Dissertationen zum Thema „Subchronic“

Thesis (M.A.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 10, 2009) Includes bibliographical references.

Thesis (M.S.P.)--University of Toledo, 2004.
Typescript. "A thesis [submitted] as partial fulfillment of the requirements of the Master of Science degree in Pharmaceutical Sciences." Includes bibliographical references (leaves 34-37).

Nitrous oxide (N2O) oxidizes vitamin B12. disrupting deoxyribonucleic acid (DNA) synthesis. Occupational exposures to subanesthetic levels of the gas have been documented that may result in suppressed proliferative cell activities. Male CD-I mice were exposed to 0, 50, 500, and 5000 parts of N2O per million parts of air (ppm) for 6 hr/day, 5 days/week for 2 and 13 weeks. Splenic lymphocytes were assayed for responsiveness to mitogens and for the ability to produce interleukin-2 (lL-2) . Tritiated-thymidine ([3H]-TdR) uptake was measured in CD-I splenic lymphocytes cultured in a mixed-lymphocyte culture (MLC). Cytolytic cell activity was measured by 51chromium release assay. Antibody-mediated immunocompetency was determined for sheep red blood cell (SRBC)-sensitized animals by plaque-forming cell (PFC) assay and sera anti-SRBC antibody titer. Deoxyuridine suppression tests (dUdRST) were performed on bone marrow cells. Serum adrenocorticotropic hormone and corticosterone levels were determined. There was significantly decreased splenic lymphocyte uptake of [3H)-TdR by cells cultured with mitogenic substances and in MLC following 2-week animal exposures to 5000 ppm. After 13-week exposures, the animals' splenic lymphocytes showed decreased [3H]-TdR uptake following low N20 dosing and nonsignificantly increased responsiveness at the higher gas exposures in both the blastogenic and MLC assays. Compared to control animals, the 5000- ppm-exposure group had significantly depressed PFC activity and circulating anti-SRBC immunoglobulin M levels following 13-week gas exposures, and all three subchronic exposure groups demonstrated both decreased liver weights and leukopenia. Bone marrow activity at these dosing levels was dose-responsively depressed following subchronic gas exposures.No hormonal effect appears to be attributable to N20 exposure.

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In order to define the safety of phytotherapeutic use of plants is important an evaluation of the toxic potential by clinical, laboratory and histopathological studies in animals after exposure to extract of parts of the plant in different intervals of time. Due to bark infusion from species of Aspidosperma is employed without proof of its toxic potential in treatment of the diabetes mellitus, hypercholesterolemia and gastric disorders, this study proposes an experimental test with ethanolic extract of Aspidosperma subincanum to verify if induces acute and subchronic toxicity in heart, liver and kidneys of mices (Mus musculus). The animals (male and female) received orally a 75 mg/kg, 150 mg/kg and 300 mg/kg of the extract for subchronic intoxication evaluation by daily exposure along of 28 days. In the acute toxicity evaluation were used female mices that received an only dose of 300mg/kg and 2000 mg/kg of the extract and observed during to 14 days. Pharmacological tests were conducted to check the possible action of the extract in central nervous system in male mices submitted to 200 mg/kg, 400 mg/kg and 750 mg/kg by oral, subcutaneous and intraperitoneal ways. The animals showed some signs of neurotoxicity whose intensity was proportional to extract concentration and died with oral dose of 2000 mg/kg. Hematological parameters did not showed any significant abnormalities. Biochemical tests did not presented any changes, except ALT and AST measures which presented significant increases in exposed groups in relation to control group. Concerning histopathological exam it was possible to detect lesions that suggest the existence of injuries in liver (microvacuolization and hyperemia) and kidney (hyperemia and hemorrhage). Thus, it can be concluded that ethanolic extract of Aspidosperma subincanum is toxic orally, both acute and subcronically, in mices and the estimated median lethal dosis (LD50) was below 2000 mg/kg.
Para se determinar a segurança do consumo de fitoterápicos é imperativo uma avaliação do pontecial tóxico por meio de exames clínicos, laboratoriais e histopatológicos de animais após exposição ao extrato de partes da planta em diferentes intervalos de tempo. Pelo fato de infusos da casca de espécies do gênero Aspidosperma serem empregados, sem comprovação de seu pontencial tóxico, no tratamento do diabetes mellitus, da hipercolesterolemia e de distúrbios gástricos, propõe-se um ensaio experimental com o objetivo de avaliar se o extrato etanólico de Aspidosperma subincanum (EEAs) induz toxicidade aguda e subcrônica no coração, fígado e rins de camundongos (Mus musculus). Os animais (machos e fêmeas) receberam por via oral a dose de 75, 150 e 300 mg/kg do extrato para a avaliação de intoxicação subcrônica por 28 dias de exposição diária ao extrato. Na avaliação da toxicidade aguda foram utilizados camundongos fêmeas que receberam a dose única de 300 e 2000 mg/kg do extrato e observados por 14 dias. Testes farmacológicos foram conduzidos para verificar a possível ação desse extrato no sistema nervoso central, sendo utilizados camundongos machos e as doses de 200, 400 e 750mg/kg por via oral, subcutânea e intraperitoneal. Os animais apresentaram alguns sinais de neurotoxicidade e os sinais tiveram intensidade proporcional à concentração do extrato, sendo letais na dose de 2000 mg/kg via oral. Dentre os exames laboratoriais realizados, o eritrograma, plaquetograma e leucograma, não apresentaram nenhuma alteração significativa. Nas provas bioquímicas não foram observadas alterações dignas de nota, à exceção de ALT e AST que apresentaram elevação significativa nos grupos expostos em relação ao grupo controle. Em relação ao exame histopatológico, observaram-se alterações compatíveis com injúrias estruturais hepáticas (microvacuolização e hiperemia) e renais (hiperemia e hemorragia). Conclui-se que o EEAs pode ser considerado tóxico quando administrado por via oral, tanto agudo como subcronicamente, em camundongos e a dose letal mediana (DL50) estimada encontra-se abaixo de 2000 mg/kg.

Akrilamid (CASR No. 79-06-1) predstavlja veoma reaktivni, hidrosolubilni monomer za koji se smatra da ima toksične i potencijalno kancerogene efekte po zdravlje ljudi. Štetne posledice akrilamida i njegovog još reaktivnijeg metabolita, glicidamida, su dokazane kod eksperimentalnih životinja i podrazumevale su neurotoksičnost, genotoksičnost i kancerogenost. Epidemiološke studije rađene na ljudima pokazale su da akrilamid izaziva neurotoksične efekte, dok se genotoksičnost i kancerogenost još smatraju potencijalnim efektima, a zasnivaju se na podacima koji su dobijeni u okviru istraživanja na laboratorijskim životinjama. Njegove štetne posledice na jetru, posebno kod mladog organizma, još uvek nisu dovoljno istražene.Akrilamid se spontano formira u hrani koja je bogata ugljenim hidratima, tokom termičke obrade namirnica na visokim temperaturama. Ovaj monomer se formira tokom tzv. neenzimatske Mallard-ove reakcije, kojom se dobijaju smeđe komponente u hrani. U namirnicama ovaj monomer se formira reakcijom između redukujućih šećera (uglavnom glukoze ili fruktoze) i aminokiseline (dominantno asparagina).Imajući na umu da je jetra centralni organ za metabolizam akrilamida, ovo istraživanje  je imalo za cilj da ispita glavne histološke i biohemijske promene na jetri juvenilnog organizma pacova, nakon njegove subhronične ekspozicije akrilamidu. Istraživanje je  rađeno na 3 eksperimentalne grupe peripubertalnih/juvenilnih mužjaka Wistar pacova, od kojih su dve bile tretirane vodenim rastvorom akrilamida u dozi od 25 ili 50  mg/kg telesne mase, dok je treća grupa služila kao kontrola i primala destilovanu vodu. Životinje su bile tretirane oralno, putem gavaže, 5 dana nedeljno, tokom 3 nedelje. Nakon 24 h od poslednjeg tretmana, životinje su uvedene u etarsku  anesteziju i dekapitovane, a zatim su prikupljeni krv i uzorci tkiva jetre.Tkivo jetre je uzeto iz srednjeg lobusa, fiksirano u 10% neutralnom puferisanom  formalinu tokom 24 h i obrađeno prema standardnom protokolu za parafinsko kalupljenje. Ukalupljeni uzorci jetre su zatim isečeni na serijske preseke tkiva debljine 5 µm, a zatim bojeni histohemijskim i imunohistohemijskim metodama. Uzorci krvi su pripremljeni za serološku analizu. Histološka analiza preseka bojenih hematoksilin-eozin (H&E) metodom nije  zabeležila prisustvo značajnih razlika u opštoj arhitekturi jetre i njenoj lobularnoj  organizaciji među eksperimentalnim grupama. Stereološka analiza je ukazala na  mikrostrukturne promene kod hepatocita i jetrinih sinusoida. Rezultati sugerišu, na dozno-zavisno povećanje volumena hepatocita, njihove citoplazme i nukleusa, i doznozavsino smanjenje volumena sinusoida, u odnosu na kontrolne uzorke jetre.Analiza glikogena je rađena na presecima jetre bojenim metodom Periodic acid–Schiff-a (PAS), gde se uočilo smanjenje količine glikogena u grupi tretiranoj nižom  dozom akrilamida, dok je u grupi tretiranoj većom dozom uočena njegova akumulacija, u odnosu na kontrolne životinje.Imunopozitivnost hepatocita na marker proliferacije, Ki-67, bila je smanjena u grupi pacova tretiranoj nižom dozom, a bila povećana u grupi tretiranoj većom dozom akrilamida pri komparaciji sa kontrolom. Stereološki nalazi su potvrdili inicijalnu histološku analizu.Imunopozitivnost hepatocita na marker apoptoze, Caspase 3, je bila smanjena kod obe grupe životinja tretiranih akrilamidom u odnosu na kontrolu. Nasuprot tome, imunopozitivnost neparenhimskih ćelija jetre, pretežno Kupffer-ovih ćelija, je bila uvećana u obe tretirane grupe pri komparaciji sa kontrolom.Imunopozitivnost Kupffer-ovih ćelija na marker CD68 je bila smanjena u uzorcima jetre kod oba tretmana akrilamidom u odnosu na kontrolu.Populacija mastocita, prikazana toluidine-blue (TB) metodom bojenja, bila je uvećana  kod obe grupe pacova tretiranih akrilamidom u poređenju sa kontrolom. Povećanje brojnosti ovih ćelija je bilo posebno prominentno kod njihove degranulisane  subpopulacije. Stereološka analiza je potvrdila histološke nalaze. Serumska analiza je pokazala uvećanu aktivnost aspartat aminotrasferaze (AST) i smanjenu aktivnost alanin aminotrasferaze (ALT) kod obe grupe životinja tretiranih  akrilamidom u odnosu na kontrolu. Aktivnost alkalne fosfataze (ALP) je bila uvećana u grupi tretiranoj nižom dozom, a smanjena u grupi tretiranoj većom dozom akrilamida, u odnosu na kontrolu. Vrednosti koncentracije ukupnih serumskih proteina kao i koncentracije C reaktivnog proteina (CRP) nisu pokazale značajnije promene među eksperimentalnim grupama.Oba akrilamidna tretmana su izazvala gubitak telesne mase kod tretiranih pacova, u odnosu na kontrolne životinje. Postojeći podaci ukazuju prominentni hepatotoksični potencijal akrilamida koji može poremetiti mikrostrukturne osobine i funkcionalni status hepatocita kod jetre mladog organizma. Akrilamid može značajno poremetiti funkcionalnost jetre, obzirom da se promene na celularnom nivou mogu relativno brzo odraziti na nivo tkiva, a kasnije ugroziti i homeostazu celog organizma.
Acrylamide  (CASR No. 79-06- 1)  is highly reactive, water-soluble monomer which is considered as toxic and potentially cancer causing chemical to humans. Adverse health effects regarding acrylamide and its more reactive metabolite,glycidamide, were detected in experimental animals, and  included neurotoxicity, genotoxicity, and   carcinogenicity.  Human epidemiological studies claim that acrylamide has neurotoxic effects, while  genotoxicity and carcinogenicity are considered as  potential human health risks only on the basis of animal studies. Its harmful effects on the liver, especially in a young organism, are still to be elucidated.Acrylamide  is spontaneously formed in  carbohydrate-rich food during high-temperature  processing. It is  formed during heat-induced non-enzymatic reaction, also known as  the Maillard browning reaction, between reducing sugars (glucose and fructose), and free amino acids (mainly asparagine).Having in mind  that  acrylamide  metabolism takes place in a liver,  the study aimed to investigate the main histological and  biochemical changes in the liver of juvenile rat following subchronic acrylamide intoxication. Study was performed on peripubertal/juvenile male Wistar rats, divided in 3 experimental groups, two of which were treated with acrylamide in doses of 25 or 50 mg/kg of body weight, while the third group served as the control and received distilled water. Animals were treated orally, via gavage, 5 days a week, during 3 weeks. Animals  were anesthetized by   ether inhalation and decapitated 24 hrs after the last treatment.Liver tissue was sampled from the middle lobe, fixed in  10% neutral  buffered formalin for 24 hrs,  routinely processed for paraffin  embedding  and cut into 5-µm thick serial sections for subsequent histochemical and immunohistochemical staining.Blood samples were collected for subsequent biochemical analysis .Histological examination of haematoxylin and eosin (H&E) stained sections did not point to any major alteration in main in liver lobular architecture or organization among the experimental groups. Stereological analysis revealed a microstructural changes in hepatocytes and liver sinusoids. The analysis detected a dose-dependant increase in the volume of hepatocytes, their cytoplasm and nuclei, and dose-dependant decrease in the volume of liver sinusoids compared to the control, respectively.Glycogen analysis was performed on Periodic acid–Schiff (PAS) stained sections which showed glycogen reduction in the low-dose group, and its accumulation in the high-dose group, compared to the control, respectively.Imunopositivity in hepatocytes for Ki-67 protein, a known marker for proliferation, showed a decrease in low-dose group, while in high- dose group was detected its increase compared to the control, respectively. Stereological analysis confirmed initial histological observation.Caspase 3 immunopositivity, a known marker for apoptosis, proved to be decreased in hepatocytes in both acrylamide-treated groups when compared to the control. One the other hand, immunopositivity was increased in non-parenchymal  cell, predominantly in Kupffer cells, in comparison to the control. Immunopositivity for CD68, a marker for Kupffer cells, proved to be decreased in both acrylamide-treated groups when compared to the control.Population of the mast cells, visualized on toluidine blue (TB) stained sections, showed its increase in both acrylamide-treated groups, in comparison to the control. The increase was especially prominent regarding a degranulated subpopulation of these cells. Subsequent stereological analysis confirmed histological findings.Serum analysis showed increased  activity of  aspartate aminotransferase (AST), and decreased  activity  of alanine aminotransferase (ALT) in  both AA-treated groups, while the  activity  of alkaline phosphatase (ALP)  was increased in low-dose, but   decreased in high- dose group compared to the control, respectively.  The concentration of total serum proteins as well as concentration of C reactive protein (CRP) did not show any major changes among the experimental groups.Body weight measurements showed that all acrylamide-treated rats lost their body weight as opposed to the control rats whose body mass increased.Present results suggest a prominent hepatotoxic potential of acrylamide which might alter the microstructural features and functional status in hepatocytes of  immature liver.  Acrylamide may cause significant perturbation in liver functionality which may be reflected from cellular to the tissue level, thereby endangering the whole body’s homeostasis.

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The objective was to evaluate the effect of probiotic on renal function in intoxicated Wistar rats by potassium dichromate (K2Cr2O7). We used 80 male Wistar rats, that were 21 to 25 days old, randomly divided into two treatments (n = 40 rats/treatment). In the DK treatment the animals consumed 0, 12, 24 and 36 mg of K2Cr2O7 added to the diet and in the DK+P treatment the rats consumed 0, 12, 24 and 36 mg of K2Cr2O7 in the diet with 0.2% probiotic. Those animals consumed their respective diets for 90 days, then, they were euthanized by exsanguination and blood samples were taken to evaluate serum creatinine and urea levels and histopathological analysis of the kidneys were realized. Serum creatinine levels in the DK+P treatment was significantly (p<0.05) lower in relation to the DK treatment in all dichromate doses analyzed. Serum urea levels did not differ significantly (p>0.05) between the DK and DK+P treatments. There was no significant difference (p>0.05) in serum creatinine and urea concentrations in the rats that consumed 0 and 12 mg of dichromate and probiotic, but in the other animals serum creatinine and urea increased significantly (p<0.05) along with increasing doses of the dichromate in both experimental treatments. The rats in DK and DK+P treatments showed hydropic degeneration in renal tubules at all doses of K2Cr2O7. The DK treatment rats showed interstitial nephritis. It was concluded that supplementation with the probiotic was beneficial for glomerular filtration in rats intoxicated with low doses of potassium dichromate, but did not prevent the tubular hydropic degeneration in Wistar rats intoxicated by potassium dichromate.
Objetivou-se avaliar o efeito de probiótico na função renal de ratos Wistar intoxicados por dicromato de potássio (K2Cr2O7). Utilizou-se 80 ratos Wistar, machos com 21 a 25 dias, divididos aleatoriamente em dois tratamentos (n=40 ratos/tratamento). No tratamento DK os animais consumiram 0, 12, 24 e 36 mg de K2Cr2O7 adicionado na dieta e o tratamento DK+P os ratos consumiram 0, 12, 24 e 36 mg de K2Cr2O7 na dieta com 0,2% de probiótico. Esses animais consumiram suas respectivas dietas durante 90 dias, foram eutanasiados por exsanguinação e colhidas amostras de sangue para realização de dosagem sérica de creatinina e uréia e realizou-se nos rins análise histopatológico. A creatinina sérica do tratamento DK+P foi significativamente (p<0,05) menor em relação ao tratamento DK em todas as doses de dicromato estudadas. A uréia sérica não diferiu significativamente (p>0,05) entre os tratamentos DK e DK+P. Não houve diferença significativa (p>0,05) na concentração sérica de creatinina e uréia dos ratos que consumiram 0 e 12 mg de dicromato e probiótico, mas dos demais animais a creatinina e uréia sérica aumentou significativamente (p<0,05) juntamente com as doses crescentes de dicromato estudadas em ambos os tratamentos experimentais. Os ratos dos tratamentos DK e DK+P apresentaram degeneração hidrópica nos túbulos renais em todas as doses estudadas de K2Cr2O7. Os ratos do tratamento DK apresentaram nefrite intersticial. Conclui-se que a suplementação com probiótico foi benéfica para a filtração glomerular nos ratos intoxicados com baixa dose de dicromato de potássio, mas não evitou a degeneração hidrópica tubular nos ratos Wistar intoxicados pelo dicromato de potássio.

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This work valued the sensibility of the acetylcholinesterase from different sources and brain regions at nicotine in vitro, and the effects of the acute and subchronic exposures at the alkaloid on the brain acetylcholinesterase and serum cholinesterase activities, body weight gain and cerebral weight of female rats. The activity of cholinesterases was determined by spectrophotometric method of Ellman (1961), using acetylthiocholine as substrate. In the nicotine in vitro study, the enzymatic analysis was performed with nicotine concentrations ranging from 0 to 1 mM and substrate concentration of 0 -1 mM. In the ex vivo enzymatic assay, 0.8 mM of acetyltiocholine was used. The results regarding at the effects of the nicotine in vitro demonstrated that the enzyme activity from rat brain, human blood and purified of Electric Eel was competitively inhibited by lower nicotine concentrations. The similar effect may be due to the predominance of the G4 molecular globular form in these three sources. The acetylcholinesterase activity from brain structures: cortex, striatum, hippocampus, hypothalamus and cerebellum, was inhibited by nicotine. Considering the IC50, the inhibitory effect was similar among the structures, although the striatum and cortex enzyme seems to be more sensitive, whereas the hypothalamus seems to be less sensitive to alkaloid. The kinetics constants calculated by Michaelis-Menten methods for striatum, cortex and hypothalamus demonstrated that the nicotine induce an increase of Km and a decrease of Vmax. These results showed that the increase of the substrate concentration was not enough for to reach the original Vmax (absence of inhibitor), even in the presence of the low nicotine concentrations. The effects of ex vivo nicotine exposure were investigated after acute or subchronic alkaloid administration. Female Wistar rats with 30 days old received one dose of 0, 0.5, 1 or 5 mg/kg (i.p.) of nicotine (acute exposure) and 10 minutes later were anesthetized and killed by decapitation. Brain was removed and homogenized, the blood was colleted and both centrifuged for obtain the S1 fraction and serum, respectively. In the subchronic exposure, female Wistar rats of 30 days old received doses of 0, 0.5 or 1.0 mg/kg (s.c.) of nicotine for 15 or 30 days, administered twice a day (0, 1 or 2 mg/kg/day). The animals were weighed every two days and killed 12 h after the last injection. The brain and the blood were prepared as previously described. The results demonstrated that the cerebral AChE and serum ChE activities were not changed by acute or subchronic exposure (15 or 30 days) at nicotine. The body weight gain and the cerebral weight also were not altered by alkaloid exposure. The absence of effect on the enzymatic activities ex vivo may be related at least the two possibilities: low levels of nicotine reached in vivo; or a possible enzymatic inhibition present in vivo induced by treatments but not apparent due to substrate excess assayed ex vivo, since the in vitro inhibitory effect of the nicotine on the AChE presents an competitive component.
Este trabalho avaliou a sensibilidade da enzima acetilcolinesterase de diferentes fontes e regiões cerebrais à nicotina in vitro, e os efeitos da exposição aguda e subcrônica ao alcalóide sobre as atividades da acetilcolinesterase cerebral e colinesterase sérica, ganho de peso corporal e peso cerebral de ratas. As atividades enzimáticas foram determinadas segundo o método espectrofotométrico de Ellman (1961), utilizando-se acetiltiocolina como substrato. No estudo in vitro, os efeitos da nicotina foram analisados em concentrações que variaram de 0 a 1 mM do alcalóide e de 0 a 1 mM de substrato. Nos ensaios enzimáticos ex vivo foram utilizadas concentrações fixas de 0,8 mM de acetiltiocolina. Os resultados referentes aos efeitos da nicotina in vitro demonstram que a atividade da enzima de cérebro de ratas, de sangue humano e purificada de órgão Elétrico de Enguia foi inibida competitivamente pelas menores concentrações de nicotina testadas. O efeito semelhante sobre as três fontes pode ser conseqüência da predominância da forma molecular G4 da enzima. A atividade da acetilcolinesterase das estruturas cerebrais: córtex, estriado, hipocampo, hipotálamo e cerebelo, mostrou-se inibida pela nicotina. De acordo com o IC50, o efeito inibitório foi similar entre as estruturas, embora a enzima de estriado e córtex tenha sido mais sensível e a de hipotálamo menos sensível ao alcalóide. As constantes cinéticas calculadas de acordo com o método de Michaelis-Menten para estriado, córtex e hipotálamo demonstraram que a nicotina induz um aumento de Km e diminuição de Vmax. Estes resultados revelam que o aumento da concentração de substrato não foi suficiente para recuperar a velocidade da reação, mesmo na presença das menores concentrações de nicotina. Os efeitos da nicotina ex vivo foram investigados administrando-se o alcalóide aguda ou subcronicamente. Ratas Wistar com 30 dias de idade receberam uma dose de 0, 0,5, 1,0 ou 5,0 mg/kg (i.p.) de nicotina (exposição aguda) e após 10 minutos foram anestesiadas e mortas por decapitação. O cérebro foi removido e homogeneizado, o sangue coletado e ambos submetidos à centrifugação a fim de obter-se a fração S1 e soro, respectivamente. Na exposição subcrônica, ratas Wistar de 30 dias receberam doses de 0, 0,5 ou 1,0 mg/kg (s.c.) de nicotina por 15 ou 30 dias, 2 vezes ao dia (0, 1,0 ou 2,0 mg/kg/dia). Os animais foram pesados a cada 2 dias e 12 horas após a administração da última dose foram sacrificados. O cérebro e o sangue foram preparados como descrito anteriormente. Os resultados demonstram que as atividades da AChE cerebral e ChE sérica não se apresentaram modificadas pela exposição aguda ou subcrônica (15 ou 30 dias) à nicotina. O ganho de peso corporal e o peso cerebral também não foram alterados pela exposição ao alcalóide. A ausência de efeitos da nicotina sobre as atividades enzimáticas ex vivo pode estar relacionada pelo menos a duas hipóteses: níveis baixos de nicotina atingidos in vivo; ou uma possível inibição enzimática presente in vivo induzida pelos tratamentos, mas não aparente devido ao excesso de substrato ensaiado ex vivo, uma vez que o efeito inibitório da nicotina sobre a AChE in vitro possui um componente competitivo.

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Universidade Federal de Minas Gerais
Both the mercury (Hg) and their compounds are recognized as important pollutants, because they are persistent, bioaccumulative and toxic. The largest sources of mercury pollution are chloride-alkaline industry and gold mining. The growing contribution of Hg in aquatic environments results in high accumulation of mercury in fishes tissue and their consumers, which poses a serious risk to humans and ecosystems. The aim of this study was to evaluate the effects of acute exposure (96 hours), via water, and a sub-chronic exposure (30 days), via food, to sub-lethal doses of inorganic mercury (HgCl2) in two species Brazilian fishes ecologically distinct, matrinxã (Brycon amazonicus) and traíra (Hoplias malabaricus). The cardiorespiratory responses to normoxia (140 mmHg) and graded hypoxia (120 - 10 mmHg), cardiac contractility in vitro, biomarkers of oxidative stress and the potential for bioconcentration and biomagnification were analyzed. The results show that exposure of these species to HgCl2 induces oxidative stress in different tissues, limiting the maintenance of cardiac contractility by reducing the force of myocardial contraction and modulates the response pattern of cardio-respiratory variables to graded hypoxia front, making the species more susceptible to environmental variations of O2. Regarding matrinxã specifically, the critical points highlighted were mainly severe oxidative stress in heart and white muscle; marked reduction of contraction force of isolated heart muscle; hyperventilation and increase the value of the critical tension of O2 in more than 100%; and intense bioconcentration in all tissue whose values exceeded the maximum allowed. In the case of traíras the results were: oxidative stress in the liver and gills; hypoventilation; decreased in metabolic rate and O2 extraction; and bradycardia with impaired electrical conduction as first degree atrioventricular block and lengthiness of the potential plateau action of cardiac muscle. Therefore, the data indicate that mercury via food or water and in environmentally relevant concentrations, can have a negative impact on behavior, health, performance and success of the species, making their survival and/or vulnerable populations.
Tanto o mercúrio (Hg) quanto seus compostos são reconhecidos como importantes poluentes, pois são persistentes, bioacumulativos e tóxicos. As maiores fontes de poluição por Hg são as indústrias de cloro-álcali e a mineração do ouro. O aporte crescente de Hg nos ambientes aquáticos resulta em grande acumulação deste metal nos tecidos de peixes e nos consumidores destes, o que representa sério risco aos seres humanos e aos ecossistemas. O objetivo do presente estudo foi avaliar os efeitos de uma exposição aguda (96 horas), via água, e de uma exposição sub-crônica (30 dias), via alimento, a doses subletais de mercúrio inorgânico (HgCl2) em duas espécies de peixes brasileiros ecologicamente distintos, matrinxã (Brycon amazonicus) e a traíra (Hoplias malabaricus). As respostas cardio-respiratórias em normóxia (140 mmHg) e hipóxia gradual (120 a 10 mmHg), a contratilidade cardíaca in vitro, os biomarcadores de estresse oxidativo e o potencial de bioconcentração e biomagnificação foram analisados. Os resultados mostram que a exposição de tais espécies ao HgCl2 induz estresse oxidativo em diferentes tecidos; limita a manutenção da contratilidade cardíaca reduzindo a força de contração do miocárdio e modula o padrão de resposta das variáveis cárdio-respiratórias frente à hipóxia gradual, tornando as espécies mais susceptíveis às variações ambientais de O2. Em relação ao matrinxã especificamente, os pontos críticos em destaque foram: estresse oxidativo severo principalmente no coração e músculo branco; redução acentuada da força de contração do músculo cardíaco isolado; hiperventilação e elevação do valor da tensão crítica de O2 em mais de 100% e bioconcentração intensa em todos os tecidos cujos valores excederam o limite máximo permitido. Já no caso das traíras foram: intenso estresse oxidativo no fígado e nas brânquias, hipoventilação, redução da taxa metabólica e da extração de O2 e bradicardia com prejuízo na condução elétrica como bloqueio átrio-ventricular de primeiro grau e prolongamento do platô do potencial de ação do músculo cardíaco. Portanto, os dados indicam que o mercúrio, via água ou alimento e em concentrações ambientalmente relevantes, pode ter um impacto negativo sobre o comportamento, a saúde, a performance e o sucesso das espécies estudadas, tornando sua sobrevivência e/ou população vulneráveis.

Cognitive dysfunction is a core symptom of schizophrenia, which is poorly treated by current antipsychotic medication. Deficits in the GABAergic system, as demonstrated by convergent genetic and [125I]-iomazenil imaging evidence from patients, are thought to underlie these cognitive deficits. The sub-chronic PCP rodent model was used as it shows cognitive and behavioural parallels to schizophrenia and therefore provides a translational model for some aspects of the disease. However the neurobiological mechanisms responsible for the behavioural alterations in this model have not been fully elucidated. The main aim of the studies presented in this thesis was to investigate the construct validity of the sub-chronic PCP model in relation to the GABAergic and sigma-1 (σ1) receptor systems. Transcriptional changes in gene markers were studied using qRT-PCR and proteomic alterations were investigated using radioligand binding, autoradiography and Western blotting. Finally, the cognitive enhancing potential of σ1 receptor modulators was tested using the novel object recognition (NOR) task. Data presented in chapter 3 shows that sub-chronic PCP treatment in rats produces an increase in GABAA receptor α5-subunit mRNA and a decrease in α3 and δ subunit mRNA levels. No differences were observed in the mRNA levels of the other studied GABAA receptor subunits (α1, α2, α4 or γ2). No alterations in benzodiazepine site- or α5-subunit-containing GABAA receptors were seen following a 7-day washout period, although increased frontal cortical levels of α5-subunit protein were observed prior to the washout period. This suggests that sub-chronic PCP treatment affects extrasynaptic cortical GABAA receptor expression, as shown by the alterations in α5- and δ-subunits, which may contribute to the cognitive deficits observed in this model. Studies in chapter 4 showed that sub-chronic PCP administration causes frontal cortical reductions in parvalbumin, GAD67, GABA transporter-1 and calretinin mRNA levels. No alterations were observed for somatostatin, GAD65, or GABA transporter-3 mRNA, although changes in the mRNA levels for the astrocytic marker glial fibrillary acidic protein were observed in the cerebellum, frontal cortex and hippocampus of sub-chronic PCP-treated animals. No differences in the frontal cortical protein levels of GAD67, GAT-1 and calretinin were observed, suggesting that any proteomic differences in these markers which are present in the sub-chronic PCP model, they are limited in a layer- or cell-type-specific manner. The NOR task is a translational cognitive test that measures recognition memory, which is known to be impaired in schizophrenia. Data in chapter 5 of this thesis showed that sub-chronic PCP-induced and delay-induced recognition memory deficits were ameliorated by acute administration of the σ1receptor agonist (PRE-084) at 1 and 3mg/kg and by the σ1receptor antagonist (NE-100) at 1mg/kg. NE-100 at 3mg/kg proved effective at ameliorating delay-, but not PCP-induced memory deficits. No procognitive effect was observed at lower concentrations of either compound or by co-administration of both compounds. These observations suggest that the improvement of recognition memory deficits is mediated, in part, by σ1 receptors in female rats. The overall results of these studies suggest that sub-chronic PCP administration causes frontal cortical transcriptional alterations in GABAergic neuronal markers which correlate to clinical findings in schizophrenia patients, although these alterations were not observed at the proteomic level following the washout period. These findings also suggest that the σ1 receptor is a potential therapeutic target for recognition memory deficits in schizophrenia, as well as other disorders.

Distribution tissulaire de l'ochratoxine marquee chez le rat et la souris (voie orale). Etude de la nephrotoxicite subchronique de doses faibles chez le rat en suivant dans l'urine et les tubules la variation des activites enzymatiques. Effet sur les enzymes de cellules renales mdck en culture. Etude de la genotoxicite de l'ochratoxine in vivo et in vitro

碩士
國立臺灣大學
職業醫學與工業衛生研究所
97
Epidemiologic studies have shown that ambient particulate matter (PM) is associated with the mortality and hospital admissions of congestive heart failure. However, the mechanism is still unclear. The goal of this study is to investigate the subchronic effects of concentrated ambient particles (CAPs) on cardiovascular toxicity in rats with myocardial injury. Male SD rats received 150 mg/kg isoproterenol by subcutaneous injection to induce myocardial injury. Ultrafine particle concentrator (UFPC) was used for subchronic CAPs exposure (Whole body inhalation exposure, 5hours/day, 4days/week for 13 weeks). Compared to filter air inhalation controls, CAPs exposure group had significantly higher fibrinogen level (190.7 ±15.0 vs 160.5 ±24.2 mg/dL, p < .05); heart rate variability parameters, HF level, was higher in CAPs exposure group than controls (0.77 ± 0.15 vs 0.31 ± 0.05ms2, p < .05). CAPs group also had higher BNP and CRP level but not significant. Our results showed that PM exposure in rats with myocardial injury had no obvious acute or chronic effect in left ventricle, but increased fibrinogen level and altered cardiac autonomic function. Our results suggest cardiovascular risk factors may increase after chronic exposure to ambient particle under present PM concentration.

碩士
國立臺灣大學
職業醫學與工業衛生研究所
106
Recently, many studies have shown that exposure to particulate matter (PM) may induce oxidative stress in the central nervous system (CNS) and contribute to neurodegenerative diseases, such as Alzheimer’s disease (AD). The major pathological characteristics of AD brain are senile plaque of amyloid-beta (Aβ) and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. Some findings have also indicated the oxidative stress would induce Tau protein hyperphosphorylation and NFTs formation and contribute to AD. Moreover, hypertension is a well-known risk factor for Alzheimer’s disease. Thus, we hypothesized that ambient particulate matter could accelerate Alzheimer’s disease-like effects in spontaneously hypertensive rats (SHR). In the experiment, we use SHR to explore the relationship between PM exposure and markers of oxidative stress and markers of AD through subchronic inhalation. 8-week-old male SHR were exposed to continuous, non-concentrated, real world PM2.5 using Taipei Air Pollution Exposure System (TAPES) for 3 and 6 months. After 3 months exposure, Morris Water Maze (MWM) was conducted. Afterwards, the rats were proceeded an another 3 months exposure of PM2.5 for a total of 6 months. After the exposure, the brain tissue, including olfactory bulb, cerebellum, hippocampus and cerebral cortex, were collected. The level of malondialdehyde (MDA), maker of oxidative stress, was determined by LC-MS/MS. Total Tau protein (t-Tau) and phosphorylated Tau protein (p-Tau), markers of AD, were assessed by Western blot. The mean mass concentration of PM2.5 was 8.6 μg/m3 from the first 3 months exposure and 10.8 μg/m3 from the second 3 months exposure. Our results showed that MWM didn’t have any differences compared to control groups after exposure for 3 months. Both control and exposure groups showed a reasonable learning curve of escape latency in acquisition phase. In MDA, the level of MDA significantly increased in olfactory bulb, hippocampus and cerebral cortex after 6 months exposure. In 3 months exposure, the MDA level in four brain regions didn’t find any significant differences between control and exposure group. The result of Tau protein expression showed that the level of t-Tau protein expression, as well as p-Tau protein expression, significantly increased in olfactory bulb in 3 months exposure. However, in 6 months exposure, the level of t-Tau and p-Tau protein expression didn’t find any significant differences between control and exposure group in four brain regions. We found no significant differences between the control and the exposure group in histopathology of lung from 3 and 6 months exposure and brain from 6 months exposure. In conclusion, our study indicated the subchronic exposure to ambient particles would induce oxidative stress and Tau protein in brain. However, exposure to ambient particles for 3 months may not impair spatial learning and memory in SHR. Our study revealed the possible mechanism of PM exposure by inhalation and neurotoxicity in CNS. Nevertheless, our study didn’t investigate many neurotoxicity-related markers in CNS. Further study should be conducted to explore the more markers related to neurotoxicity in CNS and clarify the relationship between PM and neurotoxicity in CNS.

碩士
國立高雄師範大學
生物科技系
106
Sediments have a major role in ecosystem functioning but can also act as physical or chemical stressors. The importance of sediments as stressors will depend on site ecosystem attributes and the magnitude and preponderance of co-occurring stressors. Risk assessments and restoration strategies should better consider the role of chemical contamination in the context of multiple stressors. Methods for assessing site-specific ecological impairments resulting from the physical stress of excessive sediments are not highly developed. In contrast to aquatic toxicity testing where test protocols have been standardized, testing protocols for soils still lack harmonization. Biological assays have been developed by the Organization for Economic Co-operation and Development(OECD), the United States Environmental Protection Agency(U.S. EPA), and individual researchers for use in assessing soil toxicity related to earthworms(Perionyx excavatus), plants, and bacteria. The objective of this study was to propose ecotoxicological studies to provide systematic assessment to evaluate ecological response when assessing contaminated sediment and soils. Bioassays used for sediment quality assessment typically rely on static continuous exposure of a test organism to a contaminant or contaminated sediment. Ecotoxicity testing has been used in combination with contaminant and remediated soils and sediments in site assessments. Earthworm and benthic species including Japanese swamp shrimp(Neocaridina denticulata), sludge worm(Tubifex hattai), and amphipod(Hyalella azteca) were adopted for toxicity test. Acute toxicity tests were used to assess the toxicity of contaminated sediments and soils to organisms. copper, cadmium, lead, and trichloroethylene were selected as target contaminants. Results of acute toxicity indicated that copper posed highest toxicological effects onto earthworms in soil prepared in the laboratory. However, trichloroethylene posed highest toxicity onto earthworm in contaminated soil. Also it was found that trichloroethylene posed highest toxicity onto earthworm in subchronic toxicity tests(i.e., LC10=61.19 mg/kg). Cadmium posed highest toxic effects onto larvae. Lead posed highest toxic effect onto Japanese swamp shrimp in sediment test. Sludge worm(Tubifex hattai) and amphipod(Hyalella azteca) was sensitive to copper with LC50 of 15.47 mg/kg and 1.14mg/kg, respectively. Japanese swamp shrimp was most sensitive by cadmium in sediment with mixture of target contaminants. However, sludge worm(Tubifex hattai) was most sensitive to lead. Cadmium posed highest toxicological effects onto amphiod(Hyalella azteca). This study provided base-line toxicity information of aquatic species in contaminated sediments and toxicity information of terrestrial species in contaminated soils for ecological risk assessment, including the NOEC, LOEC, LC50 and EC50. Moreover to establish ecotoxicological test protocols of contaminated sediment and soils.

碩士
國立中興大學
獸醫病理生物學研究所
103
Melamine has been used to adulterate animal food and infant formula in order to give false elevated results in the protein content of food. It’s a well-known issue that simultaneous ingestion of melamine and cyanuric acid (MCA) could induce acute renal failure and high mortality in animals or obstructive nephropathy in infants. Many investigators have been devoted to the studies of MCA combined toxicity and the mechanisms of MCA-induced renal injury. In our study, we conducted the subchronic toxicity test of nephrotoxicity of melamine and cyanuric acid combination via repeated feeding for 13 weeks, and the possible recovery effect in rats. The data were evaluated by histopathologic examination, the gold standard for assessing a compound’s effect on an animal system of nephrotoxicity, the urinary biomarkers and oxidative enzymes. The objective of our study was to evaluate the acceptable daily intake (ADI) in human by assessing the no-observed-adverse-effect level (NOAEL) in subchronic nephrotoxicity study of MCA combination via repeated feeding in rats. Sprague-Dawley rats (10 males and 10 females per dose group) were fed ad libitum for 13 weeks with 0 (control), 100, 150, 200 ppm in both sex and 300 ppm (female only). Furthermore, the recovery groups for 4 weeks were designed as 0, 200 ppm in male and 0, 300 ppm in female. The results of BUN and creatinine showed significantly increase in the F200, F300 groups of females, but not in M200 group of male rats. In the F200, F300 groups of female rats, the oxidative stress markers, SOD and catalase, showed significant decrease and inflammatory cytokine, IL-1s, were significantly increased. The data confirmed that renal tissue is overwhelmed by the oxidative stress and severe inflammatory response. The urinary biomarkers, KIM-1 and NGAL, which is considered to be more sensitive and diagnostic markers of nephrotoxicity caused by MCA, showed significant increased in both M200 and F200 group of male and female rats, but not in the lowest 100 ppm group. Otherwise, histopathologic examination showed the results that MCA crystals still could cause the MCA lesions such as deposition of MCA crystal, mononuclear cell infiltration in interstitial area, tubular dilation, regeneration and necrosis in kidney, above 150 ppm dosage group, implied that the safety risk in this dosage. Based on the results, the histopathological examination of renal lesions should be the primary consideration of renal damage in nephrotoxical evaluation. The NOEAL of 100 ppm MCA was set in rats in the 13-week repeated subchronic study. There is equivalent dosage of MCA to be about 7.2 mg/kg bw/day in both sexes. According to this result, the acceptable daily intake (ADI) in human is recommended to be 0.07 mg/kg bw/day of MCA.

Yes
Female hooded-Lister rats received either sub-chronic phencyclidine (PCP) (2 mg/kg, n=20) or vehicle (1 ml/kg, n=20) i.p. twice daily for seven days, followed by a seven-day washout period. Rats were challenged with acute PCP or vehicle and tested for locomotor activity to ensure hyperactivity was observed in the sub-chronic PCP treated rats. Rats were then tested on the elevated plus maze and in an open field for 10 minutes. Sub-chronic PCP did not significantly affect behaviour on the elevated plus maze or in the open field. In conclusion, sub-chronic PCP does not induce anxiety-like behaviour.

by Chan Po Wai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1997.
Includes bibliographical references (leaves 110-117).
Table of Contents --- p.i
Abbreviations --- p.iv
Abstract --- p.v
List of Figures --- p.vii
List of Tables --- p.xi
Chapter Chapter One: --- Introduction
Chapter 1.1 --- Objective and scope of the project --- p.1
Chapter 1.2 --- Literature review --- p.3
Chapter 1.2.1 --- Balkan endemic nephropathy --- p.3
Chapter 1.2.2 --- Chinese herbs nephropathy --- p.4
Chapter 1.2.3 --- Aristolochic acid --- p.6
Chapter 1.2.4 --- Guan-mu-tong --- p.9
Chapter 1.2.4.1 --- Plant --- p.9
Chapter 1.2.4.2 --- Traditional uses --- p.10
Chapter 1.2.4.3 --- Chemical constituents --- p.11
Chapter 1.2.4.4 --- Pharmacological study --- p.11
Chapter 1.2.4.5 --- Reported adverse cases --- p.12
Chapter 1.2.5 --- Ma-dou-ling --- p.12
Chapter 1.2.5.1 --- Plant --- p.12
Chapter 1.2.5.2 --- Traditional uses --- p.13
Chapter 1.2.5.3 --- Chemical constituents --- p.14
Chapter 1.2.5.4 --- Clinical and pharmacological studies --- p.14
Chapter 1.2.5.5 --- Reported adverse cases --- p.15
Chapter 1.3 --- Chemical analysis --- p.16
Chapter 1.3.1 --- Thin layer chromatography --- p.16
Chapter 1.3.2 --- High performance liquid chromatography --- p.17
Chapter 1.4 --- Toxicology --- p.18
Chapter 1.4.1 --- Acute toxicity --- p.18
Chapter 1.4.2 --- Subchronic toxicity --- p.19
Chapter 1.4.3 --- Reproductive toxicity --- p.23
Chapter Chapter Two: --- Materials & Methods
Chapter 2.1 --- Materials --- p.24
Chapter 2.2 --- Methods --- p.27
Chapter 2.2.1 --- "Aqueous extraction of Guan-mu-tong and Ma-dou-ling for acute, subchronic and reproductive toxicity tests" --- p.27
Chapter 2.2.2 --- Chemical analysis --- p.28
Chapter 2.2.2.1 --- Thin layer chromatography --- p.28
Chapter 2.2.2.2 --- High performance liquid chromatography --- p.28
Chapter 2.2.3 --- Assays for the toxicity --- p.30
Chapter 2.2.3.1 --- Acute toxicity --- p.30
Chapter 2.2.3.2 --- Subchronic toxicity --- p.31
Chapter 2.2.3.3 --- Reproductive toxicity --- p.32
Chapter 2.2.4 --- Statistical analysis --- p.33
Chapter Chapter Three: --- Results
Chapter 3.1 --- Chemical Analysis --- p.34
Chapter 3.1.1 --- Thin layer chromatography --- p.34
Chapter 3.1.2 --- High performance liquid chromatography --- p.34
Chapter 3.2 --- Toxicity of Guan-mu-tong --- p.42
Chapter 3.2.1 --- Acute toxicity --- p.42
Chapter 3.2.2 --- Subchronic toxicity --- p.44
Chapter 3.2.3 --- Reproductive toxicity --- p.54
Chapter 3.3 --- Toxicity of Ma-dou-ling --- p.56
Chapter 3.3.1 --- Acute toxicity --- p.56
Chapter 3.3.2 --- Subchronic toxicity --- p.66
Chapter 3.3.3 --- Reproductive toxicity --- p.89
Chapter Chapter Four: --- Discussion
Chapter 4.1 --- Chemical Analysis --- p.91
Chapter 4.1.1 --- Thin layer chromatography --- p.91
Chapter 4.1.2 --- High performance liquid chromatography --- p.91
Chapter 4.2 --- Toxicity of Guan-mu-tong --- p.93
Chapter 4.2.1 --- Acute toxicity --- p.93
Chapter 4.2.2 --- Subchronic toxicity --- p.93
Chapter 4.2.3 --- Reproductive toxicity --- p.94
Chapter 4.3 --- Toxicity of Ma-dou-ling --- p.95
Chapter 4.3.1 --- Acute toxicity --- p.95
Chapter 4.3.2 --- Subchronic toxicity --- p.97
Chapter 4.3.3 --- Reproductive toxicity --- p.105
Chapter Chapter Five: --- Conclusion --- p.107
Bibliography --- p.110
Appendix A: Procedure on determining the total urinary protein --- p.119
Appendix B: Procedure on determining the total urinary glucose using Sigma diagnostic kits --- p.121
Appendix C: Procedure on determining the activity of aspartate aminotransferase --- p.123
Appendix D: Procedure on determining the activity of alanine aminotransferase --- p.124
Appendix E: Procedure for preparing a calibration curve for the measurement of aspartate aminotransferase and alanine aminotransferase activities --- p.125
Appendix F: Procedure on tissue preparation for light microscopic study --- p.128

The etiology and pathophysiology of schizophrenia are poorly understood, although increasing evidence suggests an important role for altered GABA neurotransmission. Animal models of schizophrenic symptoms include administration of noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, such as dizocilpine (MK-801), and post-weaning social isolation. The present study tested the hypothesis that a “double-hit” model, in which subchronic MK-801 administration and post-weaning social isolation are combined, produces greater behavioural and neurochemical effects than either insult alone. As a secondary objective, the present study also assessed whether the timing of the subchronic MK-801 injections (early adolescence vs. early adulthood) influences these measures. Male Sprague-Dawley rats (N = 74) were obtained at weaning (P21) and were either socially isolated (n = 42) or group housed (n = 32) for the duration of the experiment. Subgroups received subchronic treatment with MK-801 (0.5 mg/kg ip) or saline injections (1.0 ml/kg ip) twice daily for seven days either during early adolescence (P25-P32) or early adulthood (P56-63). At P70, all groups were tested for locomotor activity and subsequently sacrificed to assess the function of the GABA membrane transport protein, GAT-1, and GABAA receptor expression in the frontal cortex and hippocampus. For animals treated in early adulthood, post-weaning social isolation, in comparison to group housing, resulted in an increase in (1) locomotor activity (2) GAT-1 activity in frontal cortex and hippocampus and (3) GABAA receptor expression in the frontal cortex. MK-801 treatment in early adulthood increased GABAA receptor expression in the hippocampus, whereas post-weaning social isolation had no effect on GABAA receptor expression in the hippocampus. Previous studies have demonstrated that increased GAT-1 activity is associated with suppression of GABA-mediated inhibitory synaptic transmission. Furthermore, increased GABAA receptor expression may be a compensatory response to decreased availability of GABA. These data indicated that combined post-weaning social isolation and subchronic MK-801 treatment do not produce additive or synergistic effects on locomotor behaviour or GABA signalling, but rather induced differential effects on GABAA receptor binding.
Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-09-01 15:21:58.474

碩士
國立臺灣大學
職業醫學與工業衛生研究所
99
part1 Zinc oxide nanoparticles have been widely used in the manufacturing for ceramics, chemicals, cosmetics, pharmaceuticals and pesticides. Previous studies have shown that the toxicity of non-soluble nanoparticles is linked with particle size and surface area. Zinc oxide nanoparticls can be dissolved in liquid and the toxicity is resulted from zinc ions in in vitro studies. However, it is not clear about the role of zinc ion in animal studies. Several methods to detect zinc ions have been used, but yet not clearly validated. In this study, we developed a centrifugation method to separate zinc ion and zinc oxide nanoparticles. Subsequently, we used this method to investigate the association between total zinc level, zinc ion level and lung inflammation in bronchoalveolar lavage fluid (BALF) in SD rats treated with zinc oxide nanoparticles. SD rats were exposed to zinc oxide nanoparticles with intratracheal instillation (10mg/ml) then sacrificed ar 6 hr (exposure-1, n=6), 24 hr (exposure-2, n=6), 48 hr (exposure-3, n=6), and 72 hr (exposure-4, n=6). PBS was used as controls (control, n=8). We collected bronchoalveolar lavage fluid and peripheral blood to analysis the lung inflammation markers, total number of cells, proportion of neutrophils, total zinc and zinc ion level to investigate the association between zinc level and lung inflammations. We found total zinc levels were highly associated with zinc ion level (r=0.98). Both total zinc (77.08±127.56 μg/L) and zinc ion (31.72±67.61 μg/L) had a peak level at 48 hr post-exposure. The inflammation markers level also reached the maximum level at 48 hr post-exposure (p＜0.05) then decreased at 72 hr. But systemic inflammations were not increased after exposure. Our results suggest zinc oxide nanoparticles may relocate after exposure into lung parenchymal tissue then reenter into the luminal side of the airway. The limitation of our study is that only BALF was used to determine the zinc levels. This may explain why we only detect an association between zinc levels and lung inflammation at 48 hr. This pioneer study shed some light on the complicated issues about the toxicity of zinc oxide nanoparticles in animals. Further studies are needed to elucidate the exact toxicological mechanism of zinc oxide nanoparticles. part2 Zinc oxide nanoparticles have been widely used in the manufacturing for ceramics, chemicals, cosmetics, pharmaceuticals and pesticides. In previous studies, the acute toxicity of zinc oxide nanoparticles has been investigated. However, the data about subchronic toxicity for zinc oxide nanopaticles were limited. Our lab has successfully constructed a nanopaticle generation system and exposed animals for acute toxicity. In this study, we used this system to investigate the zinc oxide subchronic toxicity on lung inflammation and injuries. The SD rats were exposed for 2 weeks (5 hr/day, 5 day/week) then sacrificed at 1 day (exposure-5, n=6；control-5, n=4), 7 day (exposure-6, n=6；control-6, n=4), and 30 day (exposure-7, n=6；control-7, n=4). We collected bronchoalveolar lavage to determine the lung inflammation including total number of cells, proportion of neutrophils and protein. Histopathological examination was also performed. Zinc oxide nanoparticles were produced in a furnace system and SMPS was used to monitor the size and number concentrations of particles. The average concentration during exposure was 51.47nm at 2.66×106 particle/cm3. The inflammation markers including netrophils% (31.92±8.96%), total cells (3.47×105±6.02×104) and total protein (0.52± 0.13 mg/ml) at 24 hr post-exposure increased significantly as compared to the control group (p＜0.05). The inflammation decreased at 7 days then become no difference from the control group. In this study, we have performed a subchronic inhalation studies with high concentrations and mono-dispersed zinc oxide nanoparticles. An acute but reversible inflammatory were observed. Further histological studies are needed to assess the subchronic toxicity of zinc oxide nanoparticles.

碩士
國立臺灣大學
食品科技研究所
107
Gut has been known as the second brain and affects the brain via various pathways such as neurotransmitter modulation. While the gut-brain axis is assumed to play an important role within the gut microbiota and emotion. According to WHO, depression is a common mental disorder, the leading cause of disability in the whole world. Statistics as of 2017 showed that over 300 million people suffered from depression, not only the olders, but also the youngster. Among them, more than half also suffered from anxiety, occupied more than 264 million people. Studies found that tranditional Chinese herbal medicine has potential to be a good and effective complementary therapy. Gastrodia elata Blume, is prospected to have bioactive functions such as anticonvulsion, antitumor, antioxidant, antiaging, immune modulation, memory improvement, neuroprotection, neuroplasticity, antianxiety and antidepressant. Our labortary previously showed that water extract of Gastrodia elata Blume (WGE) exerts antidepression-like effect in forced swimming test, unpredictable chronic mild stress, and chronic social defeat stress model. However, the impact of WGE on gut in subchronic and mild social defeat stress (sCSDS) model has not been studied. Thus, I investigated the latent gut-brain linked mechanisms of WGE in relieving stress-induced depression-like behaviors which may result from the tryptophan (TRP) metabolism in mice subjected to sCSDS model. Results showed that sCSDS induced both social avoidance and depression-like behaviors such as anxiety, low locomotion and exploration. In the biochemical analysis showned significantly increased of serum corticosterone (CORT) (p < 0.0001), prefrontal cortex indoleamine 2,3-dioxygenase (IDO) protein expression (p < 0.01) and colon kynurenine (KYN) production rate (p < 0.05), while significantly decreased serotonin (5-HT) production rate (p < 0.005). While treatment with WGE, significantly reversed the depression-like phenotypes, such as social avoidance and stress index, serum CORT (p < 0.05) induced by sCSDS. Also, WGE significantly normalised the deviant metabolic of KYN in colon cause by defeat stress (p < 0.01). The administration of antibiotics elevated body weights and induced high locomotion and anxiety behavior of mice, while significantly increase 5-HT production rate (p < 0.05) and decrease 5-HT turnover rate (p < 0.05) in prefrontal cortex. In addition of WGE administration, significantly normalised the deviant metabolic of 5-HT cause by antibiotics (p < 0.05). In general, high levels of 5-HT in brain induced by sCSDS with antibiotics may linked to anxiety behavior, while WGE treatment could reverse it. By analyzing the fecal microbiota, I found that 14 days of WGE treatment increased the fecal diversity (p < 0.05). After the 10-day social defeat, stress groups had a significantly different microbiome status compared with control (p < 0.05), while WGE ameliorated part of them, such as Bacteriodales S24-7 group (F), Rumminococcaceae (F), Lachnospiraceae (F), Clostridiales vadinBB60 (F), Rumminococcaceae UCG-014 (G), Rumminococcaceae UCG-005 (G) and Lachnospiraceae NK4A136 (G), implying that WGE may inhibit the inflammation and promote short chain fatty acids production in gut. Thus, WGE possessed antidepressive and anxiolytic effects via modulating the inflammation, serotonin and kynurenine pathway.

碩士
國立陽明大學
生理學研究所
102
Chronic ethanol exposure-associated oxidative stress causes various degrees of brain damage among individuals depending on their exposure to other environmental factors, such as organic environmental pollutants that activate aryl hydrocarbon receptors (AhR). This study was to investigate how the combination of subchronic ethanol ingestion and AhR ligand exposure regulates mitochondrial enzymes, the oxidative stress-associated cytochrome P450 2E1 (CYP2E1) and CYP1A1, and the anti-oxidative Cu/Zn-superoxide dismutase (SOD1) in cerebral cortical neurons. In this study, primary cultured cortical neurons were prepared from embryonic day 17 Sprague Dawley fetal rats, and cultured neurons at 9 days in vitro (9 DIV) were used in our study. The cultured neurons were subjected to subchronic ethanol treatment, i.e. 80mM ethanol for 3 days, followed by incubating with or without AhR agonists, a natural ligand tryptophan photoproduct 6-formylindolo [3,2-b] carbazole (FICZ, 20nM) or the environmental ligand 2,3,7,8-tetrachlorodibenzodioxin (TCDD, 20nM) for 24hr. Immunofluorescent staining and WST-1 assay indicated that subchronic ethanol and AhR ligand induced neuronal loss. Subchronic ethanol increased CYP2E1 protein expression as examined by Western blotting in cortical neurons, and the effect was suppressed by the AhR agonist application. The induction of CYP1A1, an AhR target gene, was enhanced when AhR agonists were applied under subchronic ethanol treatment. In contrast, subchronic ethanol does not affect the AhR ligand-induced AhR protein and mRNA downregulation. The anti-oxidative enzyme SOD1 was reduced under subchronic ethanol exposure with or without AhR ligands, whereas AhR ligands can induce SOD1 mRNA but the effect was diminished under subchronic ethanol treatment. Reciprocally, we found that subchronic ethanol - AhR agonist exposure can increase oxidative stress as determined by NADPH oxidase activity assay. Next, we used AhR antagonist TMF to block AhR activation, and found that TMF suppressed SOD1 expression and increased oxidative stress in the ethanol-free condition, but can enhance SOD1 expression without inducing oxidative stress in neurons under subchronic ethanol exposure. Finally, we established a subchronic binge ethanol animal model, and found that SOD1 and CYP1A1 mRNA expressions were both increased in the rat cerebral cortex and hippocampus, whereas the SOD1 mRNA in the cerebellum was decreased. Together, our data tend to suggest a cross-talk between the subchronic ethanol ingestion and AhR activation reciprocally enhances or attenuates their own primary effects on the expression of CYP2E1, CYP1A1, and SOD1, which may subsequently regulate oxidative stress in cortical neurons and alcoholic brains. Information obtained from this preliminary study may provide clues for the impact and mechanism of environmental confounding factors on the alcoholism-associated neurological deficits.