Faculty of Health Sciences School of pharmacy 0002280t jacquilalli@yahoo.co.uk / Despite commercial interest and ethnobotanical data, the chemical composition and pharmacological activities of a number of indigenous Pelargonium species remain unexplored. Twenty-one Pelargonium species, from the section Pelargonium, were included in this study. The volatile compounds of 13 species were extracted by hydrodistillation and their chemical compositions determined by gas chromatography coupled to mass spectroscopy (GC-MS). The essential oil data was chemotaxonomically informative confirming taxonomic relationships between P. graveolens and P. radens; P. papilionaceum and P. vitifolium and between P. panduriforme and P. quercifolium. New chemical affinities were established among P. betulinum, P. hispidum and P. scabrum; P. capitatum (provenance WSBG), P. glutinosum and P. quercifolium (provenance SBG) and among P. graveolens, P. radens and P. tomentosum. The non-volatile compounds were extracted with acetone and the extracts were analysed using high performance liquid chromatography (HPLC). The representative flavonoid patterns of the Pelargonium species indicated that P. betulinum, P. capitatum, P. graveolens, P. hispidum, P. panduriforme and P. vitifolium have numerous similarities in their chemical profiles. Pelargonium scabrum and P. sublignosum share definite chemical patterns. The HPLC fingerprints of P. papilionaceum and P. vitifolium were chemically diverse. A microdilution bioassay was performed on the acetone extracts and the essential oils to assess their antimicrobial (both bacterial and fungal) potential. The essential oils and extracts were more selective for the Gram-positive test pathogens than for the Gram-negative bacterium. The crude extracts of P. glutinosum (provenance SBG), P. pseudoglutinosum, P. scabrum and P. sublignosum exhibited considerable antimicrobial activity against the Gram-positive bacteria (B. cereus and S. aureus) with P. pseudoglutinosum exerting the highest activity (MIC = 0.039 mg/ml). The essential oils showed reduced antimicrobial activity compared to the plant extracts. Using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, exceptional anti-oxidant activity was observed for the crude extracts of P. betulinum and P. crispum (IC50 values of 4.13 μg/ml and 4.49 μg/ml, respectively, compared to ascorbic acid, IC50 = 4.72 μg/ml). The essential oils of P. quercifolium showed the greatest inhibition of 5-lipoxygenase activity (IC50 = 33.24 μg/ml v - 38.67 μg/ml). The antimalarial activity of the non-volatile extracts was evaluated against the choloroquine-resistant Gambian FCR-3 strain of Plasmodium falciparum using the hypoxanthine incorporation assay. Pelargonium panduriforme (provenance SBG) exerted the greatest activity (IC50 = 1.34 ± 0.29 μg/ml). Other species possessing similarly potent antimalarial activity included P. citronellum (provenance NBG), P. citronellum (provenance SBG), P. quercifolium (provenance SBG) and P. radens. A microculture tetrazolium salt reduction (MTT) assay was used to determine the cellular toxicity of the acetone extracts and essential oils against transformed human kidney epithelium (Graham) cells. The acetone extracts of P. sublignosum and P. citronellum (provenance NBG) displayed the highest toxicities (IC50 = 11.89 ± 1.54 μg/ml and 19.14 ± 0.98 μg/ml, respectively). Pelargonium vitifolium (IC50 = 178.48 ± 5.44 μg/ml) and P. tomentosum (provenance SBG) (IC50 = 195.13 ± 7.90 μg/ml) appeared to be non-toxic. The Pelargonium essential oils proved to be considerably toxic (IC50 ≤0.10 μg/ml - 30.30 ± 1.81 μg/ml). The flavonoid derivatives detected in the Pelargonium acetone extracts may have contributed to their positive biological activities. The results from the MTT assay suggested that the antimicrobial and antimalarial activity of the extracts may be ascribed to general cytotoxic effects. The pharmacological properties manifested by the extracts and essential oils of certain Pelargonium species substantiates their use in traditional medicines and validates their commercial exploitation in the perfumery, cosmetic, food and pharmaceutical industries; however, their toxicity profiles must be considered

The national programme of expanded access to antiretroviral therapy in the South African public health sector has resulted in hundreds of thousands of South Africans being subjected to prolonged therapy with the risk of adverse drug effects. Among the most common adverse effects are metabolic disorders one of which is mitochondrial toxicity. Mitochondrial toxicity may manifest as hyperlactatemia. The study was designed to determine the frequency with which hyperlactatemia occurs in HIV – infected adults on long-term antiretroviral therapy (ART). The objective was to determine the proportion of patients with blood lactate levels that exceed a predetermined cut-off level and to attempt to relate hyperlactatemia to a set of factors namely, gender, age, obesity, symptoms, type of ART regime and duration of ART use. The study was conducted at an ART clinic in the provincial state hospital of Bongani in the town of Welkom in Free State. The target population was male and female adult patients (18 years and above) on ART for a duration of 1 year or longer. Participants were selected by a random sampling of hospital case file numbers using random table numbers. The patients answered a set of 7 questions on symptoms, underwent weight and height measurements before having blood drawn for lactate assays Blood specimens for lactate assays were processed at the local National Health laboratory.

Faculty of Sciences School of Pharmacy and Pharmacology 0000073k moollaaneesa@yahoo.com / As part of an investigation of the biological activities of South African plants and due to their extensive traditional use and lack of scientific evidence, a phytochemical and pharmacological investigation was performed on 17 indigenous Agathosma species (19 samples). The chemical composition of the essential oils was determined using gas chromatography coupled to mass spectroscopy (GC-MS). Analysis resulted in the identification of 333 compounds. To evaluate the chemical similarities and differences, cluster analysis was used to assess the essential oil composition of the samples. The results showed qualitative and quantitative differences amongst the taxa. The essential oils of Agathosma hirsuta and A. zwartbergense are particularly rich in citronellal, hence they are tightly clustered in the dendrogram obtained from the cluster analysis. Linalool, myrcene and limonene are the major constituents of both A. capensis (Gamka) and A. capensis (Besemfontein). Qualitative and quantitative differences are noted in the chemical compositions of the leaf oils of Agathosma capensis (Gamka) and A. capensis (Besemfontein). Agathosma arida and A. lanata are united in a single cluster due to the compounds β-pinene, linalool and spathulenol being major components in both species. The presence of 1,8-cineole in large quantities in both Agathosma namaquensis (23.5%) and A. ovalifolia (9.7%), unites them in a single cluster. A wide chemical variability for the essential oils of indigenous Agathosma species has been demonstrated. There was considerable variation in the percentage oil yield of the essential oils. Agathosma hirsuta produced the highest yield (1.15%) whilst A. ovalifolia produced the lowest yield (0.16%). vi Previous studies have revealed that the coumarin and flavonoid components of Agathosma species are responsible for their biological activities. High performance liquid chromatography (HPLC) was used to document the non-volatile composition of Agathosma species and to establish if phenolic patterns were present amongst the species. All species were found to be rich in flavonoids (i.e. flavones and flavonols). Many of the compounds detected were common to most of the species. A pure coumarin, puberulin, was identified in the diethyl ether extract of Agathosma ovata (round-leaf) and detected in the dichloromethane and methanol (1:1) extract of A. namaquensis. Agathosma species have been used traditionally to treat a wide variety of infections. They has been used as a cough remedy, for the treatment of colds and flu, kidney and urinary tract infections, for the treatment of cholera and other stomach ailments. Based on the extensive use and lack of scientific evidence, a study was embarked upon to determine its bioactivity. Using the disc diffusion assay as a preliminary screening and thereafter the minimum inhibitory concentration (MIC) assay, the antimicrobial activity of the essential oils and non-volatile compounds was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gram-negative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. All of the extracts proved to be active against the four pathogens tested with the exception of Agathosma bathii which showed poor activity against Klebsiella pneumoniae (MIC value of 32mg/ml). The extracts exhibited stronger activity against the pathogens as compared to the essential oils. Both the essential oils and extracts exhibited higher activity towards the Gram-positive bacteria than the Gram-negative bacterium, with the extract of Agathosma ovata (round-leaf) displaying the greatest vii activity against Staphylococcus aureus (MIC value of 0.156mg/ml) and Bacillus cereus (MIC value of 0.125mg/ml). The extract of Agathosma parva displayed the greatest activity against Candida albicans and Klebsiella pneumoniae (MIC value of 1.5mg/ml). Amongst the essential oils, Agathosma pungens proved to be the most active against the Gram-positive pathogen, Bacillus cereus (MIC value of 3mg/ml). Agathosma collina was the most active against Candida albicans (MIC value of 3mg/ml) whilst A. zwartbergense proved to be the least active against most of the tested pathogens. The antimicrobial activity of the essential oils may be ascribed to oxygenated constituents, such as 1,8-cineole, linalool and carvacrol. The activity of the extracts may be ascribed to constituents such as flavonoids, coumarins and alkaloids. Due to the availability and accessibility of Agathosma ovata, a seasonal variation study was performed on the chemical composition of the essential oils and how this may impact on the antimicrobial activity. Furthermore, this species has recently been earmarked for commercial development by the flavour and fragrance industry and information on variability is required to establish the harvesting protocol. Ten samples were harvested in total. There was a substantial variation in the oil yield throughout the year, ranging from 0.23% in early Spring to 0.85% in late Autumn. A higher yield was observed during the flowering season as compared to the non-flowering season. Oil yields were low during Summer (0.44%-0.48%) which may have been due to the low oil content in stems and higher proportion of stems after flowering. The proportion of oil-rich green leaves also decreased markedly, hence affecting the yield. Overall the yields were dependant on the season harvested and proportion of plant parts distilled. viii The chemical composition of the essential oils was determined using GC-MS and resulted in the identification of 145 compounds in 10 of the samples. All samples contained a large number of common monoterpenes and had very similar compositions, with minor quantitative variation. Some components common to all samples include: sabinene, p-cymene, β-pinene, α-pinene, α-thujene, myrcene, limonene, linalool and terpinen-4-ol. Sabinene was found to be the most dominant component in all samples, ranging between 25.6% and 44.4%. Myrcene levels dropped sharply between the beginning of Spring and end of Summer, from 14.9% to 1.0%. β-pinene followed a similar trend, peaking during Spring and decreasing during the Summer months. The lowest levels of linalool (4.3%), myrcene (1.0%), β-pinene (3.9%), limonene (1.9%) and sabinene (25.6%), occurred during the Summer months when the temperatures were high. There was a Springtime increase in the levels of β- pinene, terpinen-4-ol, linalool, sabinene, limonene and p-cymene in the non-flowering Agathosma ovata. These changes may have been due to the higher proportion of young leaves during Spring, which may have oil compositions slightly different to those of mature leaves. A rare thiol derivative (tr) that could not be identified was detected in the March sample. Many of the changes were associated with flowering and the results obtained reveal that the chemical composition of the essential oil of Agathosma ovata is subject to seasonal variation. Using the MIC assay, the antimicrobial activity of the essential oils was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gramnegative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. The study demonstrated differences in the potency of antimicrobial activity of the essential oils distilled each month. The Winter samples were more active against Bacillus cereus, Staphylococcus aureus and Klebsiella pneumoniae. Activity in mid Spring ix was greater against Staphylococcus aureus (MIC value of 3mg/ml) and Klebsiella pneumoniae (MIC value of 3mg/ml), whilst activity decreased in Summer. There was a correlation between the concentrations of the active compounds each month and the oils antimicrobial activity. The results reveal that the antimicrobial activity of the essential oil of Agathosma ovata may not depend on the level of one component but rather the ratio of several components. ‘Buchu’ has been used traditionally as a general tonic and medicine. Tonics generally have a high anti-oxidant content in order to promote the overall well-being of the user. The anti-oxidant properties of the essential oils and non-volatile compounds was investigated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Only the non-volatile compounds exhibited activity. Their activities may be ascribed to the flavonoid components. Most of the species portrayed moderate to poor activity in the DPPH assay with the exception of Agathosma capensis (Gamka) (IC50 value of 24.08 + 4.42μg/ml) and A. pubigera (IC50 value of 35.61 + 0.86μg/ml) which were two of the most active species, although their activities were inferior when compared to vitamin C. The results from the ABTS assay differed from that of the DPPH assay. All extracts showed greater activity in this assay with Agathosma namaquensis (IC50 value of 15.66 ± 4.57μg/ml) and A. capensis (Besemfontein) (IC50 value of 19.84 ± 0.09μg/ml) being the most active species. This may be due to the ABTS assay having an additional reaction system. ‘Buchu’ has been used traditionally as an antipyretic, topically for the treatment of burns and wounds and for the relief of rheumatism, gout and bruises. The antix inflammatory activity of the essential oils and non-volatile compounds was assessed using the 5-lipoxygenase (LOX) assay. Only the essential oils exhibited activity. All proved to be active with the exception of Agathosma stipitata which was UV active and caused interference. This was due to its major compounds neral (39.9%) and geranial (10.1%) which absorbed strongly at 234 nm and hence rendered its spectrophotometric measurement impossible. The essential oil of Agathosma collina displayed the most promising activity (IC50 value of 25.98 ± 1.83μg/ml). It is well known that many herbal medicines can have adverse effects, in which case it is necessary to evaluate the benefit-risk profile. The toxic effects of Agathosma species have been poorly studied and no information is available in this regard. Hence the toxicity profile of the non-volatile compounds and essential oils was assessed on transformed human kidney epithelium (Graham) cells using the microculture tetrazolium (MTT) cellular viability assay. The extracts of Agathosma lanata (IC50 value of 26.17 ± 9.58μg/ml) and A. ovata (round-leaf) (IC50 value of 25.20 ± 6.30μg/ml) proved to be the most toxic, whilst the extracts of Agathosma bathii, A. capensis (Besemfontein), A. betulina, A. crenulata and A. namaquensis did not prove to be toxic at the concentrations tested. Serial dilutions displayed different inhibitions of cell growth and the species proved to be toxic in a dose-dependant manner. The essential oils of all 19 species proved to be much more toxic (IC50 values < 0.0001μg/ml) than a plant-derived compound that is considered relatively safe, namely quinine (IC50 value of 136.06 ± 4.06μg/ml). The toxicities of the essential oils may be due to compounds like methyl chavicol, eugenol, methyl eugenol, pulegone and methyl salicylate whilst the toxicities of the extracts may be due to the alkaloid and coumarin components.

ABSTRACT Twenty essential oil constituents (EOC′s) from seven structural groups were tested for their antimalarial, antimicrobial (both bacterial and fungal), anti-oxidant, anticholinesterase and toxicity properties. To test for their antimalarial property, the tritiated hypoxanthine incorporation assay was used, while the disc diffusion and minimum inhibitory concentration (MIC) microplate assays were employed for the antimicrobial properties. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to test the anti-oxidant property and their toxicity profile was assessed with the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay. The anticholinesterase activity was determined using the thin layer chromatography (TLC) bioautographic method. The EOC´s were found to inhibit the growth of Plasmodium falciparum with IC50 values ranging between 0.9 to 1528.8μM with E- and Z-(±)-nerolidol, (-)-pulegone, (+)-α-pinene and linalyl acetate being the most active. In combination p-cymene (the least active) and E- and Z-(±)-nerolidol (the most active) displayed the most synergistic interaction (ΣFIC = 0.09), with their antimalarial activity comparable to that of the interaction between E- and Z-(±)-nerolidol and quinine (ΣFIC = 0.01). Eugenol had the most favourable safety index and was the only EOC with anti-oxidant activity comparable to vitamin C. Combination studies showed that E- and Z-(±)-nerolidol and (-)-pulegone or quinine, p-cymene and γ-terpinene or (-)-pulegone potentiated each other′s toxicity. The EOC´s inhibited the growth of Gram-positive, Gram-negative bacteria and yeast with MIC values ranging from 1.66 to >238.4mM. When combined, synergism was observed between (+)-β-pinene and carvacrol or γ-terpinene; γ-terpinene and geranyl acetate when tested against Staphylococcus aureus, while (+)-β-pinene and (-)-menthone showed antagonism against C. albicans. The combinations of EOC′s and a standard antimicrobial resulted in synergistic interactions between carvacrol and ciprofloxacin against Bacillus cereus, eugenol and ciprofloxacin against Eschericia coli, carvacrol and amphotericin B against C. albicans. The trans-geraniol and E- and Z-(±)-nerolidol combination demonstrated an additive interaction against B. cereus, while for eugenol and E- and Z-(±)-nerolidol an indifferent interaction against E. coli was noted. These results show that the biological activities of EOC′s can vary when used alone and in combination. They do have the potential to be used as templates for novel drugs and as adjuncts to modern medicines in the combat against drug resistance.

Antes de se iniciar os estudos clínicos de uma nova droga, é necessário realizar uma bateria de testes de segurança, para avaliar o risco humano. Os radiofármacos como qualquer outra nova droga, devem ser testados levando em conta sua especificidade, duração de tratamento e principalmente a toxicidade de ambas as partes, a molécula não marcada e a sua radioatividade em si, além das impurezas provindas da radiólise. Órgãos regulatórios como o Food and Drug Administration-EUA (FDA) e a Agência de Medicina Européia (EMEA), estabelecem guias para a regulamentação de produção e pesquisas de radiofármacos, No Brasil a produção de radiofármacos não era regulamentada até o final de 2009, quando foram estabelecidas pela Agência Nacional de Vigilância Sanitária (ANVISA) as resoluções nº 63, que visa as Boas Práticas de Fabricação de Radiofármacos e a nº 64 que visa o registro do radiofámaco. Para a obtenção do registro de radiofármacos são necessárias a comprovação da qualidade, segurança, eficácia e especificidade do medicamento. Para a segurança dos radiofármacos devem ser apresentados estudos de toxicidade aguda, subaguda e crônica como também a toxicidade reprodutiva, mutagênica e carcinogênica. Hoje o IPEN-CNEN/SP produz um dos radiofámacos mais importantes da medicina nuclear, o 18F-FDG, que é utilizado em muitas aplicações clínicas, em particular no diagnóstico e estadiamento de tumores. O objetivo deste trabalho foi avaliar a toxicidade sistêmica (aguda/subaguda) do radiofármaco 18F- FDG em um sistema teste in vivo, conforme preconiza a RDC nº 64, que servirá de modelo para os protocolos de toxicidade dos radiofármacos produzidos no IPEN. Os ensaios realizados foram: os testes de toxicidade aguda e de toxicidade subaguda, estudos de biodistribuição do 18F-FDG, ensaio cometa e toxicidade reprodutiva. Na toxicidade aguda, ratos sadios foram injetados com 18F- FDG e observados durante 14 dias enquanto na toxicidade subaguda os animais foram observados durante 28 dias. Os resultados não mostraram nenhuma evidência de toxicidade na exposição ao 18F-FDG na toxicidade aguda e na subaguda. A biodistribuição demonstrou resultados semelhantes aos da literatura, onde a bexiga é o órgão que mais recebe radiação. O ensaio cometa mostrou que a radiação do radiofármaco não foi significativa para gerar danos no DNA. Na toxicidade reprodutiva, casais de ratos expostos ao 18F-FDG geraram filhotes completamente normais e saudáveis. Por fim, o 18F-FDG não evidenciou nenhuma toxicidade. / Before starting clinical trials of a new drug, it is necessary to perform a battery of safety tests for assessing human risk. Radiopharmaceuticals like any new drug must be tested taking into account its specificity, duration of treatment and especially the toxicity of both parties, the unlabeled molecule and its radionuclide, apart from impurities emanating from radiolysis. Regulatory agencies like the Food and Drug Administration - USA (FDA) and the European Medicine Agency (EMEA), establish guidelines for the regulation of production and research of radiopharmaceuticals. In Brazil the production of radiopharmaceuticals was not regulated until the end of 2009, when were established by the National Agency for Sanitary Surveillance (ANVISA) resolutions No. 63, which refers to the Good Manufacturing Practices of Radiopharmaceuticals and No. 64 which seeks the registration of record radiopharmaceuticals. To obtain registration of radiopharmaceuticals are necessary to prove the quality, safety, efficacy and specificity of the drug . For the safety of radiopharmaceuticals must be presented studies of acute toxicity, subacute and chronic toxicity as well as reproductive, mutagenic and carcinogenic. Nowadays IPEN-CNEN/SP produces one of the most important radiopharmaceutical of nuclear medicine, the 18F-FDG, which is used in many clinical applications, particularly in the diagnosis and staging of tumors. The objective of this study was to evaluate the systemic toxicity (acute/ subacute) radiopharmaceutical 18F-FDG in an in vivo test system, as recommended by the RDC No. 64, which will serve as a model for protocols toxicity of radiopharmaceuticals produced at IPEN . The following tests were performed: tests of acute and subacute toxicity, biodistribution studies of 18F-FDG, comet assay and reproductive toxicity. In acute toxicity, healthy rats were injected with 18F-FDG and observed for 14 days while in subacute toxicity animals were observed for 28 days. The results showed no evidence of toxicity at exposure 18F-FDG in acute and subacute toxicity. The biodistribution showed similar results to the literature, where the bladder is the organ that receives the most radiation. The comet assay showed that the radiation from the radiopharmaceutical was not significant to generate DNA damage. In reproductive toxicity in coupled rats exposed to 18F-FDG generated completely normal and healthy puppies. Finally, the 18F-FDG did not show any toxicity.

O bisfenol A (BPA) e o nonilfenol (NP), presentes em detergentes, pesticidas, plásticos e resinas, são conhecidos como Endocrine Disrupting-Chemicals (EDCs), Disruptores ou Desreguladores Endócrinos (DE) ou ainda Perturbadores Endócrinos ou Interferentes Endócrinos. O desregulador endócrino pode ser definido como uma substância química exógena ou mistura, que promove alterações em uma ou mais funções do sistema endócrino e na sua estrutura, causando, conseqüentemente, efeitos adversos na saúde de um organismo, ou a sua descendência. Esses compostos estão presentes nas águas de abastecimento, nos efluentes domésticos e industriais. Os desreguladores endócrinos têm ação mimética aos hormônios tanto no sítio de ligação quanto nos efeitos provocados nos seres vivos a eles expostos. Os compostos bisfenol A e nonilfenol foram identificados como desreguladores endócrinos em inúmeros trabalhos em diversos países, porém sendo pouco os efeitos em organismos tropicais. Este estudo teve como objetivo avaliar a toxicidade aguda do bisfenol A e do nonilfenol para Daphnia similis e Ceriodaphnia silvestrii, bem como, a toxicidade crônica para Ceriodaphnia silvestrii e Chironomus xanthus. Nos testes de toxicidade aguda com BPA os valores de CE(I)50; 48h foram de 10,64 mg/L para Daphnia similis e 19,9 mg/L para Ceriodaphnia silvestrii. Nos testes crônicos o valor do CEO obtido para Ceriodaphnia silvestrii foi 1,29 mg/L e para Chironomus xanthus 12 mg/L e o CENO 6 mg/L. Nos testes de toxicidade aguda feitos com NP, os valores de CE(I)50; 48h foram de 0,309 mg/L para Daphnia similis, 0,4520 mg/Lpara Ceriodaphnia silvestrii com solvente água e 0,0541 mg/L com solvente etanol, e 0,03398 mg/L para Ceriodaphnia dubia. O valor do CEO para Ceriodaphnia silvestrii foi 0,0198 mg/L e para Chironomus xanthus foi 100 \'mü\'g/g, com CENO de 50 \'mü\'g/g. Apesar das concentrações encontradas nos testes serem maiores que as encontradas nos estudos que quantificaram esses compostos no ambiente, outros estudos demonstraram que, mesmo em concentrações inferiores as obtidas nesse estudo já ocorrem problemas relacionados com o tempo da muda e com a androgenização do metabolismo de cladóceros. Além disso, a partir dos resultados obtidos com estes testes, pretende-se alertar o poder público sobre os riscos inerentes da presença destes produtos químicos nas águas e da urgência em se adotar novas técnicas no tratamento de efluentes visando à remoção eficaz desses poluentes. / Bisphenol A (BPA) and nonylphenol (NP), present in detergents, pesticides, plastics and resins, are known as Endocrine-Disrupting Chemicals (EDCs), Disruptors or Endocrine Disruptors (ED), Endocrine Disruption or Interferences Endocrine. The endocrine disruptors can be defined as an exogenous chemical substance or mixture, which causes changes in one or more functions of the endocrine system and its structure, causing, therefore, adverse effects on the health of an organism, or its progeny. These compounds are present in the water supply in domestic and industrial effluents. EDs have mimetic action to hormones in both binding site as the effects caused in living beings exposed to them. The compounds bisphenol A and nonylphenol were identified as endocrine disruptors in numerous studies in many countries, but with little effects on tropical organisms. This study aimed to evaluate the acute toxicity of bisphenol A and nonylphenol Daphnia similis and Ceriodaphnia silvestrii, as well as chronic toxicity to Ceriodaphnia silvestrii and Chironomus xanthus. In acute toxicity tests with BPA values EC(I) 50, 48 h were 10.64 mg/L for Daphnia similis and 19.9 mg/L for Ceriodaphnia silvestrii; chronic In the tests the value obtained for the CEO Ceriodaphnia silvestrii was 1.29 mg/L and 12 mg/L Chironomus xanthus CENO and 6 mg/L. In acute toxicity tests made with NP values EC(I) 50; 48h were 0.309 mg/L for Daphnia similis, 0.4520 mg/L for Ceriodaphnia silvestrii solvent and water with 0.0541 mg/L ethanol solvent and 0.03398 mg/L for Ceriodaphnia dubia. The value of the CEO to Ceriodaphnia silvestrii was 0.0198 mg/L and Chironomus xanthus was 100 \'mü\'g/g, with CENO was 50 \'mü\'g/g. Although the found concentrations in the tests are higher than those found in studies to have quantified these compounds in the environment, other studies showed that at concentrations lower than those obtained in this study have problems occur with time and the change of the metabolism of Cladocera androgenization. Also, based on the results obtained with these tests, is intended to alert the public on the risks associated of the presence of these chemicals in the water and the urgency in adopting new techniques in wastewater treatment aiming at the effective removal of these pollutants in water and in the sediment.

As atividades humanas vêm aumentando dramaticamente a quantidade de nitrogênio inorgânico liberado nos ecossistemas, seja através da aplicação de fertilizantes na agricultura, da descarga de dejetos humanos e de seus rebanhos, ou da queima de combustíveis fósseis. Os excessos de nitrogênio são eventualmente transportados para corpos d´água, onde podem, na forma de nitrato, nitrito e amônio, atingir concentrações tóxicas para organismos aquáticos. Nesta pesquisa tive dois objetivos principais. O primeiro foi testar em laboratório a toxicidade relativa dos íons nitrato, nitrito e amônio, e a variação interespecífica na sensibilidade a esses íons, em larvas de cinco espécies de anuros (Rhinella ornata, Hypsiboas faber, Hypsiboas pardalis, Physalaemus cuvieri e Physalaemus olfersii ). Para isso utilizei bioensaios seguindo protocolos internacionalmente padronizados para testes de ecotoxicidade com organismos aquáticos, e que portanto permitem máximas reprodutibilidade e comparabilidade de resultados entre compostos, espécies, e laboratórios. No entanto, estes bioensaios carecem de realismo uma vez que simulam um cenário de exposição aguda a altas concentrações de contaminantes quando na natureza o cenário de exposição tende a ser crônico e prolongado a baixas concentrações. Além disso, bioensaios usam mortalidade como principal variável de resposta, quando também efeitos subletais podem influenciar a persistência de populações ao modular o sucesso dos indivíduos. Por isso, meu segundo objetivo foi testar em laboratório se concentrações relativamente baixas e ecologicamente relevantes de nitrato, nitrito e amônio podem afetar a sobrevivência, o crescimento, o desenvolvimento e o comportamento das larvas de R. ornata, P. cuvieri e H. faber. Demonstrei através dos bioensaios de exposição aguda que nitrato, a forma mais abundante na natureza, é de baixa toxicidade quando comparada a nitrito e amônio. Demonstrei também que há significativa variação interespecífica na sensibilidade ao nitrogênio inorgânico, e que o ranqueamento de sensibilidade das espécies ao nitrato e ao nitrito foram similares, possivelmente por conta de mecanismos comuns de ação tóxica. Através de experimentos de exposição crônica demonstrei que concentrações relativamente baixas de nitrogênio inorgânico podem causar efeitos letais e subletais às larvas de anuros se houver exposição prolongada. O nitrato causou redução no desenvolvimento larval de P. cuvieri e o amônio na sobrevivência e nas taxas de atividade nos girinos de H. faber. A exposição crônica ao nitrito também reduziu significativamente a sobrevivência das três espécies testadas, o crescimento de H. faber e as taxas de atividade de R. ornata. Contudo, é improvável que as concentrações de nitrito que manipulei em laboratório sejam comuns na natureza, especialmente em condições aeróbicas. Esta pesquisa, além de fornecer importantes informações sobre os possíveis efeitos da poluição por nitrogênio em larvas de anuros, contribui para o avanço da ecotoxicologia no Brasil ao estabelecer as bases para o emprego de espécies nativas de anfíbios como sistema-modelo experimental. Estudos futuros que almejem avaliar o risco ambiental da contaminação por nitrogênio deverão por um lado monitorar concentrações em hábitats naturais e por outro avaliar as consequências das interações sinérgicas entre nitrogênio inorgânico e outros estressores físicos, químicos ou biológicos para larvas de anfíbios. / Human activities dramatically increased the amount of inorganic nitrogen released in ecosystems through the application of fertilizers in agriculture, the generation of human and livestock waste, and the combustion of fossil fuels. This nitrogen eventually reaches water bodies where it can, in the form of nitrate, nitrite and ammonium, be toxic to aquatic organisms. In this study I had two main objectives. The first was to test the relative toxicity of nitrate, nitrite and ammonium, and the interspecific variation in sensitivity to these ions, in tadpoles of five anuran species (Rhinella ornata, Hypsiboas faber, Hypsiboas pardalis, Physalaemus cuvieri and Physalaemus olfersii ). This objective was accomplished by laboratory bioassays following internationally standardized protocols for ecotoxicity tests with aquatic organisms, therefore allowing maximum reproducibility and comparability of results among compounds, species and laboratories. However, these bioassays lack realism for simulating a scenario of acute exposure to high concentrations of contaminants, while exposure in nature tends to be chronic and prolonged at low concentrations. Furthermore, bioassays use mortality as the main response variable, whereas sublethal effects may also influence the persistence of populations by modulating individual success. My second objective was therefore to test in the laboratory if low and environmentally relevant concentrations of nitrate, nitrite and ammonium affect survival, growth, development and behavior of R. ornata, P. cuvieri and H. faber larvae. Through acute exposure bioassays I demonstrated that nitrate, the most abundant N form in nature, has low toxicity when compared to nitrite and ammonium. I also demonstrated that there is significant interspecific variation in the sensitivity to inorganic nitrogen, and that the ranking of species sensitivity to nitrate and nitrite were similar, possibly due to common mechanisms of toxic action. Through chronic exposure I demonstrated that relatively low concentrations of inorganic nitrogen can cause lethal and sublethal effects on anuran larvae if there is extended exposure. Nitrate decreased developmental rate in P. cuvieri and ammonia decreased survival and activity rates in H. faber tadpoles. Chronic exposure to nitrite also significantly reduced survival of all three species tested, growth of H. faber and activity rates of R. ornata. However, it is unlikely that the concentrations of nitrite manipulated in the laboratory are common in nature, especially in aerobic conditions. This is the first study to document deleterious effects of nitrogen pollution to Brazilian amphibian species, and contributes to the development of ecotoxicology in Brazil by establishing the basis for the employment of native amphibians as model experimental system. Future studies that aim to assess the environmental risk of nitrogen contamination should monitor concentrations in natural habitats and evaluate the effects of synergistic interactions between inorganic nitrogen and other physical, chemical or biological stressors to amphibian larvae.

Normalmente, a causa da toxicidade do efluente de uma estação de tratamento de esgotos não é conhecida. Devido à complexidade dos efluentes não domésticos por ela recebidos, um programa de redução de toxicidade deve ser implementado, contemplando desde a descarga no corpo d?água até a fonte de poluição. A identificação do(s) poluente(s) que causa(m) impacto na estação e o mecanismo de toxicidade, tipicamente, não são simples de serem determinados a partir de dados de caracterização tradicional de águas residuárias. Além disso, a toxicidade pode estar ligada à presença de compostos refratários ou sub-produtos gerados na própria estação de tratamento de esgotos. Devido ao efeito sinérgico dos poluentes, a metodologia dos limites locais nem sempre pode ser aplicada. Desta forma, o presente trabalho foi desenvolvido com o objetivo de determinar as classes de poluentes presentes na água residuária de uma indústria química, que causa toxicidade e inibição na estação de tratamento de esgotos de Suzano (ETE Suzano), sistema convencional de lodos ativados, localizada na Região Metropolitana de São Paulo, e propor, a partir dos resultados obtidos, tecnologias de pré-tratamento para o referido efluente. Ensaios de fracionamento da água residuária industrial seguidos de testes de avaliação da toxicidade refratária modificados por Ferraresi (2001) foram realizados, utilizando-se um reator que simulava as condições atuais da estação e outros dois que avaliavam a condição futura, nas condições críticas de máxima vazão do efluente industrial pré-tratado e mínima da ETE-Suzano. Os resultados dos ensaios de fracionamento indicaram que compostos voláteis, adsorvíveis e material particulado eram os principais responsáveis pela inibição à nitrificação e pela toxicidade conferida ao efluente final da ETE-Suzano. Desta forma, arraste com ar, adsorção em carvão e coagulação, floculação e sedimentação ou flotação com ar dissolvido são tecnologias que podem ser utilizadas como pré-tratamento do efluente industrial estudado. / Typically, the cause of effluent aquatic toxicity is unknown. Due to this and the complex makeup and varying toxicants in individual waste streams, a toxicity reduction program must usually begin at the discharge and proceed upstream to find the source. The key toxicant(s) and the actual toxic mechanism are normally not apparent from traditional characterization data and may consist of refractory organics or by-products generated in biological treatment process. Due to the complex interaction between toxicants, threshold concentration for specific compounds cannot be universally applied. The research work was carried out to identify classes of pollutants from chemical industry effluent that causes toxicity and inhibition at Suzano Wastewater Treatment Plant (Suzano WTP), a conventional activated sludge process, located at São Paulo Metropolitan Region and to suggest based on results, pretreatment technologies to this wastewater. Methods applied in effluent treatment scenarios (e.g., air stripping, powdered carbon, coagulation, flocculation and sedimentation) and Refractory Toxicity Assessment (RTA) according Ferraresi (2001) were used. A reactor simulated the real condition of Suzano WTP and two others, future condition with maximum flow rate of treated industrial wastewater and minimum flow rate of sewage. Volatile compounds, adsorbed and particulate material present in industrial effluent caused inhibition in activated sludge process and effluent WTP toxicity. Thus, air stripping, carbon adsorption and coagulation, flocculation and sedimentation or air dissolved flotation were efficient technologies to pretreat this industrial effluent.

O tecido ósseo é o maior sítio de acúmulo de flúor (F) do nosso organismo. Parte do F ingerido pode transitar através de um mecanismo de estado estacionário entre cristais da superfície óssea e plasma, ou quando da remodelação óssea, mantendo constantes os níveis plasmáticos algum tempo após a diminuição da ingestão de F. O objetivo deste estudo foi avaliar o período de tempo para que o F acumulado no osso de ratos, após uma exposição aguda, seja removido. Três grupos experimentais com 10 ratos Wistar (70 dias de idade) receberam, por via gástrica, dose única de 50 mg F/Kg peso corporal (como NaF) e 3 grupos controle, também com 10 animais cada, receberam água deionizada. A eutanásia dos animais ocorreu após 30, 90 ou 180 dias da administração de F. Os ratos foram anestesiados, sangue e fêmures removidos e analisados quanto à concentração de F. Para o fêmur, foi analisado o F solúvel em ácido removido por biópsia da superfície óssea, além do F presente no osso total, após calcinação. As cinzas e o plasma foram analisados para o F com o eletrodo, após difusão facilitada por HMDS. Os dados foram analisados pelo teste de Kruskal-Wallis, seguido pelo teste de Dunn (p<0,05). Os níveis plasmáticos de F do grupo controle foram constantes ao longo do tempo, e similares aos do grupo experimental eutanasiado aos 30 dias. Para os grupos experimentais eutanasiados após 90 e 180 dias, houve uma redução significativa nos níveis plasmáticos de F em relação ao grupo controle. A concentração de F na superfície do fêmur para o grupo experimental foi significativamente maior em relação ao grupo controle no tempo de eutanásia de 30 dias apenas, sendo que para os demais períodos experimentais, apesar de a concentração de F ser maior no grupo experimental quando comparado ao controle, esta diferença não foi estatisticamente significativa. Para ambos os grupos pôde ser observado um aumento nos níveis de F na superfície óssea longo do tempo. Para o osso total, o grupo experimental apresentou concentrações similares de F nos diferentes tempos de morte, e estes níveis foram similares aos encontrados para o grupo controle, no tempo de eutanásia de 180 dias. O grupo controle, nos tempos de morte 30 e 90 dias, apresentou valores significativamente menores quando comparado às demais situações. Os dados sugerem que o F incorporado à superfície óssea de ratos a partir de uma exposição aguda não é irreversivelmente ligado, mas vai sendo perdido ao longo do tempo, parecendo ser completamente perdido entre 90 e 180 dias após a exposição. Já os níveis de F incorporados no osso total 30 dias após uma ingestão aguda subletal não parecem ser perdidos ao longo do tempo. A não ocorrência de aumento ao longo do tempo nos níveis de F no osso total do grupo experimental sugere que existe um limite para a incorporação de F no osso total, provavelmente relacionado ao número de sítios possíveis para ligação irreversível do F. / Bone is the major site for fluoride (F) accumulation in the body. Part of the ingested F can transit between bone surface crystals and plasma, through a steady-state mechanism, which can also occur during bone remodeling. This could maintain constant plasma F levels for some time after the F intake is reduced. The aim of this study was to evaluate the lag time required to remove F accumulated in bone of rats after acute exposure. Three experimental groups, containing ten 70-day-old Wistar rats each, received, by gastrogavage, a single dose of 50 mgF/Kg body weight (as NaF), while 3 control groups received deionized water. The animals were euthanized 30, 90 or 180 days after F administration. The animals were anesthetized, blood and femurs were collected and analyzed for F. F on the femur surface was removed through an acid biopsy and F in whole bone was analyzed after ashing. Ash and plasma F were analyzed with the electrode following HMDS-facilitated diffusion. Data were analyzed by Kruskal-Wallis and Dunn\'s tests (p<0.05). Plasma F levels in control group were constant along time and similar to the levels found for the experimental group euthanized after 30 days. For the experimental groups euthanized after 90 and 180 days, a significant reduction in plasma F levels in respect to control was found. F in bone surface for the experimental group was significantly higher than for the control group only 30 days after F administration. For the other experimental times, despite F concentration was higher in the experimental group when compared to control, this difference was not significant. For both groups an increase in bone surface F levels along time was seen. For whole bone, the experimental group had similar F concentrations for all times of euthanasia and these levels were similar to those found for control group 30 days after F administration. Control group, 30 and 90 days after F administration had values significantly lower when compared to the other situations. Data suggest that F incorporated into bone surface of rats after acute exposure does not seem to be irreversibly bound and is lost along time. It seems to be completely lost between 90 and 180 days after F administration. On the other hand, F levels incorporated in whole bone 30 days after acute exposure to F do not seem to be lost along time. The lack of increase in whole bone F levels in the experimental group along time suggests that there is a limit for F uptake in whole bone, probably related to the number sites available to bind F irreversibly.

O manganês é um elemento traço essencial, porém existe uma preocupação com seus potenciais efeitos neurotóxicos associados à exposição a níveis excessivos, podendo provocar uma síndrome conhecida como manganismo, cujos sintomas são semelhantes aos da doença de Parkinson. A maioria dos trabalhos envolvendo manganês usa espécies de Mn(II), mas sabe-se que Mn(III) é acumulado em maior quantidade no cérebro. Nesse sentido, foi feito um estudo dos danos oxidativos e de toxicidade provocados por três complexos de Mn(III): citrato, pirofosfato e salicilenodiamina (respectivamente MnCit, MnPPi e EUK8). Para tanto, as três espécies foram sintetizadas e caracterizadas por métodos espectroscópicos. Em seguida foram determinadas suas capacidades pró-oxidantes sobre os seguintes marcadores: dihidrorodamina (DHR), tirosina (Tyr), albumina (BSA) e dopamina (DA). Finalmente, seu efeito sobre células cerebelares e da cepa HeLa estressada por meio de irradiação UV também foi avaliado, e foi usado ascorbato na tentativa de tratar o dano sobre células HeLa. O teste com a DHR também foi feito em presença de H2O2 e ascorbato. A capacidade pró-oxidante testada por fluorescência da DHR sugere que o ascorbato atua como anti-oxidante. Além disso, MnCit e MnPPi (mas não EUK8), quando na presença de H2O2, são menos oxidantes. O mesmo comportamento foi percebido nas medidas de fluorescência de Tyr. A carbonilação da BSA, verificada pela absorbância do seu marcador (DNPH), seria indício de capacidade oxidante dos complexos, mas não percebeu-se variação significativa de grupos C=O na proteína após tratamento com espécies de Mn(III), mesmo em amostras com H2O2, embora notem-se as mesmas tendências apresentadas pelos complexos com DHR e Tyr. Estudos de oxidação de DA por luminescência tiveram resultados inconclusivos, mas dados mais concretos em testes com medidas de absorbância de soluções de DA e fluorescência de misturas de DA com DHR indicaram que DA é preferencialmente oxidada por todos os compostos. A viabilidade celular de culturas de células neuronais granulares (CGC) mostrou pouca diferença entre as toxicidades dos compostos, mas verifica-se uma relação inversamente proporcional entre as toxicidades e lipofilicidades dos complexos. O mesmo não ocorre nos experimentos com HeLa, cuja viabilidade foi avaliada por contagem de colônias após fixação e coloração das células, pois nesse caso o EUK8 se mostrou o mais tóxico dos três. Além disso, ao contrário do observado com a DHR, o ascorbato teve ação pró-oxidante, e, aparentemente, houve um efeito sinérgico negativo entre os complexos e a radiação UV. Tratamento com o quelante p-aminossalicilato só foi eficaz na recuperação das culturas para amostras não irradiadas. / Manganese is an essential trace element, however there is considerable concern regarding its neurological effects when in excess, giving rise to a condition termed manganism which is characterized by Parkinson disease-like symptoms. Most evaluations of manganese toxicity use poorly defined Mn(II) species, although Mn(III) is known to accumulate preferentially in the brain. Therefore, in this work we proposed a study of oxidative damage and citotoxicity of Mn(III) derivatives of citrate, pyrophosphate and salycilenediamine (respectively, MnCit, MnPPi and EUK8). The species were synthesized and characterized by spectroscopic methods. Their pro-oxidant abilities were assessed over markers of oxidant activity dihydrorhodamine (DHR), tyrosine (Tyr), albumin (BSA) and dopamine (DA). In addition, their effect over granular cerebral cells (CGC) and HeLa cells stressed by ultraviolet irradiation was studied, and treated with ascorbate. Tests with DHR were repeated treating the samples with H2O2 and ascorbate. Pro-oxidant ability tested by both DHR and Tyr fluorescence suggest that ascorbate is antioxidant towards Mn(III)-induced oxidative damage. MnCit and MnPPi (but not EUK8), when in presence of peroxide, are less oxidants. An analogous trend was observed for BSA, although without statistical significance. Evaluation of DA formation by luminescence was inconclusive, but competition studies of DA+DHR mixtures indicated that DA is preferentially oxidized by all the complexes. To CGC, little difference was observed for the toxicities of the complexes. An inverse relationship of toxicity and lipophilicity has been observed. However this was not observed for HeLa cells, to which EUK8 was more toxic. In addition, and in opposition to the DHR solution study, ascorbate was found to be pro-oxidant. A negative synergic effect was observed between complex doses and irradiation. Treatment of the cells with paraaminosalicylate was beneficial only for non-irradiated cells.