or further therapy of a poisoned patient. The quantitative determination of specific compounds in the blood allows for example an appraisal of the prognosis and helps to indicate a specific therapy after intake of acetaminophen or methanol. New designer drugs can not at all be detected by the classic screening tests for drugs of abuse. The have to be identified by chromatographic methods.