This work is focused on verification of p-Nitrotoluenu toxicity and compounds resulting from the aerobic transformation, and methyl group oxidation and reduction of nitro. Some of these products are not commercially available and had to be made soon. Toxicity of the substances was determined by toxicity tests. Were tested root growth inhibition Sinapis alba, which resulted in an inhibitory concentration IC50; test inhibition duckweed Lemna minor , which was also the determination of IC50 inhibitory concentration, and acute toxicity test Artemia salina, which was observed in mortality and immobility organisms and the test result was an efficient concentration EC50 value. Results of tests are compared in the conclusion of the work and is made evaluation of the toxicity of individual substances.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. There are numerous studies reporting developmental cardiac toxicity in multiple fish species due to PAH exposure. However, there are relatively few instances where the effects of acute PAH exposure in adult fish have been characterized. Furthermore, the majority of experiments comparing PAH toxicity with exposure route in adult fish focus on CYP1A gene expression or enzyme activity, while there is a lack of information about the possible pathophysiological effects. Therefore, the overall objective of this thesis was to characterize the sublethal effects of benzo[a]pyrene (BaP), a prototypical PAH, on adult zebrafish (Danio rerio) cardiorespiratory function and fitness following acute exposure by two different routes. In the first experiment, adult zebrafish were intraperitoneally (i.p.) injected twice (one injection/24 hr) with increasing concentrations of BaP (0.1, 10, and 1000 μg/kg) and compared to corresponding dimethylsulfoxide (DMSO) controls. In a second set of experiments, adult zebrafish were aqueously exposed to BaP (static, renewal at 24 hr; 16.2 and 162 μg/L) and compared to DMSO controls. Following 48 hr exposure, one group of fish (n=10/treatment group) were subjected to swimming performance tests to assess critical swimming speed (Ucrit), oxygen consumption rate (MO2), cost of transport (COT), standard metabolic rate (SMR), active metabolic rate (AMR), and factorial aerobic scope (F-AS). Another group of fish (n=12/treatment group) were subjected to echocardiography following 48 hr BaP exposure to evaluate cardiac function. Following echocardiography analysis, samples were collected for parent compound (BaP) body burden and CYP1A mRNA induction analysis. 48 hr BaP injection resulted in significant sublethal effects on adult zebrafish cardiorespiratory function. Oxygen consumption (MO2) was increased at three swimming speeds in injected BaP groups compared to control. In contrast, aqueously BaP-exposed fish showed increased MO2 only at the single lowest swim speed. COT was also similarly increased for both exposure routes. SMR was elevated with both exposure routes, while AMR remained unchanged. This resulted in a significant decrease in F-AS for all treatment groups compared to corresponding controls with both exposure routes. Cardiac function was significantly affected by both routes of BaP exposure. Ventricular heart rate was significantly decreased in BaP-exposed fish, both injected and aqueously-exposed. However, stroke volume was decreased only in fish aqueously exposed to BaP, which resulted in significantly reduced cardiac output with that exposure route. In contrast, the ratio of atrial to ventricular heart rate (AV ratio) was increased only in fish i.p. injected with BaP, indicating the possibility of cardiac arrhythmias occurring. Analysis of BaP body burdens in fish tissue allowed for identification of an overlapping dose group between exposure routes, through which comparisons of cardiotoxicity were then made. This comparison revealed slight differences in cardiotoxicity between exposure routes. BaP-injected fish suffered from more severe bradycardia than aqueously exposed fish. Furthermore, cytochrome P4501A (CYP1A) mRNA levels in liver and heart tissue showed more significant increases in injected fish, while skeletal muscle CYP1A was increased only following aqueous exposure. In conclusion, acute BaP exposure caused metabolic alterations and impaired cardiorespiratory function in adult zebrafish regardless of exposure route. Interestingly, the primary mechanism behind these effects appeared to differ slightly with exposure route. These results suggest that acute BaP exposure may have negative effects on adult fish survivability in the environment. Overall, this work provides valuable insight into the pathophysiogical consequences of acute PAH exposure in adult stage fish.

This thesis, which is divided into four chapters, represents an account on the isolation, identification and the assessment of bioactivity of cytotoxic compounds from the genus Centaurea (Family: Asteraceae alto Compositae), a large genus of about 500 species. The first three chapters deal with an introduction of natural products and Centaurea species, followed by the isolation and characterisation of compounds from twelve Centaurea species. The last chapter describes the bioactivities of extracts and isolated compounds from these species. A total of 45 compounds were isolated from twelve Centaurea species, and only C. americana, C. cyanus, C. dealbata and C. macrocephala had previously been studied. Four of these are novel compounds. Four lignans arctiin, matairesinoside, matairesinol and lappaol A were isolated fromthe methanol extract of C. macrocephala seeds. Arctiin and matairesinoside were also isolated from the methanol extract of C. americana, C. bornmuel/eri, C. dealbata, C. huber-morathii, C. mucronifera, C. pamphylica, C. schischkinii and C. urvillei. The methanol extract of C. americana also afforded 20-hydroxyecdysone, 24-hydroxyecdysone, lappaol A, arctigenin and a novel compound, 3"-O-caffeoyl(9"'→3")-arctiin. The methanol extract of C. cyanus produced lariciresinol 4-0-B-D-glucoside, berchemol, moschamine and cis-moschamine. Arctigenin, astragalin, afzelin, matairesinol and a novel indole alkaloids, schischkiniin, were isolated from the methanol extract of C. schischkinii. Extract from C. bornmuelleri afforded arctigenin, astragalin, afzelin and matairesinol. The methanol extract of C. mon/ana yielded berchemol, berchemol 4'-O-B-D-glucoside, p-coumaroylquinic acid, cis-pcoumaroylquinic acid, pinoresinol, pinoresinol mono methyl ether, pinoresinol dimethyl ether, pinoresinol 4-0-B-D-glucoside, pinoresinol 4,4'di-0-B-D-glucoside, pinoresinol 4-0-apiose-(1→2)-B-D-glucoside, centcyamine, cis-centcyamine, N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, cis-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, moschamine, cis-moschamine, tryptamine and two novel compounds, flavanone-apiose-glucuronic acid and montamine. C. gigantea afforded arctiopicrin, 8-0-(4-hydroxy-3-methylbutanoyl)-salonitenolide, chlorogenic acid, cirsiliol, isoquercetrin, orientin, isoorientin and 4"-hydroxybenzoyl-isoorientin. General toxicity, cytotoxicity and antioxidant activity of the extracts and isolated compounds were evaluated, respectively, by the brine shrimp lethality assay, MTT assay on human colon cancer cell line (CaCo-2) and DPPH assay. Among all the species, the methanol extract of C. bornmuelleri, C. gigantea, C. huber-morathii and C. montana were the most toxic extracts in brine shrimp lethality and MTT assay. Arctigenin (IC50=7.0 mM), matairesinol, montamine (IC50=43.9 mM) and lappol A, schischkiniin, arctiopicrin (IC50=8.5 mM) and 8-0-(4-hydroxy-3-methylbutanoyl)-salonitenolide (IC50=26.4 mM) showed higher cytotoxicity against MTT assay. Matairesinoside (IC50=2.2 x 10-3 mg/mL), matairesinol (IC50=2.0 x 10-3 mg/mL) and schischkiniin (lC50=3.8 x 10-3 mg/mL) exhibited significant free radical scavenging activities towards DPPH assay.

Class of 2010 Abstract / OBJECTIVES: To determine the clinical significance of a population of scorpions, C. sculpturatus, found in New Mexico. This includes the toxicity of the venom as well as the interactions of venom and antivenom. METHODS: This project will include a descriptive retrospective study of clinical and laboratory data obtained through a patient chart and analytical laboratory procedures to positively identify the species of scorpion responsible for the envenomation. Scorpion Collection Scorpions from the location of the reported sting will be collected for venom analysis. Specific details and directions will be obtained from the grandparent of the victim regarding the campsite at Caballo Lake State Park. Human Subject – N/A Sample Size The sample size of specimens needed should be a minimum of 50 scorpions. This is due to factors which include; extremely small volumes of venom produced by each individual, the possibility of no venom production, damaged telson during collection (anatomical feature used in the delivery of venom), size variation in specimens and short window of opportunity to collect. Since these scorpions are more active during summer months, and travel time must also be accounted for, only a few months a year are acceptable. Instrumentation and Variables This is primarily laboratory assays rather than clinical. The clinical aspect, a case study involving the victim of a scorpion envenomation, was the reason behind needing to identify this Centruroides species. The analytical laboratory findings will be what will determine the exact species of Centruroides. A number of laboratory instrumentations and tests will be used or performed. These include; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGE with hyaluronic acid, turbidimetric absorbances of hyaluronidase, reverse phase high pressure liquid chromatography (RPHPLC), enzyme-linked immunosorbent assay (ELISA), lethal dose 50 (LD%) and effective dose 50 (ED50). Variables regarding these findings will include two main factors; the human factor and the instrumentation factor. First the human factor, samples collected must remain free of contaminants up to the point of analysis. The instruments RESULTS: 104 specimens were collected at Caballo Lake State Park. Four of these specimens were sent to a taxonomist and identified as C. sculpturatus. SDS-PAGE, turbidimetric HA, and RPHPLC showed no significant difference in venom between the New Mexico and Arizona Centruroides, but did show a significant difference between these two groups and the Mexico Centruroides. SDS-PAGE/HA and ELISA assays showed no significant differences between groups. LD50 and ED50 data were similar between New Mexico and Mexico Centruroides, both being more potent and more readily reversed by antivenom than the Arizona Centruroides. CONCLUSIONS: The assays which show possible differences between species, SDS-PAGE, SDS-PAGE-HA, turbidimetric HAase, RPHPLC, and ELISA were all identical between the New Mexico Centruroides species and the Arizona Centruroides sculpturatus. These findings were opposite when comparing New Mexico Centruroides species and the Mexico Centruroides limpidus limpidus. Three of the five assays showed a significant difference. Since the Mexico Centruroides limpidus limpidus is a known different species, this was expected. With this data the scorpion specimens collected in New Mexico have been identified as Centruroides sculpturatus. An interesting difference between the New Mexico and Arizona Centruroides sculpturatus was toxicity of the venom. The New Mexico groups had close to a two fold increase in toxicity. In fact, the toxicity of the New Mexico groups was equivalent to the Mexico Centruroides limpidus limpidus which is well documented as having an increased toxicity.

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No description available.

Statins are the mainstay of cholesterol-lowering therapy and are taken by millions of people worldwide. These drugs are generally well-tolerated but can cause myopathy ranging from mild muscle pain to fatal rhabdomyolysis. The mechanism of statin-induced myopathy (SIM) is not fully understood and there is currently no convenient and reliable marker of SIM, but mitochondrial dysfunction has been implicated. We sought to investigate the effect of statins on mitochondrial DNA (mtDNA) levels in order to gain information on the mechanism of SIM and to explore the possibility of utilizing changing mtDNA levels as a marker of SIM. Several approaches were used. First, mtDNA levels were quantified in skeletal muscle biopsies collected from a previously published 8-week clinical trial of high-dose simvastatin or atorvastatin versus placebo. Forty-eight hypercholesterolemic subjects were randomly assigned to receive placebo (N=16), high dose atorvastatin 40mg/day (N=16), or high dose simvastatin 80mg/day (N=16) for 8 weeks. Muscle mtDNA content was assessed by real-time PCR atbaseline and after 8-weeks on statin treatment and found to be significantly reduced in the groupreceiving simvastatin (P=0.005) but not the other two. In addition, a significant positive correlation was observed between mtDNA and muscle ubiquinone in all groups (R=0.63, P<0.01), with the strongest association found in the simvastatin-treated subjects (R=0.75, P=0.002). Next, in an attempt to determine whether statin-induced muscle pain may be associated with muscle mtDNA depletion, archived muscle biopsies collected from statin users with muscle complaints were sought through a review of a muscle biopsy database and possible study samples were identified; however, this was put on hold as too much information was missing from the pathology reports. Third, a series of cell culture experiments were carried out in which human skeletal muscle myotubes were exposed to various concentrations of simvastatin or atorvastatin, in order to determine an appropriate dose range for subsequent mitochondrial toxicity experiments. Lastly, mtDNA content and expression was quantified in skeletal muscle biopsies collected from 10 patients with statin-induced rhabdomyolysis (SIR) and compared to 8 healthy controls to investigate whether muscle mtDNA is altered in rhabdomyolysis. No differences in mtDNA content or expression were observed between the two study groups, but this may have been be due to the SIR subjects' marked heterogeneity. Statin therapy can be associated with considerable alterations in mtDNA content, which may play a role in the aetiology of SIM. MtDNA levels alterations with statin exposure should be investigated further to explore the involvement of mitochondrial alterations in the mechanism of SIM, and determine whether these may represent a useful clinical tool for assessing statin-induced muscle toxicity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Background: A third of HIV-infected patients are co-infected with HCV in the developed world, and more of co-infected patients than ever before are dying because of liver related diseases today. Drug-related hepatotoxicity is a growing concern among human immunodeficiency virus (HIV) / hepatitis C virus (HCV) co-infected population. Nucleotide analogues containing HIV antiretroviral therapy, namely highly active anti-retroviral therapy (HAART), can induce mitochondrial toxicity. However, little is known about the effect of nucleotide analogues on the liver at the cellular and molecular level, and how it may affect treatment. Objective: To investigate whether liver tissue from HIV/HCV co-infected individuals will show greater liver mitochondrial toxicity if currently receiving antiviral HIV medication, compared to those who are not taking it. Methods: Liver biopsies were collected from 23 HIV/HCV co-infected males. Fourteen patients were on stable HAART (ON-HAART) and 9 were OFF-HAART, including 4 who stopped HAART >6 months prior and 5 who were HAART-nave. Liver mitochondrial toxicity was assessed by transmission electron microscopy-based quantitative stereological analyses of hepatocyte and mitochondrial morphometry, as well as by mitochondrial DNA (mtDNA) and mtRNA (COX1/(ß-actin) real-time-PCR quantification. Results: Hepatocytes tended to be larger in the ON-HAART group than in the OFF-HAART group (p=0.05), but they both showed similar mitochondrial volume fraction of the cell and mitochondrial crista density. Liver mtDNA and mtRNA levels were not significantly differentbetween ON-HAART and OFF-HAART. Hepatocyte lipid accumulation was significantly higher in HCV genotype 3 compared to genotype 1 infection ()=0.002), but was not associated with HAART status. Conclusions: We found no evidence or trend of increased mitochondrial toxicity in HIV/HCV co-infected individuals currently on HAART compared to those who are not. This finding could be relevant to the decision-making process with respect to initiating HCV therapy in this population. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Toxic metals are major pollutants of the aquatic environment and are able to cause survival impairment of the early life stage of the aquatic organisms. They can affect the osmoregulatory system and electrolyte balance in fishes as well as the expression of genes which are essential in the formation and development of the organs at the early embryonic stages of development. There are a lack of studies concerning the toxic effects of waterborne copper and silver on the osmoregulation, electrolytes balance and expression the genes which are responsible for the formation and development of heart and metal binding proteins in the early life stage of zebrafish. The current study aimed to assess the toxic effects of waterborne concentrations of copper as an essential trace element, and silver as a non-essential trace element, on biochemical processes and the molecular biology of the early life stages (ELS) of zebrafish. The first experiment of the current study (Chapter 3) aimed: 1. to determine the time of nkx2.5 gene expression, a gene involved in cardiac development, relative to the time of embryonic development. 2. To assess the toxic concentration of the copper and most vulnerable and sensitive stage of the embryos < 1 hour post fertilization (hpf) exposed to the copper via water route. The result of the experiment showed that the expression of the nkx2.5 gene reached a maximum at 16 hpf. The first 10 hpf of the embryonic development was the most vulnerable and critical stage of the developing embryos, and characterized by increased mortality as copper concentration increased, and delayed and decreased hatching success. Exposure of embryos for 72 hpf to a concentration of 500 µg L-1 Cu increased heart rate, whereas the exposure of the embryos at the blastula stage only, showed decreases in heart rate. The third part of the experiment evaluated the protective effect of calcium as a major cation of water hardness on Cu toxicity. Embryos age < 2 hpf were exposed to copper (0, 100, 250, and 500 µg L-1), with or without added calcium (40 mg L-1). An increase in embryonic Cu accumulation was observed in live and dead embryos exposed to Cu, with and without added calcium. Calcium concentration increased with embryonic copper tissue concentration in dead embryos. Na+ and K+ concentrations were higher in live embryos compared to dead embryos, and a 4 fold decrease in Na+K+-ATPase activity was seen in live embryos exposed to copper compared to controls. There was no effect of copper on total glutathione. Expression of nkx2.5 as one of the essential genes for the formation and development of the heart increased significantly; approximately 10 fold in the presence of Cu+Ca in comparison to the unexposed control or Cu exposure alone. Whereas expression of mt2 increased significantly 6 fold compared to the control during Cu exposure without added Ca2+. The second experiment (Chapter 4) aimed to investigate the effect of the dissolved Ag+ as AgNO3 on the survival of the early life stage of zebrafish. Embryos < 2 hpf were exposed to silver 0 (no added Ag), 2.5, 5, 7.5, 10 and 15 µg L-1 Ag as AgNO3 for up to 72 h. Although, the survival was not affected by increasing concentrations of total silver, a decrease in hatching and increase in heart beat was observed. A significant increase in embryonic silver accumulation in both live embryos (at 24 and 72 hpf) and dead embryos (at 24 hpf) was observed. The accumulation of silver in 24 hpf live embryos was more significant than in dead embryos. Dead and live embryos at 72 hpf exposed to Ag had lower Na+ and K+ concentrations. Live embryos also showed a transient increase in Ca2+ concentration at 24 h. Four fold increases in Na+K+-ATPase activity, Mt2, and total glutathione concentration were seen in embryos after 72 h of exposure to AgNO3 compared to controls. In contrast, nkx2.5 gene expression was significantly decreased by 3 fold in 24 h aged embryos exposed to silver compared to controls. Due to the lack of studies that investigate the effect of silver on protein expression profiles during the early stages of development of zebrafish, the third experiment (Chapter 5) aimed to investigate the effect of silver on the changes of the expressed proteins of zebrafish embryos at the segmentation stage (24 hpf). The proteomics analysis successfully identified total of 810 proteins in the embryonic homogenate and quantified changes in their abundance in response to silver exposure. MS analysis showed the induction of new proteins which were absent in control embryonic homogenates. Also the analysis revealed there were increased expression of proteins such as zona pellucida glycoprotein, ATP synthase subunit α and β, stressed proteins such as metal chaperones and heat shock proteins, antioxidant proteins such as catalase (CAT), superoxide dismutase (Cu-Zn), glutathione S-transferase M, and glutathione S-transferase and proteins related to muscular development such as myosin heavy polypeptide 2, actin alpha 1 skeletal muscle, slow myosin heavy chain 1, actin cytoplasmic 1, and tropomyosin proteins. Overall, the thesis confirmed that the early life stages of zebrafish are sensitive to metals and that there are critical windows of toxicity during development. Metal exposure at early stages of the development initiate several disturbances in biochemical processes as well as changes in molecular biology that affect fish survival.

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