As metalotioneínas (MTs) constituem uma superfamília de proteínas de baixo peso molecular (5-10 kDa), ricas em cisteinas. As MTs de plantas são classificadas em quatro tipos de acordo com o arranjo de cisteinas na cadeia polipeptídica. A função das metalotioneínas em plantas é ainda desconhecida, mas as evidências sugerem que teriam um papel na regulação da homeostasia de metais essenciais, detoxificação de metais pesados e proteção contra espécies reativas de oxigênio. Neste trabalho foram identificados e caracterizados seis genes de metalotioneína a partir das seqüências codificantes para MTs depositadas em banco de dados de cana-de-açúcar, mediante análise filogenética e comparações por alinhamento com os membros da família gênica de metalotioneínas de arroz (Oryza sativa) e Arabidospsis. Os seis genes MTs de cana de açúcar foram classificados como sendo dois do tipo 1 (SoMT1a e SoMT1b); dois do tipo 2 (SoMT2a e SoMT2b); um do tipo 3 (SoMT3); e um tipo 4 SoMT4 (Ec). A região promotora presumível de quatro genes (SoMT1a, SoMT1b, SoMT2b e SoMT3) foi caracterizada e os motivos cis-regulatórios identificados. A distribuição de éxons e íntrons foi estabelecida, e dois éxons separados por único íntron foram identificados na estrutura dos genes SoMT1a e SoMT2b. Um segundo íntron parece estar presente na estrutura dos genes SoMT2a e SoMT3, mas os resultados não foram conclusivos. O gene SoMT1b não foi caracterizado para composição éxons/íntrons. Foi avaliada a expressão constitutiva de SoMT1a, SoMT1b, SoMT2a, SoMT2b e SoMT3 em diferentes tecidos/órgão (colmo, inflorescência, limbo foliar, meristema e raiz). SoMT1a e SoMT1b foram mais abundantemente expressos em todos os tecidos, seguidos de SoMT2b e SoMT3. SoMT2a apresentou a menor expressão. A expressão dos cinco genes SoMTs foi também avaliada em parte aérea e raízes de plântulas de cana-de-açúcar em resposta a 100 e 500 µM de cobre, crescidas in vitro. Não houve modulação da expressão dos genes SoMTs nos tecidos examinados para as doses de cobre utilizadas, as 6, 24, 48 e 96 h após imposição dos tratamentos. A resposta dos genes SoMTs também foi avaliada para o tratamento com 200g L-1 ha-1 de paraquat, aos 15 min, 6 e 24 h após aplicação do herbicida em folhas de plantas deSP80-3280. Houve uma tendência a repressão da expressão dos genes SoMTs, significativa as 6 e 24 h somente para SoMT1a. A expressão de SoMT1b, SoMT2a e SoMT3 foi significativamente reprimida as 24 h após a imposição do tratamento, enquanto que não houve efeito significativo sobre a expressão de SoMT2b. Em relação a expressão dos cinco genes SoMTs em resposta a inoculação com patógenos, plântulas de \'SP70-1143\' e \'RB72-454\', consideradas suscetíveis e resistentes, respectivamente ao fungo da ferrugem, foram inoculadas e não inoculadas com Puccinia melanocephala, enquanto que plântulas de \'SP78-4467\' e \'SP82-1176\', consideradas suscetíveis e resistentes, respectivamente a bactéria da escaldadura, foram inoculadas ou não com Xanthomonas albilineans. As coletas de parte aérea das plântulas foram realizadas aos 15 min, 4, 8, 24, 48, 96 e 240 h após inoculação. Foi observado aproximadamente 4 vezes mais transcritos de SoMT1a em plantas de \'SP70-1143\', suscetível a Puccinia melanocephala, em relação a cultivar resistente \'RB72-454\'. Entretanto, houve uma tendência a repressão de SoMT1a nas plantas suscetíveis inoculadas com o fungo, significativamente as 8 e 24 h após tratamento, não havendo diferenças para as plantas da cultivar resistente após inoculação. A expressão constitutiva dos genes SoMT1b, SoMT2a, SoMT2b e SoMT3 foi similar entre ambas as cultivares e manteve-se inalterada após inoculação com o fungo. Não houve diferenças significativa entre as cultivares suscetíveis e resistentes em relação a expressão basal dos genes SoMTs, nem foi observado efeito significativo sobre a expressão dos genes SoMTs após inoculação das plântulas com X. albilineans. Por fim, transcritos dos cinco genes SoMTs foram clonados em vetor de expressão para expressão heteróloga em Escherichia coli e proteína presumível pode ser observada em géis de poliacrilamida SDS-PAGE. Entretanto, não foi caracterizada uma modulação importante dos cinco genes SoMT em resposta a condições de estresse bióticos (patógenos) e abióticos (Paraquat e metais), que permitisse a associação direta desses genes com a resposta a estresse oxidativo / Metallothioneins (MTs) are part of a low molecular weight (5-10 kDa) cysteine-rich protein super-family. Plant MTs are classified into four types according to cysteine location in the protein sequence. MTs function is largely unknown, but evidences suggested their role in metal homeostasis, heavy metal detoxification, and protection against reactive oxygen species. This work identified and characterized six sugarcane MT genes from expressed sequences available in databases, by alignment and phylogenetic analyses using members from the MT gene family from rice (Oryza sativa) and Arabidopsis. The six sugarcane MT genes were classified as two of type 1 (SoMT1a and SoMT1b); two of type 2 (SoMT2a and SoMT2b); one of type 3 (SoMT3); and one SoMT4 (Ec). The gene putative promoter region of four genes (SoMT1a, SoMT1b, SoMT2b and SoMT3) were characterized, with the cis-regulatory motifs identified. Exon and intron location were established, with two exons and one intron identified for SoMT1a and SoMT2b, A second intron appeared to be present on SoMT2a and SoMT3, but results were inconclusive. SoMT1b could not be characterized for exons and introns. Gene expression analyses was conducted for SoMT1a, SoMT1b, SoMT2a, SoMT2b and SoMT3 in various tissues/organs (stem; inflorescence; leaf; meristem; and root). SoMT1a and SoMT1b were the most expressed genes in all tissues, followed by SoMT2b and SoMT3; SoMT2a was the least expressed. Expression of the five SoMTs genes were evaluated in shoots and roots from in vitro plant grown on 100 or 500 µM copper. There was no modulation of expression in the evaluated tissues at 6, 24, 48 and 96 h after treatment. Gene expression was also evaluated at 15 min, 6 and 24 h after tretament with 200g L-1 ha-1 Paraquat. There was a trend to repress expression of the SoMT genes, being significan only for SoMT1a at 6 and 24 h. Expression of SoMT1b, SoMT2a and SoMT3 were significantly repressed at 24 h after treatment, while there was no significant effect on SoMT2b expression. In relation to the expression of the five SoMTs in response to biotic stress, plants from cultivars \'SP70-1143\' and \'RB72-454\', considered susceptible or resistant respectively, to the rust fungus, were inculated or not with Puccinia melanocephala, whereas plants of \'SP78-4467\' e \'SP82-1176\', considered susceptible or resistant respectively, to the sugarcane leaf scald, were inoculated or not with Xanthomonas albilineans. Shoots were sampled at 15 min, 4, 8, 24, 48, 96 and 240 h after inoculation. Plants of the susceptible \'SP70-1143\' contained 4 times SoMT1a transcripts than the resistant \'RB72-454\'. However, there was a trend to decrease SoMT1a transcripts in susceptible plants, significantly 8 and 24 h after P. melanocephala inoculation, while no difference was observed for expression in resistant plants. The constitutive expression of the other genes (SoMT1b, SoMT2a, SoMT2b and SoMT3) were similar between cultivars, and remained unchanged after inoculation. There was no significant differences for constitutive SoMTs expression between the X. albilineans susceptible and resistant cultivars, nor an inoculation effect of gene expression. Transcripts from the five SoMTs genes were cloned into vetor for heterologous expression in Escherichia coli , and putative proteins were seen in polyacrylamide gels. Therefore, the modulation of the five SoMT genes in response to various abiotic (Paraquat or metals) or biotic (pathogen) stress condition was not characterized to allow definite conclusions about the direct role of metallothioneins in response to oxidative stress

by Wai Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 123-157). / Abstracts in English and Chinese. / Acknowledegment --- p.iv / Abstract --- p.v / List of Abbreviations --- p.ix / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Parkinson's Disease (PD) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Neuropathology --- p.2 / Chapter 1.1.3 --- Clinical Symptoms --- p.3 / Chapter 1.1.4 --- Treatment --- p.6 / Chapter 1.2 --- Proposed Mechanisms of Neurodegeneration in PD --- p.11 / Chapter 1.2.1 --- Oxidative Stress --- p.11 / Chapter 1.2.2 --- Mitochondrial Dysfunction --- p.13 / Chapter 1.2.3 --- Genetic Factors --- p.15 / Chapter 1.2.4 --- Environmental Factors --- p.17 / Chapter 1.2.5 --- Ageing --- p.20 / Chapter 1.3 --- "1-Methy-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) as a PD Model" --- p.22 / Chapter 1.3.1 --- Discovery of MPTP --- p.22 / Chapter 1.3.2 --- The Mechanisms of MPTP-induced Neurotoxicity --- p.23 / Chapter 1.4 --- Antioxidants in the Central Nervous System --- p.26 / Chapter 1.4.1 --- Superoxide Dismutase --- p.26 / Chapter 1.4.2 --- Glutathione --- p.27 / Chapter 1.5 --- Metallothioneins (MTs) --- p.29 / Chapter 1.5.1 --- Characteristics of MTs --- p.29 / Chapter 1.5.2 --- Functions of Astrocytes --- p.31 / Chapter 1.6 --- Astrocytes --- p.34 / Chapter 1.6.1 --- Characteristics of Astrocytes --- p.34 / Chapter 1.6.2 --- Functions of Astrocytes --- p.35 / Chapter 1.6.3 --- Role of Astrocytes in Parkinson's Disease --- p.39 / Chapter 1.7 --- Aim of Project --- p.41 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- In Vitro Study --- p.44 / Chapter 2.1.1 --- Astrocyte Cultures --- p.44 / Chapter 2.1.2 --- Treatment Regimen --- p.46 / Chapter 2.1.2.1 --- 1 -methyl-4-phenyl-pyridinium (MPP+) Treatment --- p.46 / Chapter 2.1.2.2 --- Induction of Metallothioneins (MTs) and Glutathione (GSH) --- p.46 / Chapter 2.1.2.2.1 --- Northern Blot Analysis --- p.47 / Chapter 2.1.2.2.2 --- Immunocytochemical Staining for MTs --- p.48 / Chapter 2.1.2.2.3 --- GSH Assay --- p.49 / Chapter 2.1.2.3 --- Iron Chelation --- p.51 / Chapter 2.1.2.4 --- Combined Pretreatment --- p.51 / Chapter 2.1.3 --- Lactate Dehydrogenase (LDH) Assay --- p.51 / Chapter 2.1.4 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) Assay" --- p.53 / Chapter 2.1.5 --- Reactive Oxygen Species (ROS) Assay --- p.55 / Chapter 2.1.6 --- Protein Assay --- p.56 / Chapter 2.1.7 --- Statistics --- p.57 / Chapter 2.2 --- In Vivo Study --- p.57 / Chapter 2.2.1 --- "Administration of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)" --- p.57 / Chapter 2.2.2 --- Tyrosine Hydroxylase (TH) Immunocytochemical Staining --- p.58 / Chapter 2.2.3 --- DAT Receptor Binding Assay --- p.59 / Chapter 2.2.4 --- Dopamine (DA) and DA metabolites - High Performance Liquid Chromatography (HPLC) --- p.60 / Chapter 2.2.5 --- Statistics --- p.61 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- In Vitro Study --- p.62 / Chapter 3.1.1. --- Induction of Metallothioneins (MTs) in Astrocytes with Zinc Sulfate (ZnS04) --- p.62 / Chapter 3.1.1.1 --- Immunocytochemical changes --- p.62 / Chapter 3.1.1.2 --- Northern Blot Analysis --- p.62 / Chapter 3.1.1.3 --- The Effects of ZnSO4 Pretreatment on 1 -methyl-4-phenyl- pyridinium (MPP+)-treated Astrocytes --- p.63 / Chapter 3.1.1.3.1 --- Lactate Dehydrogenase (LDH) Activities --- p.63 / Chapter 3.1.1.3.2 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl- tetrazolium bromide (MTT) Activities" --- p.67 / Chapter 3.1.1.3.3 --- Reactive Oxygen Species (ROS) Production --- p.71 / Chapter 3.1.2 --- The Effects of NAc Pretreatment on MPP+-treated Astrocytes --- p.75 / Chapter 3.1.2.1 --- Glutathione (GSH) levels --- p.75 / Chapter 3.1.2.2 --- LDH Activities --- p.77 / Chapter 3.1.2.3 --- MTT Activities --- p.80 / Chapter 3.1.2.4 --- ROS Production --- p.83 / Chapter 3.1.3 --- The Effects of Deferoxamine on MPP+-treated Astrocytes --- p.87 / Chapter 3.1.3.1 --- LDH Activities --- p.87 / Chapter 3.1.3.2 --- ROS Production --- p.89 / Chapter 3.1.4 --- The Effects of ZnSO4 and NAc Combined Treatment on MPP+-treated Astrocytes --- p.92 / Chapter 3.1.4.1 --- LDH Activities --- p.92 / Chapter 3.1.4.2 --- ROS Production --- p.95 / Chapter 3.2 --- "Effects of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on MT-I, -II Knock-out Mice" --- p.99 / Chapter 3.2.1 --- The Effects of MPTP on Substantia Nigral (SN) Cell Loss --- p.99 / Chapter 3.2.2 --- The Effects of MPTP on Striatal (ST) and SN Dopamine Transporter (DAT) Binding --- p.99 / Chapter 3.2.3 --- The Effects of MPTP on ST Dopamine (DA) Metabolites --- p.100 / Chapter CHAPTER FOUR: --- DISCUSSION AND CONCLUSION --- p.102 / REFERENCES --- p.123

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this study, acute effects of mercury on mouse blood, kidneys and liver were evaluated. Mice received a single dose of mercuric chloride (HgCl2 - 4.6 mg/kg, subcutaneously) for three consecutive days. We investigated the possible beneficial effects of antioxidant therapy (N-acetylcysteine (NAC) and diphenyl diselenide (PhSe)2) comparing to sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), an effective chelating agent on mercury exposure in mice. We also verified if metallothionein (MT) induction would be involved in a possible mechanism of protection against mercury poisoning and if different therapies would modify MT levels and other toxicological parameters. The results demonstrated that animals treated with mercuric chloride presented a reduction in the body weight gain and therapies did not were effective in reverting this damage. HgCl2 exposure inhibited δ-aminolevulinic dehydratase (δ-ALA-D) activity in liver and only DMPS treatment prevented the inhibitory effect. Animals treated with mercury presented an increase in renal NPSH and therapies did not modify these levels. Urea concentration was increased after mercury exposure. NAC plus (PhSe)2 was partially effective in protecting against this effect of mercury . DMPS and (PhSe)2 were effective in restoring the increment in urea concentration caused by mercury. Thiobarbituric acid-reactive substances (TBARS), ascorbic acid levels, aspartate (AST) and alanine (ALT) aminotransferases were not modified after mercury exposure. Moreover, mercury poisoning caused an increase in hepatic and renal MT levels and antioxidant therapies did not modify this parameter. Our data pointed out the lack of the therapeutic effect of antioxidants tested. / Neste trabalho foram avaliados os efeitos da intoxicação aguda induzida por cloreto de mercúrio (HgCl2) em sangue, rim e fígado de camundongos. Os animais receberam uma única dose de HgCl2 (4,6 mg/Kg de peso), via sub-cutânea, por três dias consecutivos. Investigou-se o possível efeito protetor da terapia com antioxidantes (N-acetilcisteína-NAC e disseleneto de difenila-(PhSe)2) comparando ao ácido 2,3-dimercapto-1-propanosulfônico (DMPS), um agente quelante efetivo contra intoxicações por mercúrio. Além disto, foi verificado se a indução de metalotioneínas (MT) poderia estar envolvida em um possível mecanismo de proteção contra a intoxicação pelo mercúrio e se as diferentes terapias poderiam modificar os níveis de MT e outros parâmetros toxicológicos. Os resultados demonstraram que os animais tratados com cloreto de mercúrio apresentaram uma redução no ganho de peso corporal e as terapias não foram efetivas em reverter este dano. A exposição ao HgCl2 causou inibição na atividade da enzima δ-aminolevulinato desidratase (δ-ALA-D) em fígado de camundongos e somente a terapia com DMPS foi efetiva em reverter esta inibição. Os animais tratados com mercúrio apresentaram um aumento nos níveis de NPSH renal e as terapias não modificaram estes níveis. A concentração de uréia foi aumentada nos animais expostos ao cloreto de mercúrio. A terapia com NAC + (PhSe)2 foi parcialmente efetiva em proteger contra este efeito do mercúrio. Já as terapias com DMPS e (PhSe)2 foram efetivas em proteger contra o aumento nos níveis de uréia induzido pelo mercúrio. As substâncias reativas ao ácido tiobarbitúrico (TBARS), os níveis de ácido ascórbico e as transaminases (aspartato-AST e alanina-ALT) não foram alteradas após a exposição ao HgCl2. Além disso, os resultados demonstraram que a exposição ao mercúrio causou um aumento nos níveis de metalotioneínas hepático e renal e as terapias com antioxidantes não modificaram este parâmetro. Nossos dados apontam para a falta de efeito terapêutico dos antioxidantes testados.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mercury (Hg) is a divalent metal found liquid at room temperature without biological functions and anthropogenically released in industrial, agricultural activities and burning of fossil fuels. Toxic effects caused by exposure to this metal are related to the interaction of different biochemical processes due to its affinity for sulfhydryl groups (SH). This damage depends on the time of exposure and the development period in which the individuals are exposed. Thus, the aim of this study was to evaluate the effects of a single subcutaneous dose of inorganic Hg in virgin, pregnant and lactating rats, as well as the protective effect of zinc (Zn) and N-acetylcysteine (NAC). For this, three experimental protocols were used: I) Virgin female rats were treated with ZnCl2 (27 mg/kg) and/or NAC (5 mg/kg) or saline (0.9%) and 24 hours after with HgCl2 (5 mg/kg) or saline (Article 1). II) Pregnant or lactating rats were treated with ZnCl2 (27 mg/kg) and/or NAC (5 mg/kg) or saline (0.9%) and 24 hours after with HgCl2 (10 mg/kg) or saline (manuscript I). III) Renal and hepatic analysis of virgin, pregnant and lactating rats exposed to a dose of HgCl2 (5 mg/kg) or saline (manuscript II). In all protocols euthanasia was performed 24 hours after the last treatment and the tissues removed and prepared for analysis. Protocols I and II focused primarily on biochemical parameters in different tissues and protocol III in morphological evaluations and protein expression in the kidneys and liver. Virgin rats exposed to Hg showed inhibition of the δ-aminolevulinic acid dehydratase (δ-ALA-D) activity in all tissues analyzed, changes in serum markers of hepatic (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) and renal (creatinine and urea) damage, morphological damage, and changes in proteins related to oxidative stress expression, for instance, mitofusin 2 (MFN2), inducible nitric oxide synthase (iNOS), heat shock protein 27 (HSP27) and glucose regulated protein 75 (GRP75). Pregnant and lactating rats exposed to mercury showed milder changes than virgin rats, including no inhibition of hepatic δ-ALA-D or alterations of proteins related to oxidative stress and few morphological damage. Pregnant and lactating rats still showed physiologically higher levels of metallothionein (MT) in the liver and larger glomerulus diameter than virgin rats. The results suggest greater resistance of pregnant and lactating rats to Hg compared with virgin rats. This difference may be related to increase of hepatic MT levels induced by pregnancy and lactation. This protein is synthesized in the liver and plays an important chelator role,. making substances, such as Hg, less harmful. The treatment with Zn and NAC showed promising results against damage caused by Hg, probably by induction of MT synthesis caused by Zn and by chelating action of NAC. In both situations occur the capture of Hg. The metal bound to MT or NAC is neutralized and consequently has lower toxicity effects. / O mercúrio (Hg) é um metal bivalente encontrado líquido a temperatura ambiente, sem funções biológicas e antropogenicamente liberado em atividades industriais, agricultura e queima de combustíveis fósseis. Os efeitos tóxicos causados pela exposição a esse metal estão relacionados à sua interação com diferentes processos bioquímicos devido a sua afinidade por grupos sulfidrílicos (SH). Estes danos dependem do tempo de exposição e do período de desenvolvimento em que os indivíduos são expostos. Com isso, o objetivo deste trabalho foi avaliar os efeitos do Hg inorgânico, em dose única subcutânea, em ratas virgens, gestantes e lactantes, bem como, a capacidade protetora do zinco (Zn) e da N-acetilcisteína (NAC). Para isso, adotamos três protocolos experimentais: I) Ratas virgens foram tratadas com ZnCl2 (27 mg/kg) e/ou NAC (5 mg/kg) ou salina (0,9%) e 24 horas após com HgCl2 (5 mg/kg) ou salina (artigo 1). II) Ratas gestantes e lactantes tratadas com ZnCl2 (27 mg/kg) e/ou NAC (5 mg/kg) ou salina (0,9%) e 24 horas após com HgCl2 (10 mg/kg) ou salina (manuscrito I). III) Análise renal e hepática de ratas virgens, gestantes e lactantes expostas a uma dose de HgCl2 (5 mg/kg) ou salina (manuscrito II). Em todos os protocolos a eutanásia foi realizada 24 horas após o último tratamento e os tecidos retirados e preparados para as análises. Os protocolos I e II tiveram como foco principal parâmetros bioquímicos em diferentes tecidos e o protocolo III em avaliações morfológicas, expressão proteica em rins e fígado. Ratas virgens expostas ao Hg apresentaram inibição da atividade da δ-aminolevulinato desidratase (δ-ALA-D) em todos os tecidos analisados, alterações em marcadores hepáticos (alanina aminotransferase [ALT] e aspartato aminotransferase [AST]) e renais (creatinina e ureia), além de danos morfológicos e alteração na expressão de proteínas relacionadas ao estresse oxidativo, como a mitofusina 2 (MFN2), óxido nítrico sintetase induzível (iNOS), proteína de choque térmico 27 (HSP27) e proteína reguladora de glicose 75 (GRP75). Ratas gestantes e lactantes expostas ao Hg apresentaram alterações mais brandas que ratas virgens, inclusive sem inibição da δ-ALA-D hepática ou distúrbios em proteínas relacionadas com dano oxidativo, bem como poucos danos morfológicos em rins e fígado. Ratas gestantes e lactantes apresentaram altos níveis hepáticos de metalotioneínas (MT) e aumento no diâmetro glomerular em relação as ratas virgens. Os resultados sugerem maior resistência de ratas gestantes e lactantes ao Hg quando comparadas com ratas virgens. Esta diferença pode ser relacionada ao aumento nos níveis hepáticos de MT induzidos pela gestação e lactação. Essa proteína é sintetizada principalmente no fígado e desempenha importante função quelante, tornando substâncias como o Hg, menos nocivas. Os tratamentos com Zn e NAC, mostraram resultados promissores contra os danos causados pelo Hg, provavelmente pela indução da síntese de MT causada pelo Zn, e pela ação quelante da NAC. Em ambas as situações ocorre a captura do Hg. O metal ligado a MT ou a NAC é neutralizado e consequentemente apresenta menor toxicidade.

As metalotioneínas (MTs) constituem uma superfamília de proteínas de baixo peso molecular (5-10 kDa), ricas em cisteinas. As MTs de plantas são classificadas em quatro tipos de acordo com o arranjo de cisteinas na cadeia polipeptídica. A função das metalotioneínas em plantas é ainda desconhecida, mas as evidências sugerem que teriam um papel na regulação da homeostasia de metais essenciais, detoxificação de metais pesados e proteção contra espécies reativas de oxigênio. Neste trabalho foram identificados e caracterizados seis genes de metalotioneína a partir das seqüências codificantes para MTs depositadas em banco de dados de cana-de-açúcar, mediante análise filogenética e comparações por alinhamento com os membros da família gênica de metalotioneínas de arroz (Oryza sativa) e Arabidospsis. Os seis genes MTs de cana de açúcar foram classificados como sendo dois do tipo 1 (SoMT1a e SoMT1b); dois do tipo 2 (SoMT2a e SoMT2b); um do tipo 3 (SoMT3); e um tipo 4 SoMT4 (Ec). A região promotora presumível de quatro genes (SoMT1a, SoMT1b, SoMT2b e SoMT3) foi caracterizada e os motivos cis-regulatórios identificados. A distribuição de éxons e íntrons foi estabelecida, e dois éxons separados por único íntron foram identificados na estrutura dos genes SoMT1a e SoMT2b. Um segundo íntron parece estar presente na estrutura dos genes SoMT2a e SoMT3, mas os resultados não foram conclusivos. O gene SoMT1b não foi caracterizado para composição éxons/íntrons. Foi avaliada a expressão constitutiva de SoMT1a, SoMT1b, SoMT2a, SoMT2b e SoMT3 em diferentes tecidos/órgão (colmo, inflorescência, limbo foliar, meristema e raiz). SoMT1a e SoMT1b foram mais abundantemente expressos em todos os tecidos, seguidos de SoMT2b e SoMT3. SoMT2a apresentou a menor expressão. A expressão dos cinco genes SoMTs foi também avaliada em parte aérea e raízes de plântulas de cana-de-açúcar em resposta a 100 e 500 µM de cobre, crescidas in vitro. Não houve modulação da expressão dos genes SoMTs nos tecidos examinados para as doses de cobre utilizadas, as 6, 24, 48 e 96 h após imposição dos tratamentos. A resposta dos genes SoMTs também foi avaliada para o tratamento com 200g L-1 ha-1 de paraquat, aos 15 min, 6 e 24 h após aplicação do herbicida em folhas de plantas deSP80-3280. Houve uma tendência a repressão da expressão dos genes SoMTs, significativa as 6 e 24 h somente para SoMT1a. A expressão de SoMT1b, SoMT2a e SoMT3 foi significativamente reprimida as 24 h após a imposição do tratamento, enquanto que não houve efeito significativo sobre a expressão de SoMT2b. Em relação a expressão dos cinco genes SoMTs em resposta a inoculação com patógenos, plântulas de \'SP70-1143\' e \'RB72-454\', consideradas suscetíveis e resistentes, respectivamente ao fungo da ferrugem, foram inoculadas e não inoculadas com Puccinia melanocephala, enquanto que plântulas de \'SP78-4467\' e \'SP82-1176\', consideradas suscetíveis e resistentes, respectivamente a bactéria da escaldadura, foram inoculadas ou não com Xanthomonas albilineans. As coletas de parte aérea das plântulas foram realizadas aos 15 min, 4, 8, 24, 48, 96 e 240 h após inoculação. Foi observado aproximadamente 4 vezes mais transcritos de SoMT1a em plantas de \'SP70-1143\', suscetível a Puccinia melanocephala, em relação a cultivar resistente \'RB72-454\'. Entretanto, houve uma tendência a repressão de SoMT1a nas plantas suscetíveis inoculadas com o fungo, significativamente as 8 e 24 h após tratamento, não havendo diferenças para as plantas da cultivar resistente após inoculação. A expressão constitutiva dos genes SoMT1b, SoMT2a, SoMT2b e SoMT3 foi similar entre ambas as cultivares e manteve-se inalterada após inoculação com o fungo. Não houve diferenças significativa entre as cultivares suscetíveis e resistentes em relação a expressão basal dos genes SoMTs, nem foi observado efeito significativo sobre a expressão dos genes SoMTs após inoculação das plântulas com X. albilineans. Por fim, transcritos dos cinco genes SoMTs foram clonados em vetor de expressão para expressão heteróloga em Escherichia coli e proteína presumível pode ser observada em géis de poliacrilamida SDS-PAGE. Entretanto, não foi caracterizada uma modulação importante dos cinco genes SoMT em resposta a condições de estresse bióticos (patógenos) e abióticos (Paraquat e metais), que permitisse a associação direta desses genes com a resposta a estresse oxidativo / Metallothioneins (MTs) are part of a low molecular weight (5-10 kDa) cysteine-rich protein super-family. Plant MTs are classified into four types according to cysteine location in the protein sequence. MTs function is largely unknown, but evidences suggested their role in metal homeostasis, heavy metal detoxification, and protection against reactive oxygen species. This work identified and characterized six sugarcane MT genes from expressed sequences available in databases, by alignment and phylogenetic analyses using members from the MT gene family from rice (Oryza sativa) and Arabidopsis. The six sugarcane MT genes were classified as two of type 1 (SoMT1a and SoMT1b); two of type 2 (SoMT2a and SoMT2b); one of type 3 (SoMT3); and one SoMT4 (Ec). The gene putative promoter region of four genes (SoMT1a, SoMT1b, SoMT2b and SoMT3) were characterized, with the cis-regulatory motifs identified. Exon and intron location were established, with two exons and one intron identified for SoMT1a and SoMT2b, A second intron appeared to be present on SoMT2a and SoMT3, but results were inconclusive. SoMT1b could not be characterized for exons and introns. Gene expression analyses was conducted for SoMT1a, SoMT1b, SoMT2a, SoMT2b and SoMT3 in various tissues/organs (stem; inflorescence; leaf; meristem; and root). SoMT1a and SoMT1b were the most expressed genes in all tissues, followed by SoMT2b and SoMT3; SoMT2a was the least expressed. Expression of the five SoMTs genes were evaluated in shoots and roots from in vitro plant grown on 100 or 500 µM copper. There was no modulation of expression in the evaluated tissues at 6, 24, 48 and 96 h after treatment. Gene expression was also evaluated at 15 min, 6 and 24 h after tretament with 200g L-1 ha-1 Paraquat. There was a trend to repress expression of the SoMT genes, being significan only for SoMT1a at 6 and 24 h. Expression of SoMT1b, SoMT2a and SoMT3 were significantly repressed at 24 h after treatment, while there was no significant effect on SoMT2b expression. In relation to the expression of the five SoMTs in response to biotic stress, plants from cultivars \'SP70-1143\' and \'RB72-454\', considered susceptible or resistant respectively, to the rust fungus, were inculated or not with Puccinia melanocephala, whereas plants of \'SP78-4467\' e \'SP82-1176\', considered susceptible or resistant respectively, to the sugarcane leaf scald, were inoculated or not with Xanthomonas albilineans. Shoots were sampled at 15 min, 4, 8, 24, 48, 96 and 240 h after inoculation. Plants of the susceptible \'SP70-1143\' contained 4 times SoMT1a transcripts than the resistant \'RB72-454\'. However, there was a trend to decrease SoMT1a transcripts in susceptible plants, significantly 8 and 24 h after P. melanocephala inoculation, while no difference was observed for expression in resistant plants. The constitutive expression of the other genes (SoMT1b, SoMT2a, SoMT2b and SoMT3) were similar between cultivars, and remained unchanged after inoculation. There was no significant differences for constitutive SoMTs expression between the X. albilineans susceptible and resistant cultivars, nor an inoculation effect of gene expression. Transcripts from the five SoMTs genes were cloned into vetor for heterologous expression in Escherichia coli , and putative proteins were seen in polyacrylamide gels. Therefore, the modulation of the five SoMT genes in response to various abiotic (Paraquat or metals) or biotic (pathogen) stress condition was not characterized to allow definite conclusions about the direct role of metallothioneins in response to oxidative stress