3,3',4,4'-tetrachloroazoxybenzene (TCAOB), and 3,3',4,4'-tetrachloroazobenzene (TCAOB), which are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are formed as byproducts during the synthesis of industrial products. They exhibit Ah receptor binding characteristics and activities similar to those defined for TCDD in vitro at equal moral doses. However, they do not express toxicities in vivo at equal molar doses. The reduced in vivo toxicity can possibly be attributed to differences in the absorption and dispositional kinetics of TCAOB and TCAB as compared to TCDD. Thus, in this study, the absorption and dispositional kinetics of TCAOB and TCAB were examined in male F-344 rats. To address this, the animals received ¹⁴C-TCAB or ¹⁴C-TCAOB and the excretion of [¹⁴C] was monitored over 96 hr. For TCAB and TCAOB, the majority of the dose was eliminated within 48 hr regardless of the route of administration. The primary route of elimination was via the feces, and significant quantities of [¹⁴C] were eliminated in the urine. Pharmacokinetic parameters indicate that the compounds are readily cleared from the blood (TCAB: t₁/₂=4 hr, CLs=12 ml/kg(min); TCAOB t1/2=7 hr, CLs=12 ml/kg(min)). By contrast, TCDD has a half-life of 16-31 days and a elimination rate of 1-2% of the [¹⁴C] -dose per day in the bile/feces with no urinary elimination. Thus, TCAOB and TCAB are eliminated faster than TCDD. Urinary metabolite analysis following administration of TCAOB or TCAB revealed a variety of dichlorolaniline conjugates, which indicates the role of azo reduction in their formation. Several metabolites were present in the bile including glucuronide conjugates of dichloroaniline (DCA) and a putative glucuronide of TCAB (formed from TCAOB as well as TCAB). Clearly, the azo bond is responsible for the enhanced elimination of TCAOB and TCAB as compared to TCDD. The removal of the intestinal flora by the antibiotic pretreatment reduced the total reductive activity but did not eliminate it completely. This indicated that the rapid metabolism and elimination of these two compounds was a result of the combination of azo-reduction by both gut flora and mammalian tissues. The production of dichloroaniline by the in vitro liver homogenate strengthens this conclusion. Since dichloroaniline was produced over time by the liver enzymes following administration of either TCAOB or TCAB, the liver thus contributes to the reduction of the azo bond.

In lung preparations, As(V) was reduced to As(III) [first order rate constant of 0.0104/min]; As(III) was oxidized to As(V) [first order rate constant of 0.005/min], methylated to MMA [K(m) = 5.383μM, V(max) = 0.00031 μmol/liter/min/mg], and complexed with GSH; MMA was converted to DMA [K(m) = 63.4 μM, V(max) = 0.0000384 μmol/liter/min/mg]; and arsine was oxidized to As(III) and As(V) and methylated. Toxicity of As(III), As(V), MMA, DMA, and arsine was assessed by measuring effects on cell and slice viability hsp32 induction, and production of DNA single strand breaks. Because all species of arsenic did not produce effects, it was possible to deduce an "active" form of arsenic from these studies. Pulmonary arsenic metabolism was modeled using SIMUSOLV. This model indicated that arsine disposition cannot be explained solely by oxidation to As(III) before methylation or further oxidation occurs. The concentration of arsenic species present in toxicity studies were predicted with this model and correlated to observed effects. There was good correlation between reduction of As(V) to As(III) with toxicity and hsp32 induction. However, the effects observed for arsine did not correlate with oxidation to arsenite.

Studies were initiated to characterize the acute and sub-chronic pulmonary responses to inhaled Jet-Propulsion Fuel 8 (JP-8) aerosol. At 24 to 30 hours following JP-8 exposure, physiological, biochemical, cellular, and morphological techniques were used to assay for lung injury. In addition, C57BL/6 and B6.A.D.(Ahyd mice were utilized in the acute study to determine if responsiveness to aryl hydrocarbon hydroxylase (AHH) induction modulates toxicity. JP-8 contains aromatic compounds that are known substrates for AHH. Acute lung injury was evident by increased respiratory permeability that was accompanied by alveolar macrophage infiltration and activation, following exposure to occupational permissible levels of JP-8. Morphological alterations were characterized by terminal bronchiolar lesions of vacuolization and necrosis. AHH responsiveness did not appear to influence the severity of JP-8 induced lung injury. Lung injury following sub-chronic inhalation was found to be progressive in nature. Repeated exposure induced alveolar hemorrhage and alveolar macrophage cytotoxicity. Morphological changes progressed to include epithelial denudation of bronchiolar airways and vacuolization of alveolar type II epithelial cells and adjacent endothelia. The pulmonary clearance of JP-8 following inhalation exposures was determined indirectly by analysis for tetradecane content within lung homogenates from exposed mice. Clearance of JP-8 following acute exposure was determined to have a half-life of 43 minutes and increased by 14 minutes following a toxic sub-chronic exposure. The relatively rapid pulmonary clearance of JP-8 following either acute or sub-chronic exposure implies that JP-8 induced lung injury was independent of pulmonary retention. Studies were also performed to determine if non-cytotoxic concentrations of JP-8 or tetradecane could decrease bronchial epithelial barrier function. Studies showed that one hour of exposure to JP-8 or tetradecane could significantly enhance paracellular permeability to mannitol in the BEAS-2B human bronchial epithelial cell line, at two hours after exposure. Bronchial epithelial permeability appeared to be more sensitive to tetradecane than JP-8. Subsequent recovery studies determined that JP-8 and tetradecane-induced decreases in barrier function reach a maximum at 12 hours and barrier function returns to control by 48 hours post-exposure. These results indicate that JP-8 induced lung injury may be initiated by changes in airway barrier function.

Arsenic is a potent toxicant and known human carcinogen. Arsenite [As (III)], a major inorganic form of arsenic, can stimulate oxidative stress, decrease DNA repair, alter gene expression, modulate stress signaling, and damage proteins in numerous model systems. This dissertation focuses on the effects of low-level arsenite on the ubiquitin-proteasome pathway. Ubiquitin is a small protein that acts as a post-translational modification of other proteins within the cell. In most cases, modification of a protein by ubiquitin acts as a signal for its degradation by the proteasome. Studies performed in rabbit renal cortical slices and HEK293 cells demonstrated that 24 hr exposure to 0.5-10 μM As (III) caused a dose dependent accumulation of ubiquitin-protein conjugates within cells. This accumulation was shown to correlate with decreases in cellular 20S proteasomal activity. As (III) did not increase ubiquitin-conjugating activity. Depletion of cellular glutathione exacerbated the effects of As (III) on ubiquitin-protein conjugates in HEK293 cells. To characterize this perturbation of the ubiquitin pathway within As (III) exposed cells, a proteomics method was developed to purify and identify the specific proteins modified by ubiquitin. A cell line expressing epitope tagged His(6X)-Ub-GFP was developed by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using Ni-affinity chromatography and digested into peptides. Complex mixtures of peptides were separated by reverse phase chromatography and analyzed using the LCQ ion-trap mass spectrometer. In purified samples from transfected cells, ubiquitin and 22 other proteins were confidently identified using Sequest. These proteins included many of the expected carriers of ubiquitin including ubiquitin-conjugating enzymes and histone proteins. In samples from transfected cells treated with 10 μM As (III) (24 hr), a number of unique proteins were identified, including the DNA repair protein, and known ubiquitin substrate, PCNA. This proteomics method, developed for the analysis of ubiquitinated proteins in As (III) treated cells, will allow for large-scale characterization of ubiquitin-protein conjugates in numerous physiological and pathological states. These results suggest that low-level arsenic may facilitate its detrimental health effects through perturbation of the ubiquitin-proteasome pathway, and that future investigations into interactions between arsenic, ubiquitin and protein homeostasis are warranted.

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Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain the antioxidant response element (ARE) in their promoter regions, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so that this pathway can be targeted for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, other mechanism responsible for modulating Nrf2-ARE signal remains to be explored. This dissertation identifies three molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) In Chapter 2, KPNA6 was identified and characterized as a negative regulatory mechanism of the Nrf2 pathway, which mediates Keap1 nuclear import and represses the Nrf2-dependent antioxidant response at post-induction phase. (ii) In Chapter 3, I identified PARP-1 as a new transcription co-activator of Nrf2, which augments ARE-specific DNA binding of Nrf2 and enhances the transcription of Nrf2 target genes. This indicates a novel function of PARP-1 and reveals another layer of regulation of Nrf2. (iii) In Chapter 4, I demonstrated that XBP1 and SYVN1 are involved in regulating the Nrf2 pathway in a Keap1-independent mechanism. During ER stress, XBP1s upregulates transcription of SYVN1, which is an ubiquitin E3 ligase. SYVN1 accelerates the clearance of Nrf2 protein through promoting ubiquitination of Nrf2, and subsequent proteasomal degradation. Moreover, we observed an inverse correlation between XBP1s/SYVN1 and Nrf2 expression in the end stage alcoholic cirrhosis liver samples, implying a pathological role of ER stress-oxidative stress crosstalk. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, providing novel targets of chemoprevention or chemotherapy.

Cumulative evidence suggests that constitutively elevated levels of proteotoxic stress represent a specific vulnerability of malignant cells that can be targeted by pharmacological modulation of the intracellular proteotoxic stress response. According to this emerging mechanism, small molecule stress modulators may induce deviations from protein homeostasis causing cytotoxicity confined to malignant cells already at a high set point of constitutive proteotoxic stress leading to functional impairment and even cell death. In contrast, normal cells with sufficient protein degradation capacity can tolerate the extra dysfunctional protein overload. My graduate research has focused on testing the feasibility of repurposing clinically used non-oncological drugs for experimental chemotherapy targeting metastatic melanoma cells. The following specific aims were pursued: (1) To identify clinically used non-oncological drugs that preferentially induce cytotoxicity in melanoma cells but not primary melanocytes through upregulation of proteotoxic and/or oxidative stress; (2) To explore the specific molecular mechanisms underlying induction of melanoma cell apoptosis by lead compounds focusing on oxidative and proteotoxic stress modulation; (3) To explore efficacy of selected lead compounds for antimelanoma intervention in a murine xenograft model. First, we demonstrate feasibility of using the FDA-approved redox-active D-cysteine-derivative D- penicillamine for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo through induction of the unfolded protein response (UPR). Second, we demonstrate that the antimicrobial oligopeptide thiostrepton displays dual activity as a selective prooxidant and proteasome inhibitor causing proteotoxic stress that preferentially targets malignant melanoma and multiple myeloma cells. Third, we demonstrate for the first time that the clinically used 4-aminoquinoline antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced melanoma cell death. Taken together, our data indicate the chemotherapeutic potential of small molecule proteotoxic stress inducers and strongly suggest feasibility of repurposing specific non-oncological drugs for proteotoxic stress-directed antimelanoma intervention.

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In the studies presented herein, MTK1 is shown to be required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced extracellular acidification and cell migration in MCF-7 breast cancer cells. Furthermore, it was shown that HRG stimulation leads to association of MTK1 with tyrosine phosphorylated HER3 in MCF-7 and T-47D breast cancer cells. The MTK1/HER3 association was dependent on HER2 activation and was decreased by pre-treatment with the HER2 inhibitor, lapatinib. Furthermore, HER2 does not directly associate with MTK1, but phosphorylates HER3 transiently. MTK1 also has a role in the ERK1/2 MAPK signaling pathway in response to heregulin (HRG) stimulation in T-47D and MCF-7 breast cancer cells. In addition to MTK1, Shc, Grb2 and GIT1 proteins are all involved in the ERK1/2 MAPK pathway in response to growth factor stimulation. MTK1 was also shown to associate with activated ERK1/2, GIT1, Shc, Grb2 and p85 of PI3K in response to heregulin stimulation. ERK1/2 kinase activity is involved in aberrant signaling that leads breast cancer progression. GIT1 is a scaffolding protein that is linked to growth factor mediated ERK1/2 signaling in cell migration. Moreover, we also identify the actin interacting region (AIR) on MTK1 and disruption of actin cytoskeletal polymerization with cytochalasin D inhibited the interaction between HER3 and MTK1, indicating that f-actin (which is needed for cell migration) is required for the MTK1/HER3 association. Additionally, HRG stimulation leads to extracellar acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.

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