Objetivos:Determinar a concentração do Marcador de Peroxidação Lipídica: Malondialdeído (MDA) e dos Marcadores Antioxidantes: Superóxido Dismutase (SOD), Glutationa Peroxidase (GPX), Catalase (CAT); no sangue do cordão umbilical e na saliva não estimulada nas primeiras 24 e 48 horas de vida nos RNPT com e sem fatores de risco maternos para sepse neonatal precoce. Casuística/Metodologia: Foram estudados 21 RNPT nascidos na Maternidade e provenientes do Serviço de Neonatologia do Hospital Municipal de Barueri, São Paulo. Os RNPT foram classificados em dois grupos: Grupo 1- RNPT de mães com fatores de risco maternos para sepse neonatal e Grupo 2- RNPT de mães sem fatores de risco maternos para sepse. Foram coletadas amostras de sangue do cordão umbilical e da saliva não estimulada nas primeiras 24 e 48 horas de vida para dosage de MDA, SOD, CAL, GPX. Resultados: Na amostra houve prevalência do sexo feminino, de RNPT adequados para a idade gestacional. Quanto a necessidade de oxigênio (61,9%) utilizaram. Foi feito o diagnóstico de sepse presumida em 9,5%. Houve somente diferença estatística na variável GPX no grupo 1.).Conclusão: Os dados deste estudo apontam para um aumento da concentração da GPX no sangue da veia umbilical dos RNPT do grupo das mães com fatores de risco maternos para sepse neonatal precoce. Sem significância estatística na comparação da saliva e o sangue do cordão umbilical. Não houve diferença estatísticamente significante nas variáveis MDA, SOD, CAT entre os dois grupos tanto na saliva de 24 e 48 horas de vida quanto no sangue do cordão umbilical. A dosagem de GPX o sangue do cordão umbilical pode ser uma possível ferramenta diagnóstica para identificar de forma mais rápida a suspeição de sepse neonatal precoce do RNPT de mãe com fator de risco para sepse neonatal precoce sem produzir maior estresse ao paciente / Objectives: To determine the concentration of the Lipid Peroxidation Marker: Malondialdehyde (MDA) and Antioxidant Markers: Superoxide Dismutase (SOD), Glutathione Peroxidase (GPX), Catalase (CAT); in umbilical cord blood and non-stimulated saliva in the first 24 and 48 hours of life in Pre-Term Newborns (PTN) with and without maternal risk factors for early neonatal sepsis. Casuistic / Methodology: Twenty - one PTN born at the Maternity Hospital and from the Neonatal Service of the Municipal Hospital of Barueri, São Paulo, Brazil, were studied. Preterm infants were classified into two groups: Group 1 - Preterm infants of mothers with maternal risk factors for neonatal sepsis and Group 2 - Preterm infants of mothers without maternal risk factors for sepsis. Blood samples from umbilical cord and non-stimulated saliva were collected in the first 24 and 48 hours of life for dosage of MDA, SOD, CAT, GPX. Results: In the sample, there was a prevalence of female, preterm infants suitable for gestational age. Regarding the need for oxygen (61.9%) they used. The diagnosis of presumed sepsis was made in 9.5%. There was only statistical difference in the GPX variable in group 1. Conclusion: The data from this study point to an increase in GPX concentration in the umbilical vein blood of the PTNB group of mothers with maternal risk factors for early neonatal sepsis. No statistical significance in the comparison of saliva and umbilical cord blood. There was no statistically significant difference in the MDA, SOD, and CAT variables between the two groups in both saliva of 24 and 48 hours of life and in umbilical cord blood. The measurement of GPX umbilical cord blood may be a possible diagnostic tool to more quickly identify the suspicion of early neonatal sepsis of the PTN from a mother with maternal risk factor for early neonatal sepsis without producing greater stress to the patient

Magister Scientiae - MSc (Biotechnology) / Soybean have been listed as a priority commodity crop in South Africa (SA) and provide a good source of protein to the population. Therefore, soybean has been earmarked as an important food security crop and strategies are currently being discussed at governmental level to increase and sustain soybean production. However, the SA landscape poses many challenges to the agricultural sector such as prolong drought periods, flooding, nutrient poor soils, saline soils and heavy metal contaminated soils. Heavy metal (HM) contamination is becoming a serious concern and is aggravated by historical mining in SA. Indeed, SA has established itself as the number one ranked mining country in the world and is frequently mining metals such as chromium, vanadium, gold, zirconium, platinum, and antimony. Prolong rainfall near mining areas leads to acid mine drainage which lowers the soil pH to approximately two. These highly acidic soils will solubilize the metals and cause the metals to leach into river systems as well as the water table leading to increase heavy metal contamination in nearby soil sites. This increase metal content negatively affects seed germination and overall plant development. Nonetheless, plants have evolved numerous internal mechanisms that help them to survive HM toxicity; by either avoiding or tolerating the stress. Two stress-activated pathways that help the plant tolerate stress have attracted much interest i.e. the glyoxalase system and reactive oxygen species (ROS) - antioxidant system as they detoxify methylglyoxal (MG) and ROS. / 2021-08-31

Made available in DSpace on 2014-06-11T19:31:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T19:20:19Z : No. of bitstreams: 1 fonteque_jh_dr_botfmvz.pdf: 314172 bytes, checksum: a0386e439bd656f3236dc3ec523e7bea (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A produção de espécies reativas de oxigênio (ERO) é um evento presente em todas as células do organismo e pode estar aumentada em condições como hipóxia induzida pela anemia causando lesões em moléculas como DNA, lipídeos e proteínas. Com o objetivo de avaliar o estresse oxidativo na anemia induzida por perda aguda de sangue, foram utilizados 10 ovinos, mestiços da raça Texel, machos e fêmeas, com idade entre seis e oito meses, clinicamente sadios, mantidos em regime de confinamento. Os animais foram submetidos a duas flebotomias para a retirada de 30% e 20% do volume sangüíneo com intervalo de 12 horas. Amostras de sangue foram colhidas imediatamente antes da flebotomia, 6h e 12h após a primeira flebotomia, 6h, 12h, 24h, 48h, 72 horas, 4d, 5d, 6d, 10d, 14d, 21d e 28 dias após a segunda flebotomia. Foram avaliados o óxido nítrico, substâncias reativas ao ácido tiobarbitúrico, malondialdeído, cortisol e lactato séricos, hemograma e bioquímica sérica. A análise estatística dos dados foi realizada por meio do Teste de Análise de Variância de Medidas Repetidas (ANOVA) ao nível de 5% de significância. Os resultados demonstraram que o protocolo de indução de anemia foi capaz de induzir anemia 12 horas após a segunda flebotomia, estresse oxidativo e lipoperoxidação caracterizado pelo aumento das substâncias reativas ao ácido tiobarbitúrico. O cortisol elevou-se e alterou o leucograma aumentando o número de leucócitos, neutrófilos e a relação neutrófilo:linfócito. Todos os animais recuperaram-se dentro do período de 28 dias após a flebotomia. Conclui-se que a retirada de 30% e 20% do volume de sangue com intervalo de 12 horas provoca estresse oxidativo e lipoperoxidação em ovinos. / Reactive oxygen species (ROS) are produced in all cells and an increase can be associated with hypoxia induced by anemia. This results in DNA, lipid and protein damage. The aim of this work was to evaluate the oxidative stress induced by experimental acute blood loss. Ten healthy cross Texel sheep underwent two phlebotomies with 12 hours interval. Blood samples were collected before the first phlebotomy (30% of blood volume) and 6 and 12 hours after. A second phlebotomy was performed 12 hours after the first one (20% of blood volume) and blood samples were collected, 6h, 12h, 24h, 48h, 72h, 4 days, 5 days, 6 days, 10 days, 14 days, 21 days and 28 days after. Thiobarbituric acid-reactive substances, malondialdehyde, nitric oxide, lipid peroxidation, serum cortisol, serum lactate, CBC and serum biochemistry were evaluated. Statistical analysis was reformed using repeated measures ANOVA. The experimental protocol used was able to induce anemia 12 hours after the second phlebotomy resulting in oxidative stress and lipoperoxiation characterized by the thiobarbituric acid-reactive substances increase. There was also a cortisol increase causing white blood cell changes: increased number of leukocytes, neutrophils and neutrophil and lymphocyte rate. All animals were normal 28 days after the phlebotomy. In conclusion, the protocol used showed that phlebotomy causing 30 and 20% at blood loss can induce oxidative stress and, lipid peroxidation in sheep.

Made available in DSpace on 2014-06-11T19:23:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-03-03Bitstream added on 2014-06-13T20:30:49Z : No. of bitstreams: 1 machado_lp_me_botfmvz.pdf: 341603 bytes, checksum: b779869a2fd113691f68f4c66bf1832f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The objective of this study was to evaluate the oxidative erythrocyte metabolism in equines submitted to exercise on high-speed treadmill and the effect of vitamin E supplementation. Eight adults Arabian horses, males and females, were divided: control group (CG) and group supplemented with vitamin E (EG), (1000 UI/animal/day). All the equines were submitted to exercise with two incremental tests (T1 and T2). Exercise protocol for two tests started with 1.8m/s for 5 min, 4m/s for 3 min, 6m/s for 2 min and right after, periods of 1 min, challenging the equines with increasing speeds until the animals had no condition to keep the exercise on a treadmill inclined at 7%. Between the tests, a training protocol was performed for 20 days. Blood samples were taken before, during, and until 120 hours after exercise to determine erytrogram, erythrocyte reduced glutathione (GSH), superoxide dismutase (SOD), methemoglobin, erythrocyte osmotic fragility (EOF), total plasmatic protein (TPP), seric malondialdehyde (MDA) and vitamin E, seric enzymatic activity of aspartate aminotransferase (AST) and creatine kinase (CK), blood lactate and bloodgas. The results were compared by non-parametric Mann-Whitney and Friedman tests. Although differences were detected in only a few variables, it was possible to see increase in erytrogram and TPP by spleenic contraction and/or decrease of the plasma volume, and increase the erythrocyte volume by swelling. Sport anemia was observed between 24h and 120h after the exercise. The methemoglobin was compatible with the physiological levels and the EOF increased during the exercise in both groups. The MDA elevation confirm the liperoxidation by exercise effect, less intense EG by the supplementation with vitamin E. The exercise also induced increase of AST and CK enzymes, increase of blood lactate and metabolic acidosis.

Orientador : Aguemi Kohayagawa / Banca: Alexandre Secorun Borges / Banca: Maria Adriana Machado Loba e Silva / Abstract: The objective of this study was to evaluate the oxidative erythrocyte metabolism in equines submitted to exercise on high-speed treadmill and the effect of vitamin E supplementation. Eight adults Arabian horses, males and females, were divided: control group (CG) and group supplemented with vitamin E (EG), (1000 UI/animal/day). All the equines were submitted to exercise with two incremental tests (T1 and T2). Exercise protocol for two tests started with 1.8m/s for 5 min, 4m/s for 3 min, 6m/s for 2 min and right after, periods of 1 min, challenging the equines with increasing speeds until the animals had no condition to keep the exercise on a treadmill inclined at 7%. Between the tests, a training protocol was performed for 20 days. Blood samples were taken before, during, and until 120 hours after exercise to determine erytrogram, erythrocyte reduced glutathione (GSH), superoxide dismutase (SOD), methemoglobin, erythrocyte osmotic fragility (EOF), total plasmatic protein (TPP), seric malondialdehyde (MDA) and vitamin E, seric enzymatic activity of aspartate aminotransferase (AST) and creatine kinase (CK), blood lactate and bloodgas. The results were compared by non-parametric Mann-Whitney and Friedman tests. Although differences were detected in only a few variables, it was possible to see increase in erytrogram and TPP by spleenic contraction and/or decrease of the plasma volume, and increase the erythrocyte volume by swelling. Sport anemia was observed between 24h and 120h after the exercise. The methemoglobin was compatible with the physiological levels and the EOF increased during the exercise in both groups. The MDA elevation confirm the liperoxidation by exercise effect, less intense EG by the supplementation with vitamin E. The exercise also induced increase of AST and CK enzymes, increase of blood lactate and metabolic acidosis. / Mestre

Orientador: Aguemi Kohayagawa. / Resumo: A produção de espécies reativas de oxigênio (ERO) é um evento presente em todas as células do organismo e pode estar aumentada em condições como hipóxia induzida pela anemia causando lesões em moléculas como DNA, lipídeos e proteínas. Com o objetivo de avaliar o estresse oxidativo na anemia induzida por perda aguda de sangue, foram utilizados 10 ovinos, mestiços da raça Texel, machos e fêmeas, com idade entre seis e oito meses, clinicamente sadios, mantidos em regime de confinamento. Os animais foram submetidos a duas flebotomias para a retirada de 30% e 20% do volume sangüíneo com intervalo de 12 horas. Amostras de sangue foram colhidas imediatamente antes da flebotomia, 6h e 12h após a primeira flebotomia, 6h, 12h, 24h, 48h, 72 horas, 4d, 5d, 6d, 10d, 14d, 21d e 28 dias após a segunda flebotomia. Foram avaliados o óxido nítrico, substâncias reativas ao ácido tiobarbitúrico, malondialdeído, cortisol e lactato séricos, hemograma e bioquímica sérica. A análise estatística dos dados foi realizada por meio do Teste de Análise de Variância de Medidas Repetidas (ANOVA) ao nível de 5% de significância. Os resultados demonstraram que o protocolo de indução de anemia foi capaz de induzir anemia 12 horas após a segunda flebotomia, estresse oxidativo e lipoperoxidação caracterizado pelo aumento das substâncias reativas ao ácido tiobarbitúrico. O cortisol elevou-se e alterou o leucograma aumentando o número de leucócitos, neutrófilos e a relação neutrófilo:linfócito. Todos os animais recuperaram-se dentro do período de 28 dias após a flebotomia. Conclui-se que a retirada de 30% e 20% do volume de sangue com intervalo de 12 horas provoca estresse oxidativo e lipoperoxidação em ovinos. / Abstract: Reactive oxygen species (ROS) are produced in all cells and an increase can be associated with hypoxia induced by anemia. This results in DNA, lipid and protein damage. The aim of this work was to evaluate the oxidative stress induced by experimental acute blood loss. Ten healthy cross Texel sheep underwent two phlebotomies with 12 hours interval. Blood samples were collected before the first phlebotomy (30% of blood volume) and 6 and 12 hours after. A second phlebotomy was performed 12 hours after the first one (20% of blood volume) and blood samples were collected, 6h, 12h, 24h, 48h, 72h, 4 days, 5 days, 6 days, 10 days, 14 days, 21 days and 28 days after. Thiobarbituric acid-reactive substances, malondialdehyde, nitric oxide, lipid peroxidation, serum cortisol, serum lactate, CBC and serum biochemistry were evaluated. Statistical analysis was reformed using repeated measures ANOVA. The experimental protocol used was able to induce anemia 12 hours after the second phlebotomy resulting in oxidative stress and lipoperoxiation characterized by the thiobarbituric acid-reactive substances increase. There was also a cortisol increase causing white blood cell changes: increased number of leukocytes, neutrophils and neutrophil and lymphocyte rate. All animals were normal 28 days after the phlebotomy. In conclusion, the protocol used showed that phlebotomy causing 30 and 20% at blood loss can induce oxidative stress and, lipid peroxidation in sheep. / Doutor

Este estudo teve como objetivo avaliar o estado nutricional relativo ao selênio (Se) de pacientes na fase de pós-tratamento da leucemia linfóide aguda (LLA) e sua relação com o estresse oxidativo. Foram selecionados 24 pacientes no pós-tratamento da LLA (9,2 ± 1,9 anos) atendidos no Instituto de Oncologia Pediátrica da Universidade Federal de São Paulo e 60 indivíduos saudáveis (9,5 ± 1,3 anos) da Escola de Aplicação da Universidade de São Paulo. Foram coletados 10 mL de sangue venoso para análise de Se plasmático e eritrocitário, glutationa peroxidase (GPx), superóxido dismutase (SOD), α- tocoferol, malondialdeído (MDA) e 8-oxo-desoxiguanosina (8-oxo-dGuo). A urina de 24 horas foi coletada para análise da excreção de Se, e três recordatórios de consumo alimentar de 24 horas para avaliação do Se ingerido. Os resultados obtidos quanto aos parâmetros bioquímicos de avaliação de Se não apresentaram diferença significativa entre os grupos de pacientes e controles, e foram respectivamente: Se plasmático, 44,4 ± 9,0 µg/L e 48,7 ± 12,0 µg/L (p = 0,122); Se eritrocitário, 49,9 ± 15,9 µg/L e 45,0 ± 15,9 µg/L (p = 0,202); Se urinário, 19,6 ± 14,8 µg Se/g de creatinina e 18,6 ± 9,6 µg Se/g de creatinina (p = 0,820). O consumo médio de Se foi de 27,4 ± 8,7 µg/dia e 28,0 ± 1,5 µg/dia (p = 0,756), respectivamente. Os grupos estudados foram considerados deficientes em Se, considerando os pontos de corte adotados. A atividade da GPx foi significativamente menor nos pacientes do que nos controles (33,3 ± 11,1 U/g Hb e 76,9 ± 25,9 U/g Hb) (p = 0,000), e a atividade da SOD não diferiu entre pacientes e controles (1796,9 ± 257,8 U/g Hb e 1915,9 ± 473,9 U/g Hb) (p = 0,145), assim como as concentrações de MDA (1,7 ± 0,3 µmol/L e 1,8 ± 0,4 µmol/L) (p = 0,053). A concentração de α-tocoferol foi estatisticamente maior nos pacientes que nos controles (17,7 ± 4,7 µmol/L e 10,6 ± 3,2 µmol/L) (p =0,000), bem como a concentração de 8-oxo-dGuo (43,6 ± 28,0 8-oxo/106 dG e 21,3 ± 22,9 8-oxo/106 dG) (p = 0,014). Os resultados apresentados apontam que os participantes deste estudo estão deficientes em Se e, em especial os pacientes no pós-tratamento da LLA estão sujeitos a um aumento do estado de estresse oxidativo, pois apesar das concentrações de MDA serem semelhantes entre os pacientes e os controles, a atividade da GPx dos pacientes foi reduzida e a concentração de 8-oxo-dGuo e α-tocoferol estavam aumentadas em relação aos controles. / This study aimed to evaluate the nutritional status of selenium (Se) in patients in post-treatment of acute lymphoblastic leukemia (ALL) and its relationship with oxidative stress. We selected 24 patients in post-treatment of ALL (9.2 ± 1.9 years) at the Pediatric Oncology Institute of Federal University of São Paulo and 60 healthy individuals (9.5 ± 1.3 years) of the School of Application at the University of São Paulo. We collected 10 mL of venous blood for analysis of Se in plasma and erythrocytes, glutathione peroxidase (GPx), superoxide dismutase (SOD), α-tocopherol, malondialdehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-dGuo). The 24-hour urine was collected for analysis of Se excretion and Se intake was evaluated by using three non-consecutive days of 24- hour recall. The results regarding biochemical evaluation of Se did not differ significantly between patients in post-treatment of ALL and controls, and the results were respectively: Se in plasma, 44.4 ± 9.0 µg/L and 48.7 ± 12.0 µg/L (p = 0.122); Se in erythrocytes, 49.9 ± 15.9 µg/L and 45.0 ± 15.9 µg/L (p = 0.202); Se in urine, 19.6 ± 14.8 µg Se/g creatinine and 18.6 ± 9.6 µg Se/g creatinine (p = 0.820). The average intake of selenium was 27.4 ± 8.7 mg/day and 28.0 ± 1.5 mg/day (p = 0.756), respectively. Both groups were considered deficient in selenium, according to the cut-off points adopted. The GPx activity was significantly lower in patients than in controls (33.3 ± 11.1 U/g Hb and 76.9 ± 25.9 U/g Hb) (p = 0.000), and no difference in SOD activity was observed between groups (1796.9 ± 257.8 U/g Hb and 1915.9 ± 473.9 U/g Hb) (p = 0.145). MDA concentrations were not different between patients and controls (1.7 ± 0.3 µmol/L and 1.8 ± 0.4 µmol/L) (p = 0.053) and α-tocopherol concentration was statistically higher in patients (17.7 ± 4.7 µmol/L and 10.6 ± 3.2 µmol/L) (p = 0.000), as well the concentration of 8-oxo-dGuo (43.6 ± 28.0 8-oxo/106 dG and 21.3 ± 22.9 8-oxo/106 dG) (p = 0.014). These results indicate that the participants in this study are deficient in Se mainly those who are in post-treatment of ALL are exposed to an increased state of oxidative stress, because although the concentrations of MDA were similar between patients and controls, the GPx activity of the patients was reduced and the concentration of 8-oxo-dGuo and α-tocopherol were increased compared to controls.

In all organisms are rising a reactive oxygen and nitrogen species by the effects of various stress factors and these species have a negative impact on the organism. Due to this species plants have built up an efficient antioxidant system, that helps them to resist negative effects of reactive oxygen and nitrogen species. In this work was researched the effect of hydrogen peroxide and sodium benzoate on the production of hydrogen peroxide, superoxide, reactive nitrogen species and malondialdehyde, contained in the root and above-ground part of maize (Zea mays L.). By use of the fluorescence microscopy there were obtained images of cross-cut of root from which was determined the intensity of fluorescence of individual parts of the root and was examined the effect of the intensity of fluorescence markers of oxidative stress in dependence on the type of the fluorescence filter used.

(1) Background: Canine distemper virus (CDV)-induced demyelinating leukoencephalitis (CDV-DL) in dogs and Theiler’s murine encephalomyelitis (TME) virus (TMEV)-induced demyelinating leukomyelitis (TMEV-DL) are virus-induced demyelinating conditions mimicking Multiple Sclerosis (MS). Reactive oxygen species (ROS) can induce the degradation of lipids and nucleic acids to characteristic metabolites such as oxidized lipids, malondialdehyde, and 8-hydroxyguanosine. The hypothesis of this study is that ROS are key eector molecules in the pathogenesis of myelin membrane breakdown in CDV-DL and TMEV-DL. (2) Methods: ROS metabolites and antioxidative enzymes were assessed using immunofluorescence in cerebellar lesions of naturally CDV-infected dogs and spinal cord tissue of TMEV-infected mice. The transcription of selected genes involved in ROS generation and detoxification was analyzed using gene-expression microarrays in CDV-DL and TMEV-DL. (3) Results: Immunofluorescence revealed increased amounts of oxidized lipids, malondialdehyde, and 8-hydroxyguanosine in CDV-DL while TMEV-infected mice did not reveal marked changes. In contrast, microarray-analysis showed an upregulated gene expression associated with ROS generation in both diseases. (4) Conclusion: In summary, the present study demonstrates a similar upregulation of gene-expression of ROS generation in CDV-DL and TMEV-DL. However, immunofluorescence revealed increased accumulation of ROS metabolites exclusively in CDV-DL. These results suggest dierences in the pathogenesis of demyelination in these two animal models.

Ο εγκέφαλος των θηλαστικών είναι αρκετά ευάλωτος στις επιδράσεις περιβαλλοντικών τοξινών, λόγω των ιδιαίτερων δομικών και λειτουργικών χαρακτηριστικών του. Η έκθεση σε μια νευροτοξίνη εκδηλώνεται συνήθως μέσω γνωστικών και συμπεριφορικών διαταραχών, που συνοδεύουν δυσμενείς νευροχημικές αλλαγές και καθορίζονται από το είδος, την ηλικία, το φύλο, το γενετικό προφίλ, τη δόση, την οδό και τη χρονική περίοδο έκθεσης. Ωστόσο, η χρόνια έκθεση σε ένα νευροτοξικό παράγοντα είναι δυνατόν να επάγει μη αναστρέψιμη νευρωνική βλάβη και εκφύλιση. Το αργίλιο (Al), που συνιστά το τρίτο σε αφθονία στοιχείο στη φύση, ασκεί ποικίλες νευροτοξικές επιδράσεις, ανάλογα με τη χημική μορφή του μετάλλου, τη δόση, την οδό και την περίοδο έκθεσης, ενώ αμφιλεγόμενη παραμένει η εμπλοκή του στην παθογένεια της νόσου του Alzheimer. Η αφλατοξίνη Β1 (AFB1) ανήκει στην ομάδα των μυκοτοξινών (δευτερογενής μεταβολίτης των μυκήτων του γένους Aspergillus), μολύνει καλλιέργειες και ζωοτροφές και αποτελεί ισχυρή ηπατοτοξίνη και ηπατοκαρκινογόνο. Εντούτοις, η νευροτοξικότητα της AFB1 είναι ελάχιστα μελετημένη και οι λίγες αναφορές που παρουσιάζουν την εκδήλωση συμπεριφορικών διαταραχών, αφορούν την έκθεση σε αναπτυξιακό στάδιο. Εκτενής και εντατική είναι τις τελευταίες δεκαετίες η έρευνα της νευροπροστατευτικής δράσης φαρμακευτικών φυτών και των βιοδραστικών συστατικών τους, με απώτερο στόχο την πρόληψη ή αντιμετώπιση της εγκεφαλικής δυσλειτουργίας που επάγεται από γενετικούς ή/και περιβαλλοντικούς παράγοντες. Ενδιαφέρον για τον ελλαδικό χώρο, λόγω της υψηλής εμπορικής του αξίας, έχει το καλλιεργούμενο φυτικό είδος Crocus sativus L., του οποίου οι στύλοι (κρόκος ή σαφράν) χρησιμοποιούνται στη διατροφή ως άρτυμα και η φαρμακευτική τους αξία έχει αναγνωριστεί εδώ και χιλιετίες. Στόχος της παρούσας διδακτορικής διατριβής ήταν να συμβάλλει στην έρευνα της νευροτοξικής δράσης του Al και της AFB1 και του νευροπροστατευτικού ρόλου των στύλων του C. sativus, εστιάζοντας σε παραμέτρους της μνημονικής λειτουργίας ενηλίκων μυών, της χολινεργικής/μονοαμινεργικής διαβίβασης και της οξειδωτικής/αντιοξειδωτικής κατάστασης του εγκεφάλου τους. ΜΕΘΟΔΟΙ: Σε αρσενικούς ενήλικες Balb-c μύες (n=7-10/ομάδα) χορηγήθηκε δια στόματος AlCl3 (50 mg/kg σωματικού βάρους/ημέρα) διαλυμένο στο κανονικό πόσιμο νερό για 5 εβδομάδες ή ενδοπεριτοναϊκά (i.p.) AFB1 (0.3 και 0.6 mg/kg σωματικού βάρους/ημέρα) για 4 ημέρες. Η ικανότητα εκχυλισμάτων των στύλων του C. sativus και της κροκετίνης, του κύριου βιοδραστικού μεταβολίτη των καροτενοειδών συστατικών (κροκίνες) του κρόκου, να προλαμβάνουν ή να ανατρέπουν τις βλαπτικές επιδράσεις του Al και της AFB1 στον εγκέφαλο των μυών, διερευνήθηκε ακολουθώντας τα εξής σχήματα χορήγησης: α) υδατικό/μεθανολικό εκχύλισμα κρόκου (60 mg/kg σωματικού βάρους/ημέρα) χορηγήθηκε i.p. τις τελευταίες 6 ημέρες της περιόδου χορήγησης του AlCl3 (50 mg/kg σωματικού βάρους/ημέρα στο πόσιμο νερό για 5 εβδομάδες), β) αφέψημα κρόκου (0.45 mg/mL) καταναλώθηκε για 2 εβδομάδες πριν τη χορήγηση AFB1 (0.6 mg/kg σωματικού βάρους/ημέρα i.p. τις τελευταίες 4 ημέρες της περιόδου χορήγησης του αφεψήματος), και γ) καθαρή κροκετίνη (4 mg/kg σωματικού βάρους/ημέρα) χορηγήθηκε i.p. για 3 ημέρες πριν ή μετά τη χορήγηση AFB1 (0.6 mg/kg σωματικού βάρους/ημέρα i.p. για 4 ημέρες). Μελετήθηκαν επίσης, οι επιδράσεις των προηγούμενων σχημάτων χορήγησης του αφεψήματος κρόκου και της κροκετίνης στον εγκέφαλο υγιών ενηλίκων μυών. Η ικανότητα μάθησης/μνήμης των μυών αξιολογήθηκε με τη δοκιμασία παθητικής αποφυγής. Η ενεργότητα της ακετυλοχολινεστεράσης [AChE, διαλυτές σε άλας (SS)/απορρυπαντικό (DS) ισομορφές], της βουτυρυλοχολινεστεράσης (BuChE, SS/DS ισομορφές) και της μονοαμινοξειδάσης (ΜΑΟ, -Α και -Β ισομορφές) προσδιορίστηκαν στον ολικό εγκέφαλο (-ce, πλην παρεγκεφαλίδας) και την παρεγκεφαλίδα, ως δείκτες της χολινεργικής και μονοαμινεργικής διαβίβασης, αντιστοίχως. Επίσης, μετρήθηκαν οι συγκεντρώσεις της μηλονικής διαλδεΰδης (MDA) και της ανηγμένης γλουταθειόνης (GSH), ως δείκτες της λιπιδικής υπεροξείδωσης και της αντιοξειδωτικής άμυνας, αντιστοίχως, των εγκεφαλικών ιστών. Με τη χρήση φασματομετρίας ατομικής απορρόφησης μετρήθηκαν τα επίπεδα Al στους εγκεφαλικούς ιστούς, ενώ, για πρώτη φορά, η κροκετίνη προσδιορίστηκε στον ολικό εγκέφαλο (-ce) των μυών μετά την i.p. χορήγηση εκχυλίσματος κρόκου, με HPLC ανάλυση. ΑΠΟΤΕΛΕΣΜΑΤΑ: Η μακρόχρονη πρόσληψη υψηλής δόσης AlCl3 μέσω του πόσιμου νερού οδήγησε σε εξασθένηση της μάθησης/μνήμης των μυών, σημαντική μείωση της ενεργότητας της AChE και BuChE, αύξηση της ενεργότητας των ισομορφών της ΜΑΟ του ολικού εγκεφάλου (-ce), αλλά αναστολή της ΜΑΟ-Β της παρεγκεφαλίδας, σημαντική αύξηση των επιπέδων MDA στον εγκέφαλο και μείωση της συγκέντρωσης GSH στους εγκεφαλικούς ιστούς. Συσσώρευση του μετάλλου καταγράφηκε στους εγκεφαλικούς ιστούς των μυών που λάμβαναν AlCl3. Μνημονικό έλλειμμα εμφάνισαν οι μύες που έλαβαν την υψηλή (0.6 mg/kg) αλλά όχι χαμηλή δόση (0.3 mg/kg) AFB1. Επίσης, η βραχύχρονη i.p. χορήγηση της μυκοτοξίνης ανέστειλε τις χολινεστεράσες (ChEs), ενεργοποίησε τη ΜΑΟ, αύξησε σημαντικά τη λιπιδική υπεροξείδωση και μείωσε τα επίπεδα GSH στους εγκεφαλικούς ιστούς. Ωστόσο, διαφορική απόκριση στην έκθεση στην AFB1 παρουσίασαν οι ισομορφές της BuChE και ΜΑΟ των εγκεφαλικών ιστών, ανάλογα με τη χορηγούμενη δόση. Αντιχολινεστερασική και αντιοξειδωτική δράση επέδειξαν τόσο η μακρόχρονη πρόσληψη αφεψήματος κρόκου όσο και η βραχύχρονη i.p. χορήγηση κροκετίνης στους εγκεφαλικούς ιστούς των υγιών μυών, ενώ δεν μετέβαλλαν τη μνημονική τους ικανότητα. Η βραχύχρονη συγχορήγηση εκχυλίσματος κρόκου στο τέλος της περιόδου πρόσληψης AlCl3, αν και δεν είχε καμία επίδραση στη γνωστική ικανότητα των μυών, αντέστρεψε σημαντικά τις επαγόμενες από το Al αλλαγές της ενεργότητας της ΜΑΟ και των επιπέδων MDA και GSH των εγκεφαλικών ιστών. Επιπλέον, η ενεργότητα των ισομορφών της AChE των εγκεφαλικών ιστών μειώθηκε περαιτέρω σημαντικά μετά τη χορήγηση του εκχυλίσματος. HPLC ανάλυση του ολικού εγκεφάλου (-ce) των μυών αποκάλυψε, για πρώτη φορά στην παρούσα μελέτη, την παρουσία κροκετίνης μετά τη βραχύχρονη συγχορήγηση εκχυλίσματος κρόκου, η οποία δεν ανιχνεύτηκε στους μύες μάρτυρες. Η μακρόχρονη καθημερινή κατανάλωση αφεψήματος κρόκου πριν την έκθεση σε υψηλή δόση AFB1 απέτρεψε την επαγόμενη από τη μυκοτοξίνη μνημονική εξασθένηση, αναστολή της DS-BuChE του ολικού εγκεφάλου (-ce), αύξηση της ενεργότητας της ΜΑΟ-Α του εγκεφάλου και της ΜΑΟ-Β της παρεγκεφαλίδας και οξειδωτική βλάβη των λιπιδίων στους εγκεφαλικούς ιστούς. Επίσης, οι μύες που κατανάλωναν το αφέψημα εμφάνισαν περαιτέρω σημαντική μείωση της ενεργότητας των ισομορφών της AChE του ολικού εγκεφάλου (-ce), της DS-AChE της παρεγκεφαλίδας και των επιπέδων GSH των εγκεφαλικών ιστών. Αν και η βραχύχρονη i.p. χορήγηση καθαρής κροκετίνης πριν ή μετά την έκθεση σε υψηλή δόση AFB1 δεν επηρέασε την ικανότητα μάθησης/μνήμης των μυών, έδρασε αποτελεσματικά στην πρόληψη ή αντιστροφή της επαγόμενης από τη μυκοτοξίνη αναστολής της BuChE του ολικού εγκεφάλου (-ce), ενεργοποίησης των ισομορφών της ΜΑΟ του εγκεφάλου και αύξησης της λιπιδικής υπεροξείδωσης των εγκεφαλικών ιστών. Ωστόσο, μόνο η προηγηθείσα χορήγηση κροκετίνης απέτρεψε την αύξηση της ενεργότητας της ΜΑΟ-Β της παρεγκεφαλίδας και τη μείωση των επιπέδων GSH των εγκεφαλικών ιστών, που προκάλεσε η χορήγηση της AFB1. Διαφορική απόκριση (περαιτέρω αναστολή ή αύξηση) στη χορήγηση κροκετίνης εμφάνισαν οι ισομορφές της AChE των εγκεφαλικών ιστών, ανάλογα με τη χρονική ακολουθία της χορήγησης. ΣΥΜΠΕΡΑΣΜΑ: Τα αποτελέσματα της παρούσας μελέτης δείχνουν ότι η μακρόχρονη πρόσληψη AlCl3 μέσω του πόσιμου νερού και η βραχύχρονη συστημική έκθεση στην AFB1 ασκούν ισχυρές νευροτοξικές επιδράσεις στους ενήλικες μύες, όπως απέδειξαν η επαγωγή μνημονικής εξασθένησης και οι νευροχημικές διαταραχές. Η αναστολή του γνωστικού ελλείμματος από τη μακρόχρονη κατανάλωση αφεψήματος κρόκου, υποστηρίζει τη νευροπροστατευτική δράση του κρόκου έναντι της νευροτοξικότητας της AFB1 και τον αναδεικνύει ως ελπιδοφόρο διατροφικό παράγοντα στην πρόληψη της εγκεφαλικής δυσλειτουργίας. Ωστόσο, οι ευεργετικές επιδράσεις της κροκετίνης στους νευροχημικούς δείκτες της εγκεφαλικής λειτουργίας υπό συνθήκες τοξικότητας και η απόδειξη της βιοδιαθεσιμότητάς της στον εγκέφαλο, προτείνουν τη συμβολή των καροτενοειδών συστατικών του κρόκου στις νευροπροστατευτικές του ιδιότητες και ενθαρρύνουν την περαιτέρω διερεύνησή τους ως νευροπροστατευτικών παραγόντων. / Mammalian brain is quite susceptible to environmental toxins, due to its special structural and functional features. Exposure to a neurotoxin is commonly manifested through cognitive and behavioral disturbances that follow adverse neurochemical changes and are defined by the animal species in question, the age, the gender, the genetic profile, the dose, the route and the period of exposure. However, chronic exposure to a neurotoxic agent may induce irreversible neuronal damage and degeneration. Aluminum (Al), which is the third most abundant element in nature, exerts diverse neurotoxic effects, depending on the metal’s chemical form, the dose, the route and the period of exposure, while its implication in the pathogenesis of Alzheimer’s disease remains controversial. Aflatoxin B1 (AFB1) is classified to the group of mycotoxins (secondary metabolite of the fungi of Aspergillus sp.), contaminates crops and feeds and constitutes potent hepatotoxin and hepatocarcinogen. Nevertheless, AFB1 neurotoxicity is poorly studied and the few reports focus on the manifestation of behavioral disorders after exposure at developmental stage. During the last decades, extensive research on the neuroprotective action of medicinal plants and their bioactive components is carried out, with the aim of prevention or treatment of brain dysfunction that is provoked by genetic and/or environmental agents. The plant Crocus sativus L. is of particular interest in Greece ,due to its large-scale cultivation and the high commercial value of its styles (saffron); saffron is used as a spice in diet and its medicinal properties have been recognized for millenia. The aim of the present study was to contribute to the investigation of the neurotoxic activity of Al and AFB1 and the neuroprotective role of saffron, focusing on aspects of memory function, brain cholinergic/monoaminergic transmission and oxidant/antioxidant state in adult mice. METHODS: Male adult Balb-c mice (n=7-10/group) received either AlCl3 orally (50 mg/kg body weight/day) dissolved in normal drinking water for 5 weeks or AFB1 intraperitoneally (i.p.) (0.3 and 0.6 mg/kg body weight/day) for 4 days. The potential of saffron extracts and crocetin, the main bioactive metabolite of saffron carotenoid constituents (crocins), in prevention or reversal of the detrimental effects of Al and AFB1 on mouse brain, was investigated by adopting the following administration schemes: a) aqueous methanolic extract of saffron (60 mg/kg body weight/day) was administered i.p. for the last 6 days of AlCl3 treatment period (50 mg/kg body weight/day in drinking water for 5 weeks), b) saffron infusion (0.45 mg/mL) was consumed for 2 weeks prior to AFB1 administration (0.6 mg/kg body weight/day i.p. for the last 4 days of saffron infusion treatment period), and c) pure crocetin (4 mg/kg body weight/day) was administered i.p. for 3 days before or after AFB1 administration (0.6 mg/kg body weight/day i.p. for 4 days). The effects of the previous administration schemes of saffron infusion and crocetin on brain of healthy adult mice, were also studied. The learning/memory ability of mice was evaluated by step-through passive avoidance task. The activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble (DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms) and monoamine oxidase (MAO, -A and -B isoforms) was assessed in whole brain (-ce, minus cerebellum) and cerebellum, as indices of cholinergic and monoaminergic transmission, respectively. Moreover, malondialdehyde (MDA) and reduced glutathione (GSH) concentrations were determined as indices of lipid peroxidation and antioxidant defence, respectively, in cerebral tissues. Cerebral tissues’ Al levels were measured by atomic absorption spectrometry, while, for the first time, crocetin was determined in mouse whole brain (-ce) after i.p. administration of saffron extract by HPLC analysis. RESULTS: Long-term intake of high dose of AlCl3 through drinking water resulted in learning/memory impairment of mice, significant reduction of AChE and BuChE activity, increase of MAO isoforms’ activity in whole brain (-ce), but inhibition of cerebellar MAO-B, significant elevation of brain MDA levels and decrease of GSH content in cerebral tissues. Metal accumulation was recorded in brain tissues of AlCl3 treated mice. Mice receiving high (0.6 mg/kg) but not low dose (0.3 mg/kg) of AFB1 displayed memory deficit. Furthermore, short-term i.p. administration of mycotoxin inhibited cholinesterases (ChEs), activated MAO, increased significantly lipid peroxidation and reduced GSH levels in cerebral tissues. However, brain tissues’ BuChE and MAO isoforms presented differential response to AFB1 exposure, depending on the administered dosage. Both long-term saffron infusion intake and short-term i.p. administration of crocetin exerted anti-cholinesterase and antioxidant action in healthy mice’ cerebral tissues, while their memory performance remained unchanged. Although short-term co-administration of saffron extract at the end of AlCl3 treatment period had no effect on cognitive capacity of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH of cerebral tissues. Moreover, cerebral AChE isoforms’ activity was further significantly decreased following saffron extract co-administration. HPLC analysis of mouse whole brain (-ce) revealed, for the first time, the presence of crocetin after short-term saffron extract co-administration, which was not detected in control mice. Long-term daily consumption of saffron infusion prior to AFB1 (high dose) exposure prevented the mycotoxin-induced memory impairment, inhibition of whole brain (-ce) DS-BuChE, increase of brain MAO-A and cerebellar MAO-B activity, and oxidative damage of lipids in brain tissues. Also, saffron infusion pre-treated mice displayed further significant decrease of the activity of AChE isoforms in whole brain (-ce), DS-AChE in cerebellum and the levels of GSH in cerebral tissues. Although, short-term i.p. administration of pure crocetin before or after AFB1 (high dose) exposure had no effect on learning/memory ability of mice, it effectively prevented or reversed the mycotoxin-induced inhibition of whole brain (-ce) BuChE, activation of brain MAO isoforms and elevation of cerebral tissues’ lipid peroxidation. However, only crocetin pre-treatment inhibited the increase of cerebellar MAO-B activity and reduction of brain tissues’ GSH content which were provoked by AFB1 administration. Cerebral tissues’ AChE isoforms presented differential response (further decrease or increase) to crocetin treatment, depending on time course of administration. CONCLUSION: The findings of the present study show that long-term intake of AlCl3 through drinking water and short-term systemic exposure to AFB1, exert strong neurotoxic effects on adult mice, as evidenced by the induction of memory impairment and the neurochemical disturbances. The inhibition of cognitive deficit by long-term saffron infusion consumption supports its neuroprotective action against AFB1 neurotoxicity and highlights saffron as a promising dietary agent in prevention of brain dysfunction. However, the beneficial effects of crocetin on neurochemical indices of brain function under toxicity and the demonstration of its bioavailability in brain, suggest the contribution of saffron carotenoids in saffron’s neuroprotective properties and encourage their further investigation as neuroprotective agents.