O Aspergillus fumigatus é um fungo filamentoso e saprofítico, encontrado em todas as regiões do mundo, desempenhando um importante papel na reciclagem de carbono e nitrogênio do solo. A principal forma de infecção ocorre através da inalação dos conídios com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. A mitocôndria de A. fumigatus foi caracterizada em nosso laboratório, que revelou a presença de uma respiração resistente a cianeto mediada pela oxidase alternativa. A clonagem e sequenciamento deste gene foram realizadas através de screening de uma biblioteca de DNA genômico. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois introns, que após splicing codifica uma proteína contendo 352 aminoácidos, possuindo uma massa molecular estimada de 40,84 kDa e um pI teórico de 9,51. Além disso, foram identificados domínios altamente conservados (LET, NERMHL, LEEA e RADEH) que interagem com átomos de ferro e estão contidos em -hélices propostas como responsáveis pela organização estrutural da enzima. A fim de caracterizar bioquimicamente esta proteína, a sequência de cDNA do gene foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae INVSc1 como um modelo eucarioto. Após expressão, a proteína encontrou-se de forma ativa, conferindo à levedura uma respiração resistente a cianeto. Esta característica herdada provocou uma discreta diminuição na taxa de crescimento em meio não-fermentativo e uma capacidade de sobrevivência na presença de KCN. Acredita-se que a atividade das AOXs esteja diretamente relacionada com a presença de diferentes espécies reativas de oxigênio (ERO). Neste contexto, a avaliação do efeito de diferentes agentes pró-oxidantes provocou um aumento na atividade e na expressão da enzima. Paralelamente, a caracterização funcional do gene foi realizada através da técnica de interferência por RNA, utilizando o vetor de expressão pALB1. Em meio contendo maltose, as cepas pALB1/aoxAf apresentaram coloração branca devido ao silenciamento do gene alb1. Os níveis de mRNA do gene aoxAf foram determinados por Real time RT-PCR, mostrando o eficiente silenciamento do gene alvo com a construção utilizada. Devido à relação já descrita entre ERO e a atividade das AOXs, avaliou-se a produção de espécies reativas de oxigênio nas cepas silenciadas utilizando-se a sonda CM-H2DCFDA, observando maior produção na cepa pALB1/aoxAf. Além disso, a viabilidade destas cepas foi determinada por citometria de fluxo após a exposição com agentes pró-oxidantes, a qual indicou maior letalidade na cepa pALB1/aoxAf, quando comparada com CEA e pALB1. Da mesma forma, após a incubação dos conídios das cepas silenciadas com macrófagos de camundongos foi verificada uma maior atividade microbicida dos macrófagos na cepa duplamente silenciada pALB1/aoxAf, quando comparada com as outras cepas. Com estes resultados podemos concluir que a oxidase alternativa apresenta uma importante atividade antioxidante, além de contribuir nos mecanismos de defesa durante o processo de infecção de A. fumigatus. / The saprophytic species Aspergillus fumigatus is a deuteromycete fungus found worldwide, which has an essential role in recycling carbon and nitrogen. Following inhalation of conidia by the immunocompetent host, the innate cellular immune system is responsible for killing the conidia, exposing them to reactive oxygen. However, A. fumigatus is capable of surviving and replicating within the phagolysosomal compartment of immunocompromised macrophages. It was previously demonstrated that A. fumigatus mitochondria possess an alternative oxidase (aoxAf) wich is a cyanide-resistant protein. A partial genomic DNA library was screened to cloning an aoxAf gene. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 352 amino acid sequence with a calculated molecular mass of 40 kDa and a theoretical pI of 9.51. The deduced amino acid sequence revealed four regions completely conserved among the AOXs sequences (LET, NERMHL, LEEA and RADE-H), where six conserved amino acids residues are proposed to be a metal ligand site. To characterize the AOX protein, a cDNA of aoxAf gene was cloned into pYES2 plasmid and transformed in S. cerevisiae INVSc1. After the incubation of the cells in a nonfermentable medium in the presence of KCN, S. cerevisiae expressing AOX was able to grow, while it was lethal for the control yeast. These results suggest that the recombinant AOXAf is properly targeted to the S. cerevisiae mitochondria where it has functional activity. Studies with different species demonstrated that AOX is induced by a variety of treatments usually labeled as stresses. To verify the function of AOX in A. fumigatus under oxidative stress conditions, conidia were treated with different donors of ROS. These treatments caused an increase in aoxAf activity and transcription levels. To identify genetically attributes of virulence and oxidative defense in A. fumigatus, we construct a RNA interference plasmid. Two inverted repeated sequences of conserved region of an interest gene were amplified and cloned in pALB1 plasmid. In maltose medium pALB1 and pALB1/aoxAf transformants demonstrated white colonies, attributable to the reduction of alb1 gene expression. The aoxAf mRNA levels were analyzed by Real time RT-PCR, showing an efficient alternative oxidase gene silencing in pALB1 plasmid construction. It was previously demonstrated that ROS can stimulate the AOXs activity, so, we used the dye CM-H2DCFDA to measure ROS production in RNAi transformants, showing that the decrease in aoxAf gene expression caused an increase in ROS production. After incubation with ROS donors the viability of these strains was determined by flow cytometry analysis. The pALB1/aoxAf strain showed higher lethality, when compared with CEA and pALB1, suggesting the involvement of AOX in antioxidant defense in A. fumigatus. Besides, ROS produced by alveolar macrophages play an essential role in the killing of A. fumigatus conidia. In the same way, phagocytosis assay revealed that pALB1/aoxAf strain was more lethal than CEA and pALB1. With these results, we concluded that alternative oxidase is an efficient antioxidant system and might contribute with defense mechanism of A. fumigatus.

O DNA é um alvo constante de modificações químicas, as quais resultam na ativação dos programas de reparo de danos no DNA. O DNA mitocondrial (DNAmt), uma molécula circular contendo aproximadamente 16,6 kb de extensão, é constantemente exposto às espécies reativas de oxigênio (EROs) devido a sua proximidade da cadeia transportadora de elétrons, presente na membrana mitocondrial interna. Quase todas as vias de reparo de DNA presentes no núcleo atuam também na mitocôndria, entretanto, a regulação das vias mitocondriais não é bem compreendida. As proteínas p53, ATM, mTOR e PGC1α participam, dentre outros papéis, do controle do metabolismo energético e das respostas a lesões no DNA nuclear. Dessa forma, decidimos gerar linhagens celulares com níveis reduzidos dessas proteínas como uma ferramenta para o estudo dos seus papéis na manutenção do DNAmt. Para isso, foram geradas linhagens celulares de HEK293 expressando constitutivamente shRNAs alvo-específicos, cuja diminuição da expressão das proteínas alvo foi confirmada através de western blotting. Neste trabalho, também foi estudado o papel de p53 na resposta ao estresse oxidativo mitocondrial provocado por azul de metileno (AM). O AM é um corante fotoativo capaz de atravessar membranas biológicas e, em células de mamíferos, se acumula em organelas, tais como a mitocôndria. Uma vez que p53 participa de diversas funções celulares e transloca para a mitocôndria sob condições de estresse, onde pode induzir apoptose ou modular o reparo de DNAmt, nós investigamos se p53 está envolvido na indução de morte celular após tratamento com AM fotoativado. Para isso, foram utilizados 2 clones com níveis reduzidos de p53 obtidos na primeira etapa deste trabalho. Sob condições normais, foi demonstrado que o silenciamento de p53 induziu uma forte redução do número de cópias de DNAmt e estimulou a proliferação celular quando fornecemos glicose ou galactose como substratos energéticos. A depleção de p53, ou a sua inibição farmacológica, resultaram em uma ligeira proteção quando as células foram submetidas ao tratamento com AM. Também foi demonstrado que AM provoca morte celular apoptótica de uma maneira dependente de p53, uma vez que a depleção dessa proteína protegeu a população do acúmulo de células em sub-G1. Portanto, nossos resultados sugerem que AM induz morte celular apoptótica em células HEK293, de uma maneira dependente de p53. Esse efeito pode ser mediado diretamente por p53, ou ainda, pelo seu papel na manutenção do número de cópias do DNAmt. / DNA is constantly being chemically modified, which results in activation of the DNA damage response program. The mitochondrial DNA (mtDNA), a circular molecule of 16.6 kb in length, is primary target of reactive oxygen species (ROS) due its proximity to the electron transport chain, in the mitochondrial inner membrane. Almost all known DNA damage repair pathways operating in the nucleus were also found in the mitochondrion; however, their regulation remains not well understood. The proteins p53, ATM, mTOR e PGC1α have many cellular functions, including control of energy metabolism and cell fate after stress. Thus, we hypothesized that those proteins could participate in maintaining of mtDNA, through direct or indirect roles. To test this hypothesis, we generated isogenic knockdown cell lines to further use them to study their role in the mtDNA damage response. For that, were generated HEK293 knockdown cell lines that stably express target-specific shRNAs. Efficient knockdown was checked using western blotting. Here, we also studied the role of p53 in the cellular response to mitochondrial oxidative stress induced by methylene blue (MB). MB is a photoactive dye that crosses biological membranes due to its lypophylic character and, in mammalian cells, accumulates in organelles such as mitochondria; however, its cytotoxic mechanism is not well understood. As the p53 protein participates in several cellular functions and translocates to mitochondria under stress conditions, where it can induce apoptosis or modulate mtDNA repair, we investigated whether p53 was involved in MB + light-induced cell death using p53 knockdown clones selected from the cell lines generated in the first phase of this work. Under normal conditions, p53 knockdown caused a decrease in mtDNA copy number and stimulated cellular growth supported by either glucose or galactose. After MB treatment, p53-kd cells showed a slight decrease in cell death compared to scrambled shRNA controls. Evaluation of cell death after MB treatment, using flow cytometry analysis, indicated that MB was able to induce significant levels of apoptotic cell death, which was dependent on p53 levels. Taken together, our results suggest that MB induces cell death, probably via apoptosis, in a p53 dependent manner. This effect may be mediated by p53 directly or by its role in mtDNA copy number maintenance.

The carbonate radical anion (CO3•-, CR) is an important reactive oxygen species produced in vivo by one-electron oxidation of CO2 or bicarbonate, constituents of the major physiological buffer. It was demonstrated for the first time by using an HPLC-based analysis of low-molecular products of DNA damage that CRs react with DNA 2΄-deoxyribose by the hydrogen abstraction mechanism. CRs exhibit a ~ 800-fold preference for one-electron oxidation of guanine over hydrogen abstraction from DNA sugar, in sharp contrast with •OH. CRs also have, as compared to •OH, an increased preference for the H1΄ abstraction, which is the most thermodynamically favorable due to the highest stability of the respective deoxyribosyl radical but kinetically the slowest due to low solvent accessibility, by the expense of the decreased preference for the H5΄ abstraction. All these findings are in agreement with the characteristics of CR as a potent oxidant and selective hydrogen abstractor.

8-oxo-7,8-dihydroguanine (8oxoG) is one of the most important base lesions formed during oxidative damage of DNA. The aim of the present research was to investigate the effects of DNA concentration, G content, and the nature of oxidizing species on the kinetics of 8oxoG in model DNA solutions by using HPLC. The experimentally obtained yields of 8oxoG were typically in the range of 2-2.5% of total concentration of guanine. The ratios of the rate constant of hole diffusion in DNA to the rate constant of conversion of the hole into 8oxoG (kd/kr) were calculated from the experimental data using the diffusion model of charge transfer in DNA to be in the range of 200-300, in agreement with previously reported kd/kr ratios in the duplex DNA oligonucleotides (GGA)n or (GGTT)n. Our current diffusion model cannot satisfactorily explain the absence of the G content dependence of the 8oxoG yields, which indicates that a more advanced model is required.

Key Points: (1) Nutrition management for individuals infected with HIV can be helpful in maintaining lean body weight, combating oxidative stress, reducing complications from hyperglycemia and hyperlipidemia, and managing gastrointestinal function. (2) Patients may need to be individualized to meet each individual's unique requirements. (3) Consideration should be given to including the expertise of a registered dietitian/nutritionist s part of the health care team to promote wellness in the individuals infected with HIV.

Significance: Nutrition is one of the most basic of medical issues and is often ignored as a problem in the management of our chronic wound patients. Unfortunately, malnutrition is widespread in our geriatric patients even in nursing homes in developed countries. Attention to basic nutrition and providing appropriate supplements may assist in the healing of our chronic wounds. Recent Advances: Recent research has revealed the epidemiology of malnutrition in developed countries, the similarities to malnutrition in developing countries, and some of the physiologic and sociologic causes for this problem. More information is now available on the biochemical effects of nutrient deficiency and supplementation with macronutrients and micronutrients. In some cases, administration of isolated nutrients beyond recommended amounts for healthy individuals may have a pharmacologic effect to help wounds heal. Critical Issues: Much of the knowledge of the nutritional support of chronic wounds is based on information that has been obtained from trauma management. Due to the demographic differences of the patients and differences in the physiology of acute and chronic wounds, it is not logical to assume that all aspects of nutritional support are identical in these patient groups. Before providing specific nutritional supplements, appropriate assessments of patient general nutritional status and the reasons for malnutrition must be obtained or specific nutrient supplementation will not be utilized. Future Directions: Future research must concentrate on the biochemical and physiologic differences of the acute and chronic wounds and the interaction with specific supplements, such as antioxidants, vitamin A, and vitamin D.

La Galanine est un peptide ubiquitaire de 29 acides aminés chez les mammifères (30 chez l'homme) qui contrôle de nombreuses fonctions biologiques: (I) secrétions endocrines (insuline, somatostatine, glucagon); (II) comportement (prise alimentaire, nociception, apprentissage, mémoire); (III) tonicité musculaire dans le tractus digestif. Ce peptide a particulièrement été étudié dans le système nerveux central où il joue un rôle dans l'évolution de certaines maladies neurodégénératives telles que les maladies de Parkinson et d'Alzheimer. La galanine agit en se fixant sur 3 récepteurs connus, GalR1-2-3 qui appartient à la grande famille des récepteurs couplés aux protéines G (GPCR). Bien que la Galanine et ses trois récepteurs soient fortement exprimés au niveau périphérique, leur rôle dans les effets périphériques et leur implication en pathologie ont été très peu étudié. L'objectif de mon travail de thèse a été d'identifier le rôle de la galanine dans le remodelage myocardique, qui constitue un déterminant majeur dans la progression de l'insuffisance cardiaque. Dans un premier temps, nous avons démontré que la galanine possède des propriétés anti-apoptotiques et anti-oxydantes in vitro dans les cardiomyocytes. En effet, le traitement de cellules par la galanine entraîne une diminution de l'apoptose et de la production de radicaux libres oxygénés en réponse à l'hypoxie. En accord avec ces effets bénéfiques au niveau cellulaire, les études in vivo réalisées dans un modèle d'ischémie-reperfusion cardiaque, ont montré que le traitement à la galanine dans la phase précoce de reperfusion réduit l'apoptose et la nécrose myocardique. De plus, nous avons confirmé in vivo l'importance de la galanine dans la défense contre le stress oxydant associé aux lésions mitochondriales post-ischémiques. Dans un deuxième temps, nous avons mis en évidence le rôle important de la galanine dans le contrôle de l'activité des fibroblastes cardiaques in vitro. Nous avons montré que la galanine inhibe des étapes clés d'activation de la cascade pro-fibrotique dans les fibroblastes cardiaques. Les propriétés anti-fibrotiques de la galanine ont également été confirmées in vivo dans un modèle de surcharge en pression par constriction aortique chez la souris. Dans ce modèle, nos résultats montrent que le traitement chronique à la galanine s'accompagne d'une amélioration de la fonction cardiaque. Ces données nous ont permis de démontrer que la galanine joue un rôle clé dans le remodelage cardiaque et de proposer la galanine comme un candidat potentiel dans le traitement de l'insuffisance cardiaque. / Galanin is an ubiquitous 29 amino acid peptide in mammals (30 in humans) that controls many biological functions: (I) endocrine secretions (insulin, somatostatin, glucagon); (II) behavior control (food intake, nociception, learning, memory, pain); (III) muscle tonicity in the digestive tract. This peptide has been particularly studied in the central nervous system where it plays a role in the evolution of neurodegenerative diseases such as Parkinson's and Alzheimer's. Although Galanin and its three receptors (GalR1, GalR2, GalR3) are strongly expressed at the peripheral level, their role in peripheral effects and their involvement in the pathology have been poorly studied. The objective of my thesis work was to identify the role of galanin in myocardial remodeling, which is a major determinant in the progression of heart failure. Firstly, we demonstrated that galanin has anti-apoptotic and anti-oxidant properties in vitro in cardiomyocytes. Indeed, the treatment of cells with galanin causes a dose-dependent decrease in apoptosis and reactive oxygen species in response to hypoxia. In line with these beneficial effects at the cellular level, using in vivo model of cardiac ischaemia-reperfusion we showed that galanin treatment reduces apoptosis and necrosis in the early phase of reperfusion. In addition, we confirmed in vivo the importance of galanin in the defense against oxidative stress associated with post-ischemic mitochondrial lesions. Secondly, we demonstrated the important role of galanin in controlling the activity of cardiac fibroblasts. Using in vitro models, we have shown that galanin inhibits activation of key steps of the pro-fibrotic cascade in cardiac fibroblasts. The anti-fibrotic properties of galanin were also confirmed in vivo in a mouse model of pressure overload-induced heart failure. This observation is accompanied by an improvement of cardiac function. These data reveal that galanin plays a key role in cardiac remodeling and suggest galanin as a potential candidate in the treatment of heart failure.

The FHIT gene is located on the most fragile site in the human genome. FHIT gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissue. Previous work in mouse and cell culture models established FHIT to be an authentic tumor suppressor. Re-expression of FHIT in cell culture causes cell death via initiation of apoptosis, but the precise mechanism underlying this process is unclear. It is well established that cellular transition from normal to transformed occurs in multiple steps and requires the accumulation of several genetic changes. Relying on the compelling phenotype of tumor development in FHIT knockout mice, this project aimed to elucidate a mechanism through which FHIT-deficient cells are primed to survive multiple genetic and environmental stresses, and promote progression of cancer. My work indicates that FHIT expression is required for the normal cellular response to oxidative stress, and presents evidence that in the absence of FHIT, an oxidative stress response pathway is superinduced. When FHIT is depleted from cells exposed to cigarette smoke, the expression of a subset of oxidative stress response genes is enhanced. Enhanced activation of these genes can occur as an adapative response to stress induced by reactive oxygen species production, and is frequently detected in cancer. Investigation into the mechanism underlying the enhanced gene expression determined that FHIT loss is associated with decreased levels of the transcriptional repressor Bach1. In this manner, we propose that loss of Fhit supports an antioxidant program that is pivotal in establishing and maintaining carcinogenic transformation.

Orientador: Carlos Alan Candido Dias Junior / Resumo: O presente estudo teve por objetivo investigar e comparar as alterações relacionadas à reatividade vascular e aos biomarcadores de estresse oxidativo e da inflamação em ratos anestesiados com isoflurano ou sevoflurano. Para alcançar nossos objetivos, ratos Wistar machos adultos foram distribuídos aleatoriamente em três grupos experimentais: não anestesiado (grupo controle) e anestesiados com isoflurano (grupo Iso) ou sevoflurano (grupo Sevo). A responsividade vascular à fenilefrina (PHE) e acetilcolina (ACh) na presença ou ausência de éster N-nitro-L-arginina-metílico (L-NAME) foram avaliadas em anéis de aorta torácica com endotélio intacto (E +) e endotélio desnudo (E-). Os metabólitos do óxido nítrico (NO) (NOx), a peroxidação lipídica, a função antioxidante, os níveis de citocinas e a atividade das metaloproteinases de matriz extracelular (MMPs) também foram avaliados. Nenhuma diferença significativa foi observada com relação aos dados hemodinâmicos e temperatura entre os grupos anestesiados. Nos anéis com endotélio intacto, a anestesia com o sevoflurano reduziu a vasoconstrição induzida pela PHE e pelo KCL quando comparado ao grupo controle e Iso, respectivamente (P ˂ 0.05). O isoflurano aumentou significativamente a vasoconstrição induzida pela PHE e pelo KCl, nos anéis com endotélio desnudo (P ˂ 0.05). Níveis de NO reduzidos foram observados após a anestesia com sevoflurano (P = 0.01), mas não com isoflurano. O estresse oxidativo foi significativamente aumentado em ambo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The current study aimed to investigate and compare the changes on vascular reactivity, biomarkers of oxidative stress and inflammation of rats anesthetized with isoflurane or sevoflurane. Adult Wistar rats were assigned to three experimental groups (n = 6 per group): Non-anesthetized (Control group) and anesthetized with isoflurane (Iso group) or sevoflurane (Sevo group). Vascular responsiveness to phenylephrine (PHE) and acetylcholine (ACh) in the presence or absence of Nω-nitro-L-arginine-methyl ester (L-NAME) was evaluated in thoracic aortic rings with intact endothelium (E+) and denuded endothelium (E-). Nitric oxide (NO) metabolites (NOx), lipid peroxidation, antioxidant function, cytokines levels and matrix metalloproteinases (MMPs) activity were also assessed. In intact endothelium-aortic rings sevoflurane impairs PHE-induced vasoconstriction versus control group and reduces KCl-induced vasoconstriction versus Iso group (P ˂ 0.05). In denuded endothelium-aortic rings isoflurane increases vasoconstriction induced by both PHE and KCl (P ˂ 0.05). Sevoflurane, but not isoflurane, reduces NO (P = 0.01). Oxidative stress was increased in Sevo and Iso groups (P ˂ 0.05). While sevoflurane reduced IL-10 and IL-1β, isoflurane increased IL-1β. MMP-2 activity in aorta was increased in Sevo (P ˂ 0.05), but not in Iso group. We concluded that 150 minutes of anesthesia with sevoflurane, but not with isoflurane, impairs vascular responsiveness to vasoconstrictors agents. Also, while b... (Complete abstract click electronic access below) / Doutor

Parkinson's disease (PD) is a prevalent neurodegenerative disorder for which the greatest risk factor is age. Four to five percent of 85-year-olds suffer from this debilitating disease, which is characterized by the selective loss of dopaminergic neurons within the substantia nigra and the presence of protein aggregates known as Lewy bodies. While the etiology of this disease is still unknown, recent research implicates oxidative stress, activated microglia, and reactive dopamine (DA) metabolites to play a role in the initiation or progression of the disease. Activated microglia cause injury to dopaminergic neurons via a host of mechanisms, including reactive oxygen species production, release of cytokines, and phagocytic activity. Microglial activation has been detected in the brains of PD patients, but the source of this activation has not been elucidated. Previous research has shown electrophiles and endogenous neurotoxins to play a role in this microglial activation. The interaction between the neurotoxic metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and microglia has not been explored. DOPAL is a highly reactive, bifunctional electrophile produced by oxidative deamination of DA by monoamine oxidase (MAO). DOPAL is oxidized in the major metabolism pathway to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH). DOPAL has previously been shown to be 100-fold more toxic than DA in vitro and in vivo. Potent inhibition of the rate-limiting enzyme in DA biosynthesis, tyrosine hydroxylase, by DOPAL has been well-established. DOPAL-mediated aggregation of Α-synuclein, the primary component of PD-hallmark Lewy bodies, has been suggested but was further explored in this work. Results presented in this body of work include further determination of the aggregation of Α-synuclein by DOPAL, including evidence of covalent modification. The interaction of DOPAL with BV-2 microglia, an immortalized cell line, was addressed in depth through exploration of DOPAL catabolism, toxicity, and generation of an activational response. Metabolism of DOPAL to DOPAC was altered in activated microglia, with the production of DOPAC reduced by ~40%. Metabolism of DOPAL to DOPAC was also inhibited by both 4-hydroxynonenal and malondialdehyde, gold standards of lipid peroxidation. Both of these compounds were found to be significantly toxic to BV-2 cells at concentrations well below those considered toxic to dopaminergic cells. Alternatively, DOPAL and DA were found to be non-toxic to this cell line, while DOPAL was shown to be significantly toxic to dopaminergic cells at concentrations as low as 10 ΜM. Significant activation of BV-2 microglia by DOPAL was observed at 10 ΜM and above by release of TNF-Α. Morphological changes, release of IL-6, and changes in expression of COX-2 also indicated activation by DOPAL but not DA or DOPAC. BV-2-conditioned media, generated by incubation with DA, DOPAL, or DOPAC, was added to MN9D cells, and toxicity was measured by the MTT assay. BV-2 conditioned media generated by DOPAL incubation produced the greatest toxicity for MN9D cells. These results implicate DOPAL in dopaminergic cell death through microglial activation.