The toxicity of nickel compounds is most clearly demonstrated in nickel refinery workers. Nickel exposure leads to lung and nasal cancers and increased risk of acute respiratory syndromes. The toxicity of nickel compounds has been attributed to its roles in oxidative damage, changes in gene expression by an epigenetic mechanism, and inhibition of iron containing enzymes. For this reason, we are interested in the mechanisms of nickel induced inhibition of some of these enzymes. Non-heme iron enzymes carry out a broad number of essential biological reactions in mammalian metabolism, including several that are involved DNA repair and histone demethylation, and thus are involved in gene expression by an epigenetic mechanism. The enzymes involved in DNA repair (e.g., ABH2) and histone demethylation ( e.g., JMJD2 proteins) require Fe(II) and αKG for their function. The replacement of Fe(II) in the active site by other metal ions, including Ni(II), produces an inactive enzyme. Cellular DNA undergoes alkylation damage by chemicals that modify DNA bases and this damage can be inherited. Cells have evolved systems to repair this DNA alkylation damage. In human, ABH2 repairs endogenously formed 1-methyladenine (1-MeA) and 3-methylcytosine (3-MeC). Our experiment suggests that nickel inhibits ABH2 in a dose dependent manner. The inactivation of ABH2 will lead to increased DNA methylation and subsequent transcriptional silencing, which can result in caner and other developmental defects. Histone tails undergo a number of posttranslational modification including acetylation, methylation, phosphorylation and ubiquitination. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, DNA repair and transcriptional regulation. The increase in cellular nickel concentration leads to an increase in the global levels of H3K9Me1 and H3K9Me2 – not by affecting histone methyltransferases, but by inhibiting a group of Fe(II) and αKG dependent histone demethylases. Using JMJD2A and JMJD2C as examples, we show that JMJD2 family of histone demethylases is also highly sensitive to inhibition by Ni(II) ions. Isothermal titration calorimetry (ITC) has been used to understand the binding of Fe(II) and Ni(II) to ABH2. Our ITC results indicate that both Fe(II) and Ni(II) form 1:1 complexes with ABH2, but Ni(II) binds ABH2 stronger than Fe(II). X-ray absorption spectroscopy (XAS) has been used as a structural probe of the active site metal center in WT-recombinant ABH2, truncated JMJD2 proteins (1 – 350 aa) and Ni(II)-substituted WT-recombinant ABH2 and JMD2 proteins in the presence and/or absence of αKG and substrate in order to examine the reaction mechanism and ascertain the intermediate(s) affected by nickel substitution. Our XAS results indicate that in the presence of both αKG and substrate the iron site is five coordinate while the nickel site is six coordinate and thus, nickel site does not have any empty coordination site for oxygen binding and activation. Thus, using ABH2 and JMJD2 proteins as examples, we show that Fe(II) and αKG dependent enzymes are inhibited by Ni(II) ions. This is consistent with previous reports in literature on other Fe(II) and αKG dependent enzymes. Our results indicate that Ni(II) inhibits ABH2 and JMJD2 proteins by replacing the active site metal and both electronic and steric components are involved. Inhibition of some of these non-heme iron enzymes, like histone demethylases may be one way nickel can alter gene expression by an epigenetic mechanism.

In this study, a structural role of the zinc in addition to its catalytic role in the type A botulinum neurotoxin has been examined. Both secondary and tertiary structure are altered upon the removal of the bound zinc. While the binding Zn$\sp{2+}$ seems to be reversible, the structure and in biological activity of the type A botulinum neurotoxin were irreversible by a altered upon removal of Zn$\sp{2+}$. Botulinum neurotoxin is internalized into neuronal cells through receptor-mediated endocytosis. Lowering of endosomal pH has been proposed to trigger the translocation of whole or a part of the neurotoxin, possibly through a membrane channel formation by the neurotoxin's heavy chain subunit. Low pH-induced voltage gated channels were observed only with the heavy chain and not with the intact neurotoxin. In liposomes membrane channel activity, as monitored by calcein release, was observed with the intact neurotoxin as well as its two subunit chains. The heavy chain exhibited the highest Calcein release activity. The pH effect on the membrane channel formation by botulinum neurotoxin was therefore, investigated to understand the molecular basis of membrane channel formation of heavy chain. The low pH induced significant conformational change in the botulinum neurotoxin heavy chain at the tertiary structure level, which could be responsible for triggering its insertion into membrane binding. The secondary structure of the light chain was dramatically changed, which could explain its interaction with membrane lipid as well as its likely passage through the membrane channel formed by the heavy chain. Low pH-induced changes in polypeptide folding were also analyzed by monitoring thermal unfolding of the heavy chain in different pH conditions. Thermal unfolding curves indicated the presence of oligomeric structure at low pH. Binding of the heavy chain with liposomes at either pH 7.4 or 4.0 introduced dramatic changes in the unfolding pattern, suggesting major structural alternation in the protein upon binding with membrane. The heavy-chain-induced liposome aggregation as well as fusion, which could be relevant to the possible oligomeric structure of the neurotoxin and the presence of the neurotoxin translocation. With the use of specific antibodies, we examined the role of specific peptide segments of heavy chain in the membrane channel activity. During the isolation of the neurotoxin, 33 kDa protein is serendipitously purified from the botulinum neurotoxin complex. This protein is an integral part of a group of neurotoxin associated proteins which are known to protect the neurotoxin from adverse environmental conditions, including acidity and protease of the gastric juice. The purified protein exhibited hemagglutination activity which was strongly inhibited by sugars. The protein was also completely resistant to the trypsin digestion. Structural investigation indicated the hemagglutinin-33 protein is predominantly $\beta$-sheet which exists in an oligomeric form in aqueous solution. (Abstract shortened by UMI.)

Concerns over the production of disinfection by-products formed when drinking water is chlorinated that may be toxic to humans, has increased the interest in alternative disinfection procedures such as ozonation and chloramination. The research in this dissertation concerns the development of analytical methodologies for organic chloramines, that are formed during chloramination and chlorination, and dicarboxylic acids that are formed during oxidation processes especially ozonation. Organic chloramines may have detrimental human health effects and interfere with the measurement of inorganic chloramine. Chromatographic methodologies for their determination are limited by the difficulty in extracting them from water, lack of standards and the lack of a chromophore for HPLC analysis. The research focused on three main areas: investigation of the feasibility of gas chromatographing the organic chloramines, investigating the compound independence of the newly developed nitrogen 388 line for the atomic emission detector, and the development of an extraction procedure for removing the organic chloramines from water. The final method employed solid phase microextraction (SPME). The SPME technique was optimized for both aliphatic amines and organic N-chloramines. Measurement of dicarboxylic acids, particularly oxalic acid, in drinking water was also investigated. They contribute to microbial growth in water and may react further to form halogenated disinfection by-products. Like the organic chloramines, their hydrophilicity and lack of a suitable chromophore hinder their analysis. Furthermore, direct gas chromatographic analysis of the dicarboxylic acids is not possible as they are not volatile. A gas chromatographic procedure for the analysis of short chain dicarboxylic acids was developed. An extraction procedure for oxalic acid using tributyl phosphate, an organic base is presented. The use of chloroformate reagents, particularly trichloroethylchloroformate, for the subsequent derivatization of mono and dicarboxylic acids is investigated. Although, the derivatization clearly formed the appropriate reaction product, cleanup of reaction by-products and reagents is problematic. Several strategies for removing these matrix affects are presented. A study for the characterization of human and veterinary sodium hyaluronate products was conducted. A procedure for the supercritical fluid extraction (SFE) and determination by HPLC for miconazole nitrate is presented.

Two scenarios for stimulating the acute phase response (APR), subcutaneous (s.c.) injection of turpentine and s.c. injection of carrageenan, were tested for their ability to protect against a range of toxic conditions that reflect distinct mechanisms. Five hepatotoxins were included: the chlordecone/carbon tetrachloride interaction (CD/CCl4), CCl4 alone, galactosamine (GalN), allyl alcohol (AA) and thioacetamide (TA). The nephrotoxin, mercuric chloride (HgCl2) was also included. Each group of Sprague-Dawley rats was administered one of six toxins following a 24-hour prior treatment with either turpentine, carrageenan, or with no prior treatment (toxin alone). Toxicity was evaluated by serum alanine transaminase (ALT) and histology (hematoxylin-eosin) at 12-hour intervals over 48 or 72 hours. The APR was monitored by serum haptoglobin (Hp) and ceruloplasmin (Cp) at 12-hour intervals over the same periods. The temporal relationship between APR-induction and toxicant administration was modified in two additional experiments: (1) simultaneous injection of turpentine and toxicant and (2) turpentine injected 12 hours after administration of the toxin. Results indicate that turpentine or carrageenan injected 24 hours prior to toxicant profoundly reduced the hepatotoxicity (89 to 99%) of CCl4, CD/CCl4, GalN and TA when compared to toxin alone. Protection corresponds to significant, often multifold, increases in Cp and Hp. Both turpentine and carrageenan failed to protect against AA and HgCl2 toxicity, despite elevated APPs. Finally, turpentine failed to protect against toxic exposure when the temporal relationship between turpentine injection and toxicant administration was modified as in cases 1 and 2, where Cp and Hp responses had not fully developed.

Hormesis is a nonlinear dose-response characterized by biological responses at low doses that are opposite to those observed at higher doses. Studies and review articles on hormesis are being published at an increasing rate by researchers from diverse disciplines and debate has emerged over the role hormesis in risk assessment. As a result, a survey was conducted to assess toxicologists and risk assessors knowledge and attitudes about the hormesis dose response. Study goals were to: (1) ascertain attitudes towards hormesis and other dose-response models, (2) identify whether acceptance or rejection of hormesis is based on knowledge of hormesis, predisposing values, or demographic characteristics, and (3) evaluate potential for response bias. The survey consisted of 44 questions pre-tested by 25 toxicologists and risk assessors. The survey was distributed via email to the membership of the Society of Toxicology and the Society for Risk Analysis, 9,500 potential respondents. The overall response rate was 17% (n= 1,463) with a completion rate over 87%. Major findings were that 50% of respondents indicated sufficient data exist to support the view hormesis occurs across a wide range of species and endpoints, 59% indicated evaluating potential benefits due to hormesis should be included in risk assessments, and 65% are in favor of modifying hazard assessment protocols to identify the presence of hormesis. Respondent characteristics such as: years of experience, society membership, education, residence, employment (excluding government and pharmaceutical companies), and political, economic or social views had little influence on opinion. One of the largest positive influences was experience with hormesis based on actual research; 79% of subjects who reported observing hormesis commonly in their studies agreed hormesis is broadly generalizable. The influence of non-response bias was evaluated through several internal and external measures. Despite a lower than hoped for response rate, but because of robust external validity measures, it is concluded that respondents’ opinions are likely a reasonable representation of the societies of which they are members. Because this is a baseline survey, a follow-up survey is in order. Future survey design should separately evaluate the science of dose-response from the regulatory approach to risk assessment.

The developing pancreas has been identified as a potentially sensitive target for oxidative damage caused by environmental toxicant exposure in the developing zebrafish, Danio rerio. The objectives of this study were to 1) investigate the effects of exposure to a common pro-oxidizing toxicant, Mono(2-ethylhexyl) phthalate (MEHP), on pancreatic development, and 2) elucidate redox-sensitive temporal windows of pancreatic organogenesis in the zebrafish. We also investigated the involvement of Nrf2 (Nfe2l2), a transcription factor involved in the oxidative stress response, in toxicant-mediated pancreatic toxicity. In aim one, zebrafish embryos were exposed to 0 or 200 µg/L MEHP beginning at 3 hours post fertilization (hpf) through 168 hpf, and imaged live under a fluorescence microscope to visualize pancreas development from 48-168 hpf. Glutathione (GSH) and cysteine (CyS) redox couples were quantified by HPLC at 72 hpf, and gene expression was investigated at 96 hpf. This study utilized wild type (AB) Tg(ins:GFP), Tg(gcga:GFP) (endocrine islet), and Tg(ptf1a:GFP) (exocrine pancreas) zebrafish strains. We observed that MEHP exposure significantly reduced endocrine islet area and exocrine pancreas length at all timepoints (48, 72, 96, 168 hpf). No significant changes were observed in the redox potential of GSH or Cys, however MEHP exposure significantly altered expression of GSH-related genes (gsr, gstp1), as well as pancreas-specific genes (insa, sst2, ptf1a). These data indicate that the developing pancreas is a sensitive target tissue of embryonic exposure to MEHP. In aim two, we exposed transgenic Tg(ins:GFP) and Tg(gcga:GFP) zebrafish embryos to water, dimethyl sulfoxide (DMSO), N-acetyl cysteine (NAC), sulforophane (SFN), tert-butylhydroperoxide (tBOOH), or tert-butylhydroquinone (tBHQ) at 24, 48, or 72 hours post fertilization (hpf), and assessed endocrine islet morphology at 96 hpf. We found both chemical-, stage-, and cell-type specific effects of redox modulation on the endocrine pancreas. Pro-oxidant exposures resulted in decreased ß-cell cluster area and an increased frequency of islet variants, while antioxidant exposures significantly increased ß-cell cluster area. These effects were most significant at the 48 hpf exposure timepoint. a-cell cluster area was only affected by prooxidant exposure at 48 hpf. These results indicate that ß-cells are uniquely sensitive to oxidative stress, specifically at 48 hpf.

Neurotransmitters are endogenous compounds located in the brain and can occur in other parts of the body at low concentrations. They are a challenging group of compounds when it comes to analysis. Compounds with amine functionality such as dopamine and serotonin are sensitive to high pH and light and can spontaneously oxidize and degrade. Since neurotransmitters exist at low levels, it can be difficult to achieve sufficient spectrometry data and clean chromatography. In order to improve data acquisition, derivatization and the use of multidimensional liquid chromatography (2D-LC) was evaluated. Dopamine, L-3,4-dihydroxyphenylalanine (L-DOPA), serotonin, tyrosine, tryptophan, and noradrenaline were the compounds of interest with tyramine included as an internal standard. Data was collected before and after derivatization to compare the resulting chromatography. Chromatography was completed using a 6x6 grid of methods with variables of organic solvent, elution pH, loading pH, and trapping column chemistry utilizing different elution column chemistries. Derivatization was examined with Dabsyl-Cl and Dansyl-Cl at pH 8.5, 9.5, 10.5, and 14. A final method was chosen with Dabsyl at pH 8.5 for the Multiple Reaction Monitoring (MRM) scans. Before derivatization, the chromatography had poor peak shapes such as tailing, fronting, or shouldering or too much distortion to be able to distinguish a peak for all of the examined methods. Resulting chromatography after derivatization showed overall improvement in peak shape and intensity for a majority of the methods. Derivatization aided in increasing the mass and stability of the compounds which allowed for more sensitive detection. Multidimensional liquid chromatography improved the separation of structurally similar compounds and increased sensitivity. By combining the two, better analysis of neurotransmitters was possible.

Mercury (Hg) in the environment has deleterious ecological and health affects for humans and wildlife and is primarily transferred to humans through the consumption of marine biota (USEPA, 2001). These ecological and health concerns are exacerbated by the production of methylmercury (CH3Hg+; MMHg) in aquatic ecosystems. Sulfur isotopes (δ34S) are an indicator of the strength of sulfate reduction associated with MMHg production. In chapter 1, we assessed the relationship between MMHg concentrations and reduced-sulfur stable isotope δ34S signals in four coastal consumer organisms (length = 26–75 mm) from Florida's Big Bend seagrass meadows: pinfish (Lagodon rhomboides); pigfish (Orthopristis chrysopter); black sea bass (Centropristis striata melana); and shrimp (Tozeuma carolinense and T. serratum). We found a significant correlation between depleted δ34S signatures in fauna tissue (R2 = 0.27; p < 0.001; total n = 179) with higher MMHg concentrations. A correlation was observed in lower δ34S isotopic signatures and higher MMHg concentrations of consumers from the southern region of the study area indicating a "hotspot" of net mercury methylation in the sediments near Tampa Bay, Florida. To better understand the sources of Hg to coastal pinfish and to assess the contribution of pinfish annual egress to offshore food webs, chapter 2 applies isotopic tracing (C, N, S) combined with mercury (Hg) concentrations and isotope ratios in sediments, juvenile pinfish, juvenile gag grouper (Mycteropera microlepis), and adult gag grouper and pinfish to assess exposure pathways of MMHg in the northeastern Gulf of Mexico. We found that pinfish from the northern and southern Big Bend regions had distinct Hg sources. Southern pinfish had enriched δ202Hg and, when combined with lower pinfish δ34S values and lower sediment δ202Hg values, suggested elevated microbial methylation/demethylation in the southern region. The southern, coastal pinfish exhibited similar Δ199Hg as offshore gag grouper suggesting pinfish from this region represent an important Hg source to offshore reef fish species and/or these two cohorts are exposed to the same Hg source. Results suggested that estuaries can be an important source of MMHg to adjacent, offshore commercially important reef species and confirmed the utility of Hg isotope analysis to identify multiple marine Hg sources and inform our understanding of the pathways of MMHg bioaccumulation in estuarine food webs. In the northern Gulf of Mexico (nGOM), the Hg cycle is further complicated by the influence of the Mississippi River (MR) and potentially confounded, since April 2010, by the Deepwater Horizon (DWH) oil spill. Approximately 3.0-4.9% of oil-derived carbon released during the Deepwater Horizon oil spill was deposited on the seafloor (Chanton et al., 2014; Valentine et al., 2014) causing local, but persistent, oxygen depletion (Kessler et al., 2011). In these low-oxygen environments, the formation of monomethylmercury (MMHg) is promoted and may have led to higher MMHg concentrations in commercially important reef fish species. Expanding our research to include the entire nGOM shelf, slope, and the far-reaches of the deep Gulf canyons, chapter 3 explores Hg cycling using benthic and pelagic fishes as indicator-samples. Fish in the western study region appeared to rely more on benthic feeding, as indicated by enriched δ13C values and depleted δ34S values. Fish species closer to the MR plume had enriched δ15N and depleted δ202Hg and Δ199Hg values suggesting the MR influenced the nutrient and Hg cycle in the northeastern GOM. Cutthroat eels (~1,000 m) had higher MIF signatures than slope snake eels (~250 M) and, when combined with decreasing δ13C values with depth, suggested the presence of near-surface derived Hg in the deep DeSoto Canyon. Our results highlighted the importance of the deposition of Hg associated upper water column OM such as the marine snow pelagic carbon. Chapter 4 investigates whether more reducing conditions cause by DWH led to higher levels of mercury in commercially important reef associated organisms. We compared pre-spill (collected 2007-09) and post-spill (collected 2012) fish species of similar length from hard-bottom reefs on the West Florida Shelf. Light stable isotope analyses of carbon, nitrogen, and sulfur was employed to assess feeding ecologies of those species exhibiting higher MMHg concentrations following the oil spill. Some species, like porgy, showed significant increase in Hg concentration. This increase may have been associated with a shift to a more pelagic, higher trophic feeding regime. The significance of this study is that it shows that the oil spill had an effect on the mercury cycling but that the effect is not uniform for each fish species. / A Dissertation submitted to the Department of Earth, Ocean, and Atmospheric Science in partial fulfillment of the Doctor of Philosophy. / Spring Semester 2016. / January 11, 2016. / Bioaccumulation, Deepwater Horizon oil spill, Florida Big Bend, Isotope, Mercury, Pinfish / Includes bibliographical references. / Jeffery Chanton, Professor Co-Directing Dissertation; William Landing, Professor Co-Directing Dissertation; Xiaojun Yang, University Representative; Yang Wang, Committee Member; Dean Grubbs, Committee Member.

Botulinumtoxin: För och emot dess användning i skönhetsbranschen Bakgrund: Botulinumtoxin (BTX) är ett kraftfullt neurotoxin som produceras av den grampositiva och anaeroba bakterien Clostridium botulinum. BTX används i medicinskt syfte vid behandling av till exempel kronisk migrän, spasticitet efter stroke, och urinläckage vid nervskada. Utöver det används BTX inom skönhetsbranschens som då injiceras i mycket låga doser under huden i muskler för att minska rynkor i olika ansiktsregioner Syfte: Användningen och lämpligheten inom skönhetsbranschen har ifrågasatts , och syftet med detta arbeta har därför varit att utvärdera och ta ställning till om BTX fortsättningsvis bör användas i kosmetiskt syfte. Metod: Litteraturstudie där vetenskapliga artiklar söktes i databasen PubMed med kombinationer av sökord, artiklar som sedan kunde användas för att besvara den aktuella frågeställningen. Resultat: Olika studier har visats att BTX är ett effektivt och säkert toxin vid skönhetsbehandling, men flera studier visade också att BTX-relaterade biverkningar uppkom efter skönhetsbehandling. Slutsats: BTX bör inte fortsätta användas för skönhetsbehandling trots positiva effekter mot rynkor i ansiktet som visades i studierna. Injektion med BTX kan bidra till att kunder i skönhetssalonger blir svårbedömda patienter vid besök hos läkare i primärvården som inte förstår att de problem patienten söker för beror på BTX-inducerade biverkningar efter skönhetsbehandling med BTX.

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