Assessment of Particulate Matter Toxicity and Physicochemistry at the Claim 28 Uranium Mine Site in Blue Gap, AZ

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Biomedical Sciences ETDs Electronic Theses and Dissertations

Fall 11-15-2019

Jessica G. Begay University of New Mexico

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Jessica G.S. Begay

Candidate

School of Medicine, Department of Biomedical Sciences

Department

This thesis is approved, and it is acceptable in quality and form for publication:

Approved by the Thesis Committee:

Dr. Matthew Campen , Chairperson

Dr. Akshay Sood

Dr. Tione Buranda

ASSESSMENT OF PARTICULATE MATTER TOXICITY AND PHYSICOCHEMISTRY AT THE CLAIM 28 URANIUM MINE SITE IN BLUE GAP, AZ

JESSICA G.S. BEGAY

B.S. Biology, University of New Mexico, Albuquerque, NM (2015)

THESIS

Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science Biomedical Sciences

The University of New Mexico Albuquerque, New Mexico December 2019

ii DEDICATION

I would like to dedicate this thesis to my parents, Morris and Velma Begay. I have learned, and continue to learn many life lessons from you. Both of you have taught me to have the spirit of the warrior and to keep fighting and keep pushing. Thank you, also, for being understanding the past few weeks when I did not call as often. Both of you are my rock and my inspiration, and are such a huge part of my support system; you have always been from the beginning. I don’t think I would have gotten this far without the weekends without you and the animals (Neyah, Jaz, Bobby, and Bodie). I thank you all, the animals, as well for helping me de-stress by loving me the same, welcoming me home like I never left, and by showing me that it’s the little things that count.

I also would like to dedicate this thesis to my late aunt, Julie Joe. You’ve always encouraged me beyond measure to just be myself, and even believed in me more than I did myself most times. All I can say now is, we did it.

iii ACKNOWLEDGEMENTS

I would like to acknowledge and thank my thesis mentor, and head of my thesis committee,

Dr. Matthew Campen, for the guidance, advice, patience, and overall continued support of my studies as well as giving me the extra push.

I would also like to acknowledge Dr. Akshay Sood for his excitement and the work being done and his continued helpful insights. I would also like to acknowledge Dr. Tione

Buranda, for his directional support. It has been an honor to have such a trio of a committee, all of whom are brilliant and continue to push me outside of my comfort zone.

I would also like to formerly acknowledge the community members of Blue Gap Tachee,

AZ. Specifically, I would like to thank Chris Nez, Aaron Yazzie, and Sadie Bill for their enthusiasm and support of us while in Blue Gap. Sadie, thank you for the use of your cabin to house and shelter us from the unforgiving rez winter. Thank you, also for the great conversations, and being patient with what Navajo I could speak.

Special acknowledgements also go to all the Campen Lab members (past and present) who took me in as their own and have been supportive of my goals and made the Lab another home for me. I also acknowledge UNM and the Biomedical Sciences Graduate

Program for selecting me and providing further guidance. I also acknowledge the support from NIH and from Dr. Campen’s grant (NIEHS ES026673), as well as any additional financial support that was provided from the UNM College of Pharmacy and Center for

Native American Health.

iv Assessment of Particulate Matter Toxicity and Physicochemistry at the Claim 28 Uranium Mine Site in Blue Gap, AZ By Jessica G.S Begay B.S. Biology, University of New Mexico, Albuquerque, NM (2015) M.S. in Biomedical Science, Albuquerque, NM (2019)

ABSTRACT

Particulate matter inhalation has adverse effects on the respiratory and cardiovascular systems, and windblown dusts from AUMs may pose an excess health risk.

In a small Navajo community, we have undertaken this campaign by: (1) measuring the amount and composition of the PM2.5 in the region, (2) meteorological characteristics, and

(3) immunological and physiological responses of mice after PM2.5 exposures. C57BL/6

-/- and ApoE mice were exposed to concentrated ambient PM2.5 in a mobile laboratory located approximately 1 km from the Claim 28 uranium mine site, for 1 or 28 days for 4

3 hours/day to approximately 80 µg/m PM2.5. Exposure to PM2.5 showed modest evidence of overt pulmonary injury or inflammation, as seen through qPCR and BALF. There was also little evidence of ambient air contamination from uranium or vanadium from the site, which aligned with meteorological factors. However, there was evidence showing potential for toxicity coming from additional metals present.

v Table of Contents

THESIS ...... ii DEDICATION ...... iii ACKNOWLEDGEMENTS ...... iv ABSTRACT ...... v Chapter 1 — Introduction ...... 1 1.1 Growing Up Navajo: A day in the life ...... 1 1.2 Uranium Mining on the Navajo Reservation: Past ...... 4 1.3 Abandoned Uranium Mines and the Navajo Nation: Present...... 5 1.4 The impact of AUMs on the present state of the health and well-being of the Navajo People ...... 7 1.5 Lung Physiology and Function ...... 12 1.6 Inflammation: the immunological aspects of inhaling fine particulates ...... 16 1.7 Metals contribution to toxicity and airborne particles ...... 19 1.8 Particle size and its effect on lung function ...... 22 1.9 Description of the Mobile Air Research Lab (AirCARE1) and its intended use ...... 24 1.10 AirCARE1 on the Navajo Reservation ...... 26 Chapter 2 — Materials and Methods ...... 28 2.1 Location selection ...... 28 2.2 Mouse models ...... 30 2.3 AirCARE1 Trailer and exposure settings ...... 30 2.4 Wind Speed and Direction Analysis ...... 32 2.5 Bronchoalveolar lavage fluid (BALF) collection and analysis ...... 32 2.6 Gene expression analysis ...... 32 2.7 Inductively coupled plasma mass spectrometry (ICP-MS) of metals collected on exposure chamber filters ...... 33 2.8 Statistics ...... 34 Chapter 3 — Results ...... 35 3.1 Wind Speed and direction over AirCARE1 and Claim 28 mine site ...... 35 3.2 PM Distribution ...... 37 3.3 BALF Analysis ...... 39

vi 3.4 Lung and aorta qPCR analysis results ...... 41 3.5 Trace Metals Analysis using inductively coupled plasma-mass spectrometry (ICP-MS)... 45 3.6 ICP-MS Literature Comparison ...... 46 3.7 ICP-MS of Metal Concentrations in the Lung ...... 48 Chapter 4 — Discussion ...... 49 4.1 Particulate Matter Toxicity ...... 49 4.2 Thoughts on trace metals data ...... 53 4.3 Caveats and Future Directions ...... 59 Chapter 5 — Conclusions ...... 60 References ...... 62

vii List of Figures

Figure 1. Claim 28 Uranium Mine Site...... 27 Figure 2. AirCARE1 Mobile Research Laboratory ...... 28 Figure 3. Map of mine sample sites on Navajo Nation ...... 29 Figure 4. Diagram of exposure time lines ...... 31 Figure 5. Daily Snapshot of Principal Wind Speed Results ...... 35 Figure 6. Blue Gap Wind Distribution ...... 36 Figure 7. Average and Daily PM Distributions ...... 38 Figure 8. BALF Cell Differential Analysis ...... 40 Figure 9. Lung qPCR Analysis Results ...... 42 Figure 10. Aorta qPCR Analysis Results ...... 44 Figure 11. ICP-MS analysis ...... 46 Figure 12. Literature comparison with references ...... 47 Figure 13. Metal concentrations measured in lungs ...... 48

viii List of Tables

Table 1. ICP-MS analysis raw data table ...... 45

Assessment of Particulate Matter Toxicity and Physicochemistry at the

Claim 28 Uranium Mine Site in Blue Gap, AZ

By Jessica Begay

Chapter 1 — Introduction

1.1 Growing Up Navajo: A day in the life

Mother gently wakes you, but it is still dark outside. The warmth from your blanket and the sinking feeling of the mattress begs you to stay just five more minutes, but then you hear her walking heavily down the hall, so you quickly jump up—like ripping off the band aid—and pull on some sweats, your high school cross country pullover, a beanie, and old tennis shoes. As you try to comprehend the time of day (or night), you stand on the front porch and look to the east, towards the iconic red rocks; but it’s so dark still, that they are nothing but black masses blocking the warmth of the sun. Behind those giant masses are specks of fading white lights, fighting to still be seen in the soon to be blue sky.

Finally, you wake enough to run towards the east (a tradition instilled in every

Navajo) and along with your blood flow, the landscape, too, comes to life. The more the sun rises and overcomes the peaks of the rocks, it ever so slightly kisses the frost-bitten landscape to life. You return to begin the day, which may include sheepherding, loading water and hay, yard work, and regular school activities. To experience this is normal. However, to non-tribal members, this is something from a Hollywood set. This is the Navajo Nation.

1 The Navajo Nation is the largest Native American tribe, with its reservation

(legal designation of land allotted back to federally recognized Native American tribes, in 1868 for the Navajo, that is managed by the tribe under the United

States government) spanning over the states of Arizona, New Mexico, and Utah with an area of 27,413 square miles and just over 300,000 enrolled tribal members (Washington & van Hover, 2011). According to a 2010 tribal census, about 74% of enrolled tribal members live on the reservation (Development,

2018). The social and economic dynamics of the tribe could be considered almost third world, with the average household income being less than $27,000 a year (Development, 2018). The majority of towns on the reservation are rural areas, where the closest neighbor may not be for miles around. Because of the rural nature of reservation towns, access to life’s necessities and electricity does not come easy. Many families have to travel over an hour to the nearest grocery store, or further to large cities for cheaper items in bulk. Another issue is access to electricity and clean water. About 10% of residents live without electricity and

40% have to actively seek out a water supply and use outdoor plumbing

(Development, 2018; Lewis, Hoover, & MacKenzie, 2017).

Blue Gap Tachee (BGT), AZ is such a town. It is located about 40 miles west from Chinle, AZ (a relatively large town of 4,500), and about 113 miles from

Gallup, NM. On a map, BGT appears to be located in the center of the Navajo

Reservation with a population of 1,882 community members (Harmon et al.,

2017; Lewis et al., 2017). Many families have resided here for generations, living off of and cultivating the land and livestock, going back to before the Long Walk

2 of 1864 (Goble, 2002). For many, this rural “John Wayne backdrop” of a town is home, and there is no other place worthy of the title. For generations, the community has depended on the land. Even now, in the midst of environmental and climatological pressures that continue to impact the land, have also impacted a resilient and sacred people to speak for the land, and for future generations.

14 miles north from the intersection of US-191 N and Indian Route 4 lie the physical remains of what can only be described as a ghost of the past. To the untrained eye, it appears as normal as the surrounding hills with sand spilling over the edge. However, this invisible and unmarked monument is what remains of what used to be a fully functional uranium mine in the 1940s, named the Claim

28 site. Sadie Bill, a concerned community member, owns a cabin located about

1km south-southwest of the site and tells of the time when the mine was first operational: “We were kids running along the hills, and we would hear the explosions from the dynamite go off all day, and there would be a cloud of dust at the top of that hill.” Among the many conversations, much had to be said about the experiences the communities have had over the past few decades since the opening of Claim 28. Each story overlapped in painting a strong image of innocence of the hazards and legacy to be left behind. Post-World War II, uranium mining immediately ceased and several hundred mines, including the

Claim 28 site, were left open, unmarked, and in shambles.

3 1.2 Uranium Mining on the Navajo Reservation: Past

Uranium was first discovered in 1789 (Goble, 2002), was initially used to decorate glass and ceramics for its yellow to green hues, and was not found to be radioactive until over a decade later. Naturally occurring uranium (with far greater abundance of 238U than 235U) itself is not especially radioactive, instead it is the broken form that can cause detrimental effects. From 1942 to 1945,

Uranium’s radioactive materials were used for nuclear fission (Jones, 2014), or the splitting of atoms to release large amounts of energy. The U.S. saw that this could be an advantage in World War II event and seized the opportunity to seized the opportunity to pursue the advancement in nuclear weapon production.

Soon after, the U.S. Atomic Energy Commission priced uranium-ore mining for radium and vanadium, focusing on the Navajo Nation’s geographic regions, which are located on top of a Uranium-rich mining belt (Blake et al., 2015;

Harmon et al., 2017; Hoover, Gonzales, Shuey, Barney, & Lewis, 2017; Lewis et al., 2017).

Life on the reservation was physically challenging enough with lack of modern amenities (i.e. electricity and running water, specifically), but the Navajo

Nation was also trying to recover from the financially devastating aftermath of the mid-1930’s livestock “reduction”: a government issue where families’ livestock were gathered and killed because of the concern of overgrazing on tribal lands.

Over a decade later, the U.S. acknowledged the use of uranium products, mostly in nuclear weapon production and declared a price for uranium mining. A call for miners was put out, as well as the additional motives of pay and on-site housing.

4 Navajo men accepted these jobs without knowledge of the potentially harmful effects of exposure to uranium, details of which has been established by the

Europeans back in the 1930’s (Goble, 2002; Sigismund, 1939).

4000 Navajo men were either blasters, timbermen (those who created the pillars that would hold up mine shafts), muckers, transporters, or millers (Goble,

2002); all of whom were being exposed first-hand to uranium-mining dust without any provision of protective equipment. For hours and days on end, Navajo men would enter the mines and hack away at the earth, inhaling and exhaling with every hack, only to return home to their families covered in mining dust. Many repeated this schedule every day for a time period of anywhere between a few months to up to ten years. The need for uranium mining remained a priority, even past the end of World War II, through the 1960s when demand was beginning to subside. During this time, over 750 operating uranium mines were abandoned, hence the term “abandoned uranium mines” (AUMs). By the end of the Uranium rush, there would be about 1000 open across the U.S., with over 500 on Navajo land (Harmon et al., 2017; Lewis et al., 2017; Rock et al., 2009) that would unknowingly leave the surrounding community members chronically exposed to unreclaimed mine waste, the effects of which would not come into question until years later.

1.3 Abandoned Uranium Mines and the Navajo Nation: Present

Uranium mines were shut down in the 1980s, largely coinciding with the end of the cold war, but there are currently more than 160,000 abandoned hard

5 rock mines (EPA, 2008; GAO, 2008; Lewis et al., 2017) spread throughout the western U.S., including Arizona, New Mexico, California, Colorado, Nevada,

Idaho, Montana, Utah, Washington, Oregon, Wyoming, and South Dakota. 4000 of those hard rock mines are abandoned uranium mines (EPA, 2008; Lewis et al., 2017). Interestingly, many of these mines overlap with American Indian tribal reservations, where each AUM left has distributed a variety of metal exposures and chronic illnesses throughout tribal communities (Lewis et al., 2017). Along with expected routes of heavy metal exposure such as inhalation, tribal communities experience a wider variety of exposures, which can be dependent on a traditional cultural lifestyle and daily practices—e.g., harvesting and ingesting wild-growing plants that absorb metal contaminated water or consuming livestock that feed in or around contaminated sites (Lewis et al.,

2017).

Currently, there has been a rise in concern for the lasting damage done to both the people and the land and there are four areas that the Environmental

Protection Agency (EPA) is focusing on to restore the security and health of the

Navajo people: (1) Working with communities, (2) providing safe drinking water,

(3) addressing contaminated structures, and (4) cleaning up abandoned mines

(Agency, 2019).

Although it has been decades since the mines were shut down, it was not until within the last 20 years (Agency, 2019; Goble, 2002) that communities and individuals who have been affected are being recognized. The first case of cancer-related problems, for example, was noted in the 1980s and the number of

6 AUM-related cases has been increasing since (Jones, 2014; Zychowski et al.,

2018). It was also known that the relationship between uranium mining and respiratory disease was more prevalent in Native American miners (Mapel DW,

1997). Previously, an association between mine waste exposure and hypertension progression has been found (Lauren Hund, 2015). AUM proximity, relation of distance to mine (also relative to size of mine) and severity of exposure, was also taken into account when assessing the circulating inflammatory potential of individuals within close proximity to a large mine site; the individuals’ serum induced inflammatory response in vitro (Harmon et al.,

2017). We also previously confirmed that inhalable particles in the mine region sediments are contaminated with metal elements, specifically uranium and vanadium (Zychowski et al., 2018). This will be discussed in further detail.

1.4 The impact of AUMs on the present state of the health and well-being of the Navajo People

A link between hard rock mining and respiratory disease was first observed in 1879 by two physicians from the mining region of Sachsen, Germany

(Martensen, 1998). In their research, they established lung cancer (or “mountain illness”) as an end result of exposure to hard rock mining for heavy metals, mainly attributed to the arsenic exposure (Goble, 2002; Härting FH, 1879;

Martensen, 1998). They also noticed a 20-year latency period for lung cancer.

Although the health effects of hard rock mining were initially attributed to arsenic, the hazards of mining-related uranium exposure were well documented by the

7 1930s (Goble, 2002). Despite this, Navajo miners were not warned and, therefore, workers could not make informed decisions about working in the uranium industry.

In 2011, Dawson and Madsen interviewed former uranium workers and, in some cases, their surviving family members, about health and psychosocial issues after working in the mines as well as the conditions and routines of working in the mines. Many people mentioned that they noticed respiratory- related health issues years, sometimes even decades, after working in the mines. This included shortness of breath, consistent coughing, pulmonary fibrosis, chronic obstructive pulmonary disease, emphysema, and lung cancer

(Dawson & Madsen, 2011; Jones, 2014).

Lung cancer, non-malignant obstructive lung disease, a decreased FEV1, and some have also reported a higher rate of pneumoconiosis in Native

Americans than non-Native Americans (Dawson & Madsen, 2011; Mapel DW,

1997). Moreover, about 70% of cancer incidence are not due to smoking—the reason being that the majority of the Navajo people, specifically, have reported they do not participate in casual smoking in a study conducted in miners from the southwestern US (Lewis et al., 2017; Mapel DW, 1997). The hypothesized effect, therefore, is due to the constant, higher than expected exposure to a variety of metals present within the environment (Lewis et al., 2017). The most prominent effects to be discussed here, however, will be lung disease and respiratory complications.

8 The history of AUMs and their impact on the people and land of the

Navajo is a long and complicated true-crime story (Pasternak, 2011). From an academic perspective, the “legacy” of AUMs is an extensive list of health adversities including kidney disease, cardiovascular disease, increased cancer mortality, and hypertension (Blake et al., 2015; Dashner-Titus et al., 2018;

Dawson & Madsen, 2011; Erdei et al., 2019; Jones, 2014; Lewis et al., 2017;

Zychowski et al., 2018). Although lung cancer and respiratory disease prevalence appears to be higher in miners who smoke cigarettes, researchers found both smokers and nonsmokers had statistically elevated rates of respiratory disease (Dawson & Madsen, 2011).

As one might assume, life on the Navajo reservation is far from the typical idea of modern living in the United States. As described earlier, many are without running water and/or electricity and have little access to clean drinking water.

Sanders, AZ is another small Navajo Reservation town, and their community members continue to fight for clean drinking water, with current levels of uranium in their drinking water that are demonstrably above the allowable EPA threshold

(L. J, 2016). However, for other unsaid reasons, it has been not been resolved and labeled by the EPA as “not concerning and still drinkable” (L. J, 2016). A study done in 2017 spanned over the entire Navajo Nation and compared water quality information for unregulated sources. They found that the samples exceeded the drinking water standard for arsenic and uranium (Hoover et al.,

2017). They also found that the water sources that were within close proximity of an AUM, yielded higher concentrations of arsenic and uranium in drinking water

9 (Hoover et al., 2017). This backs up the claim that exposure to metal contaminants from AUMs could extend further than direct and indirect contact from waste that are present in the dirt or water (Basha, Yasovardhan,

Satyanarayana, Reddy, & Kumar, 2014; Blake et al., 2015; Dashner-Titus et al.,

2018; Harmon et al., 2017; Hoover et al., 2017; Zychowski et al., 2018).

Although soil and water are very concerning routes of exposure, life on the reservation poses certain risks from following a unique traditional lifestyle. This requires utilizing as many available natural resources as possible that are being provided by the land, specifically the use of plants for dietary and medicinal consumption (Lewis et al., 2017). An example of this is Ch’il gowéhi (the Navajo wild tea). As the name implies, this plant grows wild throughout the reservation lands. Many Navajo collect these wild weed-looking plants to boil and use as a dye or to drink as tea when one catches a bad cold and sore throat. It is also used to soothe and treat other bodily ailments, such as stomach pains. The idea is that many of the traditional resources are contaminated via the uptake of contaminated sources found within the soil (Hoover et al., 2017). The resources are then collected and consumed. In 2017, Lewis et al. noted this unique route of exposure as it increases what already is a higher than expected exposure to

AUM waste.

Expecting mothers within close proximity to an AUM are also thought to especially be at risk because babies are born with congenital anomalies and are later affected by developmental disorders (Dashner-Titus et al., 2018). It is also thought that metal exposures enhance the risk of developing diabetes mellitus, a

10 disease that the highest prevalence in Native Americans and also effecting

Native Americans in having death rates that are 3-times higher than the general

US population (Chow, Foster, Gonzalez, & McIver, 2012). Many of the overall health issues that could be heavily influenced by AUMs, however, are not completely understood and we risk complications of treatment options because of unknown mechanisms.

In 2017, we previously investigated the inflammatory potential of serum from 145 volunteers from across the Navajo Nation with various residence proximities (a surface-area weighted distance metric) to an AUM or its waste sites (Harmon et al., 2017). The results showed that closer proximity to an AUM associates with an increase in inflammatory potential (as seen with increases in

CCL2, VCAM-1, and ICAM-1) (Harmon et al., 2017). Additionally, we have investigated the potential hazards of dust samples from Claim 28 compared to background dust. A cascade impactor separated dust sample to a size less than

10µm (PM10) and instilled into mice. Additionally, the samples were analyzed for concentrations of uranium and several of its cooccurring metals. TEM imaging also showed an unexpected agglomeration and nanoscale ultrastructure to the particulates (Zychowski et al., 2018). Collectively, the particulates were shown to high contents of uranium and vanadium, compared to background dust; and, when aspirated into mice, showed enhanced cytotoxicity, lung inflammation, and vasoconstriction (Zychowski et al., 2018). As a result, Claim 28 in BGT was of interest for follow-up and further investigation because the particles were found to be in the respirable range and also confirmed suspicions of having an

11 inflammatory potential. It was then questioned if the wind was a potential route for exposure. Although there are other routes of exposure, the wind has potential because the particulates of the mine surface, being of respirable size, can easily be redistributed into the air and available for inhalation.

1.5 Lung Physiology and Function

As humans, we inhale an average of about 23,000 times per day

(Knudsen & Ochs, 2018). With every inhalation, oxygen (O2) from the surrounding environment enters our lungs and is carried throughout the rest of our body. Then we exhale to remove carbon dioxide and other waste gases, and repeat. However, this process is a little more meticulous than we make it out to be. In perspective, our lungs are thin, elastic extensions of our throat that, through muscular contractions, are engineered to keep us alive. At the top of the throat is the trachea, which is where O2 enters and is led down into the bronchi that split into branches in the lung (Freeman, 2010; Knudsen & Ochs, 2018).

From here, the negative intrapleural pressure created by the downward extension of the chest bring the O2 molecules further into the lungs, to the bronchioles (Freeman, 2010; Knudsen & Ochs, 2018). The bronchioles are smaller branches that divide the bronchus into much narrower tubes. At the end of each bronchiole are alveoli, consisting of tiny sacs. On average, the human lungs contain approximately 1.5 million alveoli (Freeman, 2010; Knudsen &

Ochs, 2018). The high number of alveoli increases the overall surface volume of the lung, and, therefore, increases the amount of O2 available for distribution to

12 the blood for other tissues and organs. From the amount of O2 being inhaled, only 2/3 diffuse into the circulation while the remaining 1/3 fills in the dead space of the lungs. In short, the design of the human lung is to optimize gas exchange between the air and our circulation (Freeman, 2010; Knudsen & Ochs, 2018).

The alveoli—making up about 90% of total lung volume (Bachofen &

Schurch, 2001; Knudsen & Ochs, 2018)— are the key players in gas exchange as surrounding the alveoli are capillaries that bring O2 and hemoglobin into close proximity. The alveoli are important because the efficiency of gas exchange occurring depends on their overall structure. Any physical abnormalities could affect the total surface area of the lung and therefore reduce overall lung function. The alveoli are also what inhaled particulates, if small enough to by pass the nose and upper airway system, first come into contact with (Yost, 2010).

We, therefore, tend to consider the particles in the deep lung as the most concerning.

There are two types of epithelial cells, type I and type II within the alveoli, type I and type II. Type I epithelial cells have a thin and flat physical structure, and they mainly contribute to the structure of the alveoli (Bachofen & Schurch,

2001; Knudsen & Ochs, 2018; Respiratory Toxicology). Type II epithelial cells, on the other hand, have more of an active role in the alveoli as they participate in alveolar repair and renewal as well as surfactant secretion (Bachofen & Schurch,

2001; Knudsen & Ochs, 2018; Respiratory Toxicology). The secreted surfactant lines the inner walls of the alveoli and contribute to the stability of the alveoli. The surfactant secreted by the type II epithelium is also key to lung function because

13 it could also have an immunomodulatory function in type I recycling and macrophage circulation (Knudsen & Ochs, 2018; Respiratory Toxicology).

Adjacent the alveolar epithelium is capillary endothelium that brings the O2 of the airspace and hemoglobin of red blood cells into close proximity, where the resulting gas exchange occurs, driven by a concentration gradient. Within the interstitial space of the alveoli and capillaries, exists an interwoven extracellular network of fibers, and include fibroblasts that support growth, wound healing and remodeling (Bachofen & Schurch, 2001; Knudsen & Ochs, 2018; Olvera Alvarez,

Kubzansky, Campen, & Slavich, 2018; Ophir, Bar Shai, Korenstein, Kramer, &

Fireman, 2019).

A normal, healthy human lung ideally has a large surface area and an optimal elasticity. However, lungs that are constantly under stress develop difficulties coping with volume changes—such as inelasticity—and become subject to cellular deformation. Additional complications include disrupted plasma membrane, blebbing (a bulge in the plasma membrane of a cell), stripped basal lamina, narrowed diameter of alveolar ducts (can be related to a reduced size of alveolar entrance rings), induced lipid-trafficking, and ultimately a reduced efficiency of gas exchange. Two important effects of lung stress laid out in a

2018 review by Kudson and Ochs are “deformation-induced lipid trafficking” and the interfacial stress caused on the surfactant being secreted.

“Deformation-induced lipid trafficking” essentially emphasizes the important role of the surfactant secreted by the type II epithelium cells (Knudsen

& Ochs, 2018). In addition to having a role in macrophage circulation, the type II

14 epithelium secretion also act in lipid trafficking. When the alveoli become damaged in any way, either at the acute or chronic level, the resulting cellular deformation causes an increase of endogenous lipid vesicle production. This is due to both the damaged epithelium barrier, stretching and pulling each other to compensate for the lack of membrane coverage, and its resulting damaged plasma membrane (Knudsen & Ochs, 2018; Respiratory Toxicology). The damaged plasma membrane then becomes endocytosed and a membrane patch forms. Although the membrane patch is a means of repairing membrane disruptions, it can be detrimental to gas exchange efficiency by increasing the layer through which gas exchange occurs.

The interfacial stress caused on the surfactant is due to the stress induced on the type II epithelium cells (Knudsen & Ochs, 2018). It is thought to be due to the fibers that are interwoven between the alveoli and the capillaries. When the membrane of the epithelium is disrupted, it becomes similar to pulling a thread from a fabric, loose and bunched together. In the alveoli, these fibers bunch together and concentrate into piles within the alveoli. This in turn sets off a chain reaction of increased surface tension, leading to increased alveolar ducts as well as a decrease in surface area, and bringing it full circle to decreased gas exchange. Here, we will look at the damage done specifically the inflammation caused via fine particles.

15 1.6 Inflammation: the immunological aspects of inhaling fine particulates

Generally speaking, inflammation is the localization of a cellular response to damage or injury and is characterized by redness, swelling, heat, and, often, pain (Merriam-Webster, 2019). Inflammation is a part of the human immune system for defense against foreign material. The rabbit hole here, is that the defense mechanisms, depending on the pathogen, can act in one of two ways, immediately (innate) or adaptively (adaptive), which can then act in a cascade of many other pathways (Murphy, 2012). From previous results (described in a later section), we’ve seen inflammatory effects occur after particle inhalation with respect to a classical innate response (Katherine E. Zychowski et al., 2019).

The body’s innate response happens immediately upon a pathogenic invasion, but is not specific for that single pathogen (Murphy, 2012). Typically, when a foreign substance, such as with a viral or bacterial infection, enters into the body system, the first step for the immune system is to recognize and distinguish this foreign substance as “self” or “not self.” If the substance is “self,” no damage control needs to happen; if the product is “not self,” then the infection needs to be contained before it can get any worse. This is where immune effector functions are called into action and include phagocytic white blood cells, such as macrophages and neutrophils, to ingest and kills the foreign pathogens by producing degradative chemicals and enzymes that essentially eat away the ingested pathogen (Murphy, 2012). A nice analogy for this process is the movie,

The Blob, where it eats everything it comes into contact with, but the immune

16 system, however, has a more directed target than simply everything The Blob comes into contact with.

Coincidently, humans were designed with intent; meaning that macrophages and neutrophil activation can be seen as a co-existing relationship.

Macrophages are recruited to the initial site of infection or injury. Once here, two signals are sent: (1) recruitment of neutrophils, and (2) release of cytokines and chemokines (Murphy, 2012). Chemokines are chemicals that are secreted by macrophages that send for and bring neutrophils, that are already circulating through the blood stream, to the site of infection. Neutrophils are a part of the acute inflammatory response, and are usually first to arrive with the macrophages (Kolaczkowska & Kubes, 2013). Typically, neutrophils that are available within the blood system, and upon signaling and recruitment, they

STOP (or slowdown), TETHER, and ROLL (Kolaczkowska & Kubes, 2013).

Essentially, they are slowed down enough to a crawling pace until they come into contact with an opening between endothelial cells (Kolaczkowska & Kubes,

2013). Neutrophils are thought to have at least three killing mechanisms: phagocytosis (classic view), degranulation (usually an extracellular process used against bacterial), or NET casting (secreting a net out to capture and engulf microbes) (Kolaczkowska & Kubes, 2013).

The other secretion that macrophages release are cytokines. Cytokines are different signals secreted by the macrophage in that they are more local for any cells that are within a certain area of the infection—calling cells to prepare for battle, essentially. This preparation via cytokine release can have a physical

17 effect on the endothelium of the blood vessels, by inducing changes in the adhesive properties and overall physical architecture of the cells(Murphy, 2012).

The adhesin junctions between the endothelial cells loosen, enough to let by passing leukocytes pass in between the barrier to reach the site of infection or injury (Murphy, 2012). And, the architecture of the cells changes in a way that

“catches” the bypassing leukocytes, such as neutrophils as previously described, and slows them down enough to enter in between the cells (Murphy, 2012). The resulting inflammation that is seen and felt is the result of all these processes occurring at once to protect the body from further infection. However, the inflammation process needs to be regulated, or genetic, cellular, and overall tissue damage may occur if this process is prolonged (Arick, Choi, Kim, & Won,

2015; Kolaczkowska & Kubes, 2013).

In a healthy individual, the mucus that coats the lungs, however, acts as a line of defense and foreign substances that reach the lungs can usually be expelled out by becoming trapped in the mucus and the sweeping motion of cilia pushes it out (Murphy, 2012). In an unhealthy individual, the mucus lining can thicken and become less effective, or ineffective, and therefore unable to remove foreign substances (Murphy, 2012). When respirable particulates are inhaled, surfactant protein (SP), type D and A, flag the substance for phagocytosis and removal by the macrophages in the alveoli (Arick et al., 2015). However, regardless of the efficacy of the mucosal lining, the inhaled particulates can overload the system and the macrophage defense can be rendered

18 overwhelmed and ineffective (Arick et al., 2015). Power in numbers can affect the outcome of particulate toxicity, but there are other factors to consider.

1.7 Metals contribution to toxicity and airborne particles

Factors that can contribute to particulate matter (PM) toxicity are particulate size and morphology, which largely determine the depth and retention of PM. However, another contributor is chemical composition of the particulate.

Metals are more than decorative, shiny substances or electrical conductors.

Chemically speaking, metals are given the general definition of elements that readily form positive ions and have metallic bonds. Metals are also naturally present within the environment and contribute to approximately 25% of the earth’s crust (Sandatlas, 2019), 8% of which is naturally occurring aluminum (Al)

(Krewski et al., 2007b). It can be found in volcanic ash and areas with clay materials (Li, He, Wu, & Xu, 2012; Theng & Yuan, 2008; Wada, 1987; Willhite et al., 2014). However, as Paracelsus stated, the dose is what makes the poison.

The cation, Al+3, can form free radicals and induce oxidative stress at high concentrations in biological systems, which then triggers the intrinsic apoptosis pathway (Willhite et al., 2014). However, the toxicity of insoluble metals is dependent on the metals as particulates as well as the route of exposure and dose.

The deposition of inhaled particulates in the lung varies with the particle’s size, shape, and density in addition to its chemical properties. When a particle is inhaled, it enters the airways and settles in one of four ways: interception,

19 impaction, sedimentation, or diffusion (also known as Brownian motion) (Safety,

2019; Tsuda, Henry, & Butler, 2013). Interception, or diffusional deposition, usually occurs with larger particles (>10µm in diameter) and can be seen within the nasopharyngeal region (nose, mouth, and upper region of the throat) (Tsuda et al., 2013). Interception also typically occurs with elongated fibrils, such as those seen with asbestos, and is described as a particle traveling along the respiratory route in a linear path until it comes into close contact with a surface

(Tsuda et al., 2013). Interception can be described as the first line of defense to keep large particles from entering the lungs. Impaction refers to particles (also relatively larger with regard to size and mass) sticking to the surface of the lung.

Impaction is highly dependent on air velocity and particle mass (Tsuda et al.,

2013). Sedimentation refers to a curved-linear path of particle travel typically occur with particles <10µm in diameter where gravity overrules the buoyancy of the particle. As the particle travels down the tracheobronchial region, the particle randomly settles on the surface of the lung (Tsuda et al., 2013). Lastly, the most important deposition mechanism for submicron particles is diffusion, also known as Brownian diffusion. This mechanism occurs with smaller particles (<1µm)and is based on impulsive, random movements and collisions with other particles within a certain space (Tsuda et al., 2013). This can be visualized as similar to how gas moves, or disturbed dust in a room with a window of sunlight. Because it is based on random movements, the settlement of the particles is random, and is also dependent on size—the smaller the particle, the more random movements occur (Tsuda et al., 2013). Specifically, the alveoli, where convective air flow is

20 negligible, are where Brownian motion would be most relevant, and would be most likely used by PM2.5 exposures.

The second contributor to metal toxicity is the particle’s morphology, which can also contribute to density and can affect the overall aerodynamic and diffusive behavior of the particle (Tsuda et al., 2013). When visiting the Claim 28 mine site, fine silicate dust could be seen along the ridges attracting the eye because of its subtle sparkle in the sun. Crystalline silica is a common by product of uranium mining, and its toxicity depends on the form of the silica (Arick et al.,

2015; C. J, 2012). It has a strong geometric structure that is visually similar to home-grown overnight sugar crystals from a child’s science kit and is fragile enough to feel like carbon fiber material breaking. Natural processes, such as wind erosion, can cause silica dispersion forming respirable silicate dust.

Inhaling small sharp crystalline pieces of silica can cause progressive fibrotic lung disease (silica-induced fibrosis), or silicosis (Lakatos et al., 2006;

Sellamuthu et al., 2017). The specific structure and size of silica particulates make them targets for macrophages to engulf upon entry into the lung. However, it is nearly impossible for the particle to later be broken down by the phagosome, leading to swelling and destabilization of the phagosome (Leung, Yu, & Chen,

2012). Phagosome destabilization triggers intracellular lysosomes to fuse with the phagosome. The phagosome is eventually digested and the internal contents of the phagosome are released into the cytosolic compartment, which then results in lysis of the macrophage and the activation of pro-inflammatory molecules and ROS generation (Leung et al., 2012). Ultimately, inhaled silica

21 increases fibroblast and myoblast stimulation, as well as collagen production, and the end result is a progressive disease with very few treatment options

(Lakatos et al., 2006; Leung et al., 2012; Sellamuthu et al., 2017)).

1.8 Particle size and its effect on lung function

PM can be as large as 100 µm to less than 10 nm, but are placed in two categories when referring to air pollution regulation: PM10 or PM2.5 , meaning smaller than 10 µm or 2.5 µm, respectively (Assessment & NC), 1996; Institute,

2002; Valavanidis, Fiotakis, & Vlachogianni, 2008). As previously described, the size of the PM is important because it determines its distribution, or lack thereof, in the lung. PM2.5 is of specific interest because these are the inhaled particles that can reach deep into the lung, contacting and interacting with the surfactant of the alveoli (Yost, 2010). PM2.5 are also most present in ambient air, higher in number and surface area compared to larger particles, and retained within the airways and alveoli more than larger particulate sizes (Brauer, 2000; Valavanidis et al., 2008). China is a modern example of a country with high concentrations of

PM2.5, and although concentrations appear to be gradually decreasing (from

3 3 59.1µg/m in 2013 to 35.8 µg/m in 2017), PM2.5 is still a concern as several

3 areas have shown an increase in PM2.5 concentration (>10.0 µg/m ) from 2013 to

2017 (Zou et al., 2019). Moreover, in non-priority regions, despite implementation of air pollution intervention policies China continues to experience a high number of premature deaths annually related to PM2.5 exposure (an estimated 1.05 million in 2017 reduced from 1.20 million in 2013) (Zou et al., 2019).

22 In a recent study, Yang et al. 2019, looked specifically at PM2.5 presence in exhaust fumes from a parking garage. In their study, they utilized ambient air quality monitoring systems to expose one group of mice to the parking garage air over the course of three months, and another group of mice to clean air. They then looked at the lung ultrastructure post-exposure and found changes in the pulmonary architecture as well as lung tissue damage, as seen by intracellular edema and increases in microvilli and lamellar bodies in the exhaust fumes exposure group (Yang, Chen, Yu, Ding, & Ma, 2019). Another interesting detail, confirming the findings of Valavanidis et al. 2008, through electron microscopy, was an increase in accumulation of PM2.5 in the lung airways (Yang et al., 2019).

Another recent study was done by Huang et al. 2019, where traffic-based PM2.5 was instilled into mouse trachea as a PM2.5-induced injury. In this paper, the authors found lung injury to be the result of oxidative damage and induction of inflammatory processes (Huang et al., 2019). These are just a few current studies, but PM2.5 continues to be a popular area of environmental research.

A novel approach was used in 2007 to investigate the urban atmospheres in terms of air pollution within Detroit and Grand Rapids, Michigan (Keeler,

Morishita, Wagner, & Harkema, 2007), and then again in 2009 to investigate the hazards of diesel exhaust fumes in a parking garages (Harkema JR, 2009). In

2007, they concluded that exposure to polluted areas increases allergen response in the lungs of their oval albumin-sensitized rat models (Keeler et al.,

2007). Similar findings were found in the 2009 study, but the allergen response was found to be increased with increasing inhalable concentrations (Harkema

23 JR, 2009). For both investigations, they used a state-of-the-art mobile inhalation laboratory (AirCARE1) to expose animals in a concentrated air particulates

(CAPs) chamber to concentrated ambient air. Here, we have obtained the trailer to further investigate the inhalation risks that Claim 28 may endue.

1.9 Description of the Mobile Air Research Lab (AirCARE1) and its intended use

The Mobile Air Research Lab is a 16.2m semi-trailer, equipped with electrical power, water, and HVAC systems to support real-time environmental animal exposures through fine ambient particle-concentration (Image 2). The trailer consists of three rooms on the inside: (1) a monitoring room, (2) an animal housing and exposure room, and (3) a necropsy and tissue preparation lab. Each of these rooms has electric outlets to support equipment as needed. The trailer also has the capability of maintaining a certain room temperature and humidity for each of the rooms, as well as HEPA filters for each room. The FA animals receive air that has been passed through a HEPA filter and the PM exposed animals receive air passed through a HEPA filter during non-exposure times which is switched to non-HEPA filtered ambient air that has been passed through a particle concentrator. On both ends of the trailer, within the monitoring and necropsy and tissue preparatory rooms are also steel fume hoods.

The top of the trailer is a mezzanine platform that provides space for equipment, which can then be fed through ports from the roof to the inside of the trailer. This is also where the meteorological (MET) tower station is placed to

24 monitor humidity and wind direction and speed. A PM2.5 inlet is also mounted on the roof of the trailer. This inlet is where the initial air jet stream brings in surrounding air particles through a stainless-steel duct and to the concentrator on the inside of the trailer. The concentrator, located in the middle of the trailer, first filters out large particles and the smaller particles continue to follow the streamline through three stages. Each stage continues to allow particles of a certain size (pre-determined and set according to experimental procedure) through, while concentrating incoming PM to three times more than the previous stage. The resulting concentrated ambient particles (CAPs) can be up to 30 times more concentrated and is then distributed into an exposure chamber

(Sioutas C, 1997).

There are two inhalation exposure chambers inside the trailer, one connected to and receiving particles from the concentrator (exposed), and the other is a control that provides filtered (non-exposed) air. Each chamber has a volume of about 0.32m2 and can hold up to 40 mice during exposures. Above each chamber, water carboys can be placed and connected to chambers to feed water to animals during exposure to collected ambient air. In 2007, Keeler et al. used the mobile research lab (known as AirCARE1) to expose ovalbumin- sensitized rats to the urban atmospheres of Detroit and Grand Rapids, Michigan.

They concluded that ambient PM exposure induced increased airway hypersensitivity and pulmonary injury (Keeler et al., 2007). They also concluded that these observed health effects were due to the chemistry of the particulates, or the ambient PM exposure was source-specific (Keeler et al., 2007). Therefore,

25 it can be assumed that any particulate exposure and the outcomes are both source-specific, meaning PM in urban regions such as Detroit may differ in toxicity from those seen in rural desert regions.

1.10 AirCARE1 on the Navajo Reservation

As discussed in detail, inhalable PM is known to have adverse effects on the respiratory and cardiovascular systems, especially when it comes to the remaining legacies of AUMs (Harmon et al., 2016; Zychowski et al., 2018).

However, when investigating these effects in the lab, exposures are usually a single chemical or component and given at high concentrations (Sioutas C,

1997). This is not an accurate representation of what is encountered in the real world. Previously, we found particles within the sediments of the BGT region physically sized less than 2.5µm – potentially small enough to enter the deep lung – and these small particles appeared enriched in metallic elements relative to larger PM (Zychowski et al., 2018; K. E. Zychowski, C. R. S. Tyler, et al.,

2019). However, it was not evident to what degree such particles could be resuspended in to the air and pose an exposure risk to nearby residents. Here, we intended to use AirCARE1 as a means of investigating the potentially harmful effects of an AUM on the Navajo Nation.

The relative risk of PM2.5 from AUMs-induced pulmonary dysfunction and obstructive diseases is currently unknown. To investigate the inflammatory potential of metals present in the ambient air near the Claim 28 AUM, we exposed mice to either filtered air or concentrated ambient PM. Here, we will

26 present information on (1) the amount of the metal-rich PM in the ambient air, (2) wind direction, (3) daily PM distributions, (4) metal content of PM intake, (5) and immunological and physiological responses of mice after PM exposures. We also will use this as the foundation to later determine the relative toxicity of the ambient PM.

Figure 1. Claim 28 Uranium Mine Site. View of the mine site from the trailer. To the untrained eye, it appears to be a normal hill. However, looking to the right end of the center part of the image, is an overspill from the mine site. Like many AUMS, there are no markings, fences, or warnings.

Figure 2. AirCARE1 Mobile Research Laboratory

Image of AirCARE1 parked at Blue Gap location. The mobile lab was placed approximately 1km southwest of the mine site. Within it, the mice were housed and exposed daily to either FA or concentrated PM with one of two chambers inside. The smaller trailer assists with chamber function and particulate collection.

Chapter 2 — Materials and Methods

2.1 Location selection

In order to place the trailer at a specific location near the mine site, we reached out to community members in BGT. As a result of these conversations, we came into contact with a community member who owns land about 1km southwest from the mine site location (see maps below) and had serious concerns about the potential for the mine site to negatively affect her health and that of her livestock.

Permissions were obtained from the BGT chapter leadership and from the

Navajo Tribal Utility Authority to install the trailer and connect to a single phase electrical grid on the land next to her seasonal cabin, which we could also as a home base while operating AirCARE1. The location of the study site is indicated

28 on the map as shown (Figure 3), about 253 miles from the University of New

Mexico.

Colorado Utah

Arizona New Mexico

Blue Gap Claim 28 Tachee DiNEH Study Area Mine Sample Location

Background PM Sample Location > 500 abandoned mines 4 mill sites 1100 exposure features EPA and IHS 2010 100s of contaminated wells (Arsenic, Uranium)

Figure 3. Map of mine sample sites on Navajo Nation

(A) Map of mine sites on Navajo Nation. Red dots indicate AUM sites with AUM regions outlines in orange. Areas circled in blue are designated cleanup areas. Grey regions indicate Chapter boundaries. (B) Aerial view of AirCare1 Laboratory with relation to Claim 28 site.

2.2 Mouse models

Male and female C57BL/6 and Apolipoprotein E-deficient (ApoE-/-) mice were purchased from Jackson Laboratories (Bar Harbor, Maine) at 6-8 weeks old.

C57BL/6 mice served as a healthy, wildtype model and ApoE-/- mice served as a model of hypercholesterolemic vascular disease, which has been previously shown to be a vulnerable phenotype (PMID: 16414948, 24377210). In order to develop high cholesterol and promote early stage vascular lesions, ApoE-/- were fed a high fat chow diet, while C57BL/6 were fed normal chow (non-high cholesterol). Mice were acclimated to the chambers for three days prior to exposure. Day one acclimation was for 1 hour, the next day for 2 hours, the third day for the full 4 hours. Exposures began the next day. All procedures were approved by the UNM Institutional Animal Care and Use Committee, and the

AirCARE1 mobile laboratory was inspected prior to deployment by the UNM institutional veterinarian and Animal Resources Facility Staff

2.3 AirCARE1 Trailer and exposure settings

The AirCARE1 particle concentrating system derived PM from a PM2.5 cut-point tower on the roof of the trailer, ensuring that all exposures were concentrated

PM2.5. The concentrator used three stages of PM concentration to cause a roughly 10X concentration of ambient PM. Due to the remote location, ambient

PM measures were not readily available nor determined, thus the concentrating efficiency was not calculated for the BGT campaign. Mice were then exposed for

4 hours/day for either 1 day or over 28 consecutive days. The cages for the mice

30 held up to 40 mice each and included free access to water. Mice were also on a

12h light cycle (6am-6pm is light, and 6pm-6am was dark) and housed at 21-

25°C. Mice were transported back to the University of New Mexico the day after the final 4-hour exposure (Figure 4).

Mice were rapidly euthanized for collection of tissues. Bronchoalveolar lavage fluid (BALF) was taken. Lungs and aorta were excised and weighed. Organs were then snap-frozen with RNA-later for future bioassays and stored in -80ºC.

Figure 4. Diagram of exposure time lines

Diagram summarizes the exposure timeline from transporting to terminal endpoints of the mice. Day 0 includes transport of mice from the University of New Mexico (UNM) to Blue Gap Tachee, Arizona site. They were then acclimated for three days before being exposed to environmental particulates. Mouse strains included ApoE-/- and C57BL/6. ApoE-/- and C57BL/6 mice were exposed 4 hr/day to either FA or PM for 1 day or 21/28 consecutive days (C57BL/6/ApoE-/-, respectively). Mice were then transported back to UNM for terminal endpoints.

31 2.4 Wind Speed and Direction Analysis

Met stations were placed on top of the Claim 28 mine site and on the trailer where particles were being collected. Both MET stations logged data at 15min intervals for 24hrs/day.

2.5 Bronchoalveolar lavage fluid (BALF) collection and analysis

Following exposures and transport to the laboratory, mice were humanely euthanized with isoflurane (24hours following the last exposure day). BALF analysis was done to examine the influx of macrophages and neutrophils into the lung. BALF was taken from mice by injecting 1ml of PBS into the lungs, massaging the lungs with the PBS inside, and removing remaining fluid after inflation. The total volumes received were recorded. BALF cells were then centrifuged at 1800 x g for 5 minutes to form a cell pellet. The cells were resuspended, stained with trypan blue for a viable cell count with a hemocytometer, and seeded to a number of 10,000 cells/slide. Slides were then visualized under a light microscope and the number of neutrophils and macrophages were manually counted. The supernatants from spun down samples were taken for total protein analysis via Bradford’s assay, the same day.

2.6 Gene expression analysis

RNA from the lungs and aorta was extracted and purified using the Qiagen

RNeasy Fibrous Tissue Mini Kit (Cat. No. 74704). UV spectroscopy was then performed to obtain the total concentration of RNA collected from samples. In

32 order to maintain pure samples, we targeted the 260/280 values to be approximately 2.0 and the 260/230 values to be approximately 1.0. We mainly focused on the 260/230 values, and if they were below 1.8, the samples were re- purified. The cDNA was created by converting 1000 ng/ul RNA using Applied

Biosystems cDNA Reverse Transcription Kit (Cat. No. 4368814) in a total volume of 20μl. The thermocycler settings were set to step 1: 25ᵒC for 10 minutes; step 2: 37ᵒC for 120 minutes; step 3: 85ᵒC for 5 minutes; and step 4:

4ᵒC for “infinity” (mostly held at 4ᵒC for no more than overnight). qPCR was performed with a goal of cDNA with a concentration of 1000ng to determine overall gene expression within samples using Applied Biosystems TaqMan Gene

Expression Master Mix (Cat. No. 4370074). Samples were probed for inflammatory markers TFG-β, TNF-α, CXCL1, IL6, VCAM1, CCL2. TBP and

HPRT1 were used as housekeeping genes for the lungs and aorta, respectively.

Relative gene expression was calculated using the 2-ΔΔCt threshold method.

Relative expression of transcripts from PM2.5-exposed mice were compared to data from FA -exposed mice.

2.7 Inductively coupled plasma mass spectrometry (ICP-MS) of metals collected on exposure chamber filters

Two 47-mm quartz filters and a teflon filter were attached to outlets on the back of the CAPs chamber to captured particles from exposures. Approximately 1 m3 of air was assessed for each 4h exposure day (Harkema JR, 2009).

33 Samples were processed and analyzed in Class 100 and 1000 ultraclean rooms at the Michigan State University Exposure Science Laboratory. Particle samples collected on Teflon filters were wetted with ethanol and extracted in 1% nitric acid solution. The extraction solution was sonicated for 48 h in an ultrasonic bath, and then allowed to passively acid-digest for two weeks. Extracts were then analyzed for a suite of trace elements using high-resolution inductively coupled plasma-mass spectrometry (ELEMENT2, Thermo Finnigan). 15 trace metals were selected, and they included: molybdenum (Mo), cadmium (Cd), gadolinium

(Gd), tungsten (W), lead (Pb), uranium (U), magnesium (Mg), aluminum (Al), vanadium (V), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), and selenium (Se). This analysis method incorporates daily quality assurance and quality control measures including field blanks, Type I waterblanks, replicate analyses and external standards as described elsewhere (e.g., Harkema et al.,

2009).

Because of the single site exposure campaign, ICP-MS data were compiled and compared to literature-reported levels of each metal in different regions of the world. The goal was to find reported levels from at least 5 different sources and compare them to the levels that were then captured on the exposure chamber filters. Comparisons were then graphed using Graph Pad.

2.8 Statistics

Two-tailed Student’s t-tests were performed to compared two groups to each other, mainly PM-exposed to FA-exposed. This was done manually using calculations on Excel.

34 Chapter 3 — Results

3.1 Wind Speed and direction over AirCARE1 and Claim 28 mine site

The MET stations collected data averaged at 15 minute intervals 24 hours a day

for the majority of the exposure period. Several consistent trends for this region

were noted, such as increased wind speed during the afternoon hours (Figure 5).

Average wind speed, percent of time, and wind Bblowing direction over AirCARE1

and Claim 28 were represented using a rose wind graph (Fig. 6a and b). We

observed that the average wind direction was relatively consistent. At the trailer –

lower in the canyon - wind typically blew from the southwest during the daytime

and from the north-northwest during the evening. Importantly, however, the

typical large gusts came from the southwest and they came during the hours of

the mouse exposures. During the night, the wind generally moved from the

northwest to southeast past the trailer, following the direction of the Tachee

canyon. The wind rose plot for the mine site location was similar, but with wind

typically traveling from the south to north most days. In terms of trailer placement

WindSpeed (mph)

0 0.0 0.2 0.4 0.6 0.8 1.0 Time of Day (Fraction of 24h) Figure 5. Daily Snapshot of Principal Wind Speed Results

Averaged over 24h for every day of exposure, the typical wind speed accelerated during the daytime and subsided in the evening. Each point denotes a 15 min average speed.

35 to Claim 28, the wind moves from the position of the trailer placement towards

the Claim 28 mine site. More poignantly, the wind typically moves from the

community to the mine site, and infrequently from the mine site towards the

community.

A B

C D

the...)

(From

Direction

Wind

0.0 0.2 0.4 0.6 0.8 1.0 Time of Day (Fraction of 24h)

Figure 6. Blue Gap Wind Distribution

A and B are wind rose plots that show that the overall average wind current, average speed, and percent of time that wind was blowing. Note that the average gust direction goes from SW to NE over the AirCARE1 site (above), and travel from NW towards SSE over the Claim 28 site (above right). C. A topographic map of the Blue Gap site that summarizes the wind directions during the day (orange) and night (blue). A graphical interpretation is also given in D.

36 3.2 PM Distribution

Inside the trailer exposure chambers the concentration of incoming PM was being calculated via the SidePak monitor. While the concentration system is on, the

SidePak records changes in the amount of PM in the chamber. Raw data values were reviewed and graphed on GraphPad. As both strains were exposed separately from each other, the average PM levels for each strain varied based on the SidePak measurements (Figure 7). The overall exposures (averages of all 4h exposure periods) for ApoE-/- mice (Figure 7A) were roughly 30% higher than those of C57BL/6 mice (Figure 7B). Daily variations were notable, as determined by other climatological factors, as shown in Figure 7C,D.

A. ApoE-/- Exposures B. C57BL/6 Exposures

0.5 0.5

28-day 1-day

) )

3 0.4 3 0.4

(mg/m 0.3 0.3 21-day 1-day

PM Levels (mg/m Levels PM 0.2 Levels PM 0.2

DustTrak 0.1 DustTrak 0.1

0.0 0.0 0 100 200 300 0 100 200 300 Time (min) Time (min)

-/- C. ApoE Exposures D. C57BL/6 Exposures 1.0 1.0

)

0.8 ) 3 0.8 3

0.6 0.6

PM Levels (mg/m Levels PM 0.4 (mg/m PM Levels 0.4

DustTrak DustTrak DustTrak 0.2 0.2

0.0 0.0

3 4 5 6 7 8 9 2 2 2 2 3 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 ------3 6 7 9 0 - - - - Day- - - - - 0 1 2 3 4 5 6 7 0 1Day2 3 4 5 6 7 8 9 0 1 2 3 A A A A A A A - - - - - M M M M M M M M M ------p p p p p p p A A A A A a a a a a a a a a M M M M M M M M A A A A A A A A A A A A A A r r r r r r r p p p p p y y y y y y y y y a a a a a a a a p p p p p p p p p p p p p p r r r r r y y y y y y y y r r r r r r r r r r r r r r

Figure 7. Average and Daily PM Distributions

Four-hour PM concentration average in each exposure campaign for ApoE-/- (A) and C57BL/6 (B) as measured by the SidePak personal PM monitor. Average daily measurements for ApoE-/- (C) and C57BL/6 (D) are also shown. The red symbols in C and D indicate the single-day exposures for each strain.

3.3 BALF Analysis

Macrophage, neutrophil, and total cell counts were assessed from the BALF of

C57BL/6 mice exposed to FA or PM (Fig. 8). Cell counts were compared between FA and PM groups, then further compared to length of exposure, either

1- or 28-day. A general trend in elevated macrophage and neutrophil cell counts in the BALF of PM-exposed mice compared to FA-exposed mice was observed

(Fig. 8A,B, and D), except in the neutrophils of ApoE-/- 28-day group (Fig. E). 1- day PM exposures significantly increase neutrophil and macrophage cell counts, compared to the FA exposure in C57BL/6 groups (Fig 8A and B). Neutrophil counts in these animals also revealed a very significant increase at the 21-day

PM exposure, compared to the 1-day exposure (Fig. 8B). There was no significant change in macrophages or neutrophils between FA 1-day and FA 21- day of the ApoE-/- (Fig. 8D and E).

Analysis of BALF from ApoE-/- mice exposed to PM showed no significant changes in macrophage or neutrophil counts or lavage protein levels across all exposure durations compared to FA controls (fig 8). The cell counts from PM- exposed ApoE-/- mice show no significant differences compared to FA-exposed mice at either exposure duration (Fig 8).

A. C57BL/6 B . C57BL/6 C. C57BL/6

0.20 0.008 p=0.009 2.0

)/ml 0.007

6 0.15 0.006 1.5

)/ml (x10 6 0.005

(mg/ml) 0.10 0.004 1.0 0.003

0.05 0.002 0.5 PMN (x10 0.001 Protein

Macrophages 0.00 0.000 0.0

, , , 2 2 2 2 2 2 P . 1 P . 1 P . 1 P . P . P . M 5 d M 5 d M 5 d M 5 M 5 M 5

D. ApoE-/- E. ApoE-/- F. ApoE-/-

0.10 0.008 2.0

)/ml 0.007

6 0.08 0.006 1.5

)/ml 6 0.005 0.06 0.004 1.0 (x10 0.04 0.003

0.002 0.5 0.02 PMN

0.001 Protein (mg/ml)

Macrophages (x10 Macrophages 0.00 0.000 0.0

, , , 2 2 2 2 2 2 P . 1 P . 1 P . 1 P . P . P . M 5 d M 5 d M 5 d M 5 M 5 M 5

Figure 8. BALF Cell Differential Analysis

BALF cell counts and protein from both C57BL/6 (A-C) and ApoE-/- (D-F) exposures groups are shown. There was an increase in macrophage counts in PM-exposed C57 mice, but not ApoE-/- mice. C57BL/6 mice also exhibited a significant neutrophilia after a single day of exposure. No protein elevations were seen in either strain due to PM exposures. n=8-10/group

3.4 Lung and aorta qPCR analysis results

Next, to analyze the potential of the concentrated PM to induce an inflammatory response in the whole lung, we quantified several pro-inflammatory markers from mRNA of isolated lung tissue, comparing PM versus FA. Lung mRNA expression levels of TGF-beta, TNF-alpha, CXCL-1 and IL-6 were analyzed for both strains and both timepoints. Lung tissue from ApoE-/- mice exposed for 1 day was probed for VCAM, CCL2, and IL-6. The probes differ because these were the initial probes selected, but technical issues arose and they could not be re-run because samples were unfortunately lost. There are no significant changes in the

1-day exposures of the C57BL/6 groups (Fig. 9). The only significant change seen within the C57BL/6 group is at 28-days of exposure via a decrease in TGF-

ß (p= 0.01) (Fig. 9). In terms of the ApoE-/- groups, a significant decrease in

VCAM is seen after the 1-day exposure, and then again in TNF-alpha after a 28- day exposure (Fig.9C and D).

A B C 5 7 B L / 6 1 d a y e x p o s u r e s C 5 7 B L / 6 2 8 d a y e x p o s u r e s

2 .5 1 .5

)

F 2 .0 M F C ) M

(

e 1 .0 g

1 .5 a

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n io s s r e p x E e n e G in M e a n F o ld C h a n g e ( M F C ) M e a n F o ld C h a n g e ( M F C ) 0 .0 0 .0 I

I - L V C L C X C C T G F C L b e T N F 1 6 A L 6 - ta a lp h a M 2

Figure 9. Lung qPCR Analysis Results

qPCR analysis of the lungs. C57BL/6 showed no significant changes in mean fold expression after 1-day exposures (Fig.9A). The mean fold change (MFC) in gene expression of TGF-b after PM-exposure was statically reduced in C57BL/6 group (p- value of 0.01). (Fig.9B). ApoE-/- showed statistically significant decreases (p-value of 0.02) in VCAM (Fig. 9C). In the 28-day ApoE-/- group, TNF-a was statistically reduced (p-value of 0.01) (Fig. 9D). n=10/group

We also investigated the extrapulmonary inflammatory effects of the aorta via vasoreactivity done with myography (K. E. Zychowski, C. R. S. Tyler, et al.,

2019). Here, our interest in the aorta is to determine the inflammatory response at the cardiovascular level. Markers for the C57BL/6 mice 1-day and 28-day exposures were TGF-beta, TNF-alpha, CXCL1, and IL6. Markers for the ApoE-/-

1-day and 28-day exposures are TGF-beta, CCL2, CXCL1, and IL6. Exposures were then graphed for each mouse strain with respect to their exposure times, and they were then graphed to compare FA versus PM.

C57BL/6 1-day exposures showed a small decrease in mean fold change (MFC) in TGF-beta, while TNF-alpha and CXCL1 showed a very subtle increase. IL6, however, showed a more obvious increase in MFC. However, there was no significant increases or decreases seen after a 1-day exposure in the C57BL/6 mice.

C57BL/6 28-day exposures showed minor decreases in MFC of TGF-beta and

TNF-alpha, a slight increase in IL6, and more of an increase in CXCL1. However, similar to the 1-day groups, there was no significant increases or decreases seen after a 28-day exposure of the C57BL/6 mice.

ApoE-/- 1-day exposures showed an overall decrease in MFC for TGF-beta,

CCL2, and IL6; and there was an increase seen in CXCL1. Similarily, the ApoE-/-

28-day exposures saw in a slight decrease in TGF-beta, but obvious increases in

MFC of CCL2, CXCL1, and IL6. However, none of these changes in both ApoE-/-

1-day or 28-day exposures were statically significant. Collectively, this data tells

us that the concentrated air from Claim 28 had no effect.

A B C 5 7 B L / 6 2 8 d a y e x p o s u r e s C 5 7 B L / 6 1 d a y e x p o s u r e s 2 .0

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2 2 Figure 10. Aorta qPCR Analysis Results

Results of the qPCR analysis of the aorta. The top row of figures show C57BL/6 mice and the bottom show the cardiovascular disease model representative group (ApoE-/- mice) after one day or 28 day exposures. Although there were slight differences seen between FA and PM exposures, there was no overall change seen in inflammatory markers of ApoE-/- mice (C-D). N=10/group

44 3.5 Trace Metals Analysis using inductively coupled plasma-mass spectrometry (ICP-MS)

In the CAPs chamber, Teflon filters were used to capture PM and we used ICP-

MS to selectively measure 15 crustal and trace metals. We selected molybdenum (Mo), cadmium (Cd), gadolinium (Gd), tungsten (W), lead (Pb), uranium (U), magnesium (Mg), aluminum (Al), vanadium (V), iron (Fe), nickel

(Ni), copper (Cu), zinc (Zn), arsenic (As), and selenium (Se). These values were then graphed together the raw data of the metals, and their concentrations (in ng/m3) found present (in measurable levels) within filters from the exposure chambers, showing the mean, minimum (min.), maximum (max.) and standard deviations (Table 1). The values were then graphed to visualize these values against thesis values in a violin graph (Fig. 11).

Table 1. ICP-MS analysis raw data table

Table shows the raw, compiled data in terms of mean, minimum (min.), maximum (max.), and standard devaitions for each trace metal measured for. The visual for these values are seen in Fig. 11.

Figure 11. ICP-MS A 1 0 0 0 0 0 analysis

)

3 1 0 0 0 0

showed the metals m

/ 1 0 0 0 that were captured in g

n the filters of the (

1 0 0 n exposure chambers.

io The table shows the

t 1 0

a mean, minimum, tr 1 maximum, and n

e standard deviation of

c 0 . 1

n the metal’s

o 0 . 0 1 concentration, in C 3 ng/m . 0 . 0 0 1

M e a s u re d E le m e n ts

3.6 ICP-MS Literature Comparison

We assessed the total concentration for select trace metals, but in order to know the meaning of these values, and whether or not they are of concern to the BGT community, we did an extensive literature search for concentrations reported in environmental literature. We aimed to find at least five references for each metal and to graph their averages, but it should also be noted that some metals are more commonly measured and reported than others. The average concentration of each metal was compared to reported levels (also in ng/m3) found in the literature (Fig 12). Based upon several references each, the levels of each metal found were generally in line with the reported levels. U and V, which were of specific interest due to their abundance in sediments from the Claim 28 mine site

(Zychowski et al, 2018) were shown to be no different in the exposure chambers

46 than the environmental concentrations reported in other regions (Fig 12). The average concentration of other metals of concern, such as Cd, Pb, Ni, Cu, As, and Se also show no difference compared to previously reported concentrations

(Fig 12). W, Mg, Al, and Fe show potential for significant changes, however there was a high degree of variability in the reported values and we did not conduct statistical tests for this comparison. Collectively, there is potential for some of the metals in our exposures to be higher than the average of the reported values, but the majority of the selected metals do not appear to be meaningfully higher or lower than the global reported concentrations.

1 0 0 0 0 0

)

1 0 0 0 0 3

1 0 0 0 g

(

1 0 0 n

t 1 0

tra 1 n

c 0 . 1 n

C 0 . 0 1

0 . 0 0 1

I Z L N U r C A C a T u A lu V a o ic o i rs S e e ra M a g k M o ly d m n g m in n a d p n e le n n iu n e s b d e n iu m s te u m iu m n e p n iu m u m n a m iu m l e r c ic M e a s u re d E le m de n ts

Figure 12. Literature comparison with references

This figure compiles the comparison of reported levels of the metals to the levels found present in BGT exposures. For each metal, the bars to the right are the concentrations measured from CAPs filters, and the bars to the left are the compiled averages of concentrations reported in published literature.

47 3.7 ICP-MS of Metal Concentrations in the Lung

Lastly, because we saw some genetic changes in the C57BL/6 groups, we measured the metal content in the lungs of the PM- and FA-exposed groups also using ICP-MS. We then graphed them from smallest to largest concentration.

Surprisingly, V and U levels were low. However, Fe and Mg concentrations in lung show now difference here, compared to the differences seen in Fig. 12.

Interestingly enough, the statistics for Al gave a p-value of 0.06 when comparing

PM-exposed to FA-exposed. Considering that the Al values seen from the filter

ICP-MS analysis are about as high as Mg and Fe, this may be the reason as to why there was almost a significant difference after PM-exposure.

100000

)

µg 10000

(in

1000

100

concentration 10

elemental

0.1

N W U V A S Z F i l e n e

Figure 13. Metal concentrations measured in lungs

This figures shows the concentrations of trace metals in the lungs between FA- and PM- exposures. Notably, Here, they are graphed from lowest to largest concentration.

48 Chapter 4 — Discussion

4.1 Particulate Matter Toxicity

Our previous findings showed proximity to an AUM in the southeastern region of Navajo Nation associated with circulating inflammatory potential

(Harmon et al., 2017). Furthermore, contaminated sediments of the Claim 28 mine contain respirable metal-containing particulates and elevated concentrations of U and V that induced inflammatory responses and increased aortic vasoconstriction (Zychowski et al., 2018). These studies led to the concern that the metals contained at the surface level of the mine could also exist as windblown PM and, therefore, this exposure route could adversely impact the neighboring community.

PM naturally exists in BGT, varying in concentration based on seasonal and climatological factors. This of concern because the smaller metal-bearing particles (PM2.5) that could travel further (and remain aloft longer) than the large particles previously seen in sediments, which, at small amounts at a time over decades of exposure, could pose pathological substrates if inhaled (Yang et al.,

2019). From the measurements during our campaigns in the Spring of 2018, we see variation in the (concentrated) PM levels that reflect both long-range particle transport and short-term disturbances from human activity (specifically, vehicle travel on the dirt road). In the daily accounts of PM level distribution, there is wide variation seen over both ApoE-/- and C57BL/6 exposures. Some days had higher wind than others, and this is very typical of this region, especially for an area that

49 exists within a canyon/valley area. Though not reported from the present exposure campaign, preliminary work in the winter found high levels of PM during periods of relatively still winds, which we predict is due to partial weather inversions and build-up of wood smoke PM. For this area of the Navajo Nation, the winds can be unpredictable and can depend on season, but spring is usually windier than the colder months.

For ambient particle exposures, both mouse strains were exposed separately and, therefore, the exposures had different concentrated PM levels

(i.e. more particulates were being blown between April 23-May 17, the time of

ApoE-/- exposures seen in Fig7C). What is interesting, however, is the ApoE-/- mice inhaled higher concentrated PM levels than the C57BL/6 mice, but the results do not indicate any statistically significant changes with either the 1-day or

28-day exposures. The C57BL/6 mice demonstrated mild, but statistically significant elevations in BALF macrophages and neutrophils. The reason for vulnerability in the presumably health model is not known, but the ApoE-/- mice were chosen for vulnerability to vascular lesions and not necessarily pulmonary injury. Although not much inflammation could be deduced, it is very important to think about the geographical and spatial area in which these short term PM2.5 exposures were done because seasons could affect the heterogeneity of the particles available (Hoover et al., 2017).

The overall wind direction we gathered was blowing toward the northern end of the canyon (Fig.6C). Wind blowing from the southwest – presumably blowing Claim 28 PM away from the trailer – during the spring and early summer

50 season, may prove to be beneficial for most of the community in BGT located west and south of the mine site because of reduced risk of being exposed to harmful particulates. However, there are a few homes up the canyon to the east and downwind of the mine site, and others may also reside on the northeastern end of the canyon that could be at high risk of exposure to respirable particulates. A lack of electrical facilities in that area limited our ability to install sampling equipment to address this concern.

Macrophages and neutrophils are known to be responders to physical and chemical damage, specifically through the innate immune system via a pro- inflammatory cascade system (Kolaczkowska & Kubes, 2013). The cell differential counts found from the ApoE-/- exposures were surprising because the

ApoE-/- mice were used as a cardiovascular disease model for their systematic proinflammatory status (Sasso et al., 2016) and, therefore, we expected them to show a higher inflammatory response than the control C57BL/6 mice. This unexpected absence of response in the ApoE-/- mice could also be because of the variable PM distribution seen in Fig.7A and B. In order to see the inflammatory damage through BALF, the PM concentrations may need to be more consistent, or longer duration exposures may need to be explored. This unexpected outcome could also be because this strain of mouse is already predisposed to inflammation, and we may need to use another model to examine cardiovascular effects more closely. Alternatives to the ApoE-/- strain include the low density lipoprotein receptor -deficient mice (LDLR-/-), and along with the

51 dietary difference of being fed a high fat chow diet (our mice were all fed regular chow diet), this strain may be a possible substitute in the future.

Another surprising result was the reduced difference seen in the number of neutrophils of the ApoE-/- when comparing the 1-day PM and the 28-day PM.

Although there is variability seen from the wide standard error mean bar at the 1- day PM, resulting in an overall non-significant decrease, this is still intriguing because it is a rather large difference. Again, the argument could be made that this mouse strain is already pre-disposed to inflammation. However, another possible explanation could be that over the course of a 28-day exposure, the mice are acclimating to the air being given. Therefore, a more chronic exposure

(increased time of exposure of increased concentration) should be taken into account for an effect to be seen.

In contrast to the ApoE-/- BALF findings, the C57BL/6 mice were exposed to a more consistent, albeit lower, concentration of PM. For both exposure durations for this mouse strain, the PM distribution averaged about 0.15 mg/m3 for both the 1-day and 21-day exposures (Fig7A and B). The physical effects of damage-induced inflammation were most prominent with the C57BL/6 exposures groups. Collectively, the C57BL/6 group showed a significant increase in both macrophages and neutrophils when exposed to PM2.5 versus FA. This portion of the data was most consistent with previous findings Claim 28 dust-induced damage (Zychowski et al., 2018)

In previous findings, we found that exposure to Claim 28 dust, and with

AUM proximity in mind, inflammatory potential increased in mice models

52 (Harmon et al., 2017). Specifically, the ligands CCL2, VCAM-1, and ICAM-1 mRNA expression levels in serum treat in vitro models increased with increase proximity (Harmon et al., 2017); and IL-1ß, IL-6, TNF-alpha, IL-10, IL-2, and IL-5 were increased after exposure to Claim 28 dust, as seen in BALF samples

(Zychowski et al., 2018). After exposure to concentrated ambient particulate exposures from Claim 28, for 28-days, the C57BL/6 groups showed a total significant decrease in TGF-beta. Overall, however, it is concluded that the inflammatory effects of PM for both models were mild and consistent with other reports using similar approaches (PMID: 23951156)

4.2 Thoughts on trace metals data

These results did bring to light the possibility of other metals being at the foundation of inflammatory and cardiovascular effects being reported in this area, rather than just the presence of U and V alone, as previously thought. It has been demonstrated that, specifically, mixtures of Fe, Ni, and V, when given in the soluble fraction of residual oil fly ash, produces acute pulmonary toxicity

(Campen et al., 2002; Dreher, 1997). Another contributing factor for these three specific metals is because that their acute toxicity is due to mediation through oxidative mechanisms (Albretsen, 2006; Campen et al., 2002; Dye, 1999;

Jaishankar, Tseten, Anbalagan, Mathew, & Beeregowda, 2014). In a 2002 study,

Fe, V, and Ni (alone and in combination) were instilled into the lungs of (MCT)- treated rats, using monocrotaline, as a cardiovascular disease model (also characterized by lung inflammation, oxidant stress, pulmonary hypertension, and

53 acute pulmonary respiratory distress syndrome) (Campen et al., 2002). End result measurements included heart rate and measured incidences of arrhythmias. Their data showed that the metals, when instilled as a single substance, did produce effects in the healthy versus unhealthy models—some more than others. Both their Ni-only and V-only dose showed an acute response of decreased heart rates and increased incidences of arrhythmias in their unhealthy models; however, Ni + V doses caused a synergistic increase in arrhythmias (Campen et al., 2002). Even more interesting, the addition of Fe to their Ni + V dose, seemed to attenuate the cardiac effects (Campen et al., 2002).

A more recent study done by our group characterized sediment samples that were obtained from near a tungsten-molybdenum ore-processing complex found that metal concentrations were 10X higher than those found in control sediment (K. E. Zychowski, A. Wheeler, et al., 2019). Interestingly, a correlative effect was also seen with lead and cadmium; therefore, the noted influence on pulmonary and systemic effects are thought to be due to the elemental composition of the particulates being inhaled (K. E. Zychowski, A. Wheeler, et al.,

2019).

The data shown in our trace metals analysis from ambient PM2.5 exposures near Claim 28 – especially U and V – appear to be no different from reported levels in the rest of the world. However, it does appear that W, Mg, Al, and Fe show a 10-fold difference. Tungsten in the BGT region shows to be well below reported concentrations, although there were very few published studies reporting this element. Mg, Al, and Fe, on the other hand, seem to be higher

54 than their reported concentrations. These are important factors because, it highlights the contribution of pathogenicity to also be dependent on PM composition, which, in turn contribute to the solubility and reactive oxidation species (ROS) production that may contribute to human health hazards (S. Jena,

& Singh, G. , 2017; S. Jena, Perwez, & Singh, 2019; Spurny, 1998). Each metal has factors that could have the potential for human hazard. Tungsten, for example—at the center of use for many consumer products, technology, and ammunition production, as well as an implication for acute childhood leukemia development(Walker, Pritsos, & Seiler, 2012)—has a variety of oxidation states that allow it to have enhanced mobility when coming in contact with organic and inorganic solutions (Koutsospyros, Braida, Christodoulatos, Dermatas, & Strigul,

2006; Lassner & Schubert). However, this metal has not been at the center of investigations and, therefore, have no set regulations or standards, making it problematic for environmental interactions (Koutsospyros et al., 2006). In regard to the BGT data we have compiled, the trace metal, tungsten, is well below the reported levels but it can be noted that the levels present were high enough to be measured but should not be of concern.

Of the three metals that appeared to be above reported levels, iron stands out because it has been at the center of several inhalation toxicology studies.

Mostly studied in relation to welding and iron-ore mining, the inhalable iron from fumes has been shown to promote lung cancer (Falcone et al., 2018). The industrial mining involves alteration of the iron-containing products to produce a pure substance (Billings & Howard, 1993; Kornberg et al., 2017). A simple study

55 of rabbits exposed to Fe in high and low concentrations for 6 hours/day for 2 months showed increased amounts of alveolar macrophages, enlarged lysosomes, and an enhanced phagocytic capacity (Johansson, Curstedt, Rasool,

Jarstrand, & Camner, 1992). In 2018, Falcone et al. noted that Fe was accessible from welding fumes promoted lung cancer, marking it as a carcinogenic metal.

Excess iron exposure increases the risk of cancer and, possibly, susceptibility to asbestosis, hemochromatosis, and endometriosis (Falcone et al., 2018). Another study done in 2018 evaluated urine samples from industrial workers, who have at least 10-year experience and, therefore, a prolonged exposure, and found elevated oxidative stress and inflammatory markers. Overall, Fe is of concern because of its known indication as a driver for inflammatory response and increased lung cytotoxicity (Kornberg et al., 2017; Zeidler-Erdely et al., 2008).

A possible mechanism for Fe has been indicated, mostly, because of its variable oxidation states (Pauluhn, 2012). Iron oxides tend to have a high electrostatic interaction and the ability to catalyze ROS pathways that can result in excess cellular damage and increased promotion of cellular death in tissues

(Pauluhn, 2012). Although, an essential to life, Fe in excess and in the wrong places or the wrong form (such as in particulate, rather than soluble) can be a threat to cells and tissues, causing particle overload to the point where more than

6% of the available phagocyte’s volume becomes particle volume (Crichton &

Ward, 2003; Pauluhn, 2012).

Therefore, it would be interesting to look more into the possibility of Fe being an antagonist in particulate form in Blue Gap, AZ. Because iron in the

56 environment most exists as magnetite or lodestone in its mineral form (Pauluhn,

2012), it could be a possibility that particulates from these forms are being resuspended in the air, along with additional trace metals, and that the resulting pulmonary assault is due to both the particle’s mineral compilation and the individuals overall resulting lung burden.

Aluminum and magnesium were additional metals highlighted in our literature comparison. Aluminum, like the other metals mentioned, has several oxidation states—each of which exhibit different surface properties—and, therefore, the toxicity of aluminum may have varying effects (Krewski et al.,

2007b). The soluble form of Al, when ingested, can eventually accumulate within the blood stream and has been seen to eventually cross the blood brain barrier, relating its toxicity to Alzheimer’s Disease and other related neurodegenerative diseases (Krewski et al., 2007a). Inhalable Al, on the other hand has been seen to cause alveolar proteinosis, lipid pneumonia, nodular fibrosis, and other foreign body reactions (Krewski et al., 2007b). Other reports continue to list an influence on overall human health and hormonal status, including: increased susceptibility to bacterial infection, asthma-like symptoms, increased lung and bladder cancer, loss of coordination, and memory deficits (Krewski et al., 2007b).

Magnesium, on the other hand, has been used for the development of novel commercial, technological, and medical applications (Mazaheri, Naghsh,

Karimi, & Salavati, 2019). Additionally, it was recently investigated for use to counteract phosphate toxicity in chronic kidney disease (Sakaguchi, Hamano, &

Isaka, 2018), and is thought to have a positive antioxidant status that can prove

57 to be beneficial (Kiranmai & Reddy, 2013). However, many of the current studies lack the dosage toxicity and possible detrimental effects. It is curious to think that in Blue Gap, Mg could be beneficial at the levels seen, posing the question of whether or not Mg could try to counteract the effects being seen, and known, from the other metals. Or, unlike other reports on Mg, it could also be possible that Mg is contributing further to the previously reported inflammatory effects.

This could be a potential avenue for additional future studies.

Additionally, the metals found in the lungs showed differing values between Al, Fe, and Mg. In Fig. 12, we see that the three are at similar concentrations during the PM-exposures. In Fig. 13, we see that Al concentrations in the lung are lower than Mg and Fe. In doing the statistics between FA- and PM-exposed, the Al values gave a p-value of 0.06 (close to significant). The fact that Al concentrations in the PM-exposure chamber are high, could have contributed to this difference.

Another interesting aspect to look into is the possibility of the residual particulates from BGT inducing an autoimmune response. Autoimmunity occurs when there is a failed balanced between effector and functional T-regulatory cells that essentially results in a defective elimination of self-reactive lymphocytes

(Bluestone, Tang, & Sedwick, 2008; Rosenblum, Remedios, & Abbas, 2015).

Characteristics of an autoimmune disorder result in on and off phases between clinical remissions and systemic inflammation episodes, or flares (Rosenblum et al., 2015). It was noted in Rosenblum et al. 2015 that development of an autoimmune disorder could be initiated by a combination of an individual’s

58 genetic factors and their environmental exposures. However, the issue of what specific environmental factors those are remain to be determined. Claim 28 at

BGT may exhibit some of these specific environmental factors and could be subject to further study in the future by investigating the presence of autoantibodies in exposed models or the community.

4.3 Caveats and Future Directions

Using the AirCARE1 trailer had important advantages for these real-world PM exposure studies. However, the remote nature of the work posed numerous challenges, such as when electrical power failed, forcing us to reduce the exposure time of the C57BL/6 mice from 28-days to 21-days. Regardless, the mice were taken back to the University for sacrifice 24h after the final exposure, as was consistent for all other groups.

Another issue is that, due to the different diets and genetics between the

C57BL/6 mice and the ApoE-/- mice, it is not possible to truly compare them head-to-head. The ApoE-/- mice were chosen due to the disease model, not the genetic deletion, per se. The high fat diet promotes the hypercholesterolemia in this mouse and ultimately drives substantial vascular lesions. The present study relied on mRNA markers in the aorta to identify potential vascular inflammatory outcomes of PM exposure, but future work may need to utilize histopathological approaches to better detail inflammatory and morphological changes of vascular lesions.

59 Chapter 5 — Conclusions

Claim 28 has been, and continues to be, at the center of research for various groups at our institution. We had justifiable concerns that potentially contaminated PM2.5 derived from the mine site could exist within the surrounding region and possibly be a health concern for residents in BGT. The effects of exposure to these respirable particles and their toxicity (as seen through our previous findings) can be a result of their existing chemical composition, as well as the total chemical composition and shape of the particulate being inhaled—as any particulate most likely exists in part alongside other trace metals.

However, we did not see any meaningful elevations in the PM levels of uranium or vanadium in the airborne PM2.5, with AirCARE1 placed 1km southwest of

Claim 28 mine site. Importantly, the majority of the BGT population resides further south from our trailer, thus our findings reflect a good approximation of a

“worst-case scenario”. We also did not see any consistent pulmonary or vascular toxicity from the concentrated PM, even at ~10X the background levels, at the parked location. In our final assessment, the predominant wind blows from the community of BGT towards the mine site and is therefore largely protective for the BGT community members. It was further observed that even on days with the winds blowing in opposite directions, there was no discernible spike in uranium or vanadium levels. Therefore, future attention should focus on the addition or presence of other trace metals, and continue to assess the potential for

60 groundwater and soil contamination around the Claim 28 region (PMID:

26158204)

61 References

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