The following arsenite methyltransferase activities (U/mg) were measured in untreated mice: liver, 1.42 ± 0.17 (mean ± SEM); kidney, 0.62 ± 0.18; lung, 0.33 ± 0.08; testis, 1.21 ± 0.01. Arsenite methyltransferase metabolites were not detectable using guinea pig liver, kidney, lung or testis cytosol as the source of enzyme. A twofold increase in liver arsenite methyltransferase activity was observed in mice exposed to 28.6 mg sodium arsenite/L drinking water after 24 hr compared to control. Trivalent arsenic species were separated from pentavalent arsenicals in liver homogenates of hamsters injected 15 hr priorly with [⁷³As]arsenate by (CCl₄)-20 mM (DDDC) extraction and both phases analyzed by HPLC. Metabolites of inorganic arsenate were observed in the following concentrations (ng/g liver ± SEM); MMAᴵᴵᴵ, 38.5 ± 2.9; DMAᴵᴵᴵ, 49.9 ± 10.2; arsenite, 35.5 ± 3.0; arsenate, 118.2 ± 8.7; MMAᵛ, 31.4 ± 2.8; and DMAᵛ, 83.5 ± 6.7. Neither in vitro nor in vivo arsenite methyltransferase activity could be detected in S. cerevisiae . Attempting to induce enzyme activity, yeast were grown in 0-100 mM, 8 μCi [⁷³As]Na₂HAsO₄. No methylated metabolites were detected in cell lysate or media under these experimental conditions. The dissociation constants for guinea pig and rabbit cytosolic arsenite binding proteins were determined to be 59.62 ± 11.97 and 120.4 μM Asᴵᴵᴵ, respectively, and the respective specificities, 53.83 ± 3.67% and 59%. Guinea pig arsenite binding protein was fractionated using bioaffinity and size exclusion chromatography to yield partially purified protein(s) with an approximate molecular weight of 115 kDa. Arsenite methyltransferase activity was purified >7000-fold from rabbit liver and identified as 1 cys peroxiredoxin, a conserved family of antioxidant proteins characterized by non-selenium dependent glutathione peroxidase and calcium-independent phospholipase A2 activities. Murine 1 cys peroxiredoxin was cloned and expressed in E. coli and S. cerevisiae and expressed in reticulocyte lysate. Recombinant proteins had neither arsenite methyltransferase nor glutathione peroxidase activities. Antibodies directed against 1-cys peroxiredoxin immunoprecipitated a ∼29 kDa protein from guinea pig kidney cytosol which was identified as 1-cys peroxiredoxin by LC-MS/MS. Under these experimental conditions, guinea pig kidney cytosol did not catalyze the reduction of peroxides

1,2-Dichlorobenzene (1,2-DCB), an industrial solvent, is a potent hepatotoxicant in Fischer-344 (F-344) rats. Bioactivation of 1,2-DCB by cytochrome P450 enzymes in the liver and subsequent activation of Kupffer cells (KC), the resident liver macrophages, are required for the development and progression of liver damage. The mechanisms by which KC cells become activated in chemical induced liver injury are unknown. The studies described in this dissertation utilized 1,2-DCB as a model hepatotoxicant to test the hypothesis that KC activation following 1,2-DCB induced liver injury is triggered by molecular and cellular events in hepatocytes that lead to the expression of proteins known to activate inflammatory cells. The administration of 1,2-DCB (3.6mmol/kg, i.p.) to F-344 rats or incubation of 1,2-DCB (3.6 umol) with primary cultured hepatocytes of F-344 rats produces an intracellular oxidative stress as assessed by the production of glutathione disulfide. The binding activity of transcription factors associated with oxidative stress (AP-1, EpRE and NF-kB) were also enhanced in F-344 rat hepatocytes by 2 to 6 hr of incubation with 1,2-DCB. After 12 hr of incubation with 1,2-DCB, the production and release of CINC was 2.2-fold greater in F-344 rat hepatocytes than control cells. The mRNA expression of the chemokine MIF was also increased by 1,2-DCB in F-344 rat hepatocytes. In contrast, these molecular and cellular events were delayed or significantly reduced in the hepatocytes of the Sprague-Dawley rat, which is much less sensitive to 1,2-DCB induced hepatotoxicity. Furthermore, KC incubated with conditioned media from F-344 rat hepatocytes treated with 1,2-DCB for 24 hr showed enhanced NF-kB binding activity. Interestingly, the receptor for CINC, CXC Receptor 2, was expressed on KC as determined by immunohistochemical analysis. These data suggest that hepatocellular oxidative stress may trigger a cascade of molecular processes in hepatocytes that promote the expression and release of oxidant sensitive chemokines. These products signal the activation of KC and the upregulation of the inflammatory response leading to the progression of 1,2-DCB induced liver damage.

The occupational chemicals 4-vinylcyclohexene (VCH) and butadiene diepoxide (BDE) have shown an ovarian effect in small pre-antral follicles in mice and rats (Flaws et al., 1994b; Doerr et al. , 1995). VCH exists as two enantiomers, S-VCH and R-VCH. Daily dosing with the enantiomers affected small primary follicles and wet tissue weights in B6C3F₁ mice. The S-enantiomer decreased small primary follicle numbers (P < 0.05) and both enantiomers reduced the weights of the ovary and uterus (p < 0.05). The results suggest the S-VCH enantiomer may have a greater ovotoxic effect as compared to the R-VCH enantiomer. In F344 rats, daily dosing with BDE reduced overall body weight (p < 0.05). There was no affect on ovarian follicle numbers. However, other ovarian effects were seen: decreased ovarian weights, delayed vaginal opening, decreased CL numbers and decreased caspase-3 activity in large antral follicles. Overall, BDE had a general toxic effect with a more specific effect in the ovary.

The cytotoxicity of water-soluble and water-suspendable nanoparticles is a sensitive function of their surface derivatization or surface coating, particle size or specific surface area, and crystalline phase (for nanocrystals). In many different human cell lines, the cytotoxicity of a nanomaterial has been shown to change up to 8 orders of magnitude with relatively minor alterations in its structure. Cellular viability was determined through live/dead staining and LDH release. If the nanomaterial was deemed cytotoxic, further biochemical endpoints were tested. In almost all cases, when the nanoparticles were brought into the aqueous phase reactive species were formed. Ex vivo chemiluminescent analysis qualitatively assessed the presence of RS, and evaluated the effect of choice of solvent, suspension agitation, and light exposure. Liquid atomic force microscopy (AFM) was utilized to examine the interaction of nanomaterials with artificial membranes, illustrating the use of in situ AFM to directly observe the interactions of a model nanoparticle with a model cell membrane. By using in vitro, ex vivo, and in situ techniques, this work demonstrates both a strategy for enhancing the toxicity of nanomaterials for certain applications such as cancer therapeutics or bactericides, as well as a remediation for the possible unwarranted biological effects of nanoparticles.

Toxic effects have been reported for positively-charged liposomes containing stearylamine (SA-liposomes). We have observed that hemolysis and turbidity changes in native plasma were induced by the presence of SA-liposomes. In order to study the mechanism of the SA-liposomes toxicity to blood, we have examined the interaction of SA-liposomes with erythrocyte ghosts (EG) by resonance energy transfer for the lipid mixing and by Terbium/Dipicolinate (Tb/DPA) assay for the mixing of aqueous contents. The results demonstrate that SA-liposomes and EG, after aggregation, have a tendency to mix their lipid before mixing of their internal contents. This interaction was reduced by the presence of 10$ sp{-3}$ or 10$ sp{-2}$% (w/v) carboxymethyl chitin (CMC). / We have also explored the properties of the association of SA-liposomes with CMC, using fluorescein labeled CMC (Flu-CMC). The polarization value of Flu-CMC increased upon SA-liposomes binding, the amount of Flu-CMC on the liposome surfaces increased with increase in the concentration of phospholipid and SA, and it decreased by 20-40% in the presence of 10 mM Ca$ sp{2+}.$ These results indicate that association of SA-liposomes to CMC reduces the propensity of SA-liposomes to interact with blood components and this association is due to predominantly electrostatic interactions and possibly some hydrophobic interactions between SA-liposomes and CMC. / Finally, using the resonance energy transfer assay, preliminary results indicate that there is lipid mixing between SA-liposomes and platelets. This is not inhibited by phagocytosis inhibitors such as EDTA, 2,4-dinitrophenol with iodoacetate and cytochalasin B, suggesting that the lipid mixing is not accompanied by phagocytosis of SA-liposomes by platelets.

The cholinergic system has been shown to play an important role in cognitive function. Previous work has demonstrated that lesions to the cholinergic pathways originating in the basal forebrain, namely the nucleus basalis magnocellularis (NBM), have profound effects on the working memory function. / The present experiments were designed to assess the manner in which nicotine, a nicotinic cholinergic agonist, administration reverses the cognitive deficits resulting from lesions to the NBM. / The first experiment used the radial arm maze to examine working versus reference memory. Next, delays were imposed between the first four radial arm maze choices and the last, in order to examine short-term memory. Finally, pre-pulse inhibition of acoustic startle was assessed to determine the effects of nicotine on sensorimotor gating in NBM lesioned animals. / The results convincingly show that the cognitive and sensory motor gating impairments of MBN lesions are reversed by nicotine administration.

<p> DNA damage is a ubiquitous challenge to replication in cells, as damage causes replicative polymerase stalling. However, once DNA has been unwound at the replication fork, replication must proceed in the presence of damage to prevent more deleterious and almost assuredly mutagenic consequences. Alleviation of replicative polymerase stalling is accomplished by specialized DNA polymerases that can synthesize across from DNA lesions using the damage as a template, a process termed translesion synthesis (TLS). DNA polymerase &eta; pol &eta; is the best understood of these polymerases, and lack of pol &eta; synthesis activity in the human cancer prone syndrome Xeroderma pigmentosum variant (XPV) leads to cancer susceptibility upon sunlight exposure. XPV cells display higher mutation rates when exposed to UV light. This prevention of mutagenesis occurs despite pol &eta; having fidelity that is thousands of fold lower than replicative polymerases when copying both damaged and undamaged DNA. Pol &eta; has been implicated in replication past the UV induced <i> cis-syn</i> cyclobutane pyrimidine dimers (CPD) and the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG) in cells. We sought to better understand the molecular basis of efficient but moderate to low fidelity bypass by pol &eta;. We have examined polymerase properties as well as replication fidelity opposite these 2 lesions and with undamaged DNA. </p><p> To this end, we have created and purified a set of single amino acid substitution mutants in and surrounding the active site of the protein, utilizing the truncated catalytic core of the protein as a model. We assessed these mutants for overall synthesis activity as well as bypass fidelity opposite both T-T CPD and 8-oxoG. Our results show that several residues are critical for polymerase function, and altering these amino acids have multiple effects on polymerase properties. The R55A mutant abolishes polymerase activity while the Q38A, Y52E, and R61A mutants display altered fidelities. Y52E increased fidelity at both lesions and undamaged DNA, while R61A increased fidelity when copying T-T CPD. Also notable, Q38A increased fidelity opposite 8-oxoG, while it decreased fidelity opposite a T-T CPD. </p><p> One proposed means of increasing pol &eta; fidelity is interaction with replication accessory proteins that assist in replication at the replication fork. We purified the full-length form of pol &eta;, containing known protein:protein interaction domains in the C-terminus, and examined the effect of adding RPA to the bypass reaction. We saw no change in fidelity when examining fidelity opposite T-T CPD or 8-oxoG. We sought to confirm our results by also expressing two previously identified mutants with specific fidelity signatures, Q38A and Y52E. These full-length mutants recapitulated the fidelity effects seen in the truncated mutants when copying damaged DNA, and these fidelity signatures were unchanged with the addition of RPA. </p><p> Taken together, these results indicate that the major determinant of pol &eta; fidelity is the active site structure of the protein. The active site sequence is robust and certain amino acids play a critical role in the molecular mechanism of synthesis by the enzyme. Further clues as to the effects of altered polymerase function could be addressed by experiments expressing these mutant proteins in cells lacking pol &eta;. Additional investigation is necessary to recapitulate a more complete set of proteins with known functions in TLS, as interaction with other proteins could possibly alter fidelity. This work emphasizes that TLS is a damage tolerance process that could potentially cause mutations if perturbed. In order to avoid the certainly mutagenic consequences of strand breaks, cells utilize damage tolerance at the cost of potential mutagenesis. This balance between tolerance and mutagenesis has implications for multiple disease processes and human health. </p>

Effects of acute and chronic cocaine on the respiratory pattern, and ventilatory responses to hypoxia and hypercapnia, were studied in awake and anesthetized adult rats. In addition, effects of the drug on vagal reflexes, respiratory mechanics and gas exchange were investigated in anesthetized animals. / Acute cocaine elicited rapid and shallow breathing in awake rats. Ventilatory excitation, but not tachypnea, was present in anesthetized animals. In untreated rats, respiratory rate (f) increased during 10% O$ sb2$ and 5% CO$ sb2$ exposures; f decreased after cocaine, but in absolute terms, it was still higher after than before treatment. In anesthetized animals, augmentation of minute ventilation and f recorded in response to low O$ sb2$ and high CO$ sb2$ were higher with than without acute cocaine. / Chronic cocaine depressed ventilatory responses to both stimuli in awake, but not in the anesthetized chronic rats. Neither acute nor chronic cocaine affected respiratory mechanics and diffusion capacity of the lungs. Finally, strength of the expiratory-promoting reflex increased following acute cocaine. / The data suggest that while acute cocaine acts as ventilatory stimulant triggering behavioral polypnea, it depresses ventilation in chronically treated rats.

Mercury is an environmental contaminant of concern, particularly for fish eating populations. The objective of this study was to assess the effects of selenium, vitamin E, and phytate on methylmercury (MeHg) toxicity and kinetics in rats. Results show that increased selenium increases McHg in the liver, kidney, and frontal lobe of the brain, while increased vitamin E increases MeHg in the kidney but lowers McHg in the liver. Increased phytate resulted in a significant increase in MeHg in the frontal lobe. Methylmercury-treated rats on all diets showed an increased trend in muscarinic acetylcholine receptor (mAChR) binding in comparison with untreated rats. There was no change in monoamine oxidase (MAO) activities in all treatment groups. These results suggest that nutrients can alter the toxicokinetics of MeHg but none of them show clear protection in neurotoxicity in adult rats.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0930. Advisers: Shelley A. Tischkau; Paul S. Cooke. Includes bibliographical references (leaves 96-110) Available on microfilm from Pro Quest Information and Learning.