Xenobiotics and their metabolites are capable of covalent modification of proteins that may lead to cellular injury. Determining the identity of modified proteins, however, has to date been hindered by available analytical instrumentation. Advances in mass spectrometry now allow for routine analysis of large biomolecules. This has given rise to the field of proteomics , which uses mass spectrometry in conjunction with various separation techniques to gain information about large numbers of proteins. This dissertation describes the development of a new proteomic methodology to identify unknown protein targets and its application to identifying protein targets and metabolites of 1,1-dichloroethylene (1,1-DCE). 1,1-DCE undergoes P450 bioactivation in hepatocytes to produce 1,1-DCE oxide and 2-chloroacetyl chloride. These either modify proteins directly, or form the glutathione conjugate S-(2-chloroacetyl)glutathione, which in turn is capable of protein cysteinyl sulfhydryl alkylation. In animals, exposure to 1,1-DCE results in selective injury to the biliary canalicular membrane. Biochemical and physiological evidence suggests damage to the canalicular membrane transport proteins. Putative 1,1-DCE metabolite adducts, including S-carboxymethylated and 2-chloroacetylated peptides and GSCOCH₂-S-cys-peptide adducts, were synthesized using model peptides containing one or two cysteines. The adducts were analyzed by ESI-MS-MS and resulted in fragmentation patterns characteristic of the adduct moiety, including fragment ions, losses from the parent ion and pairs of ions separated by the mass of the modified cysteine residue. The data reduction algorithm SALSA was developed to search for user-specified fragmentation characteristics in MS-MS data dependent scans. Bile samples obtained from animals exposed to 1,1-DCE were analyzed by ESI-MS-MS. SALSA was then used to search the data for spectra containing adduct-specific fragmentation. Five hepatic S-carboxymethylated proteins were identified, as were the 1,1-DCE metabolites S-carboxymethylglutathione, S-(cysteinylacetyl)glutathione and the cyclic product of the intermolecular rearrangement of S-(2-chloroacetyl)glutathione. This work demonstrates the use of mass spectrometry to characterize unknown, modified proteins in complex mixtures without the use of radio- or immunochemical labeling.

Factors affecting the deposition of cocaine into hair and the relationships between those concentrations, survey questionnaire results and natural killer cell activities were investigated. First, two and 18 month old mice were infected with LP-BM5, injected with cocaine, and hair cocaine content measured. There was no difference in cocaine concentration between the age groups. Retrovirally infection mice had greater amounts of cocaine in their hair. Next, a group of mice were fed an ethanol diet and injected with cocaine. There was a decrease in cocaine recovered in the hair of the ethanol-fed mice. In a group of human drug users no correlation was found between survey questionnaire answers and hair cocaine concentrations. There was a correlation between natural killer cell activity and hair cocaine concentrations. It was concluded that hair analysis for cocaine may have a place in clinical settings, but a predictive correlation between drug intake and hair concentration may not be possible.

<p> Millions of people are exposed to arsenic in the United States and worldwide. Commonly found arsenic species in human urine are AsIII (arsenite), AsV (arsenate), MMA (monomethyl arsenic acid), DMA (dimethylarsinic acid) and AB (arsenobetaine). Evidence has shown that these species vary in toxicity, and since each of these metabolites can be detected through analysis, they have the potential to be used as biomarkers for human exposure. For human exposure assessments in areas that have naturally occurring arsenic contaminated sources, or those who live or work near contaminated environmental sites where arsenic has been used, it is important to fully understand what species of arsenic residents are being exposed to in order to grasp the risk of arsenic exposure specifically and in its entirety.</p><p> Since it is difficult to determine direct human exposures, a swine model was used as a surrogate. Swine urine was collected from two different swine studies where animals were given non-toxic doses of arsenic contaminated soil and another group receiving a soluble reference dose using sodium arsenate for comparison. The urine samples from these studies were used to modify an arsenic speciation method using high-performance liquid chromatography and inductively coupled plasma mass spectrometry (LCICPMS). It is evident that when comparing the percent of arsenic species found in swine urine samples with what is found in humans a correlation can be made. There was a range of 64&ndash;74% DMA in swine samples for all test soils where a range of 60&ndash;75% DMA has been reported in human urine samples. This further illustrates the importance of arsenic speciation in swine urine since it does appear that it could correlate to human exposure. If proper measurement systems are utilized to quantify As species of health concern, dosed swine can be used to assess and predict human toxicological effects of arsenic exposure.</p>

<p> Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-binding proteins that are thought to play key roles in detoxification and Zn homeostasis. Four metallothionein isoform families, termed MT 1-4, have been identified in mammalian species. The majority of studies on MT have focused on the biochemical properties of the widely expressed MT-1 and MT-2 and, in comparison, few studies have investigated the metal binding characteristics of the neuronal-specific MT-3. While the function of MT-3 in neurons is not fully understood, a better understanding of the biochemical properties of MT-3 and its relationship with the other MT isoforms may aid in determining the role of this isoform in Zn homeostasis in the brain. In the current study, different MT isoforms, each labeled with a unique stable isotope of Zn, have been used to study the interaction of Zn between the MT isoforms and to study their ability to donate metal in transfer reactions.</p>

<p> Environmental pollutants can act as endocrine disruptors to affect the biology of organisms including, development, reproduction, behavior, and overall health. Many endocrine disrupting chemicals (EDCs) can mimic or inhibit functions of sex steroids which are critical for the development and maintenance of vertebrate reproductive systems. Sex steroids also interact with arginine vasotocin (AVT)/arginine vasopressin (AVP; mammalian homologue) systems to mediate vertebrate social and sexual behaviors, including vocalizations in male anurans. I used the Western clawed frog, <i>Xenopus tropicalis, </i> as an amphibian model species to evaluate the effects of exposure to EDCs on AVT-regulated behaviors and on brain AVT and sex steroid signaling systems. </p><p> In order to understand the role of neurohormones and social stimuli in behavior of <i>X. tropicalis,</i> I studied the effect AVT and human chorionic gonadotropin (hCG) administration and the influence of different social contexts on sexual behaviors, including calling, in males. I found that AVT and hCG treatment alone and in combination induces male sexual behavior, and the presence of a female is necessary to stimulate this behavior. I also analyzed the expression of genes related to AVT and sex steroid signaling in the brain of male and female <i>X. tropicalis</i> to identify genes that are expressed sexually dimorphically and could be potentially altered by EDC exposure. </p><p> Then I utilized the behavior and gene expression assays I developed to study the effects of developmental exposure to the synthetic androgen 17&beta;-trenbolone (17&beta;-TB) on behavior, fecundity, morphology, and brain gene expression in adult <i>X. tropicalis.</i> Developmental 17&beta;-TB exposure reduced sexual behavior and inhibited female egg release during breeding trials. Exposure to 17&beta;-TB also blocked oviduct development and altered AVT-related brain gene expression in females. </p><p> In another study I evaluated the effects of adult exposure to the common EDCs bisphenol A (BPA), nonylphenol (NP), and triclosan (TCS) or a mixture of the three on sexual behavior, morphology, and brain gene expression in adult male <i>Xenopus tropicalis.</i> Behavior and brain gene expression were not significantly affected, but TCS-exposed animals had a lower normalized liver weight than animals exposed to BPA or the chemical mixture. Males exposed to BPA, NP, and the mixture also exhibited increased oviduct development compared to TCS-exposed males, and the source of the animals influenced oviduct development. </p><p> Results from my dissertation research demonstrate that exposure to endocrine disruptors can affect multiple levels of physiology. Gender, timing of exposure and nature of the chemical are also critical factors in determining these physiological effects. Importantly, because there is evolutionary conservation in vertebrates for the role of sex hormones in regulating the reproductive system as well as the AVT/AVP system and related social behaviors, my work has important broader implications for endocrine-disrupting effects from environmental chemical exposure for all wildlife and humans.</p>

<p> Gene expression of the biotransformation enzymes, glutathione S-transferase (GST) glutathione peroxidase (GPx) and glutathione synthesizing enzyme glutamyl-cysteine-ligase (GCL), was investigated in zebrafish (Dani orerio) exposed to arsenate, perchlorate, a mixture of these two chemicals, and thyroxine (T4). The exposure persisted for 30 days for perchlorate, 7 days for arsenate and 7 days for thryroxin. To test the mixture toxicity of arsenate and perchlorate, arsenate was added for 7 days in addition to 30 days exposure to perchlorate. Arsenate is known to induce oxidative stress, and GCL, GPx, and GST are involved in the oxidative stress response. Thyroid hormone is involved in mediating the oxidative stress response, and perchlorate is a known thyroid gland disruptor. Therefore, this research was designed to test the hypotheses that 1) thyroid hormone affects the gene expression of GCL, GPx, and GST enzymes and 2) thyroid hormone modulates As-induced expression of these genes. Fish were exposed to the mixture of arsenate and perchlorate to examine the effect of thyroid hormone on As-induced gene expression of GCL, GPx, and GST. The findings indicate that arsenate up-regulates the transcription of GCL and GST pi genes in the gills and GSTpi gene in liver, and this induction was not observed in presence of perchlorate. Therefore, it can be concluded that thyroid hormone inhibits As-induced gene expression of GCL and GSTpi.</p>

Alcohol abuse and addiction are among the most costly and prevalent behavioural disorders due to the drug's reinforcing potential and ubiquity in society. Studies suggest an interaction between alcohol and the endogenous opioid system: manipulation of endogenous opioid activity affects alcohol intake, and alcohol administration alters endogenous opioid activity. However, the data are inconclusive given the discrepancy between pharmacological and genetic knockout studies on alcohol intake, and the difficulty in interpreting and replicating findings determined postmortem. / There were two major objectives in the present study. First, to use estradiol valerate to induce selective lesions of endorphinergic perikarya in adult rats in order to assess its effect on alcohol intake. Voluntary alcohol consumption was significantly increased in rats treated with estradiol valerate compared to vehicle, but was not associated with changes in basal levels of anxiety or hypothalamic beta-endorphin content. / Second, to use microdialysis to determine the effect of alcohol on extracellular levels of opioid peptides and catecholamines in the nucleus accumbens of awake freely-moving animals. The nucleus accumbens is heavily implicated in alcohol abuse. Four sets of experiments were conducted: (1) Various doses of alcohol (0.0-2.4 g ethanol/kg body weight) were tested on beta-endorphin and catecholamine release in the nucleus accumbens. Only the 2.4 g/kg dose significantly increased levels of dopamine and beta-endorphin. (2) The release of beta-endorphin was assessed in response to stress and alcohol in the nucleus accumbens and hypothalamus. The results showed a different profile for both stimuli: stressors had an immediate effect in the hypothalamus, while alcohol had a latent effect in the nucleus accumbens. (3 & 4) Various doses of alcohol (0.0-3.2 g ethanol/kg body weight) were assessed on extracellular levels of Met-enkephalin and dynorphin A in the nucleus accumbens. While the 1.6 g/kg dose induced the greatest release of Met-enkephalin, the 3.2 g/kg dose induced the greatest release of dynorphin. / In conclusion, although estradiol valerate affected alcohol preference, the results did not support the involvement of beta-endorphin. On the other hand, opioid peptides are differentially released in the nucleus accumbens in response to alcohol. The findings lend support to the hypothesis that endogenous opioids mediate some of the effects of alcohol.

Cyclophosphamide is a bi-functional alkylating agent, which is used primarily in cancer chemotherapy. Cyclophosphamide acts by binding to nucleophilic ekes and causes DNA adducts, stand breaks and cross-links. / The working hypothesis in our laboratory is that cyclophosphamide treatment causes specific DNA damage in male germ cells, and that cells at different stages of spermatogenesis are affected to varying degrees. / In order to examine this hypothesis, the following objectives were put forth: (1) To develop the competitive PCR technique (QPCR) for the measurement of DNA damage in rat spermatozoa, (2) To examine the DNA damage incurred by CPA treatment(s) in rat spermatozoa. / This work introduces the quantitative polymerase chain reaction (QPCR) assay for the measurement of DNA damage in a highly developed cell type, the spermatozoa. Cyclophosphamide treatment effectively inhibits Taq polymerase in the amplification of the spermatozoal DNA target. Two treatment regimens were used: an acute high dose of cyclophosphamide that is pharmacologically relevant to the treatment of certain malignancies and a chronic low dose regimen that results in male mediated teratogenicity. (Abstract shortened by UMI.)

Chronic occupational exposure to styrene is associated with a number of adverse effects on the nervous system, including sensory neuropathy and neurophysiological alterations. In order to test styrene for neurotoxic potential and investigate its mechanism of action, primary co-cultures of murine spinal cord-dorsal root ganglia (DRG)-skeletal muscle were used in a simple in vitro neurotoxicity screen. The neurotoxicity of styrene and its major metabolites, styrene oxide and styrene glycol was evaluated after both short and long-term exposure. Endpoints used to characterize neurotoxicity were both morphological and neurophysiological, and included: (1) chromatolysis, (2) axonopathy, and (3) interference with action potential generation. The major findings of this study were: (1) styrene and oxide were acutely cytotoxic to all cell types at concentrations in excess of 2.0 mM and 0.2 mM respectively. (2) There was no evidence of neurotoxicity attributable to styrene or its metabolites, with the exception of a slight chromatolytic effect induced by 0.2 mM styrene oxide in DRG neurons after long-term exposure. It is therefore possible that cytotoxic mechanisms rather than effects on neuron specific processes, underlie styrene's damage to cells of the nervous system. (3) Surprisingly, dimethyl sulfoxide (DMSO), a common in vitro solubility vehicle, was neurotoxic after long-term exposure, producing both chromatolysis and axonopathy. This study, therefore also established the upper limit of DMSO recommended for use as a solubility vehicle in long-term in vitro tests.

3,4-methylenedioxymethamphetamine (MDMA/ecstasy) is a serotonin neurotoxin in animals. Human MDMA abusers exhibit evidence of disturbed serotonin transmission. Altered serotonergic neurotransmission in MDMA abusers might underline susceptibility to developing depressed mood. The acute tryptophan depletion (ATD) challenge was used to study altered serotonin levels in MDMA abusers. Sixteen MDMA abusers and nineteen age- and gender-matched controls were tested. In a double-blind crossover study, subjects ingested a 100g amino acid mixture with or devoid of tryptophan. Mood was measured before and after the ingestion of the amino acid, a total of three times throughout the day. Impulsivity was measured with the Go/No-Go task. ATD lowered mood (p < 0.01) and increased anxiety (p < 0.05) in female MDMA abusers. The present design does not permit a distinction between effects of MDMA use or a pre-existing trait. If the effects are due to neurotoxicity, they could reflect MDMA abuse alone or an effect of a combination of drugs.