Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.

The ability of NSAIDs to delay bone healing has been long known, although the extent and exact mechanism remains elusive. The present study evaluates the effect of short duration NSAID on bone healing in dogs following experimental tibial osteotomy. Carprofen was administered twice daily for either 0, 2, or 8 weeks following surgery. Bone healing was evaluated radiographically using RUST scoring at 4 and 8 weeks postop. Postmortem, quantitative CT of for bone mineral density analysis, histologic cartilage:callus ratio of the fracture, and biomechanical testing were performed. Biomechanically, stiffness and maximum stress were higher in dogs that received no carprofen than those that received 8 weeks. Radiographic healing scores were the same for dogs which did not receive carprofen and those receiving a short course, but both were more healed than dogs which received 8 weeks of carprofen. There was no treatment effect on cartilage:callus ratio or bone mineral density.

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The overall objective of this research was to investigate the status of tumor necrosis factor-?? (TNF-??), and molecules associated with its signaling, in the pathological state of hepatic steatosis. The effect of NSAID piroxicam, a cancer preventive agent also known to affect TNF-?? signaling on hepatic steatosis, was also investigated. The biological state of the tissue was assessed by examining the expression of TNF-?? signaling molecule in whole tissue, as well as in hepatic lipid raft. Lipid rafts are dynamic assemblies of cholesterol and sphingolipids, microdomains that form in the exoplasmic leaflet of the biological membranes shown to play a role in compartmentalization, modulation and integration of the cell signaling. In the present research, Zucker obese rats were used as a model of human obesity and insulin resistant state. These rats exhibit hepatic steatosis in adulthood similar to those noted in obese individuals. Female Zucker obese and lean rats (5 weeks old) were fed a semisynthetic diet with or without piroxicam (150 ppm). Zucker lean counterparts served as control. After 8 weeks of feeding, rats were euthanized and liver from each animal was collected. Liver tissue from each animal was processed for histology and biochemical analysis which included lipids and proteins (COX-1 and 2, TNF-??, TNF-RI and RII, IKK-??, I??B-?? and NF-??B). Liver histology and the level of total lipids confirmed that Zucker obese rats had hepatic steatosis, which was further augmented by piroxicam treatment. Whole tissue protein expression, using western blot, showed that the steatotic liver differed from non-steatotic livers by having lower levels of TNF-RII. TNF-RII showed a trend which was inversely proportional to the pathological state of the tissue. The obese-piroxicam liver had the lowest level of TNF-RII and lean livers had the highest (p<0.05). The total NF-??B level was higher in the obese and obese-piroxicam groups compared to the lean or lean-piroxicam groups (p<0.05). Piroxicam treatment lowered the level of NF-??B in obese and lean livers. I??B-?? was higher in obese livers than in lean livers. The nuclear level of NF-??B by western blot analysis showed the same pattern as noted in the whole tissue homogenate. However, the difference in the level between obese and lean was marked. The obese nuclei contained two to three fold higher levels of NF-??B protein than the lean liver nuclei. I??B-?? level was significantly higher in the obese liver tissues and nuclei than their lean counterparts. While transcriptionally active NF-??B was higher (p<0.05) in the obese livers than in the lean livers, the difference between obese and lean groups was not as significant as that noted for the level of NF-??B assessed by western blot. This suggests that the proportion of active NF-??B present in the nuclear fraction is much higher in the lean than in the obese nuclei. Lipid raft was extracted and identified successfully from obese and lean livers. The total caveolin and flotillin levels were significantly higher in the liver lipid rafts of the obese-piroxicam than that of the other groups. This is the group that also exhibited higher steatosis. Piroxicam treatment significantly decreased the level of caveolin in the lean liver and significantly increased the level of flotillin in the obese liver. While COX-1 was not detectable, however, the level of COX-2 and TNF-RII in lipid raft was opposite to the level noted in the whole tissue homogenate. TNFRII was highest in the obese-piroxicam lipid raft and lowest in the lean-piroxicam lipid raft. TNF-RII, COX-2, I??B-?? and NF-??B proteins were the molecules profoundly affected by the pathological state of the tissue and piroxicam treatment. This research is the first to report the presence of I??B-?? in the nuclear compartment with a higher level in the nuclei and whole tissue in the obese liver than in the lean liver. This research demonstrates that TNF-?? to NF-??B axis is altered in steatotic liver, and analysis of lipid rafts in steatotic and non-steatotic liver demonstrates that lipid rafts play a distinct role in modifying the biological availability of key proteins in the pathological state of liver steatosis.

Beta-blockers and non-steroidal anti-inflammatory drugs (NSAIDs) are often avoided in asthma over risk of bronchospasm which may vary according to drug selectivity and duration of administration. This thesis attempts to quantify the risk of beta-blocker and NSAID exposure in asthma by synthesising clinical trial evidence and conducting observational studies using linked electronic medical records. As part of this thesis, three systematic reviews of clinical trials were conducted evaluating: the prevalence of aspirin-exacerbated respiratory disease (AERD); risk of selective NSAIDs/COX-2 inhibitors in people with AERD; and risk of acute beta-blocker exposure in people with asthma. Electronic primary care data from the Clinical Practice Research Datalink (CPRD) was used to define a cohort of people with active asthma, measure the prevalence of beta-blocker and NSAID prescribing, and perform a series of nested case control studies evaluating asthma death, asthma hospitalisation and primary care asthma exacerbations (PCAE). A self-controlled case-series was performed for PCAE as well. Based upon work in this thesis, the prevalence of AERD in people with asthma was around 9%. Selective NSAIDs triggered respiratory symptoms in 8% of people with AERD whilst no significant changes in lung function or symptoms occurred with COX-2 inhibitors. Acute non-selective beta-blocker exposure caused a significant mean fall in FEV1 of 10%, a significant increase in respiratory symptoms in around 1 in 13 and a non-significant increase in falls in FEV1 of ≥20% in around 1 in 9. Acute selective beta-blocker exposure caused a significant mean fall in FEV1 of 7%, significant falls in FEV1 of ≥20% in around 1 in 8 and a non-significant increase in respiratory symptoms in around 1 in 33. The prevalence of selective beta-blocker prescribing in asthma rose by around 200% over the 12 year period whilst the prevalence of non-selective beta-blocker prescribing rose by around 90%. Changing trends in NSAID prescribing occurred over the 12 year period with COX-2 inhibitors now rarely prescribed. Using the nested case control design, both incident and high-dose non-selective beta-blocker exposure was associated with significantly increased risk of asthma morbidity (hospitalisation and PCAE). In contrast, no significant increased risk of asthma morbidity occurred with any type of selective beta-blocker exposure. Consistent findings were seen for PCAE using the self-controlled case series. No significantly increased risk was seen with different oral NSAIDs apart from weak evidence of an association between asthma death and non-selective NSAID exposure which is unlikely to be causal. Significant numbers of people with asthma are prescribed beta-blockers and NSAIDs. Evidence from clinical trials and observational studies demonstrate that non-selective beta-blockers significantly increase asthma morbidity with risk appearing to vary according to dose and duration of administration. Although selective beta-blockers have the potential to cause significant changes in lung function, no significant increase in asthma morbidity was observed in observational studies. Although around 9% of asthmatics may be susceptible to NSAIDs, no strong evidence was found to suggest that the current practice of NSAID prescribing increases asthma morbidity. At the same time, COX-2 inhibitors are infrequently prescribed despite apparently being well tolerated by people with AERD.

<p>Validerat; 20101217 (root)</p>

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs prescribed to relieve pain, fever and inflammation, and are among the most commonly consumed medications in Ontario. Approximately 70% of the ingested dose is excreted unchanged or as an active metabolite, much of which reaches the surface waters of lakes and rivers. NSAIDs function through the inhibition of cyclooxygenase (COX), an enzyme present in two isoforms in the body; the constitutively expressed COX-1 and the inducible COX-2. Traditional NSAIDs like ibuprofen inhibit both isoforms with little selectivity while newer variants such as naproxen preferentially inhibit COX-2. Both COX isoforms share a high similarity between humans and fish creating a potential for off target effects to exposed aquatic organisms. This research investigated the chronic effects of waterborne exposure to 0, 0.1, 1, 10 and 100 μg/L of a nonselective and selective NSAID (ibuprofen and naproxen, respectively) on Florida flagfish (Jordanella floridae) over one complete life-cycle. Chronic exposure concentrations were selected by performing a short term experiment which examined the hatchability of flagfish eggs using continuous semi-static exposure conditions. Growth, survivability and reproductive endpoints were assessed in the life-cycle study. A concentration-response relationship for both NSAIDs was detected during the first 28 days post-hatch, resulting in increased body length for F1 fish and their offspring with increasing concentrations. Exposure to 0.1 μg/L of both ibuprofen and naproxen resulted in a decrease in egg fertilization providing an experimental LOEC (lowest observable effect concentration) of 0.1 ug/L and NOEC (no observable effect concentration) of < 0.1 ug/L for both ibuprofen and naproxen based on the reproductive endpoint. This indicates that either NSAID has the potential to affect the reproductive success of flagfish at concentrations at or below those commonly found in the environment. / UOIT

This dissertation focuses on chemical and biological evaluation of diazeniumdiolate based nitrogen oxide releasing prodrugs. Three projects are described. i. Synthesis and biological evaluation of a series of new nitroxyl (HNO) releasing non-steroidal antiinflammatory drugs (NSAIDs) and comparison to related nitric oxide (NO) releasing NSAIDs A series of HNO releasing isopropylamine-based diazeniumdiolate adducts of NSAIDs and the NO releasing diethylamine diazeniumdiolate counterpart were synthesized. The aspirin derivatives were evaluated for antiinflammatory, cardioprotective and anticancer effects. Both prodrugs demonstrated similar antiinflammatory properties to aspirin but significantly lower gastrointestinal ulceration, which is a common side effect of aspirin. The HNO adduct also improved cardiac contractility. The chemotherapeutic potential of the prodrugs was assessed in vitro and in vivo. Both the prodrugs inhibited growth of cultured carcinoma cells without inducing cytotoxicity towards non-tumorogenic cell lines. The higher cytotoxicity of the HNO adduct was in part due to increased production of reactive nitrogen and oxygen species leading to oxidative damage to DNA, inhibition of glyceraldehydes-3-phosphate dehydrogenase and upregulation of signaling pathways leading to caspase-3 mediated induction of apoptosis. The NO adduct is a promising candidate for reduction of metastasis by increasing E-cadherin levels, which influences cellular adhesion. Both derivatives showed significantly reduced angiogenesis in cultured cells and tumor volume in nude mice. ii. Synthesis and characterization of primary amine based cyclic amine diazeniumdiolates and comparison to their acetoxy methyl ester derivatives. A series of HNO releasing cyclic amine diazeniumdiolates were synthesized to expand upon the few examples of primary amine diazeniumdiolates. An ester derivative of cyclopentylamine NONOate was also synthesized, to increase decomposition half-life and to improve HNO production and better cellular uptake. This modification increased its cytotoxicity compared to ionic NONOates. iii. Evaluation of mechanism of action of JS-K. JS-K (O²-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1-yl]-diazeniumdiolate) has previously been found to be highly cytotoxic in many cancer cell lines compared to ionic diazeniumdiolates. Thus, the role of NO in cytotoxicity of JS-K was explored. A low intracellular NO flux in combination with a lack of any effect on cyclic guanosine monophosphate (cGMP) dependent pathway suggests that NO is not directly responsible for the cytotoxicity of JS-K.