The effect of 2,4-D on gene expression in cultured cells

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2’,7’-dichlorofluorescin diacetate (2’,7’-DCFH-DA), and real time–polymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the ‘inert ingredients’ in the commercial formulation.The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans

The effect of acyclovir on the tubular secretion of creatinine in vitro

<p>Abstract</p> <p>Background</p> <p>While generally well tolerated, severe nephrotoxicity has been observed in some children receiving acyclovir. A pronounced elevation in plasma creatinine in the absence of other clinical manifestations of overt nephrotoxicity has been frequently documented. Several drugs have been shown to increase plasma creatinine by inhibiting its renal tubular secretion rather than by decreasing glomerular filtration rate (GFR). Creatinine and acyclovir may be transported by similar tubular transport mechanisms, thus, it is plausible that in some cases, the observed increase in plasma creatinine may be partially due to inhibition of tubular secretion of creatinine, and not solely due to decreased GFR. Our objective was to determine whether acyclovir inhibits the tubular secretion of creatinine.</p> <p>Methods</p> <p>Porcine (LLC-PK1) and human (HK-2) renal proximal tubular cell monolayers cultured on microporous membrane filters were exposed to [2-¹⁴C] creatinine (5 μM) in the absence or presence of quinidine (1E+03 μM), cimetidine (1E+03 μM) or acyclovir (22 - 89 μM) in incubation medium.</p> <p>Results</p> <p>Results illustrated that in evident contrast to quinidine, acyclovir did not inhibit creatinine transport in LLC-PK1 and HK-2 cell monolayers.</p> <p>Conclusions</p> <p>The results suggest that acyclovir does not affect the renal tubular handling of creatinine, and hence, the pronounced, transient increase in plasma creatinine is due to decreased GFR, and not to a spurious increase in plasma creatinine.</p

A comparison of roles played by men and women in public relations with an examination of possible influencing factors

Call number: LD2668 .T4 1982 M93Master of Scienc

Methanol adducts leading to the identification of a reactive aldehyde metabolite of CPAQOP in human liver microsomes by ultra-high-performance liquid chromatography/mass spectrometry.

The incubation of compound AZX (1-[(2R)-2-[[4-[3-chloro-4-(2-pyridyloxy)anilino] quinazolin-5-yl]oxymethyl]-1-piperi-dyl]-2-hydroxy) with human liver microsomes generated several metabolites that highlighted the hydroxyacetamide side chain was a major site of metabolism for the molecule. The metabolites were derived predominantly from oxidative biotransformations, however two unexpected products were detected by LC-UV-MS and identified as methanol adducts. It became apparent that a metabolite formed in the microsomal incubation reacted with methanol in the mobile phase when no methanol adducts were detected in the analysis where acetonitrile was used. This observation prompted further investigations into the metabolic modification of the parent. Although this reactive metabolite could not be isolated or structurally characterised by LC-MS, several metabolic indications enabled the proposal of a reactive aldehyde. Experiments using methoxyamine post-incubation showed the disappearance of the two methanol adducts and appearance of a methoxyamine adduct, confirming the presence of an aldehyde group. The proposed structure of the reactive aldehyde derived from oxidation of the terminal hydroxyl group on the hydroxyacetamide side chain, leading to the formation of the diastereoisomeric methanol adducts detectable by LC-UV-MS

The role of strain in the response of rapidly growing young male rat bones to parathyroid hormone

Abstract Human parathyroid hormone (hPTH 1-34) stimulates an anabolic response in human and animal skeletons; however, it is unclear if the effect is strain dependent. To determine if the anabolic response to hPTH (1-34) was dependent upon strain in rats we used 2 outbred strains (Sprague Dawley, Wistar), 2 inbred strains (Fischer 344, Wistar spontaneously hypertensive:SHR), and 2 mutant strains (Zucker obese, Zucker lean) of rats. Male rats, 5 weeks of age, from each strain were treated subcutaneously with 80ug/kg body weight hPTH (1-34) or vehicle for 12 days. The response to PTH was similar in all strains whereby PTH exerted an anabolic effect on femoral bone mass and cancellous bone histology that was independent of strain differences. Histomorphometric indices of bone volume, mineralized surface and bone formation in lumbar vertebrae increased in all PTH-treated rats. Additionally, femur bone mineral content and bone mineral density measured by dual energy X-ray absorptiometry (DEXA), and ash weight increased in all PTH-treated rats. These increases occurred regardless of strain. In summary, PTH exerted comparable anabolic effects on bone mass, bone mineral density and bone formation in all rat models tested demonstrating that the skeletal responsiveness to PTH was not dependent upon strain

The impact of oat structure and β-glucan on in vitro lipid digestion

Oat β-glucan has been shown to play a positive role in influencing lipid and cholesterol metabolism. However, the mechanisms behind these beneficial effects are not fully understood. The purpose of the current work was to investigate some of the possible mechanisms behind the cholesterol lowering effect of oat β-glucan, and how processing of oat modulates lipolysis. β-Glucan release, and the rate and extent of lipolysis measured in the presence of different sources of oat β-glucan, were investigated during gastrointestinal digestion. Only a fraction of the original β-glucan content was released during digestion. Oat flakes and flour appeared to have a more significant effect on lipolysis than purified β-glucan. These findings show that the positive action of β-glucan is likely to involve complex processes and interactions with the food matrix. This work also highlights the importance of considering the structure and physicochemical properties of foods, and not just the nutrient content

Holmes tremor in a patient with progressive multifocal leukoencephalopathy.

BACKGROUND: Progressive multifocal leukencephalopathy (PML) is a rare, sometimes fatal viral disease in patients with primary or secondary immunosuppression. CASE DESCRIPTION: A 57-year-old immunocompetent female with intractable Holmes tremor and elongated unique brainstem lesion reported to our hospital. The cerebrospinal fluid (CSF) screening for John Cunningham virus was negative and the diagnosis was established by brain biopsy. The course was rapidly fatal. CONCLUSION: This atypical presentation of PML in an immunocompetent patient illustrates that diagnosis can be missed without brain biopsy