Arsenic is a metalloid present throughout the world, and the primary sources of exposure are through air, soil, and water. Arsenic is currently ranked as the most hazardous substance among environmental toxicants, and is well recognized as a human carcinogen, as well as a contributor to metabolic and cardiovascular diseases. However, cardiovascular effects have been mostly evaluated in epidemiological studies, and the direct mechanisms of pathogenesis remain largely unknown. The scope of studies described in this dissertation characterizes the cardiovascular pathophysiology associated with exposure to environmentally-relevant arsenic concentrations (100 µg/L), and attempts to elucidate the molecular mechanisms behind impaired vascular function. The effects of chronic arsenic exposure on blood pressure regulation were examined using a mouse model exposed to 100 µg/L for 22 weeks. Chronic exposure to arsenic results in the development of hypertension and concentric left ventricular hypertrophy. Furthermore, data presented here demonstrates that in utero exposure contributes to the development of metabolic syndrome throughout adulthood. Results indicate the development of hyperglycemia, hypercholesterolemia and nonalcoholic fatty liver disease. Mechanistic studies demonstrate the effects of arsenicals on endothelial nitric oxide synthase (eNOS) and its role in arsenic-induced vascular relaxation impairment. Biochemical assessment of eNOS conclude that decreased nitric oxide availability does not occur through alterations in protein levels or phosphorylation changes; however, decreased activity is likely a result of protein dimer stability through alterations in zinc tetrathiolate binding.

Arsenic is common metalloid that is found globally. Its ubiquitous nature means that large portions of the global population are exposed through a variety of pathways. Arsenic is a known human carcinogen and its role in the development of cardiovascular and metabolic disease has become more completely characterized in the past decades. However, the examination of arsenic exposure during embryonic development at relatively low level exposures is an emergent area where lots of questions remain unanswered. As arsenic is difficult and costly to remove from water, the investigation of exposures in vulnerable populations at relevant concentrations is necessary to justify remediation efforts. This dissertation work examines fetal arsenic metabolism contributing to the understanding of tissue specific arsenic effects during embryonic development. Following that, the focus shifts to fetal and early life exposure to arsenite at 100 parts per billion in drinking water (10 times higher than the EPA mandated limit in municipal water but common in well water globally) and how it contributes to the severity and incidence of diet-induced non-alcoholic fatty liver disease (NAFLD). NAFLD is the most prevalent chronic liver disease in the United States and it contributes to increased cardiovascular morbidity and mortality. NAFLD also results in alterations in hepatic drug metabolism and disposition which contributes to adverse drug reactions. We demonstrate similar effects in arsenic potentiated NAFLD in addition to changes in transporter expression with arsenic exposure alone independent of triglyceride accumulation associated with NAFLD. Taken together, this work highlights deleterious health effects of low level arsenic exposure during development and demonstrates the need for further investigation of developmental arsenic effects.

The aryl hydrocarbon receptor (Ah-receptor) is a member of the bHLH-PAS protein family. The Ah-receptor is a ligand dependent transcription factor, which activates a wide range of genes, most notably the xenobiotica metabolising genes, CYP1A1 and CYP1A2. The biological function of the Ah-receptor is still unknown and an endogenous ligand has yet not been identified. A possible Ah-receptor ligand is 6-formylindolo[3,2-b]carbazole (FICZ). FICZ has a high affinity for the Ah-receptor and is rapidly metabolised by CYP1A1, CYP1A2 and aldehydeoxidase (AOX). To try to trap FICZ or other possible endogenous Ah-receptor ligands, the metabolising enzymes CYP1A1, CYP1A2 and AOX were blocked. This was achieved through chemical blockage of CYP1A1 and CYP1A2 by ellepticin and through silencing with siRNA directed against CYP1A1 and CYP1A2. Successful blockage would be seen as an increase in Ah-receptor dependent XRE-luciferase activity. Chemical blockage of AOX with tungstate did not affect FICZ-dependent XRE-luciferase activation which could indicate that HepG2 cells lack AOX. The chemical blockage of CYP1A1 and CYP1A2 with ellepticin modified the XRE-luciferase response, but did not completely block Ah-receptor activation. In addition it is possible that ellepticin is a ligand for the Ah-receptor. The blockage of CYP1A1 by siRNA was successful; a silencing of CYP1A1 mRNA by at least 50 percent was detected. However due to lack of time it was not tested if the blockage of CYP1A1 and CYP1A2 was sufficient to trap Ah-receptor ligands.

Syftet med studien är att belysa hur barn i åldrarna tre till fem år tänker kring genus. Barnen fick lyssna på sagan ”Prinsessan Papperspåse” (Robert N. Munsch, 1980) och intervjuades sedan med utgångspunkt från sagan. Resultatet visar att barnen tänker relativt lika kring genus oberoende av kön och ålder. Både flickorna och pojkarna hade tydliga föreställningar om vad som är ett acceptabelt uppträdande av det motsatta könet. Framför allt hade de klara tankar om hur en flicka ska se ut om hon ska gifta sig. Att vara smutsig är för barnen inte acceptabelt, vare sig man är flicka eller pojke. Utan undantagsvis lade flickorna fokus på relationen mellan prinsessan och prinsen medan pojkarna lade fokus mer på draken och prinsen.

Breast cancer is one of the deadliest forms of cancers in the world. About 15–30% of all breast cancers are HER2-positive, which involves overexpression of HER2-receptors on the surface of tumor cells. HER2-positive breast cancer is viewed as an aggressive form of cancer because the overexpression of the HER2-receptor causes dimerization with other receptors of the same family; together they bind to ligands and become activated. The activation causes fast, uncontrolled cell proliferation that often results in the formation of a tumor. Cancer can be divided into different stages, at stage 4 the cancer cells from the original tumor breaks away, follows the bloodstream, and forms metastases in other places of the body. When cancer evolves and becomes metastatic the prognosis drastically worsens, and the treatment options are limited. Patients often need several lines of cancer treatment. The first line of treatment is usually trastuzumab in combination with pertuzumab and a taxane, second line is usually trastuzumab emtansine. There is no conclusive third line treatment for HER2-positive metastatic breast cancer (MBC). Due to the development of new anti-HER2 treatments over the last two decades, less patients are dying from breast cancer, however most patients diagnosed with HER2-positive MBC are estimated to face an early death.  The objective of this study was to analyze the pharmaceutical advances that has improved the prognosis for patients diagnosed with HER2-positive MBC. The material, on which this study was based on, was obtained from the Pubmed database via the Linnaeus University Library. Five articles were chosen based on criteria relevant to the topic. The articles were published between 2001 and 2021; all of them were randomized controlled trials (RCT).  The subject of the articles was to compare the efficacy and safety of different forms of anti-HER2 treatments, using patient populations diagnosed with HER2-positive MBC. Mainly the patients' disease progression, treatment response and survival time was analyzed. Pharmaceutical safety was assessed by the rate of adverse events. A total of 2513 patients participated in the studies. Among all the different treatment options that were analyzed in the articles, one treatment combination yielded some of the best results. Pyrotinib in combination with capecitabine increased the disease progression-free time, had the highest proportion of patients who responded to treatment as well as the highest proportion of patients with size-reducing lesions for the longest time. However, the patient group receiving pyrotinib also had the highest incidence of serious adverse events and had the largest percentage of patients who chose to discontinue the study due to adverse events.  Analysis of the five articles concludes that the prognosis for patients diagnosed with HER2-positive MBC has been improved by pharmaceutical advances regarding tyrosine kinase inhibitors, pan-HER inhibitors, combination therapy with monoclonal antibodies (single, mixed, modified or conjugated with other drugs) and chemotherapy with different mechanisms of action. The results from the studies indicated that treatment with a single anti-HER2 drug had the lowest effectiveness, and that some drug combinations had better synergistic effects than others, reflecting on patient survival data. Despite the pharmaceutical advances of the past two decades, the prognosis for HER2-positive MBC can still be considered bleak due to its high death rate. Resistance to anti-HER2 drugs is an ongoing concern that requires more research and development of new treatments.

The risk of breast cancer transiently increases immediately following pregnancy. Hispanic women have one of the highest rates of postpartum breast cancers of all racial/ethnic minority groups in the US. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. In this study, we measured the miRNA expression of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n=12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n=44). We identified 15 miRNAs that are differentially expressed between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression; miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 were overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were under expressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Taken together, the results of this study show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.

Adverse drug reactions (ADRs) are a pervasive complication in the realm of pharmacotherapy. At the root of ADRs lies interindividual variability in drug response, which can range from allergic reactions, to genetic variability, to any factors that influence the pharmacokinetics of a drug. Nonalcoholic steatohepatitis (NASH) is the late-stage of non-alcoholic fatty liver disease (NAFLD), characterized by fat deposition, oxidative stress, inflammation, and fibrosis. Over the last several years, alterations in drug metabolizing enzymes and transporters have been broadly characterized through NAFLD progression. Multidrug resistance-associated protein 2 (MRP2) is a canalicular efflux transporter that directs the biliary elimination of a wide variety of xenobiotics and metabolites. In NASH, MRP2 is mislocalized away from the canalicular membrane in a post-translational event. The mechanism and extent of this mislocalization has yet to be elucidated. While transporter misregulation has been shown to influence the disposition of a variety of substrates, the direct impact of MRP2 mislocalization on its overall transport capacity, and pharmacologic consequence of this change, is unknown. The purpose of this study was to elucidate the mechanism behind, and functional consequence of, MRP2/Mrp2 mislocalization in NASH, predominantly using the rodent methionine-and-choline-deficient (MCD) dietary model.To identify the mechanism of MRP2/Mrp2 mislocalization, a comparison of the activation status of various mediators of MRP2/Mrp2 retrieval was conducted between healthy and NASH livers. Results in rat samples and human NASH samples indicate that activation changes of these mediators, including radixin, PKCα, PKCδ, and PKA, are consistent with a shift toward active retrieval of MRP2/Mrp2 from the membrane, and some evidence of impaired membrane insertion is also present. Measurement of Mrp2 transport capacity was completed using pemetrexed, a novel Mrp2 probe substrate. Comparison of biliary excretion of pemetrexed between wild-type and Mrp2^(-/-) rats shows a 100% decrease, confirming that it relies upon Mrp2 for biliary excretion. NASH rats exhibited a 60% decrease in pemetrexed levels in the bile compared to their control counterparts, indicating that Mrp2 transport capacity is severely impaired in NASH rats. Finally, to ascertain the pharmacologic consequence of impaired Mrp2 transport, a study was conducted measuring the effects of the active morphine glucuronide on control and NASH rats. NASH rats exhibited a decreased biliary excretion, and increased systemic retention, of M3G. While they did also exhibit increased antinociception of M6G, the definitive impact of altered disposition on pharmacologic response was masked due to the interference of an MCD dietary effect on antinociception. Overall, the data reported herein identify active membrane retrieval as a mechanism of MRP2/Mrp2 mislocalization in NASH, and that mislocalization results in a 60% decrease in overall Mrp2 transport capacity. This decrease significantly hinders biliary excretion of Mrp2 substrates, and may result in ADRs by contributing to interindividual variability in drug response.

Uranium has been mined for many years and used for fuel for nuclear reactors and materials for atomic weapons, ammunition, and armor. While the radioactivity associated with uranium mining has been linked to the development of lung and kidney cancers, and leukemia, little is known about the direct chemical genotoxicity of uranium. The overall hypothesis of the current research is that uranium can produce DNA damage by chemical genotoxicity mechanisms. Three specific aims were tested. In Aim 1, specific DNA lesions caused by direct interaction of uranium and DNA were investigated. Chinese Hamster Ovary cells (CHO) with mutations in various DNA repair pathways were exposed to 0 – 300 μM of soluble depleted uranium (DU) as uranyl acetate (UA) for 0 – 48 hr. Results indicate that UA readily enters CHO cells, with the highest concentration localizing in the nucleus. Clonogenics assay shows that UA is cytotoxic in each cell line with the greatest cytotoxicity in the base excision repair deficient EM9 cells and the nuclear excision repair deficient UV5 cells compared to the non-homologous end joining deficient V3.3 cells and the parental AA8 cells after 48 hr. This indicates that UA is forming DNA adducts that may be producing single strand breaks through hydrolysis rather than double strand breaks in CHO cells. Fast Micromethod® results indicate an increased amount of single strand breaks in the EM9 cells after 48 hr UA exposure compared to the V3.3 and AA8 cells. In Aim 2, the role of oxidative stress in producing DNA lesions was determined. Cellular oxidative stress has been implicated in the genotoxicity of many heavy metals as a mechanism of induced DNA damage. To investigate this possible mechanism, human bronchial epithelial cells (16HBE14o⁻) were exposed to 30 ppb (0.13 μM U) UA for 2 – 24 hr. UA did not significantly induce oxidative stress compared to untreated cells at 3 – 4 hr time points. These results suggest that cellular oxidative stress is not a major pathway of DU genotoxicity at low concentrations. In Aim 3, DNA damage response to uranium-induced DNA damage was investigated. It has been widely reported that metals can be genotoxic by inhibiting DNA repair. Cultured cells were co-exposed to 0.13 μM UA in the presence of 0 – 25 μM of etoposide for 0 – 48 hr. Results indicate that UA inhibited double strand break repair. Coexposures of etoposide and UA synergistically induced cytotoxicity compared to individual treatments and untreated cells. Co-exposed UA and etoposide treated 16HBE14o⁻ cells exhibited a decrease in phosphorylation of DNA repair proteins compared to etoposide treatments. Untreated and UA-treated 16HBE14o⁻ cells did not induce phosphorylation of DNA repair proteins. These results suggest that DU inhibits double strand break DNA repair at low concentrations in the presence of a known DNA double-strand damaging agent, etoposide. The inhibition of DNA repair by DU at environmentally relevant concentrations suggests a novel means by which uranium may exert its genotoxic effects. Results found at low dose exposures are not consistent with alterations seen with radioactivity, suggesting that the effects of uranium at low doses are due to its chemical genotoxic effects. Understanding how uranium reacts with DNA is important to better understand how this suspected carcinogen induces cancer and to help to elucidate mechanisms that produce cancers in people exposed to uranium.

Chronic environmental exposure to inorganic arsenic is widely associated with human disease. Low human arsenic secondary methylation efficiency (SME), represented by high urinary monomethylarsonic acid (%uMMA) and low urinary dimethylarsinic acid to monomethylarsonic acid ratio (uDMA/uMMA), has been consistently associated with increased risk of arsenic-related diseases. Therefore the determination of factors modulating arsenic SME acquires particular importance. The aims of the present study are to identify novel factors of variability in arsenic secondary methylation, and to test for potential factors influencing arsenic SME for which there is equivocal literature support. A population of 808 subjects was recruited from northwest Mexico environmentally exposed to arsenic. The mean total urinary arsenic in the population was 171 μg/L. Great interindividual variability in %uMMA excretion was observed (0.85% - 40.5%). Three intronic polymorphisms in arsenic (3+ oxidation state) methyltransferase (AS3MT), the key gene in the metabolism of arsenic, were confirmed to be associated with increased arsenic SME in this study. Further analysis of this genomic region showed a large block of linkage disequilibrium (LD) comprising these three genetic variants and other 43 intronic polymorphisms within AS3MT and four additional genes. Genetic association analysis showed that all linked polymorphisms in this region except one were significantly associated with higher arsenic SME. The existence of this long region of LD associated with arsenic SME underscores the complexity of association studies involving any of these linked polymorphisms since there is no certainty of which polymorphism or gene is the causative of the association. In addition, a strong positive association between body mass index (BMI) and arsenic SME was observed in females but not in males. This association was replicated in two independently recruited populations of adult women. Moreover a unique finding of this study is the association between higher genetically estimated indigenous American (AME) ancestry and increased arsenic SME in this ancestrally admixed Mexican population. These results establish the importance of genetic and phenotypic factors in the efficiency of arsenic secondary methylation. Furthermore this study has identified several arsenic-associated risk factors that should be carefully considered in future studies seeking to better understand disease susceptibility in arsenic-exposed populations.

The immune system is a target of arsenic toxicity. Epidemiological data have shown that arsenic exposure is associated with characteristics of immunosuppression. Human B-lymphoblastoid cell lines (LCL) were used as an in vitro model of immune cell targeting by arsenic to investigate the mechanism of arsenic-induced cytotoxicity, which could provided insight into the mechanism underlying arsenic-induced immunotoxicity leading to the immunosuppression observed in humans. In LCL arsenite-induced cytotoxicity was not associated with apoptosis, but associated with hallmarks of autophagy, a cell stress-responsive process that facilitates the removal of cellular components through lysosome-mediated degradation. At environmentally relevant concentrations, arsenite-induced toxicity resulted in a decrease in cell proliferation that was correlated with hallmarks of autophagy including expansion of acidic vesicles, global induction of lysosomal gene expression, increased flux of the autophagosome marker LC3-II, and increased enzymatic activity of the lysosomal hydrolase cathepsin D. Investigation of the upstream cellular damage leading to the induction of autophagy revealed that arsenite induces proteotoxic damage leading to an accumulation of protein aggregates that may be targeted to the lysosome for degradation. In addition, global gene expression data showed an enrichment of ER stress responsive genes after arsenite exposure. Further evaluation of global gene expression data indicated that the global induction of lysosomal genes occurs before the activation of ER stress genes, suggesting that the induction of autophagy may occur before the generation of ER stress. To investigate the effect of arsenite-induced proteotoxicity and autophagy on normal immune function, the ability of LCL to process and present exogenous antigens onto MHC class II molecules was evaluated. Arsenite decreased antigen presentation of the exogenous antigen HSA. This decrease was associated with decreased lysosomal degradation of the model substrate DQ-Ova, suggesting that arsenite is disrupting lysosome-mediated degradation. In addition, arsenite exposure was associated with an increase in MHC class II protein aggregates, which could render them unavailable to bind peptide fragments. Through the identification that arsenite induces proteotoxicity and autophagy in LCL, it provides novel insight into the mechanisms of arsenic-induced immunotoxicity that could lead to a better understanding of the mechanisms underlying arsenic-induced immunosuppression observed in humans.