Zugl.: Hamburg, Univ., Diss., 2003. / Zsfassung in engl. Sprache.

Universiẗat, Diss., 2003--Frankfurt (Main).

A radioimmunoassay has been developed for the major metabolite of melatonin, 6-suIphatoxymelatonin (aMT6s). The assay is specific, sensitive and is direct for both urine and plasma samples. Classical validation procedures have been employed and the urinary assay has been compared with an established gas chromatographic/mass spectrometric assay. A number of physiological studies have been carried out. A marked diurnal rhythm in both urinary and plasma aMT6s excretion was found which correlated closely with plasma melatonin values. There were large inter-individual variations in aMT6s excretion but its production was consistent for any one volunteer over a four-day period. No evidence was obtained for the episodic secretion of melatonin or aMT6s when blood samples were taken every thirty seconds for ten minutes. Administration of a peripheral B-blocker to volunteers, resulted in the abolition of the night-time rise in aMT6s excretion. In a seasonal study, a small but highly significant change in the acrophase of the aMT6s rhythm was found during the year with a phase advance in summer compared to winter. Changes in aMT6s excretion with age were also investigated. Total urinary aMT6s excretion was relatively constant in forty children aged 2-20 years. However, when aMT6s excretion was expressed as a function of body weight highly significant age-related changes were observed. In ninety adult volunteers aged 20-80 years there was a significant decline in total 24h aMT6s excretion with age, with significantly lower excretion in elderly subjects. No relationships were found between total 24h aMT6s excretion and body weight, height or pineal calcification. In addition to the above physiological studies, the pharmacokinetics of melatonin and aMT6s were investigated following the oral administration of melatonin to normal volunteers.

Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.

The purpose of this project was to investigate the role of melatonin in the pathophysiology of anxiety in humans. The literature study confirmed the intimate relationship between serotonin and melatonin. Melatonin is not only able to act as an agonist (in physiological concentrations) and an antagonist (at higher concentrations) on serotonin receptors but via control of brain pyridoxal kinase activity might have an effect on GABA, serotonin, dopamine and norepinephrine synthesis. A clinical trial to investigate melatonin's effect on anxiety in humans was conducted as a pilot study. Thirty patients complaining of anxiety participated in a liN of 1" double blind placebo controlled trial. During the experiment each subject was thus exposed to melatonin and a placebo for a week at a time on two occasions. During the first phase of the experiment, (Pair '1) patients showed a statistically significant reduction in their anxiety levels during the first period (P1P1), which was not the case during the second period (P1P2). The improvement however continued during the second phase of the experiment (Pair 2) so that there was also a statistically significant improvement during P 2 P 2 (Period 2 / Pair 2) when placebo was administered. It could not conclusively be shown that melatonin was responsible for the improvement in the patients' anxiety. The explanation for these results suggests thelt the improvement was due to a: 1) placebo effect throughout, 2) psychotherapeutic effect due to contact with a clinician, 3) melatonin induced phase shift in the patient's endogenous melatonin response curve, 4) combination of all 3 options. This pilot study lays the groundwork for a much more exhaustive study in which the melatonin of the patients is determined before melatonin is administered, the role of the clinician is clarified and the most appropriate time for melatonin administration is sought .

The dose- and time dependence of melatonin and the effective window of melatonin administration were determined in a mouse model of myocardial infarction. When mouse hearts were subjected to 60 min of occlusion of the left anterior descending artery (LAD) followed by 4 h of reperfusion, melatonin pretreatment for 30 min significantly reduced the infarct size/risk area. The most effective dose was found to be 150 μg/kg intraperitoneally, and the effective period of protection lasted up to 2 h after melatonin administration. Melatonin administration 45 min after LAD ligation or right before reperfusion was as effective as administration 30 min before ligation; however, melatonin administered after the release of occlusion was not protective. Melatonin's effect was still present in mice deficient for the Mel1a melatonin receptor. 8-Methoxy-2-propionamidotetralin, a melatonin receptor agonist with no antioxidant activity, offered no protection, suggesting a lack of involvement of melatonin receptors. Finally, the effects of melatonin were similar in rats and mice. Our results demonstrate that melatonin is an effective cardioprotective agent when administered either before or during coronary occlusion at a very low dose.

Most research evaluating the effects of the reproductive tract microbiota on reproductive performance has been done in humans, thus far. In bovids, reproductive microbiota research is not as advanced, with preliminary conclusions, not supported by contamination checks or repeatability. Our studies concluded that endogenous reproductive hormones, days of gestation, and pregnancy status does not change the overall vaginal microbiota composition. Although, the overall composition did not change there were species level differences. These differences could have implications in reproductive performance and fertility in heifers. Heifers that undergo nutrient restriction have similar vaginal microbiota to adequately fed heifers with no species differences. The most impactful finding is that exogenous supplementation of melatonin was associated with changes in the vaginal microbiota in Brangus heifers during late gestation. The implications of this finding are not yet clear, but to date, this is the first hormone, in bovids, determined to change the composition of the vaginal microbiota.

No description available.

The multiple physiological effects of the indoleamine hormone melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Our group has shown an upregulation of melatonin receptors following treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and Trichostatin A, in cultured cells and/or in the rat brain. VPA increases histone H3 acetylation at the MT1 gene promoter region in rat C6 glioma cells, indicating that this epigenetic mechanism underlies its upregulation of MT1 expression. Since HDAC inhibitors can also alter DNA methylation, the possible involvement of this second major epigenetic mechanism in the regulation of MT1 expression, was examined. C6 cells were treated with the DNA demethylating agent, azacytidine (AZA, 1 - 25 µM), for 24 or 48 hours. Treatment of C6 cells with AZA caused a significant upregulation of MT1 mRNA expression, as compared with controls (DMSO 0.05%). Moreover, treatment with AZA (10 or 20 µM) for 24 or 48 hours, suppressed or abolished DNMT1 protein expression, and inhibited DNMT1 mRNA expression, which indicates inhibition of the DNMT1 enzyme activity. A combination of VPA and AZA caused a trend toward additive upregulation of the MT1 receptor. These results show that DNA demethylation plays a role in the regulation of the MT1 receptor, consistent with the well-known effects of this epigenetic mechanism on gene transcription. Epigenetic regulation of melatonin receptor expression could provide a novel strategy for modulating the therapeutic effects of this hormone and its clinically relevant agonists, such as agomelatine, and could also provide avenues for enhancing the antioxidant, neuroprotective, oncostatic and other benefits of this hormone and its agonists. / Thesis / Master of Science (MSc) / The hormone, melatonin, is involved in maintenance of the sleep cycle, and has many neuroprotective effects, initiated by its binding to specific proteins called receptors. Epigenetic or reversible chemical modifications which alter DNA, without changing its sequence, can alter the levels of these receptors. This process can be modulated by drugs, which can increase levels of the melatonin receptor. In this study, the drug 5-Azacytidine (AZA) was used to cause specific chemical changes to DNA, termed demethylation. This thesis shows for the first time, that AZA causes an increase in melatonin receptors. AZA’s ability to cause demethylation was confirmed by observing decreased levels of the protein responsible for DNA methylation, DNA methyltransferase. Melatonin receptors in the brain exhibit changes in disorders such as Alzheimer’s and Parkinson’s disease. Understanding the mechanisms underlying the regulation of these receptors could provide avenues for enhancing the neuroprotective benefits of melatonin and related drugs.

published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy