Insect repellent N,N-diethyl-m-toluamide (DEET) and sunscreen oxybenzone are commonly incorporated into commercially available repellent and sunscreen preparations. Both compounds have demonstrated an increased percutaneous permeation and systemic disposition after concurrent application in vitro and in vivo. The permeation enhancement between DEET and oxybenzone not only compromises their respective protective efficacy against biting insects and UV radiation, but also potentiates toxicological properties in susceptible subjects. The pharmacokinetic and toxicological profiles from concurrent use of DEET and oxybenzone were evaluated and compared in this thesis. DEET and oxybenzone were administered by intravenous and topical routes in rats, either alone and/or in combination, to compare the pharmacokinetics of parent compounds and their primary metabolites in vivo. To evaluate toxicological characteristics, rat primary cortical neurons and astrocytes, and rat hepatoma 1548 cells were exposed to DEET, oxybenzone and their metabolites in vitro, and cell viability was analyzed. Various behavioral testing protocols were also performed to assess arousal, locomotion, habituation, and motor coordination of rats over a 30-day study period. Concurrent topical application of DEET and oxybenzone enhanced the disposition of DEET and its metabolites in rats, but did not consistently affect the distribution of oxybenzone and its metabolites. The disappearance of DEET from skin application site was accelerated; its apparent elimination half-life was decreased while its plasma and tissue concentrations were predominantly increased. Cellular toxicity occurred at 1 μg/ml for neurons and 7-day exposure for both astrocytes and neurons. Viability of hepatoma cells was also reduced when treated with DEET, oxybenzone and their metabolites, either alone or in combination, most notably after 72 hours of exposure. However, no overt signs of toxicity were observed from behavioral testing in rats after a 30-day topical study. The pharmacokinetic data obtained was beneficial in understanding and elucidating absorption and biodistribution of DEET and oxybenzone in vivo. The toxicological data suggested that the risk for increasing adverse effects from concurrent skin application of repellents and sunscreens would be low and marginal in healthy individuals. Nevertheless, further studies should be carried out to assess the long-term health impact of these compounds in susceptible subjects, especially at higher application doses.

Insect repellent N,N-diethyl-m-toluamide (DEET) and sunscreen oxybenzone are commonly incorporated into commercially available repellent and sunscreen preparations. Both compounds have demonstrated an increased percutaneous permeation and systemic disposition after concurrent application in vitro and in vivo. The permeation enhancement between DEET and oxybenzone not only compromises their respective protective efficacy against biting insects and UV radiation, but also potentiates toxicological properties in susceptible subjects. The pharmacokinetic and toxicological profiles from concurrent use of DEET and oxybenzone were evaluated and compared in this thesis. DEET and oxybenzone were administered by intravenous and topical routes in rats, either alone and/or in combination, to compare the pharmacokinetics of parent compounds and their primary metabolites in vivo. To evaluate toxicological characteristics, rat primary cortical neurons and astrocytes, and rat hepatoma 1548 cells were exposed to DEET, oxybenzone and their metabolites in vitro, and cell viability was analyzed. Various behavioral testing protocols were also performed to assess arousal, locomotion, habituation, and motor coordination of rats over a 30-day study period. Concurrent topical application of DEET and oxybenzone enhanced the disposition of DEET and its metabolites in rats, but did not consistently affect the distribution of oxybenzone and its metabolites. The disappearance of DEET from skin application site was accelerated; its apparent elimination half-life was decreased while its plasma and tissue concentrations were predominantly increased. Cellular toxicity occurred at 1 μg/ml for neurons and 7-day exposure for both astrocytes and neurons. Viability of hepatoma cells was also reduced when treated with DEET, oxybenzone and their metabolites, either alone or in combination, most notably after 72 hours of exposure. However, no overt signs of toxicity were observed from behavioral testing in rats after a 30-day topical study. The pharmacokinetic data obtained was beneficial in understanding and elucidating absorption and biodistribution of DEET and oxybenzone in vivo. The toxicological data suggested that the risk for increasing adverse effects from concurrent skin application of repellents and sunscreens would be low and marginal in healthy individuals. Nevertheless, further studies should be carried out to assess the long-term health impact of these compounds in susceptible subjects, especially at higher application doses.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD. It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH. Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.

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