Pre-systemic elimination by local enzymatic degradation can play a key role in limiting the bioavailability of intranasally administered drugs. Despite remarkable advancement in the characterization of the nasal biotransformative enzymes, knowledge of the role of the nasal mucosa in limiting bioavailability of therapeutic agents is still inadequate. The aim of this work was to evaluate the expression and substrate biotransformation activity of cytochrome P450 enzymes in the nasal mucosa using bovine olfactory and respiratory explants as in vitro models. Gene expression and localization of major CYP450 isoforms in the nasal mucosa were examined using RT-PCR and immunohistochemistry. The bovine nasal mucosa showed abundant expression of CYP2A6 and 3A4 genes whereas 1A1, 1A2, 2C9, and 2C19 isoforms were expressed at much lower levels. The CYP450 proteins were observed to be present in the epithelial layer and in submucosal glandular cells. The diffusion of melatonin, a CYP1A2 substrate, and the appearance of 6-hydroxymelatonin, its primary metabolite, across bovine olfactory and respiratory explants was measured, and nasal olfactory and respiratory microsomal preparations were used to quantify the kinetic parameters for melatonin 6-hydroxylation. Results indicated that bovine olfactory and respiratory CYP450 isoforms were metabolically active towards melatonin metabolism, and the respiratory mucosa demonstrated the greatest melatonin 6-hydroxylation activity. Numerical simulations were used to probe the effects of the relative magnitudes of the permeability coefficient and enzymatic parameters on net substrate mass transfer across nasal mucosal tissues. The simulations indicated that the concentration gradient of the drug coupled with its permeability coefficient were the most significant factors controlling the transport of drugs across the mucosal tissue. Enzymatic degradation decreased the flux of drugs across the mucosa and had the greatest impact on low permeability compounds. The results from these studies show that the bovine nasal mucosa possesses significant metabolic activity, and the flux of a metabolically labile substrate across the nasal mucosa can be significantly reduced by its enzymatic degradation within the tissue. Use of kinetic modeling to characterize of the extent of biotransformation in the nasal mucosa enables the identification of metabolism-limited bioavailability of intranasally administered drug compounds.

The use of mobile phones is continually increasing throughout the world, with recent figures showing that there are currently more than 2 billion mobile phone users worldwide. However, despite the recognised benefits of the introduction and widespread use of mobile phone technologies, concerns regarding the potential health effects of exposure to the radiofrequency electromagnetic fields emitted by mobile phone handsets have similarly increased, leading to an increase in demand for scientific research to investigate the possibility of health effects related to the use of mobile phones. An increasing amount of radiofrequency bioeffects research related to mobile phone use has focussed on the possible effects of mobile phone exposure on human brain activity and function, particularly as the absorption of energy in the head and brain region is much higher than in other body regions, which is a direct result from the close proximity of the mobile phone to the head when in normal use. In particular, the use of sleep research has become a more widely used technique for assessing the possible effects of mobile phones on human health and wellbeing, and is particularly useful for providing important information in the establishment of possible radiofrequency bioeffects, especially in the investigation of potential changes in sleep architecture resulting from mobile phone use. A review of the previous literature showed that a number of studies have reported an increase in the electroencephalogram spectral power within the 8 � 14 Hz frequency range in both awake and sleep states following radiofrequency electromagnetic field exposure. In regards to sleep, the enhancements reported have not been entirely consistent, with some early studies failing to find an effect, while more recent studies have reported that the effect differs in terms of particular frequency range. However, in general the previous literature suggests that there is an effect of mobile phone emissions on the sleep electroencephalogram, particularly in the frequency range of sleep spindle activity. In addition to changes in spectral power, changes in other conventional sleep parameters and the production and secretion of melatonin have also been investigated, however, there has been little or no consistency in the findings of previous studies, with the majority of recent studies concluding that there is no influence of mobile phone radiofrequency fields on these parameters of sleep or melatonin. Following a detailed review of the previous research, the current study was developed with the aim to improve on previous methodological and statistical limitations, whilst also being the largest study to investigate mobile phone radiofrequency bioeffects on human sleep. The principle aims were thus to test for the immediate effects of mobile phone radiofrequency electromagnetic fields on human sleep architecture and the secretion of the pineal hormone, melatonin. The experiment included 50 participants who were randomly exposed to active and sham mobile phone exposure conditions (one week apart) for 30 minutes prior to a full night-time sleep episode. The experimental nights employed a randomised exposure schedule using a double-blind crossover design. Standard polysomnography was used to measure subsequent sleep, and in addition, participants were required to provide urine samples immediately following exposure and upon waking in the morning. A full dosimetric assessment of the exposure system was also performed in order to provide sufficient details of the exposure set-up used in the current thesis and to account for the lack of detailed dosimetric data provided in the majority of previous studies. The results of the current study suggest that acute exposure to a mobile phone prior to sleep significantly enhances electroencephalogram spectral power in the sleep spindle frequency range compared to the sham exposure condition. The current results also suggest that this mobile phone-induced enhancement in spectral power is largely transitory and does not linger throughout the night. Furthermore, a reduction in rapid eye movement sleep latency following mobile phone exposure was also found compared to the sham exposure, although interestingly, neither this change in rapid eye movement sleep latency or the enhancement in spectral power following mobile phone exposure, led to changes in the overall quality of sleep. Finally, the results regarding melatonin suggested that, overall, overnight melatonin secretion is unaffected by acute exposure to a mobile phone prior to sleep. In conclusion, the current study has confirmed that a short exposure to the radiofrequency electromagnetic fields emitted by a mobile phone handset immediately prior to sleep is sufficient to induce changes in brain activity in the initial part of sleep. The consequences or functional significance of this effect are currently unknown and it would be premature to draw conclusions about possible health consequences based on the findings of the current study.

Graduation date: 1994

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) act as second messengers in intracellular signaling cascades to influence neuronal responses. Hippocampal cAMP signaling is thought to underlie the pathophysiology of major depressive disorder (MDD) and antidepressant action; however, little is known about the possible role of cGMP signaling. Furthermore, circadian rhythm disturbances can occur as part of the clinical symptoms of MDD and resolve with antidepressant therapy. The pineal gland is relevant to circadian rhythms as it secretes the hormone melatonin following activation of cAMP signaling and the rate-limiting enzyme for its synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Little is known about the contribution of the phosphodiesterase (PDE) system to antidepressant-induced alterations in pineal cAMP signaling and melatonin synthesis. There is a need to clarify the trajectory of cAMP and cGMP concentrations, their synthesis by cyclases, and degradation by PDEs to understand the role of cyclic mononucleotide signaling in the effect of chronic antidepressant therapy. Using quantitative real-time PCR and enzyme immunoassay, we systematically studied elements of intracellular signaling in the hippocampus of rats chronically treated with imipramine, fluoxetine, and amitriptyline and in the pineal gland of rats treated chronically with fluoxetine. In the hippocampus, we found chronic imipramine downregulated cAMP signaling with decreased cAMP, increased PDEs and decreased adenylate cyclase mRNA. In contrast, repeated fluoxetine and amitriptyline increased hippocampal cGMP signaling, with increased cGMP and decreased PDE mRNA. We conclude that in contrast to the assumption of antidepressant-mediated increases in cAMP levels, increased hippocampal cGMP signaling might underlie the efficacy of chronic antidepressant treatment. A follow up study using cultured embryonic rat hippocampal cells in vitro treated with the PDE type 5 inhibitor, sildenafil, demonstrated increased cAMP content following acute and chronic treatment, indicating either crosstalk between cAMP and cGMP pathways or a non-specific inhibitory effect of sildenafil on other PDEs. In the pineal gland, we found elevated melatonin synthesis with increased pineal AA-NAT mRNA and daytime plasma melatonin and downregulated cAMP signaling with increased PDE and unchanged AC pineal mRNA, and decreased pineal cAMP. We conclude that chronic fluoxetine increases daytime plasma melatonin and pineal AA-NAT mRNA despite downregulated pineal cAMP signaling.

Melanophores are dark brown pigment cells located in the skin of fish, amphibia, reptiles, and many invertebrates. The color of the animal can change via rearrangement of pigment granules, melanosomes, in the cells. The dark melanophores can either hide colorful cells so that the animal appears dark, or let through colors from underneath. The animal regulates its colors and patterns via communicating nerve cells and hormones in the blood stream. It is nowadays well established that melatonin-stimulation of melanophores results in aggregation of melanosomes to the cell center and that the evident outcome is more transparent cells. It has previously been shown that the activity of serine and threonine kinases as well as phosphatases regulates the distribution of melanosomes in the cells. We wanted to study if tyrosine phosphorylations were involved in the regulation of melanosome aggregation. Melatonin-stimulated signaling in the African clawed frog, Xenopus laevis, melanophores was examined. Melansome aggregation was accompanied by tyrosine phosphorylation as shown by immunoblots. Inhibition of tyrosine phosphorylation reduced melanosome aggregation by melatonin, and the phosphorylation most likely regulated pigment aggregation. Tyrosine phosphorylation of the protein was mediated via a Gi/o protein coupled receptor, probably the melatonin receptor Mel1c. The phosphorylation was most likely not a result of the classical Gi/o protein pathway, as Src-kinase and mitogen-activated protein kinase seemed required for phosphorylation and melanosome aggregation. Two candidates for the phosphorylated protein were presented, talin and β-spectrin. The possible involvement of nitric oxide in melanosome aggregation by melatonin was investigated. Nitric oxide appeared to be necessary for melanosome aggregation. The effect of nitric oxide synthase inhibition on melanosome aggregation was not mediated via changes in the tyrosine-phosphorylated protein. We speculated that nitric oxide could affect melanosome distribution via modifications of the actin cytoskeleton. The use of recombinant melanophores as a biosensor has also been examined. A human G protein coupled receptor, opioid receptor 3, was inserted into melanophores by electroporation. The transfected melanophores responded dose-dependently to opioids and an inhibitor of opioid receptors reduced the aggregation response. Future melanophore biosensors migh detect a variety of substances, such as narcotics, pheromones, odors, and tastes.

Graduation date: 1993

published_or_final_version / abstract / toc / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Background: Long-term night shift work has been associated with multiple cancer sites, including breast, prostate, colon and endometrial. The mechanism for this effect is hypothesized to include the hormone melatonin; where increased light at night exposure during shift work reduces melatonin production and decreased melatonin levels are associated with increased cancer risk. In addition, physical activity has been shown to reduce cancer risk and existing laboratory studies indicate it has the potential to influence melatonin levels. Methods: A cross-sectional study of light intensity exposure, physical activity and melatonin levels was conducted among 61 rotating shift nurses at Kingston General Hospital. Light intensity exposure was measured using a light intensity data logger and melatonin concentrations were measured from urine and saliva samples, collected over a 24-hour period. Physical activity was assessed from a study questionnaire and one-day diary. Results: A statistically significant inverse association between light exposure and urinary melatonin levels was observed; however, the relationship was no longer significant when stratified by shift group. Analysis of salivary melatonin levels demonstrated that circadian rhythms of melatonin production in night workers were not altered in timing, such that peak melatonin production occurred at night. No association between light exposure and the magnitude of salivary melatonin variation was observed. The relationship between recent physical activity and melatonin differed by shift group, with a positive association seen among day workers, while an inverse relationship was seen among night workers. There was no association between usual physical activity and melatonin in either shift group. Finally, no significant correlation was observed between sleep duration and melatonin among either day or night workers. Conclusions: While this study demonstrated an inverse relationship between light intensity and melatonin, the comparison of functional time points between day and night workers meant that differences in urinary melatonin levels between shift groups could be attributed to differences in the time of day when urine samples were collected. No consistent relationship between recent or usual physical activity and melatonin levels was observed in either shift group. Sleep duration was not correlated with urinary melatonin levels, suggesting it cannot be used as a proxy for melatonin production. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-06-26 08:39:21.645

Background: In 2007, the International Agency for Research on Cancer (IARC) classified long-term shift work as a probable human carcinogen; however, the mechanism through which shift work potentially increases cancer risk is not known. One hypothesis is that diminished melatonin production may be involved, possibly as a result of exposure to light during night work. Experimental studies suggest a link between melatonin and peptides in the insulin-like growth factor (IGF) family, also implicated in carcinogenesis. This research aimed to describe the distributions of circulating concentrations of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) and their associations with urinary melatonin as possible intermediates in the pathway between work at night and breast cancer. Methods: A cross-sectional study was conducted among 85 premenopausal nurses working a rotating shift pattern of two 12-hour days, two 12-hour nights, and five days off. Once during both the summer and winter seasons, melatonin metabolites were measured in urine samples and circulating concentrations of IGF-I and IGFBP-3 were determined from serum samples. Weight and height were measured by the study coordinator, while a questionnaire and study diaries were used to collect all other covariate information. Predictors of IGF levels were identified using multivariate mixed effects modeling and relationships between melatonin and the IGFs were investigated using Spearman’s rank correlation and multivariate mixed effects modeling. Results: Both age (β = -3.6, p < 0.0001) and current OC use (β = -40.8, p = 0.003) were associated with decreases in circulating IGF-I, while levels of IGF-I were increased in the winter months (β = 26.3, p = 0.02). A positive relationship between recent alcohol consumption and serum IGFBP-3 was also suggested (β = 197.8, p = 0.05). Neither Spearman’s rank correlations nor mixed effects modeling indicated that urinary melatonin was a determinant of serum IGFs. Conclusions: Age, season, and current OC use were observed to predict circulating IGF-I, while recent alcohol consumption was a determinant of IGFBP-3 levels. A relationship between melatonin and IGFs, theorized as a component of the mechanism linking shift work and cancer, was not supported by the results of this project. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-05-30 15:29:31.253