Jonathan A. Doorn

Humans are exposed to a myriad of environmental pollutants, with recent evidence indicating several of these toxicants serve as risk factors for neurodevelopmental disorders and neurodegenerative diseases. Given this, there is a need for both interventional and protective strategies; however, of concern, the mechanistic targets of these environmental pollutants are variable or unknown in some cases. A prior report indicated that analogs of the natural product fraxinellone act as potent NRF2 activators, mitigating excessive reactive oxygen species (ROS) generation and Glu toxicity in vitro. Using one of the most effective fraxinellone analogs (i.e., analog 2) for NRF2 activation identified, we sought to determine the range of protection, in vitro, against a panel of neurotoxicants with varying mechanisms for adverse effects, including 6-hydroxydopamine (6-OHDA), organochlorine pollutants, and a fungicide. The data for analog 2 were compared to those for a structurally similar but inactive analog (i.e., analog 1). The dose–response for each toxicant with PC12 and SH-SY5Y cell lines was determined. Interestingly, the fraxinellone analog provided significant protection against all agents screened: 6-OHDA, dieldrin, benomyl, PCB52 hydroxy and sulfate metabolites, and rotenone. The extent to which the fraxinellone analog mitigated toxicity varied for each toxicant. In all cases, pretreatment with analog 2 significantly decreased total cellular ROS production, and in addition, generation of mitochondrial ROS via rotenone was mitigated. Furthermore, analog 2 provides some degree of restoration of cell viability following rotenone insult. In summary, our data indicate that an analog of the natural product fraxinellone potently inhibited ROS production and toxicity, thereby protecting cells against a panel of agents with varying mechanisms from adverse outcomes.

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Polychlorinated biphenyls (PCBs) are industrial chemicals that are ubiquitously found in the environment. Exposure to these compounds has been associated with neurotoxic outcomes; however, the underlying mechanisms for such outcomes remain to be fully understood. Recent studies have shown that astrocytes, the most abundant glial cell type in the brain, are susceptible to PCB exposure as well as exposure to human-relevant metabolites of PCBs. Astrocytes are critical for maintaining healthy brain function due to their unique functional attributes and positioning within the neuronal networks in the brain. In this study, we assessed the toxicity of PCB52, one of the most abundantly found PCB congeners in outdoor and indoor air, and two of its human-relevant metabolites, on astrocyte mitochondria. We exposed C6 cells, an astrocyte cell line, to PCB52 or its human-relevant metabolites and found that all the compounds showed increased toxicity in galactose-containing media compared to that in the glucose-containing media, indicating the involvement of mitochondria in observed toxicity. Additionally, we also found increased oxidative stress upon exposure to PCB52 metabolites. All three compounds caused a loss of mitochondrial membrane potential, distinct changes in the mitochondrial structure, and impaired mitochondrial function. The hydroxylated metabolite 4-OH-PCB52 likely functions as an uncoupler of mitochondria. This is the first study to report the adverse effects of exposure to PCB52 and its human-relevant metabolites on the mitochondrial structure and function in astrocytes.

Numerous insults, both endogenous (e.g., glutamate) and exogenous (e.g., pesticides), compromise the function of the nervous system and pose risk factors for damage or later disease. In previous reports, limonoids such as fraxinellone showed significant neuroprotective activity against glutamate (Glu) excitotoxicity and reactive oxygen species (ROS) production in vitro, albeit with minimal mechanistic information provided. Given these findings, a library of novel fraxinellone analogs (including analogs 1 and 2 described here) was synthesized with the goal of identifying compounds exhibiting neuroprotection against insults. Analog 2 was found to be protective against Glu-mediated excitotoxicity with a measured EC50 of 44 and 39 nM for in vitro assays using PC12 and SH-SY5Y cells, respectively. Pretreatment with analog 2 yielded rapid induction of antioxidant genes, namely, Gpx4, Sod1, and Nqo1, as measured via qPCR. Analog 2 mitigated Glu-mediated ROS. Cytoprotection could be replicated using sulforaphane (SFN), a Nrf2 activator, and inhibited via ML-385, which inhibits Nrf2 binding to regulatory DNA sequences, thereby blocking downstream gene expression. Nrf2 DNA-binding activity was demonstrated using a Nrf2 ELISA-based transcription factor assay. In addition, we found that pretreatment with the thiol N-acetyl Cys completely mitigated SFN-mediated induction of antioxidant genes but had no effect on the activity of analog 2, suggesting thiol modification is not critical for its mechanism of action. In summary, our data demonstrate a fraxinellone analog to be a novel, potent, and rapid activator of the Nrf2-mediated antioxidant defense system, providing robust protection against insults.

The disposition and toxicity of lower chlorinated PCBs (LC-PCBs) with less than five chlorine substituents have received little attention. This study characterizes the distribution and metabolomic effects of PCB 52, an LC-PCB found in indoor and outdoor air, three weeks after intraperitoneal exposure of female Sprague Dawley rats to 0, 1, 10, or 100 mg/kg BW. PCB 52 exposure did not affect overall body weight. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis identified PCB 52 in all tissues investigated. Hydroxylated, sulfated, and methylated PCB metabolites, identified using GC-MS/MS and nontarget liquid chromatography-high resolution mass spectrometry (Nt-LCMS), were primarily found in the serum and liver of rats exposed to 100 mg/kg BW. Metabolomic analysis revealed minor effects on L-cysteine, glycine, cytosine, sphingosine, thymine, linoleic acid, orotic acid, L-histidine, and erythrose serum levels. Thus, the metabolism of PCB 52 and its effects on the metabolome must be considered in toxicity studies. Highlights: PCB 52 was present in adipose, brain, liver, and serum 3 weeks after PCB exposure Liver and serum contained hydroxylated, sulfated, and methylated PCB 52 metabolites Metabolomics analysis revealed minor changes in endogenous serum metabolites Levels of dopamine and its metabolites in the brain were not affected by PCB 52

Exposure to polychlorinated biphenyls (PCBs) is associated with developmental neurotoxicity and neurodegenerative disorders; however, the underlying mechanisms of pathogenesis are unknown. Existing literature has focused mainly on using neurons as a model system to study mechanisms of PCB-mediated neurotoxicity, overlooking the role of glial cells, such as astrocytes. As normal brain function is largely astrocyte-dependent, we hypothesize that astrocytes play an important role in PCB-mediated injury to neurons. We assessed the toxicity of two commercial PCB mixtures, Aroclor 1016 and Aroclor 1254, and a non-Aroclor PCB mixture found in residential air called the Cabinet mixture, all of which contain lower chlorinated PCBs (LC-PCBs) found in indoor and outdoor air. We further assessed the toxicity of five abundant airborne LC-PCBs and their corresponding human-relevant metabolites in vitro models of astrocytes, namely, the C6 cell line and primary astrocytes isolated from Sprague-Dawley rats and C57BL/6 mice. PCB52 and its human-relevant hydroxylated and sulfated metabolites were found to be the most toxic compounds. No significant sex-dependent cell viability differences were observed in rat primary astrocytes. Based on the equilibrium partitioning model, it was predicted that the partitioning of LC-PCBs and their corresponding metabolites in biotic and abiotic compartments of the cell culture system is structure-dependent and that the observed toxicity is consistent with this prediction. This study, for the first time, shows that astrocytes are sensitive targets of LC-PCBs and their human-relevant metabolites and that further research to identify mechanistic targets of PCB exposure in glial cells is necessary.

Polychlorinated biphenyls (PCBs) are a class of legacy, and also current inadvertently produced, toxic persistent chemicals found in building materials and consumer products. PCB sulfates are metabolic products derived from hydroxylated metabolites of PCBs (OH-PCBs). Both OH-PCBs and PCB sulfates exert multiple toxicological effects on human health such as disrupting thyroid hormone transport and inhibiting steroid sulfotransferases (SULT1E1 and SULT2A1). Although PCB 11 sulfate has been previously detected in human serum samples, the lack of a generally applicable method for a broad range of PCB sulfates in human serum has limited our understanding of their prevalence and importance. This thesis describes studies on how we successfully developed a method for extraction and quantification of PCB sulfates from human serum. Our method employs acetonitrile extraction of the PCB sulfates from serum followed by differential analysis with, and without, hydrolysis to OH-PCBs catalyzed by a purified sulfatase from crude Helix pomatia juice using a tyrosine ethyl ester (TEE)-Sepharose affinity column. Our results on the specificity of the purified enzyme for PCB sulfates indicate the feasibility of its use for their quantitative hydrolysis. OH-PCBs are quantified by GC/MS/MS after derivatization to their corresponding methoxy PCBs. In a pooled human serum sample, we identified ten PCB sulfate congeners above the limit of quantitation with statistical evidence to validate their significance, with three PCB sulfate congeners exhibiting the highest concentrations from 1200-3970 pg/g of serum. Application of our method in serum samples collected from 24 individuals (6 mother-daughter and 6 mother-son dyads) from a rural community (Columbus Junction, Iowa) and 24 subjects (6 mother-daughter and 6 mother-son dyads) from an urban community (East Chicago, Indiana) also brought us valuable information. We found 8 PCB sulfate congeners that contributed the most to the total PCB sulfates in these samples, and these were all lower-chlorinated congeners: 4-PCB 2 sulfate, 4’-PCB 2 sulfate, 2’-PCB 3 sulfate, 4’-PCB 3 sulfate, 4-PCB 11 sulfate, 4’-PCB 18 sulfate, 4’-PCB 25 sulfate, and 4-PCB 52 sulfate. The urban community was known to have a history of PCB-utilizing industries, whereas the rural community had no known current or past industrial uses of PCBs. We detected significantly higher levels of total PCB sulfates in the urban group (110-8900 ng/g fresh weight of serum, mean = 3351 ng/g, S.E.= 300, N=24) than in the rural group (530-6700 ng/g fresh weight of serum, mean = 1785 ng/g, S.E.=500, N=24), however, based on previous studies, the total serum PCB levels of individuals from these two areas have no significant difference. This suggests that the serum levels of many PCBs do not fully reflect the actual composition of PCB exposures in humans. Our work contributes to understanding of PCB metabolism in humans as well as guiding future research on the relationship of PCB exposure to their metabolite concentrations from biological samples. Also, the results from our study provide clues for future research directions such as PCB metabolite determination in human urine samples and how this may affect steady-state concentrations and toxicities of OH-PCBs and PCB sulfates in serum and tissues.

Metal oxide nanomaterials (NMs) have been used in various applications and they possess unique properties that differ from their bulk materials; however, there are risks associated with inadvertent exposure, especially through inhalation, that need to be assessed and understood. Toxicity assessment of inhaled metal oxide NMs has been widely studied to investigate pulmonary toxicity and inflammation. Copper oxide (CuO) NMs are one of the most toxic metal oxide NMs and are widely used in several fields. Therefore, CuO NMs were chosen as a candidate to further study in terms of biodistribution, translocation to secondary organs, and immunomodulatory effects.The first part of this research was to perform characterizations and in vitro toxicity screening of metal oxide NMs independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3, and MgO. NMs that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser-explored WO3 NMs were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide NMs in a nose-only exposure system, and toxicity outcomes (cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 hr after the start of exposure were assessed. Among the NMs studied, the highest toxicity was observed for CuO NMs and ZnO NMs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there were significant increases in levels of neutrophils and proinflammatory cytokines, and a loss of macrophage viability, in the bronchoalveolar lavage fluid from mice exposed to ZnO NMs and CuO NMs at both 4 hr and 24 hr but not for mice exposed to V2O5 NMs or WO3 NMs. These effects were observed in both female and male mice. The cell viability assays performed after in vitro exposure to NMs using viable cell counts with propidium iodide staining, and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict NM acute toxicity. It has been shown that CuO NMs induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NMs from the respiratory tract. Therefore, the second part of this dissertation aimed to observe the kinetics of pulmonary toxicity during and after CuO NM sub-acute inhalation exposure and investigate copper (Cu) biodistribution and clearance rate from the exposure site as well as homeostasis of selected trace elements in secondary organs of BALB/c mice. Our studies demonstrated that sub-acute inhalation exposure to CuO NMs led to pulmonary toxicity represented by increases in lactate dehydrogenase (LDH), total cell counts, neutrophils, macrophages, inflammatory cytokines, and iron (Fe) levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed that Cu concentrations increased steadily during exposure and gradually declined after exposure. Cu elimination from the exposure site showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart, indicating that inhaled Cu could be translocated into the bloodstream and heart and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs. Furthermore, concentrations of selenium (Se) in kidneys and Fe in the spleen were decreased, pointing to disruption of trace element homeostasis. The next part of this dissertation was to develop a HDM-induced airway inflammation model and a vaccine (allergen immunotherapy, AIT) protecting against HDM-induced airway inflammation to be used as tools to investigate the immunomodulatory effect of CuO NMs. First, we established a HDM-induced airway inflammatory mouse model that resulted in increased TH2-dominant immunological responses and also boosted the number of TH17 cells. Therefore, it can be used to study allergic airway inflammation and potential treatments. Then, we developed HDM vaccines comprising 50 μg CpG nanoparticles with either 50 or 100 μg Der p1/Der p1 mixture (80:20). Both vaccine doses showed no significant reductions in total cell number, eosinophils, total IgE, and HDM allergen-specific IgE in the HDM-induced airway inflammation model. Nonetheless, a trend of greater IgG2a:IgG1 ratios in the vaccination groups compared to the HDM allergen-exposed group suggested a probable activation of TH1-immune responses. Furthermore, the vaccine 100 μg showed a trend toward fewer TH2-type immune responses compared to the vaccine 50 μg, although the differences were non-significant. Therefore, the vaccine 100 μg was used to investigate the effect of CuO NMs aerosols on immune responses. Lastly, we investigated the immunomodulatory effect of CuO NM aerosols under different health conditions, including naïve, asthmatic, and AIT-treated asthmatic mouse models. CuO NM-exposed naïve mice did not show a significant change in CD4+ T cell subtypes in the lung tissues. However, certain immune cell types such as neutrophils, CD11b+ DCs, and alveolar macrophages (AMs) significantly increased compared to naïve mice, indicating the presence of lung inflammation induced by CuO NM exposure. When asthmatic mice were exposed to CuO NM, increased TH1 and Treg cell levels and decreased TH2 cell levels in the lung tissue were observed compared to the non-exposed asthmatic mice. In AIT-treated asthmatic mice, the effect of CuO NM inhalation exposure had no noticeable effect on any of CD4+ T cell subtypes. The effect of CuO NM exposure on airway hyperresponsiveness was investigated in all 3 models, and there was no significant effect on airway sensitivity. However, there was a trend toward lower resistance and elastance in the asthmatic and AIT-treated asthmatic mice. In...

Tissue engineering offers new treatments for disorders such as temporomandibular joint disorder which among others frequently afflicts patients. In this thesis, tissue engineering strategies are explored with an emphasis on addressing bone tissue repair. The primary technique used was gene therapy in conjunction with a matrix to provide therapeutic growth factors and a foundation for new bone to form on, respectively. The first chapter describes the previous research of tissue engineering to address TMD; the next four chapters then describe optimizations and combination of gene therapy to improve current bone tissue engineering outcomes. The final chapter looks at the future of tissue engineering in the field of bone tissue repair with a focus on TMD.Damage or disease in the TMJ adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This chapter outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder. In the subsequent chapters, the goal was to investigate different bone tissue engineering approaches with gene therapy. The first was to identify if calcium ion (Ca2+) concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene‐activated matrices designed to promote bone growth. The results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI‐pDNA complexes in terms of cell viability and transfection efficiency, and a Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP‐2). In the third chapter, gene-activated matrices (GAMs) mineralized with a simulated body fluid (SBF) were prepared and implanted in rat calvarial defects. The optimal GAM consisted of a collagen sponge with PEI-pDNA complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared to 19% for control samples. A follow up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was 5 parts of pBMP-2 to 3 parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding FGF-2 and BMP-2 can optimally improve bone regeneration outcomes. The next chapter focuses on the development of a system to control the formation of bone to complement developments that have enabled potent regeneration of bony tissue. Scaffolds were fabricated with chemically modified RNA encoding for bone morphogenetic protein-9 (cmBMP9) and capped with salicylic acid (SA)-containing polymer (SAPAE) with the goal to determine if SAPAE could inhibit the formation of bone in a pilot animal study since cmBMP9 has been demonstrated to be highly effective in regenerating bone in a rat calvarial defect model. The results indicated that cmBMP9 increased bone formation (30% increase in area covered compared to control) and that SAPAE trended toward reducing the bone formation. These results suggest SAPAE could be useful as a chemical agent in reducing unwanted bone formation in implants loaded with cmBMP9. The final chapter investigates synthesis of chemically modified RNA and the application of these cmRNAs to bone defects. EGFP cmRNA was first synthesized to measure transfection efficiency and viability of BMSCs transfected with PEI-cmRNA complexes. Then BMP-2 and FGF-2 cmRNAs were created from plasmid DNA. These cmRNAs were demonstrated to increase protein expression in vitro. The cmRNAs were then loaded into GAMs and used to treat rat calvarial defects. In conclusion, gene therapy is a promising method for improving outcomes related to bone tissue engineering, and the techniques and data described in this thesis are paramount in furthering this field. The main takeaways from this thesis are calcium can improve gene therapy efficiency, and chemically modified is a more potent form of gene therapy as compared to plasmid DNA when target bone tissue.