Doctor of Philosophy

dissertationNanomaterials are promoted as a promising technology in highly diverse applications, but concerns about risks of these materials have stimulated extensive research on the adverse effects of manufactured nanoparticles. Our research utilized ex vivo tissues to study quantification of unlabeled nanoparticles in biological media and in vitro cultures of human cells, treated with suspensions of low-solubility metal oxide materials, to study uptake, cell signaling, and gene regulation. The effects of nanoparticles on the cardiovascular system occur because inhaled nanoparticles may enter systemic circulation, or are directly injected into systemic circulation for medical applications, causing adverse health effects. Silicon dioxide (Si02) particles with sizes between 20-100 nm were used in our studies because they have many commercial and medical applications and can be readily modified with surface functional groups. Our hypothesis is two-fold. First, we hypothesize that tissue uptake and deposition of Si02 nanoparticles can be measured by sedimentation field-flow fractionation (SdFFF) after particle isolation using enzyme digestion. We also hypothesize that the adverse effects associated with nanoparticle exposures are greater in vascular cells than pulmonary and colorectal cell lines, due to the formation of reactive oxygen species, which may stimulate apoptosis. The results presented here demonstrate the cellular uptake of nano and submicron-sized Si02 using the traditional methods of microscopy. Additionally, the method of SdFFF, which is typically used to separate macromolecules, colloids, and particles, was used in combination with enzyme digestion to provide a novel, useful method to simultaneously measure both the size and concentration of particles in tissues. The toxic effects of metal oxide particle treatment on vascular, pulmonary and colon cells were evaluated. Significant cytotoxicity and inflammation were observed with vascular endothelial cells, but little to no adverse effects were seen in pulmonary and colon cancer epithelial cells at concentrations of 1-316 pg/cm2. Nano-sized SiC>2 caused apoptosis in vascular endothelial cells that was ameliorated by pretreatment with 5 mM of the antioxidant, N-acetyl-L-cysteine. Our results show that exposures of endothelial cells to high concentrations of nano-sized Si02 may have serious adverse cardiovascular consequences. Furthermore, these results provide insights into treatments, such as the use of antioxidants, for persons exposed to significant levels of metal oxide nanoparticles

TRPA1 gene polymorphisms and childhood asthma.

BACKGROUND: Animal data have suggested that the transient receptor potential ankyrin-1 (TRPA1) ion channel plays a key role in promoting airway inflammation in asthma and may mediate effects of paracetamol on asthma, yet confirmatory human data are lacking. To study associations of TRPA1 gene variants with childhood asthma and total IgE concentration, and interactions between TRPA1 and prenatal paracetamol exposure on these outcomes. METHODS: We analysed associations between 31 TRPA1 single nucleotide polymorphisms (SNPs) and current doctor-diagnosed asthma and total IgE concentration at 7.5 years in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort. We sought to confirm the most significant associations with comparable outcomes in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) and Generation R birth cohorts. In ALSPAC, we explored interactions with prenatal paracetamol exposure. RESULTS: In ALSPAC, there was strong evidence for association between six SNPs and asthma: rs959974 and rs1384001 (per-allele odds ratio for both: 1.30 (95% CI: 1.15-1.47), p = 0.00001), rs7010969 (OR 1.28 (1.13-1.46), p = 0.00004), rs3735945 (OR 1.30 (1.09-1.55), p = 0.003), rs920829 (OR 1.30 (1.09-1.54), p = 0.004) and rs4738202 (OR 1.22 (1.07-1.39), p = 0.004). In a meta-analysis across the three cohorts, the pooled effect estimates confirmed that all six SNPs were significantly associated with asthma. In ALSPAC, TRPA1 associations with asthma were not modified by prenatal paracetamol, although associations with IgE concentration were. CONCLUSION: This study suggests that TRPA1 may play a role in the development of childhood asthma. (249 words)

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na⁺ and Ca2 + influx via TRPV1 channels, which leads to mitochondrial Ca2 + accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist- induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid- sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau- type Ca2 + signals, mitochondrial Ca2 + accumulation and Na⁺- and Ca2 +-dependent membrane disorganization. Moreover, long- term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic <i>MUC5AC</i> Expression by Vasaka (<i>Adhatoda vasica</i>; Malabar Nut) Tea

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy