According to the Centers for Disease Control and Prevention, diarrheal diseases are the second leading cause of death in children under the age of 5 globally. Cryptosporidium and Giardia are two microscopic parasites that cause diarrhea, infecting both humans and animals. The current gold standard field method for collecting water samples for parasite testing (Environmental Protection Agency (EPA) 1623), is not practical for remote sample locations as it requires 10-L of water to be kept on ice during transport to testing facilities. A pilot study by the UW Center for One Health Research will implement a new water sampling system, known as Bag-Mediated Filtration System (BMFS), to look at how these parasites impact low resource communities. My goal was to determine if BMFS could be used in conjunction with EPA 1623 to give recovery rates consistent with the current EPA 1623 method. I went to 4 different locations in Washington to retrieve surface water samples using the BMFS kit. Sites were chosen based on the likelihood that there would be parasites present: sources with the possibility of animal waste and areas with turbid water. The environmental samples, along with 10 parasite-spiked laboratory samples were analyzed using the EPA method - after filtration using the bag. Parasite numbers were measured as oocysts per liter using immunofluorescence, and recovery rates of spiked samples were compared to acceptance criteria from the EPA. This study will be useful to water quality researchers working in rural or remote field sites, or areas where climate conditions make sample storage challenging.

Protein glutathionylation is a post-translational modification that can take place under mild oxidative stress. It occurs when the antioxidant glutathione (GSH) forms mixed disulfide bridges with low pKa cysteine residues on proteins. This may play a role in reversibly regulating the activity of proteins that have key cysteine residues or protecting those cysteines from further irreversible oxidation if oxidative stress worsens. GSH itself is synthesized in a two-step process, and the rate limiting step is catalyzed by glutamate-cysteine ligase (GCL), a heterodimer composed of the catalytic subunit (GCLC) and modifier subunit (GCLM). GCLM acts to increase the activity of GCLC. Thus, in the absence of GCLM, GCLC is less efficient in making GSH. We have generated Gclm null mice and these mice have liver GSH levels that are about 10% of normal, resulting in an oxidized thiol redox environment. An important question that has not been answered is whether Gclm null mice have different levels protein glutathionylation than Gclm wild-type mice. The oxidized thiol redox status of Gclm null mice is predicted to increase the level of protein glutathionylation. On the other hand, there may in fact be less protein glutathionylation in Gclm null mice because they have less GSH to begin with and there is a compensatory increase in sulfiredoxin and thioredoxin reductase expression, both of which are known to reverse glutathionylation. Using assays of protein glutathionylation, this project seeks to provide confirmation of the hypothesis that Gclm deficiency results in adaptive regulation of glutathionylation under different degrees of oxidative stress.

Intestine is responsible for the biotransformation of many orally-exposed chemicals. The nuclear receptor constitutive androstane receptor (CAR/Nr1i3) can be activated by various drugs and environmental chemicals, and in turn it is known to up-regulate the expression of many drug-processing genes (DPGs) in liver to facilitate xenobiotic biotransformation. However, relatively less is known regarding the effect of CAR on the basal and inducible expression of DPGs in various sections of intestine. To fill this critical knowledge gap, sixty-day-old wild-type and CAR-null mice were administered the CAR-ligand TCPOBOP (3mg/kg, i.p.) or vehicle once daily for 4-days. CAR-null mice had altered basal expression of many DPGs in a section-specific manner in intestine. Consistent with the liver data (Aleksunes and Klaassen, 2012), TCPOBOP up-regulated many DPGs in specific sections of small intestine in a CAR-dependent manner. In contrast, the mRNAs of certain DPGs were previously known to be up-regulated by TCPOBOP in liver but were not altered in intestine. Interestingly, many known CAR-target genes were highest expressed in colon where CAR is minimally expressed, suggesting that additional regulators are involved in regulating their expression. In conclusion, the present study has shown that CAR is critical in both maintaining the basal expression as well as the pharmacological regulation of certain DPGs in a section-specific manner of the intestine, and the CAR activation in liver and intestine produces overlapping but not identical results. Therefore, Identification of the xenobiotic responses to CAR activation in intestine is critical for further understanding the adverse drug-drug interactions for many orally-exposed chemicals.

People in agriculture commonly use pesticides to prevent insect damage on crops. One common pesticide is called acetamiprid (ACP). While this pesticide has low toxicity to mammals, there have been cases of acute toxicity from overexposure to this pesticide. In this study, we focused on pesticide handlers, people who apply pesticide to crops in the fields, from the Yakima valley and analyzed their urine samples to measure their exposure to the pesticide ACP. The primary goal of the study was to quantify urine biomarkers to ACP exposure, including the parent compound ACP, and two metabolites: acetamiprid-N-desmethyl (ACPDM) and 6-chloronicotinic acid (6-CNA). We collected urine samples from 10 pesticide handlers in the 24 hours after applying ACP in an orchard. Each person was given a liter sized sampling vial to collect each void. We collected 34 human samples in total. I identified and quantified the biomarkers in the human urine samples collected. I did this by processing the samples using Solid Phase Extraction (SPE) and then quantifying them by using high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). We expected to see small amounts of each biomarker in each sample, but found relatively high concentrations of the metabolite ACPDM. The results of this study will help to improve our ability to measure urinary markers of exposure, better estimate exposure and risk to pesticide handlers, understand the kinetics of ACP in the human body, and think of means to minimize exposure to prevent acute toxicity.

Wildland firefighters are trained to combat forest fires by prescribed burning or wildfire suppression. Prescribed burning is a planned fire lit intentionally by firefighters to reduce fire hazard and to provide greater safety for firefighters and the public. Wildland firefighters often work on controlled burns using one of two work tasks: lighting, which consists of laying a strip of fire on the forest floor; and holding, keeping fire inside specified boundaries. It is the exposure inherent to these work tasks that our research is primarily concerned with. During these operations, firefighters are exposed to various combustion products, including fine particulate matter (PM2.5), carbon monoxide, and polycyclic aromatic hydrocarbons (PAHs). Many PAHs are carcinogens that undergo metabolism and are excreted in urine. In order to measure the PAH exposure severity, urine samples from wildland firefighters were collected before and after prescribed burn operations as well as on non-burn days. Urine samples were hydrolyzed by treatment with sulfatase and glucuronidase enzymes, and cleaned up with solid phase extraction. High performance liquid chromatography with fluorescence detection was used to analyze the samples for PAH metabolites. Our data analysis compares PAH exposure by work task, by season, and between burn and non-burn days. We anticipate that urinary concentrations of PAHs in firefighters will be higher after the prescribed fire operation compared to pre-burn levels. Findings of this research can be used to measure PAH exposure not only in wildland firefighters, but also in other occupational or residential settings where people are exposed to high concentrations of PAHs to reduce risk factors of developing cancer.

Polio is on the cusp of worldwide eradication but remains endemic in Afghanistan and Pakistan. The Global Polio Eradication Initiative has implemented an environmental surveillance program to detect poliovirus (PV) in environmental samples such as sewage. Systematic environmental sampling provides supplemental data to disease-based clinical surveillance data. However, traditional PV detection methods from environmental samples, cell culture and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), are time-consuming and expensive. Development of a screening test has potential to streamline environmental surveillance of PV. Reverse transcriptase recombinase polymerase amplification (RT-RPA) is an isothermal molecular amplification technique with potential as a screening test. In this technique, virus particles are lysed to release RNA, which is converted to cDNA and amplified using enzymes. Amplified PV cDNA can then be detected through lateral flow detection. Collaborators at PATH have developed a RT-RPA protocol that has shown promise in rapid PV detection in stool samples. The aim of this research is to adapt PATH’s clinical PV RT-RPA protocol for use with environmental concentrates. Concentrates were obtained by filtering raw sewage from a Seattle wastewater treatment plant. Results of the RT-RPA test and subsequent visualization on lateral flow detection strip were compared to cell culture and RT-qPCR. We anticipate that RT-RPA will show similar PV recovery rates when compared to traditional methods and will be suitable as a rapid screening test for environmental concentrates. Future work will include designing primers to expand PV serotype detection and evaluating PV detection in environmental samples from existing sampling sites. Rapid PV detection with RT-RPA may streamline environmental surveillance methods, improve outbreak detection, and provide data for certification of polio-free status and global eradication.

Xenobiotic biotransformation is a vital process in the body that detoxifies various drugs, dietary factors, and environmental toxicants. This process is mediated by various drug-metabolizing enzymes in Phase-I (oxidation, reduction, and hydrolysis) and Phase-II, as well as transporters. The hepatic nuclear factor 4alpha (HNF4α) is a master regulator that transcriptionally up-regulates a wide spectrum of drug-metabolizing enzymes and transporters in liver. HNF4α also plays an important role in liver development and nutrient homeostasis. Previously, it has been shown that HNF4α can trans-activate the constitutive androstane receptor (CAR), which is a major ligand-activated xenobiotic-sensing nuclear receptor. However, very little is known about the effect of CAR activation by xenobiotics on the regulation of HNF4α in liver. In the present study, male mice at either 5-day-old neonatal age or 60-day-old adult age in wild-type, CAR-null, or humanized CAR-transgenic (hCAR-TG) genotypes were treated with vehicle (corn oil, which served as the control), or a species-appropriate CAR ligand (TCPOBOP for mouse CAR and CITCO for hCAR), once daily for 4 days. A novel truncated coding exon 9 was observed for the HNF4α transcript variant in mouse liver (RNA-Seq). Isoform-specific RT-PCR and gel electrophoresis confirmed the presence of the novel HNF4α isoform. During development, both the known and the novel isoform had increased expression with age. The mouse CAR ligand, TCPOBOP, down-regulated HNF4α in a mouse CAR-dependent manner only at mice at age 60, whereas hCAR did not appear to regulate the expression of HNF4α at any ages. In conclusion, the present study has identified a novel HNF4α transcript variant in the mouse liver, and discovered that both the known and novel isoforms were regulated by CAR in a species and age-specific manner.

Exposure to air pollution has been linked to increased risk of cardiovascular morbidity and mortality, with special concern about fine particulate matter and traffic-related air pollution such as diesel exhaust. One potential mechanism for this association is an impact on the smallest blood vessels, the microvasculature, which regulate blood pressure. The microvasculature can be directly visualized in the eye’s retinal vessels, and some studies have observed narrowing of retinal arterioles with exposure to pollutants. This project investigates changes in human retinal vessel diameter in response to diesel exhaust exposure. We hypothesize that retinal arterioles will contract in response to diesel exhaust exposure. For this study, we recruited healthy, young adults, who visited the lab on four study days—two with diesel exhaust inhalation and two with filtered air inhalation, in a double-blind controlled trial. We collected retinal photographs before and after exposure for 17 participants on study days. We use semi-automated grading of retinal vessel diameters from the photographs to determine the arteriolar diameters assessed as central retinal arteriolar equivalents (CRAE). We use paired t-tests to determine if diesel exhaust exposure is associated with temporary reduction in CRAE. We expect to see reductions in CRAE in response to diesel exhaust exposure and no change in CRAE in response to clean air exposure. These study results would support the idea that retinal arterioles contract with diesel exhaust exposure. Under the assumption that retinal arterioles model a change in the microvascular bed throughout the body in response to this traffic-related air pollutant mixture, the results would ultimately suggest that a microvascular effect is involved in the contribution of air pollutants to the burden of cardiovascular morbidity and mortality.

Low wage (<$15 per hour) jobs are often structured in ways that expose workers to dangerous chemical and physical agents, as well as policies and practices (e.g., volatile work schedule, low job control, etc.) that may result in adverse stress-related health outcomes. These outcomes are compounded when social disparities such as race, immigration status, and gender are also present. This study will provide a comprehensive assessment of working conditions among low-wage workers and examine the relationship between work conditions, stress and health, and further, if this relationship is affected by union status. Two hundred and fifty low-wage workers from local community-based organizations and labor unions were recruited to participate. Each participant completed a survey describing his or her workplace exposures and health. A composite index of work-related psychosocial stressors was created by combining previously validated scales relating to job strain (control and demand), security, benefits, and overall satisfaction, and each worker was assigned individual scores of work-related psychosocial exposures. We described work-related stressors and health status by union status and other demographic variables. We then examined the association between stress and health, and between union status and health. We further assessed the interaction of belonging to a union and stress on health outcomes. We anticipate that these psychosocial exposures will be associated with stress-related health outcomes. Further, we anticipate that unionized low-wage workers will show less psychosocial exposures to workplace conditions and in turn, less stress-related health outcomes due to better job security, workplace empowerment, and less volatile work schedules. The study information collected from this population may also provide baseline data on which a future longitudinal study proposal can be developed to evaluate the effect of the increase in the minimum wage laws over the next few years.

As environmental monitoring technology advances, people will increasingly have access to new data about their surrounding environmental quality. But, a challenging issue is the question of how can people best access the information? In this research project I focused on data collected from a new neighborhood air pollution monitoring study in Oakland, CA. Our research question is: How can we best express complex air pollution data so that it can be understood and acted upon by people with different educational backgrounds and community interests? I employed user experience research and design methods to answering this question by having the local community members at organizations like West Oakland Environmental Indicators Project, Communities for a Better Environment, La Clinica, and Bay Area Air Quality Management District respond to questionnaires and interviews related to their interest and concerns about air pollution. After gathering this information, I analyzed the air pollution data our research group and the government are currently collecting in the region. Later I used the sorted data to design personas and wireframes through Illustrator and prototyping tools for how the interface should look and have the community members test the prototypes by responding to surveys and interviews as to whether they can use the given data with ease. Lastly, I reported the results of our iterative interviews and questionnaires. I anticipate that the outcome of using user experience research and design methods will be an interface that is highly usable for the community partners in Oakland, CA. 

Newborns and children are more vulnerable to xenobiotics (foreign chemical substances) partly because little is known regarding the developmental regulation of drug-processing genes (DPGs) by drugs and environmental chemicals. The xenobiotic-sensing nuclear receptors, namely the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), are well-known to up-regulate many DPGs in liver. However, relatively less is known regarding the effect of developmental exposure to PXR- or CAR-activators on the expression of DPGs in intestine, where the biotransformation of orally-exposed xenobiotics takes place. Therefore, the goal of this study was to determine both the acute and the potential long-term effects of neonatal exposure to PXR- and CAR-activators on the regulation of DPGs in neonatal and adult intestine. Wild-type mice were pharmacologically administered the PXR-ligand PCN (the activator of PXR in mice), the CAR-ligand TCPOBOP (the activator of CAR in mice), or vehicle, at 3-day-old neonatal age. Intestines were collected 24h post-dose (whole intestine) or at 60-day-old adult age (duodenum, jejunum, ileum, and colon). Neonatal exposure to TCPOBOP and PCN up-regulated many DPGs 24h postdose. Interestingly, at 60-days adult age, neonatal exposure to TCPOBOP persistently up-regulated many DPGs especially in small intestine, but down-regulated particularly in colon (large intestine). Neonatal exposure to PCN persistently up-regulated 6 DPGs but down-regulated 11 DPGs in specific sections of intestine. In conclusion, this study has demonstrated that neonatal exposure to xenobiotics that activate PXR and CAR not only produce acute induction effect on the DPG expression in newborns, but also lead to persistent alterations on the expression of certain DPGs in adult intestine, and this may potentially alter the pharmacokinetics of orally-exposed chemicals in adults.

This project will inform a systems biology platform for identifying adverse responses to environmental compounds on testicular development in mice, a commonly used model system in the field of Toxicology. In order to validate the organotypic culture system, we have investigated the adverse effects of the known testicular toxicant, cadmium. To initiate the culture model, we isolate and plate testicular cells from immature mice on postnatal day (PND) 9 in order to capture a developmental window of susceptibility. Based on a literature search derived developmental timeline, we chose to treat the culture on days in vitro (DIV) 2, 5 and 15 with cadmium and quantify effects 24 hours later. We hypothesized that dosing with cadmium during these time points would correspond to impacts on steroid regulation, proliferation, and spermatogenesis processes. In order to evaluate a dose response relationship in our testicular culture, we treated the testis cells with 2.5, 5.0 and 10 uM concentrations of cadmium. To quantify the effects of cadmium in our cell culture model we evaluated; total protein, testosterone production, cell type specific proteins with western blots, LDH cytotoxicity, 3 color assay for live and dead cells and immunofluorescence to capture morphology for DIV 3,7 and 16, 24 hours post treatment for all three doses. Our initial studies have observed that cadmium treatment for all three doses impacted testosterone production, germ cell morphology and proliferation on DIV 7 and 16.  

Multiple strains of rats and mice are used to evaluate differences in response after exposures to various toxicants at the molecular or cellular levels. However, neuronal toxicity in different strains of species has received much less attention. Therefore, the objective of our research was to compare and characterize fetal mouse micromass culture system using two different strains of mouse—C57BL/6 and A/J—which may be used to screen and prioritize variety of neurodevelopmental toxicants. Our mouse midbrain micromass culture system, a high-density in vitro cultures of primary midbrain cells, is an alternative method to in vivo neurodevelopmental testing because of the ability of cells within the cultures to differentiate in vitro without additional stimulants. Differentiation is purely driven by high cell density. For our 3D in vitro micromass culture system, we isolated the primary midbrain cells from fetus at gestational day 11 (for C57BL/6 mice) or 12 (for A/J mice). The embryos were surgically removed, and midbrain cells of fetal mice were digested into single neuronal cells. These primary in vitro neuronal cells were plated onto plates and cultured up to 22 days in vitro. When morphology was examined throughout 22 days in culture with a 400x phase microscope, cell proliferation, differentiation, and migration were observed for both strains of mice. Proliferation (PCNA, Nestin) and differentiation (B-tubulin, EN1, MAP-2) related protein expression levels were examined using western blot for both C57BL/6 and A/J mouse strains. Dynamics of protein expression levels in western blot indicated that proliferation may be at its highest point between DIV 4-6 while differentiation is between DIV 8-14. If midbrain cells from multiple mouse strains can be harvested at similar neurodevelopmental stages, differences in response due to genetic variability may be studied using our 3D fetal mouse micromass model.