The goal of these studies was to investigate the potential for low dose ionizing radiation to modify Bax induced oncogenesis. Bax is a regulator of apoptotsis as well as a regulator of prooxident function and outer mitochondrial membrane permeability. Transgenic mice which over express one or two copies of Lck-Bax gene were treated with low doses (0, 10 and 100 cGy) of low LET radiation. The insertion and overexpression of the Bax gene appears to dominate the effect upon the phenotype in that the higher the gene dose the faster and more likely is the onset of thymic lymphoma. Mice grouped by radiation dosage potentially offer some insight. The double transgenic mice appear to be unaffected by the exposure to the radiation used in this study. While not statistically significant, there appears to be a trend in the single transgenic mice that the exposure to radiation at either level (10 or 100 cGy) will shorten the life span of the mice. Paradoxically, the shortest survival times in the Lck-Bax 1 mice appear with the lowest radiation dose implying some adaptive response. Further investigation demonstrated this effect is gender specific in the Lck-Bax 1 mice. A pairwise comparison between the 0.1 Gy and 0 Gy irradiated female Lck-Bax 1 mice show a statistically significant acceleration of lymphoma formation. This difference is not seen in any of the other pairwise comparisons of female mice. Furthermore , this trend is not present in the Lck-Bax 1 male mice.

Arsenic and atrazine are two water contaminants of high public health concern in Iowa. Arsenic is released into drinking water from soil and atrazine is the most heavily used herbicide. My hypothesis was that people in Iowa have high risks of certain cancers from exposure to arsenic and atrazine via drinking water. This study was performed to examine this hypothesis with three study aims: 1) to quantify the contamination of arsenic and atrazine in Iowa drinking water, 2) to analyze patterns of major cancers in Iowa and the US, and 3) to evaluate the association between arsenic exposure from drinking water and prostate cancer. First, I investigated the occurrence of arsenic and atrazine in drinking water from Iowa private wells and public water systems over several decades. Percentages of detection and violation of regulations were compared over region, season, and water source. Factors affecting detection and concentration of arsenic and atrazine, and correlations among atrazine and its degradation products, were analyzed using a mixed effects model. I found that detection and concentration of atrazine in drinking water decreased over time (all samples were below the drinking water standard of 3 ppb). However, the percentage of arsenic detections and concentrations higher than the drinking water standard (10 ppb) increased over time in the public water systems. Therefore, I focused on arsenic as a water contaminants of concern for the further study. Second, the patterns of trends in cancers related to arsenic and atrazine in Iowa were analyzed and compared to the US. Cancer is the second leading cause of death in the USA. However, cancer rates vary by different regions. In this study, the cancer trends in Iowa were investigated and compared to 8 other states for white individuals aged over 20. Temporal trends in age-standardized cancer rates were evaluated using joinpoint regression analysis by gender. Results of analysis indicate that overall cancer incidence and mortality were lower in Iowa than the US, and different trends of major cancers were found between Iowa and the US. However, prostate cancer was the most frequent type of cancer in men in both Iowa and the US and I focused on prostate cancer for further study. Lastly, based on what I found from previous studies, I conducted an ecologic study to evaluate the association of prostate cancer and arsenic in drinking water in Iowa, where exposure levels are low, but duration of exposure can be long. Spatial Poisson regressions were conducted to estimate the risk ratios of prostate cancer by tertiles of arsenic level at a county level, adjusted for demographic and risk factors. The county averages of water arsenic levels varied from 1.08 to 18.59 ppb across the counties, with three counties above the 10 ppb limit. Based on the tertiles of their arsenic levels, counties were divided into three groups: low (1.08-2.06 ppb), medium (2.07-2.98 ppb), and high (2.99-18.59 ppb). Spatial Poisson regression model analysis showed that the risk ratios of prostate cancer were 1.16 (95% CI, 1.10-1.23) and 1.28 (95% CI, 1.21-1.34) in the medium and high groups (p-trend < 0.001), compared to the low group after adjusting for risk factors. The results were similar when analyses were restricted to aggressive prostate cancers. These data show a significant dose-dependent association between low-level arsenic exposure and prostate cancer. These findings need to be confirmed from more in-depth individual studies. Overall, the results showed that 1) the detections of arsenic increased in Iowa drinking water in the last decade, 2) there were disparities of trends in major cancers between Iowa and the rest of US, and 3) the risk of prostate cancer increased with arsenic exposure via drinking water. This study approach allows for the identification of possible health issues caused by water contaminants.

Polychlorinated biphenyls (PCBs) are synthetic organic compounds that resist natural breakdown in the environment. Various toxic metals are found naturally in the earth and may become concentrated as a result a human activity. PCBs and toxic metals are a major public health concern due to human exposure from both natural and anthropogenic sources. Humans may be exposed to these chemicals from occupational sources, ingestion through food, drinking water and some medications. The main risk arising from environmental pollution by toxic metals and PCBS is damage to the nervous system. This thesis set out to answer two questions 1) does co-exposure to toxic metals and PCBs happen from occupational sources since these chemicals are present in various building materials? 2) Does co-exposure to Pb and the most abundant and neurotoxic PCBs (such as PCB153 or PCB95) generate more toxicity on neurons as compared to exposure to a single toxicant? To investigate whether co-exposure occurs from occupational sources, toxic metals and PCB (in progress) levels of 84 construction workers in eastern Iowa were assessed. Levels of DNA damage and antioxidant proteins associated with these exposures were also evaluated. The results revealed a wide range of Pb, Cd, and Hg levels in construction workers. Levels of DNA damage and levels of 8-Oxo-dG in these workers were influenced by whether they handled fluorescent light ballasts, whether they ate fish and whether they wore protective masks. To answer the second question a PC12 cell line was used, a well-known model for dopaminergic (DA) neurons, to study the effect of single and co-exposure on cell viability, DA level, DA metabolism, DA synthesis and packaging enzyme expression and antioxidant enzyme expression. The results illustrate that effects of single exposure to Pb, PCB153 and PCB95 and the combination of Pb and PCB153 or PCB95 following 12 and 24h exposure are variable and the effects are based on different mechanisms. Likewise, PCB153 and PCB95 which are both non-dioxin like PCBs were found to have different effects on PC12 cells based on the structure of the congener. This thesis demonstrates for the first time that PCB153 or PCB95 synergistically increase Pb toxicity to neurons, however, more research is needed to explore the interactions and the mechanisms which are involved in the influence of Pb in the presence of PCB.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that are carcinogenic and cause a variety of adverse health effects to the nervous, reproductive and endocrine systems. They bind to cytochrome P450 (CYP) enzymes to undergo phase I biotransformation to form hydroxylated metabolites. The present study investigates the hypothesis that the binding of multiple ortho substituted PCBs depends on the number of chlorine substituents and the chlorine substitution pattern. It also explores if hydroxylation alters the binding of these compounds. To test this hypothesis the spectral interactions of hepatic microsomal CYP enzymes with 2,2',3,4',6-pentachlorobiphenyl (PCB91), 2,2',3,5',6-pentachlorobiphenyl (PCB95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB132), and 2,2',3,4',5',6-hexachlorobipheny (PCB149) and their 5-hydroxylated (5-OH) metabolites were investigated. Hepatic microsomes were prepared from rats treated with phenobarbital (PB), dexamethasone (DEX), corn oil (CO) and saline (SL). The difference spectra of these microsomes in the presence of increasing concentrations of PCBs were recorded. All four PCB congeners and their metabolites (except for 5-OH PCB 149 with the DEX microsomes) elicited type I difference spectra with the PB and DEX microsomal preparations. No difference spectrum was observed with the CO and SL microsomes. With the parent PCBs, there was no distinct trend with the maximal absorbance change, δA max with the PB and the DEX microsomes. The spectral dissociation constant (K ss app) was lowest for PCB 91 and it had the highest relative binding efficiency (= K ss app / δA max) with PB and DEX microsomes. This suggests that PCB congeners and metabolites that have more unsubstituted carbon atoms at the meta position bind better to the CYP enzymes. The highest binding efficiency was observed with the PB microsomes both with the parent PCBs and their metabolites. This suggests that all the four PCB congeners and metabolites bind preferentially to the CYP2B enzymes (induced by PB) than to the CYP3A enzymes (induced by DEX). Also the binding efficiencies of the parent compounds were found be higher than their metabolites implying that hydroxylation of the parent PCB congener reduces its binding to the CYP enzymes. From the structure activity relationships and the effect of chlorination studied on PCB-CYP interactions in this project, it was found that the chlorine substitution pattern, but not the number of chlorine substituents play an important role in the binding of multiple ortho chlorine substituted PCBs to CYP enzymes.

Polychlorinated biphenyls (PCBs) are well known as classic environmental pollutants. Lower halogenated PCBs are mainly metabolized to mono- and di-hydroxyl compounds, and the latter further oxidized to quinones through a semiquinone intermediate. These quinone or semiquinone metabolites of PCBs can induce cytotoxicity by conjugating with functional protein and DNA as well as leading to oxidative stress in cells. Glutathione (GSH) is involved in lower chlorinated PCBs metabolites induced toxicities. Previous studies have shown that GSH reacts readily with PCB-quinones through Michael addition. The goal of this thesis was to investigate the effect of GSH on the reaction kinetics of PCB-hydroquinone autoxidation. Quinones produced during the autoxidation of hydroquinones serve as catalysts of the oxidation reaction. Therefore, it is hypothesized that GSH will suppress the autoxidation of PCB-hydroquinone and inhibit the formation of PCB-quinone. One of the PCB-hydroquinones, 2',5'-dihydroxy-4-chlorobiphenyl (4-CB-2',5'-OH), was employed as a model compound. Methods were established with 1,4-hydroquinone (1,4-H2Q). The reaction rate of 4-CB-2',5'-OH autoxidation showed pH dependency from my study. Superoxide dismutase (SOD) accelerated the reaction rate of 4-CB-2',5'-OH autoxidation dramatically at neutral pH. I used SOD in the studies to explore the effect of GSH on the autoxidation of 4-CB-2',5'-OH at near-neutral pH. It was found that GSH suppressed the autoxidation of 4-CB-2',5'-OH by inhibiting the formation of corresponding 4-CB-2',5'-Q and 4-CB-2',5'-SQ*−. A mathematical model simulating the inhibiting effect of GSH on the autoxidation of 4-CB-2',5'-OH was constructed. The mathematical model reproduced my experimental data well, supporting my proposed mechanism for the autoxidation of the PCB-derived hydroquinones. I also explored the reaction kinetics of 4-CB-2',5'-OH autoxidation in cell culture medium with fetal bovine serum (FBS). FBS showed a parallel inhibiting effect on 4-CB-2',5'-OH autoxidation as GSH. In summary, GSH and other thiols present in FBS can suppress the autoxidation of lower halogenated PCB-hydroquinone and decrease the rate of PCB-quinone formation. This finding suggests an unrecognized role for GSH in the toxicity of lower chlorinated PCBs.

In this dissertation, concentrations of 209 polychlorinated biphenyl (PCB) congeners and 64 hydroxylated PCB (OH-PCB) congeners were determined in 97 adolescents and 86 mothers living in East Chicago, Indiana and Columbus Junction, Iowa. Sera of the participants were collected between April 2010 and March 2011. PCBs and OH-PCBs in human sera were extracted, separated and quantified using gas chromatography tandem mass spectrometry (GC-MS/MS). A quality control protocol was established to ensure data quality during extraction and analysis processes and to affirm the data were representative, accurate, reproducible and precise. Total PCB concentrations in 164 participants’ sera ranged from 0.021 – 4.683 ng/g fresh weight (f.w.). Associations between the concentration of total 209 PCBs, along with the concentration of an additional 30 individual PCB congeners, and socio-demographic characteristics (age, gender, ethnicity and community of residence), body mass index and breastfeeding history were examined. Total PCB concentrations were significantly higher in older mothers and significantly lower in mothers who experienced longer breastfeeding duration. Columbus Junction adolescents had significantly higher total PCB concentrations than East Chicago adolescents. Associations were also found to be congener-specific. Lower-chlorinated congeners were significantly associated with environmental factors such as community of residence. Host factors such as age, gender, body mass index and breastfeeding history were significantly associated with higher-chlorinated PCBs. Non-Aroclor PCBs, PCBs that were not found in Aroclor commercial mixtures, were measured in sera of the participants. Concentration of total non-Aroclor PCBs among 175 participants ranged from none-detected to 0.288 ng/g f.w. Their concentrations were found to account for an average of 10% (up to 50%) of the overall concentration of total 209 PCBs in human serum. Moreover, an average of 50% of these concentrations may be due to exposure of paint pigments. PCBs 11, 14, 35 and 209 were the major dominating and most frequently detected non-Aroclor PCB congeners in the samples. Adolescents had significantly lower concentrations of total non-Aroclor PCBs than mothers regardless of their community of residence. In addition, total non-Aroclor PCBs were significantly higher in Columbus Junction community. Among the 64 OH-PCB congeners, 58 of them were detected in serum of 159 participants and ranged from 0.017 – 0.324 ng/g f.w. Total OH-PCB concentrations were significantly lower in adolescents in both communities. Lower-chlorinated OH-PCBs were found to be less frequently detected in serum. Besides that, a few rarely reported OH-PCBs (4, 4’-diOH-PCB 202, 4’-OH-PCB 208, 4-OH-PCB 163, and 3’-OH-PCB 65) were measured an highly detected in the samples. Apart from that, 3’-OH-PCB 65 was discovered for the first time in human serum. Evidence showed possible direct environmental exposure of this congener instead be the result of regular PCB metabolism. Furthermore, concentrations of 4-OH-PCB 107 and 4-OH-PCB 187 in human serum changed significantly over 3-year evaluations. The research findings of this dissertation indicate the importance of congener-specific PCB analysis to examine their association with different host and environmental factors. Results of these studies further emphasize the importance of assessing potential toxicity of non-Aroclor PCBs and their adverse health effects to the general population. Thus, studies have significant implications for future human risk assessment.

Exposure of mammalian cells to ionizing radiation is believed to affect mitochondrial metabolism, which can lead to increased levels of superoxide (O2*-). Sirtuin3 (Sirt3) is a key mitochondrial protein deacetylase thought to play a significant role in the maintenance of mitochondrial integrity and oxidative metabolism in response to radiation. Here I show evidence that Sirtuin3 regulates mitochondrial oxidative metabolism in unirradiated mouse embryonic fibroblasts (MEFs) as well as regulates survival and steady-state levels of O2*- following exposure to radiation. This work also identifies novel, small molecule, superoxide dismutase mimetics as being capable of ameliorating the alterations in mitochondrial oxidative metabolism and cell toxicity following exposure to radiation. When compared to WT MEFs, unirradiated Sirt3-/- MEFs demonstrate increased levels of reactive oxygen species (ROS), hyperpolarized mitochondrial membranes, increased oxygen consumption, higher glycolytic flux and lower ATP levels demonstrating that the loss of Sirt3 profoundly regulates mitochondrial metabolism. Furthermore, increased levels of mitochondrial fragmentation were also observed in irradiated Sirt3-/- MEFs, relative to un-irradiated controls. When exposed to 2 Gy of X-rays, Sirt3-/- MEFs also demonstrated increased steady-state levels of O2*- as indicated by DHE oxidation using flow cytometry and confocal microscopy. Sirt3-/- MEFs irradiated with 1-2 Gy also showed significantly increased clonogenic cell killing as well as a compromised ability to induce an adaptive response following exposure to a 0.1 Gy priming dose, as compared to Sirt3 WT MEFs. Increased levels of O2*- seen following irradiation of Sirt3-/- MEFs were ameliorated by the use of two superoxide dismutase mimetics (GC4401 and GC4403) but not the catalytically inactive form of the compound (GC4404) suggesting the causal role of the metabolic production of O2*- in radiation-induced toxicity. In addition, alterations in the activity of Complex II of the electron transport chain were also observed in the Sirt3-/- MEFs suggesting that Sirt3 may be essential in the maintenance of mitochondrial electron transport chain (ETC) activity following exposure to radiation. Collectively, this work demonstrates that Sirt3 plays a key role in regulating the levels of mitochondrial O2*- that may affect the overall maintenance of mitochondrial oxidative metabolism and survival following exposure to ionizing radiation. (Supported by DOE DE-SC0000830).

Vatalanib is a novel, potent, orally bioavailable angiogenesis inhibitor which can block all known vascular endothelial growth factor (VEGF) receptor tyrosine kinases. It has been shown to have potential in treating hematological malignancies, including myelodysplastic syndromes (MDS). Previous studies have revealed substantial pharmacokinetic variability that is further increased by auto-induction of its metabolism. And safety and tolerability are of great importance for such anti-angiogenic treatment. Therefore, the objectives of thesis was to characterize the pharmacokinetics and auto-induction process of vatalanib using population pharmacokinetic approaches, and to evaluate the relationship between systemic exposure to vatalanib and its toxicity in order to achieve optimal therapy in the treatment of MDS with standard fixed vatalanib doses. In this thesis, we characterized the time dependent pharmacokinetics of vatalanib employing both the standard two-stage approach with maximum a posteriori Bayesian estimation method in Chapter 2 and the nonlinear mixed effects modeling approach in Chapter 3 with sparse sampling data from a phase II clinical study, in which adult MDS patients received vatalanib orally once daily on a 28-day cycle of therapy. A one compartment model with a time dependent oral clearance term and lagged first order absorption was determined to be the best structural model for both approaches. The mean parameter estimates from both approaches were similar, probably due to similar model structure and the same underlying model assumptions. The mean apparent oral clearance was estimated to increase to approximately 2-fold after the enzyme auto-induction. The relationship between pharmacokinetic parameters and subject specific covariates was assessed using simple linear regression analysis in Chapter 2. In Chapter 3, graphical exploration, generalized additive models, and stepwise forward addition and backward elimination approaches were utilized for covariate modeling. Results from both chapters revealed that none of the available covariates was found to significantly influence the pharmacokinetics of vatalanib. Finally, exposure toxicity analysis was carried out to characterize the relationship between the systemic exposure to vatalanib and its toxicity. Both simple logistic regression and multinomial logistic regression approaches were utilized. Results from both analyses showed no correlation between the vatlanib exposure and the magnitude of its toxicity.

Basic health science research, which includes cell culture, typically underpins clinical and toxicological research. The results are used to predict biological effects of xenobiotics, e.g. environmental toxins and drugs, in humans. A goal of this research program was to apply aspects of quantitative redox biology to three separate, but interrelated projects that address improvements that can be made to evidence-based biological and toxicological research. This includes using absolute quantitation: to improve the specification of dose of xenobiotics added to cell culture systems; to determine absolute differences between the antioxidant capacity of tumor and normal cells; and to predict the implications of this new knowledge on the use of pharmacological ascorbate as an adjuvant in cancer therapy. Dose is a central parameter in determining the biological consequences of a xenobiotic; however, the dose of a xenobiotic at which these consequences are observed is dependent not only on biological variables, but also the physical aspects of cell culture experiments (i.e. cell number, medium volume). This is often overlooked due to the unrecognized ambiguity in the dominant metric used to express dose, i.e. initial concentration of xenobiotic. We hypothesized that specifying the dose of xenobiotics absolutely (as moles of xenobiotic per cell; mol cell-1) will reduce this ambiguity and provide additional information that is difficult to discern when traditional dosing metrics (initial concentration) are used. We investigated the use of mol cell-1 as an informative dosing metric using two model compounds: 1,4-benzoquinone and oligomycin A. When the dose of these two compounds was specified as mole cell-1, the toxicity observed was independent of the physical conditions used (i.e. number of cells, volume of medium). This makes it a scalable dosing metric that reduces ambiguity between experiments having different physical conditions; allows direct comparison between different cell types; addresses the important issue of repeatability of experimental results, and could increase the translatability of information gained from in vitro experiments. We utilized quantitative methods to explore the absolute differences in the ability of tumor vs. normal cells to remove H2O2 and how this impacts the use of pharmacological ascorbate as an adjuvant in cancer therapy. Ascorbate (AscH-, vitamin C) functions as a versatile reducing agent. At pharmacological doses (P-AscH-, plasma levels ≥ 20 mM), achievable through IV delivery, the oxidation of ascorbate can produce a high flux of H2O2 in tumors. We hypothesized that the increased sensitivity of tumor cells to P-AscH- compared to normal cells (i.e. non-transformed) is due to their lower capacity to remove H2O2. The rate constants (kcell) for removal of H2O2 revealed a differential in the capacity of cells to remove H2O2, with the average kcell for normal cells (N = 10) being twice that of tumor cells (N = 15). The ED50 of P-AscH- correlated directly with the capacity of cells to remove H2O2. Quantitation made it possible to make comparisons across very different cell lines on an absolute basis. These results indicate that the capacity of cells to remove H2O2 varies widely and in vivo measurement of this may predict which tumors may respond best to P-AscH- therapy. By designing experiments that begin with a quantitative dosing metric and utilize quantitation to produce absolute information from the results of experiments, we can better leverage data. We propose that this will lead to better predictions from such experiments. These enhancements to in vitro cell culture studies will increase the success in translation of data from in vitro experiments to in vivo animal studies and ultimately impact the success of extrapolation of basic science research to human clinical studies.

3,5,6-trichloro-2-pyridinol, when administered in the diet, increases the feed efficiency (the ratio of weight gain to feed consumed) in various species of domestic animals and also appears to be retained in the liver at low concentrations, possibly by binding to a specific macromolecule. Because of its structural similarities to the outer ring of the thyroid hormones, trichloropyridinol and three structural analogs (2,4,5-trichlorophenol, 4-bromo-2,5- dichlorophenol, or 4-i odo-2,5-dichlorophenol) were tested for ability to compete in vitro with 3, 5, 3' -triiodothyronine (T ) for nuclear recept ors specific for the thyroid hor- 3 manes . All four of the halogenated compounds were found to be weakly competitive for the receptor, indicating a passible anti-thyroid effect. Weanling male rats were fed diets containing 5, 50, and 500 ppm trichloropyridinol for 30, 60, and 90 days. Other groups received a diet containing 200 and 2000 ppm 2-thiouracil, a known thyroid toxicant. Both chemicals significantly reduced serum thyroxine (T4) levels in a dose-related manner. It was of interest that trichloropyridinol was about as levels. potent Serum T3 as thiouracil in suppressing serum T 4 and 3,3'5'-triiodothyronine (rT3) levels were depressed by thiouracil, but generally not with trichloropyridinol. Nuclear T binding capacity was not 3 changed in the liver of rats ingesting trichloropyridinol. Body weight, feed efficiency, and organ weights and histology were not significantly altered by the chronic ingestion of trichl oropyridinol. In conclusion, the effects trichloropyridinol upon animal growth and feed efficiency may involve various mechanisms surrounding thyroid hormone expression including reduction of serum T4 levels and competition with T3 for the nuclear receptor.