Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.

Idiosyncratic drug reactions (IDRs) are unpredictable adverse drug reactions. Their exact mechanisms are unknown but most appear to be immune-mediated. Mechanistic studies require valid animal models, but there are very few available and none for the study of drug-induced agranulocytosis. Thus, the first part of my thesis has focused on the development of an animal model of agranulocytosis. We pursued many attempts to develop one in rabbits, guinea pigs, and rats by treatment with aminopyrine, amodiaquine, and clozapine and manipulating the factors hypothesized to be involved in the mechanism of IDRs such as reactive metabolite formation/detoxication and immune stimulation. Clozapine-induced agranulocytosis is not associated with immune memory, which suggests that it may not be immune-mediated. Therefore, other factors, specifically selenium and vitamin C deficiencies, were assessed as possible risk factors for clozapine-induced agranulocytosis. Despite many attempts, we were not able to develop an animal model of idiosyncratic drug-induced agranulocytosis. The second part of this thesis was focused on investigating the effects of clozapine on neutrophils. It is known that the reactive metabolite of clozapine increases neutrophil apoptosis in vitro; however, it was not clear that the conditions of these experiments reflect in vivo conditions. Therefore, the effect of clozapine on neutrophil kinetics in vivo was examined. We found that clozapine treatment decreased the half-life of circulating neutrophils and increased the rate of release of neutrophils in rabbits. Thus, even though these animals did not develop agranulocytosis clozapine did appear to cause neutrophil damage that was compensated for by an increased production of neutrophils. Failure of the bone marrow to keep up with the increased rate of neutrophil destruction in certain individuals could result in agranulocytosis. Alternatively, damage to neutrophils could lead to an immune response in some patients that results in agranulocytosis. The failure to develop an animal model of drug-induced agranulocytosis despite many attempts using interventions based on the current mechanistic hypotheses suggests that these hypotheses are wrong. However, it is also possible that we are just unable to overcome the default response of immune tolerance; future studies will examine this possibility and the mechanism of clozapine-induced neutrophil damage.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.

Idiosyncratic drug reactions (IDRs) are unpredictable adverse drug reactions. Their exact mechanisms are unknown but most appear to be immune-mediated. Mechanistic studies require valid animal models, but there are very few available and none for the study of drug-induced agranulocytosis. Thus, the first part of my thesis has focused on the development of an animal model of agranulocytosis. We pursued many attempts to develop one in rabbits, guinea pigs, and rats by treatment with aminopyrine, amodiaquine, and clozapine and manipulating the factors hypothesized to be involved in the mechanism of IDRs such as reactive metabolite formation/detoxication and immune stimulation. Clozapine-induced agranulocytosis is not associated with immune memory, which suggests that it may not be immune-mediated. Therefore, other factors, specifically selenium and vitamin C deficiencies, were assessed as possible risk factors for clozapine-induced agranulocytosis. Despite many attempts, we were not able to develop an animal model of idiosyncratic drug-induced agranulocytosis. The second part of this thesis was focused on investigating the effects of clozapine on neutrophils. It is known that the reactive metabolite of clozapine increases neutrophil apoptosis in vitro; however, it was not clear that the conditions of these experiments reflect in vivo conditions. Therefore, the effect of clozapine on neutrophil kinetics in vivo was examined. We found that clozapine treatment decreased the half-life of circulating neutrophils and increased the rate of release of neutrophils in rabbits. Thus, even though these animals did not develop agranulocytosis clozapine did appear to cause neutrophil damage that was compensated for by an increased production of neutrophils. Failure of the bone marrow to keep up with the increased rate of neutrophil destruction in certain individuals could result in agranulocytosis. Alternatively, damage to neutrophils could lead to an immune response in some patients that results in agranulocytosis. The failure to develop an animal model of drug-induced agranulocytosis despite many attempts using interventions based on the current mechanistic hypotheses suggests that these hypotheses are wrong. However, it is also possible that we are just unable to overcome the default response of immune tolerance; future studies will examine this possibility and the mechanism of clozapine-induced neutrophil damage.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.