<p> In selected locations offshore of urban southern California, male hornyhead turbot <i>(Pleuronichthys verticalis)</i> exhibit elevated plasma concentrations of the female sex steroid, 17&beta;-estradiol (E2). Males sampled from Santa Monica Bay have consistently elevated E2 concentration (>1000 pg/ml), as much as 10 times higher than males from locations offshore of Orange County. Since estrogens, including E2, are at undetectable levels in the ocean discharge of regional WWTPs, it was of interest to determine whether testicular expression of steroidogenic enzymes and other proteins impacting estrogen production may be altered and linked to endogenous E2 production. In this study, proteomics was used to screen for changes in testicular protein expression. In addition, proteomes of fish experimentally treated with E2 were compared to a control. These experiments resulted in the molecular characterization of 30 unique proteins, and provided some insight into potential underlying molecular mechanisms associated with this aberrant E2 phenotype.</p>

Thesis (Ph.D.)--Indiana University, School of Public and Environmental Affairs, 2009. / Title from PDF t.p. (viewed on Feb. 10, 2010). Source: Dissertation Abstracts International, Volume: 70-05, Section: B, page: 2888. Adviser: Diane S. Henshel.

Inorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are lacking. Presently, 17 species of non-human primates were screened using an in vitro assay to determine their arsenic methylation ability as a predictive tool for better understanding the presence, and in some instances, deficiency of arsenic methyltransferase activity among animal species. Four of the 17 species investigated had arsenite methyltransferase activity, three of which were from the genus Macaca. This suggested a phylogenetic component to an animal having arsenic methyltransferase activity. That all of the hepatic tissues were viable was demonstrated by their all having arsenate reductase activity. These data suggested that arsenic methylation cannot be a detoxification mechanism for many non-human primates and that alternative methods of detoxifying inorganic arsenic must be considered and investigated. Biomethylation of inorganic arsenic has classically been considered its primary mechanism of detoxification, although results of recent investigations disagree. As arsenic methylation may represent a bioactivation process leading to cancer development, it now becomes critical to identify the methyltransferase protein, its endogenous function, as well as additional substrates and inhibitory molecules. Using a combination of chromatographic and isoelectric focusing protein separation methods, rabbit liver arsenite and methylarsonic acid methyltransferase activities were purified 19,000- and 604-fold, respectively. These activities co-purified during all steps. The arsenic methyltransferase protein had a molecular mass of approximately 46.5 kDa and a pl of 5.7 to 6.0 and is inhibited by Sadenosyl-L-homocysteine, sinefungin and trivalent antimony. The final purified fraction contained a heterogeneous protein mixture as assessed by SDS-PAGE analysis. Proteins in this fraction were identified using tandem and matrix-assisted laser desorption/ionization mass spectrometry techniques against databases of known proteins. Identified protein sequences were searched for putative S-adenosylmethionine binding motifs conserved among small molecule methyltransferases. Homologs of the identified proteins were obtained commercially, where available, and screened for arsenite methyltransferase activity. The arsenic methyltransferase was not identified among the obtained homologs and cloning efforts must be considered to screen the other proteins identified in the final purified fraction for arsenic methyltransferase functionality.

Protein modifications by reactive intermediates may have a causal role in cellular toxicity but information about which proteins are modified is limited. In order to address identification of protein targets a novel method for detection of adducted peptides based on adduct-specific fragmentations in tandem mass spectrometry (MS/MS) was developed. This method consists of characterizing the MS/MS fragmentation of adducted-model peptides to identify adduct specific features and screening MS/MS spectra for characteristic features of adduction. Benzoquinone (BZQ) and glutathione-conjugated benzoquinone (GS-BZQ) were selected as model electrophiles to develop this method and adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in MS/MS experiments. BZQ-adducted peptides fragmented to give adduct-derived fragments of ion pairs of 141/142 and 211 and a neutral loss of 142. GS-BZQ-adducted peptides fragmented to give adduct-derived fragments of neutral losses of 74, 129, 273, and 447, charged losses of 274 and 448 and ion pairs of 515 and 129. We suggest that these adduct-specific fragments can be used to detect adducted peptides. Subsequently, the data reduction algorithm SALSA was developed to screen MS/MS spectra for spectral characteristics including neutral losses, charged losses and ion pairs in order to facilitate adduct detection. SALSA scores multiple types of spectral features simultaneously and reports a combined score for each spectrum, and search criteria can be arranged in a hierarchal manner for more selective searching. The SALSA algorithm was used to screen spectra of a BSA digest treated with GS-BZQ for fragment characteristics of GS-BZQ-adduction, and spectra from six GS-BZQ modified peptides were ranked among the top twenty highest scoring spectra. Detection of unanticipated peptide modifications is illustrated using the motif-searching algorithm of SALSA which is described and searches for patterns of product ions without regard to precursor m/ z or position along the m/z axis. Finally, because the effects of adduction may depend on its abundance in the cell, a new stable isotope label for differential quantitation of peptide adducts is described. Relative quantitation using the label is linear across a 10,000 fold range of concentration ratios, standard deviation is less than 20%, and quantitation of multiple peptides in a BSA digest is reported. Styrene oxide adducts of hemoglobin are differentially quantified using the label and a concentration/adduct curve for the formation of eight peptide adducts is plotted.

Arsenic is a toxic metalloid and is ubiquitous in our environment. In ancient cultures it was valued as a poison and today is becoming an increasing public health problem. Chronic arsenic exposure has a broad range of toxic effects including cancer. Currently millions of people are exposed to higher levels of arsenic in their food and drinking water than is considered safe by the World Health Organization. Although arsenic metabolism is not completely understood, it is known that inorganic arsenate is reduced to arsenite which can then be methylated and excreted in the urine. It is also known that some arsenic is retained in the body, presumably by binding to cellular proteins. To better understand how arsenic is metabolized, our approach was to identify and characterize proteins that are involved in arsenic metabolism. Using biochemical approaches we demonstrated that arsenate reductase activity from human liver was purine nucleoside phosphorylase (PNP). We were able to demonstrate that calf spleen PNP has arsenate reductase activity in vitro in the presence of inosine and dihydrolipoic acid, and that the reaction exhibits Michaelis-Menten kinetics. This identifies an enzymatic route for arsenate reduction. We also demonstrate that ferritin, an iron storage protein containing phosphate, can bind arsenic both in vitro and in vivo. In addition, we demonstrate that ferritin can oxidize arsenite to arsenate, and then interact with arsenate as it does with phosphate. We also establish that arsenate can inhibit ferritin's ability to store iron in vitro. Our results combined with data reported by others, suggest that DNA damage and enzyme inactivation associated with arsenic challenge may occur via reactive oxygen species generated by arsenic-iron redox reactions in ferritin, and that iron may augment arsenic toxicity. The interaction between ferritin and arsenate has two important implications. First, it suggests that iron exposure may be an important parameter to examine in epidemiological studies of arsenic sensitivity. Second, it suggests that iron chelation therapy might be beneficial in conjunction with arsenic chelation therapy for patients suffering from acute arsenic poisoning.

Arsenic is an environmental toxicant and a human carcinogen. The kidney is a known target organ for arsenic (As) and is critical for both arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a model for low level exposures of the human kidney to arsenic. Subcytotoxic concentrations of arsenite (≤ 10 μM) and arsenate (< 100 μM) were determined by leakage of LDH from cells exposed for 24 hours. Threshold concentrations of arsenite (1-10 μM) and arsenate (10-25 μM) were found to affect mitochondrial MTT processing. Biotransformation of arsenite or arsenate was determined using HPLC-ICP-MS to detect metabolites in cell culture media and lysates. Analysis of media revealed that arsenite was minimally oxidized to arsenate and arsenate was reduced to arsenite. Only arsenite was detected in cell lysates. Pentavalent methylated arsenicals were not detected following exposure to either inorganic arsenical. This demonstrates that the HK-2 cell line is capable of biotransforming inorganic arsenic, primarily reducing arsenate to arsenite. In addition, the mitochondria are a primary target for low-level arsenic toxicity. Previous studies show that at high doses (ppm) inorganic arsenic is toxic to mitochondria primarily by affecting cellular respiration. Mitochondrial injury was further assessed in HK-2 cells by examining mitochondrial membrane potential. Subcytotoxic arsenite caused mitochondrial depolarization, which could subsequently lead to permeability transition and apoptosis. Arsenite also induced translocation of phosphatidylserine to the outer layer of the plasma membrane, indicative of early apoptosis. To confirm whether subcytotoxic arsenite induces cellular and/or mitochondrial morphological alterations consistent with apoptosis, HK-2 cells were evaluated with both light and transmission electron microscopy. Classic morphological changes indicative of apoptosis were not observed at either the light microscopic nor the electron microscopic level; however, evidence of necrotic changes in cytoplasmic structure and morphology--particularly in the mitochondria--were apparent. Therefore, HK-2 cells appear to initiate apoptosis following subcytotoxic arsenite insult, but fail to complete apoptosis and undergo necrosis instead. Subcytotoxic arsenite can be sufficiently toxic to mitochondria that they lose their ability to keep the cell on course for apoptosis.

In the lateral choroid plexus, Mn was sequestered from the blood against a concentration gradient. Intrathecal administration of Mn²⁺ showed that the choroid plexus does not remove Mn from the cerebrospinal fluid and that intrathecal Mn²⁺ rapidly caused neurological abnormalities. Either 1 or 2 mg Mn²⁺/kg intrathecally resulted one day after dosage in a 91% and 95% decrease in the spontaneous motor activity of rats, respectively. Control activity was unchanged by intrathecal NaCl. Five days after administration, dopamine was decreased 70%; and Mn in the ventral mesencephalon was increased 14-fold as compared to controls. Mn in the central nervous system reached a maximum and dopamine reached a minimum 6 hours after 250 μg Mn²⁺/rat, intrathecally. The ventral mesencephalon contained more Mn than any other brain region (p < 0.001). Neurotransmitter reuptake carriers were inhibited in an effort to reduce the Mn concentration in the CNS. To inhibit neurotransmitter reuptake, rats were given cocaine i.p. and later Mn intrathecally. Mn²⁺ caused the ventral mesencephalon Mn concentration to increase from 0.57 μg/g to 31.8 μg/g. Cocaine prior to Mn decreased the concentration from 31.8 μg/g to 3.3 μg/g. To decrease neurotransmitter concentration, rats were given reserpine i.p. and later Mn intrathecally. Mn2+ caused the ventral mesencephalon Mn concentration increased from 0.77 μg/g to 29.9 μg/g. Reserpine prior to Mn decreased the concentration from 29.9 μg/g to 3.7 μg/g. Cocaine or reserpine decreased the Mn concentration in the occipital pole, frontal lobe and caudate putamen but not in the cerebellum. Manganese, cocaine or reserpine decreased the dopamine concentration in the caudate putamen from 10.5 to 6.3, 4.1 or 0.8 μg/g, respectively. The results suggest that intrathecal Mn²⁺ can rapidly cause dopaminergic specific neurotoxicity resulting in: (1) decreased spontaneous motor activity, (2) increased Mn concentration in the ventral mesencephalon and (3) decreased dopamine concentration in the caudate putamen. Mn concentration in the CNS is related to neurotransmitter uptake carriers since inhibiting reuptake or reducing concentrations of neurotransmitters resulted in decreased Mn concentrations in many brain regions.

Intake and serum levels of β-carotene have inverse associations with cancer risk. Previous research indicates chemopreventive actions may be due to antioxidant properties. Photooxidation reactions are an important source of reactive oxygen species. Photosensitizers can damage tissue by catalyzing the formation of oxyradicals and singlet oxygen (¹O₂). β-Carotene efficiently quenches ¹O₂ catalytically via a physical reaction. However, concomitant chemical reactions during photosensitized oxidations consume β-carotene. This dissertation is a study of β-carotene antioxidant mechanisms in solution and phospholipid membrane models of photooxidation. Photosensitized oxidation of β-carotene in solution yielded products analyzed by reverse-phase HPLC, UV-vis spectrophotometry, and mass spectrometry. These products were identified as β-ionone, β-apo-14'-carotenal, β-apo-10'-carotenal, β-apo-8'-carotenal, and the novel product β-carotene-5,8-endoperoxide, which was determined to be a specific marker for ¹O₂ oxidation of β-carotene. To study the effect of β-carotene and other agents on photooxidation, an isotope dilution GC-MS assay was developed which quantitatively distinguishes ¹O₂-mediated and radical-mediated lipid peroxidation products resulting from photosensitized oxidation of dilinoleoylphosphatidylcholine liposomes. This unique assay utilizes quantitative standards of 9- and 10-hydroxyoctadecadienoate and was used to generate "photooxidation profiles" of the photosensitizers methylene blue, Rose Bengal, and tetraphenylporphine. These profiles indicate a shift from Type II to Type I photooxidation mechanisms in later stages. In the liposome system, β-carotene successfully inhibited both ¹O₂-mediated and radical-mediated lipid peroxidation at early stages but was less effective at later stages. Production of radical-mediated products increased faster than ¹O₂-mediated products at later stages even though 40% of the initial β-carotene was present after 4 h. β-Carotene-5,8-endoperoxide was not detected in this system. Equimolar α-tocopherol was ineffective in inhibiting lipid peroxidation, however, a 10-fold increase in α-tocopherol concentration inhibited almost all radical-mediated lipid peroxidation as well as early-stage ¹O₂-mediated lipid peroxidation. Cumene hydroperoxide stimulated radical-mediated lipid peroxidation. Type II photooxidation products may enhance Type I mechanisms. β-Carotene was shown quantitatively to suppress photooxidation by inhibiting Type II mechanisms alone. Since β-Carotene may prevent tissue damage due to photooxidation, an understanding of the mechanisms involved will be important in maximizing its protective effects.

A high-performance thin-layer chromatographic (HPTLC) separation system coupled with a scientifically operated charge-couple device detector (CCD) has been developed to improve the identification and quantitative analysis of pharmaceutical products and natural toxins. The combination of the fast, easy and high throughput properties of TLC, high speed, sensitivity, wide dynamic range of the CCD detector, and the high reproducibility and accuracy of the micro-nebulization sampler can be used to facilitate pharmaceutical industrial quality control and food industrial safety control. The application of this system in these areas has shown significant improvement in sensitivity, precision, and accuracy.

The surface of indium phosphide single crystals exposed to synthetic lung fluid (Gamble solution) has been investigated. An oxygen depth profile, obtained by employing the 3.035 MeV resonance in the elastic scattering of alpha particles from 16O, detected oxygen at maximum concentrations of 24% in a layer approximately 1000 A thick. O:In and O:P ratios were found to increase with prolonged exposure time as determined by XPS, although binding energies and peak fwhm's remained relatively constant. The surface concentration of indium was found to be decreased in this 1000 A layer. Indium was detected in the Gamble solution, confirming that indium was leached from the InP surface. The similarity of P:In ratios at all lengths of exposure suggests that phosphorus was also leached by the Gamble solution. An efficient method for determining arsenic species As III, As V, methylarsonic acid and dimethylarsinic acid in groundwater samples has been developed. Ion exchange chromatography using a tandem SCX/SAX column was used to separate the four species which were analyzed using hydride generation atomic absorption spectroscopy (IEX/HGAAS). The method produced excellent separation, and off-column arsenic fraction total concentrations were generally within 10% of measured total arsenic concentrations. IEX/HGAAS was used to speciate arsenic in groundwater samples collected from a suspected arsenic contamination site at an industrial facility in Missouri. Results of the speciation analysis provided evidence to substantiate the presence of high naturally-occurring arsenic in groundwater at the site. Trivalent arsenic was the predominant species detected. The occurrence of trivalent arsenic was consistent with the reducing conditions of the alluvial aquifer and the high iron oxide concentrations detected in soil at the site. Though sensitive, the traditional IEX/HGAAS method for speciating arsenic is tedious and time-consuming. Analysis of a single sample can require as much as eight hours. A microscale SPE/PIXE analysis method was investigated. This method allows for preconcentration of arsenic species by extraction onto an SPE column containing ion exchange resin. Because PIXE is not species-sensitive, the arsenic-containing resin was analyzed directly, thereby eliminating digestion and reduction requirements imposed by the HGAAS method. Preliminary results gave excellent retention for As V and DMA over the concentration range analyzed. As III and MMA were not significantly retained on the SAX resin under any of the extraction conditions tested.