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Day 8: Ping-Pong Pathologists

For my last day at the National Toxicology Program in the National Institute of Health, I spent my time in the morning attending some interesting toxicology technical briefings on different chemicals recently tested for carcinogenesis. The most interesting of the studies were focused on the hookah and electronic cigarettes, primarily to find out if nicotine is a carcinogen. The study also aimed to determine how to classify the variety of additives used with electronic cigarettes as tobacco related products or not. During the study, I found it interesting to be one of two high school students in the room as the researchers (there were probably around thirty) around us discussed tobacco use in teens; I probably received a couple of awkward looks, but nevertheless, I learned a lot.

Later, Grace and I met with a molecular pathologist to discuss how the work was done by Dr. Malarkey as a veterinary pathologist differs from molecular pathology. We learned that molecular pathology is primarily concerned with how a cancer is created (the gene expression of the cancerous cells and RNA or DNA defects), while veterinary pathology is concerned with identifying tumors through histology. Then, Grace and I presented our cell phone study presentations, in which we drew our own conclusions on whether cell phone radiofrequency radiation proposes a carcinogenic risk.

Finally, to finish off our time at the NIEHS, we joined an office ping-pong tournament, where we witnessed some of the ping-pong regulars during their practice session; they take a break from their labs for ninety minutes every day at 2:30 pm to hone their skills. In the end, I enjoyed my time at the NTP and am glad to have had the opportunity to learn so much in just two weeks!

The NIEHS as seen from the lake and the ping-pong room!

Day 6: Archives

In the morning, Grace, Dr. Malarkey, and I traveled to another National Institute of Environmental Health Sciences building, known as the National Toxicology Program Archive. At the NTP Archive, we joined some of the staff there on a tour of all the different storage units there. First, we visited a fire and vacuum sealed room containing all the NTP project files, dating from 1980 to present. Next, we visited the room that contains all the histopathology slides from projects; government policy requires that all slides must be archived. The NTP archive staff is currently in the process of scanning all histopathology slides to put on a public atlas for easy access; it is currently in a pre-release phase only accessible by NIEHS scientists. Later, we toured the wet tissue archives, which houses all the tissue that has been studied in previous projects. Each tissue is preserved in chemicals and vacuum sealed into a plastic container. Finally, we had the opportunity to travel into the massive freezer warehouse, which houses dozens of minus eighty degrees Celsius refrigerators, and for extreme cooling, roughly twenty liquid nitrogen tanks for samples. Grace and I also, briefly, walked into the minus twenty degrees Celsius freezer about the size of a school bus.

After our tour, we sat down with Dr. Malarkey at a microscope to review some of the archived slides from the GSM and CDMA Modulated Cell Phone Radio Frequency Radiation study. We went through a couple of different histopathology slides from male rats and attempted to identify what lesions if any, the radiation caused on the tissue. We identified a particularly interesting malignant oligodendroglioma in the brain of one mouse, which is indicatory of an adverse effect from radiation. The challenge, though, is determining if an unusual brain tumor in one rat out of ninety is enough to justify cell phone radiation as a carcinogen. Further studies are required and are currently in planning at the NTP, as present data is inconclusive. We also found some cataracts in the lens of the eye, a tumor in the liver, and extramedullary hematopoiesis (the creation of blood cells outside of bone).

During lunch, Dr. Malarkey challenged Grace and me to take our food up to one of the conference rooms where he would be reviewing tumors and lesions with veterinary pathology students. I tried my best to stay focused on the screen while eating, but some of them made me look away while trying to stomach my meal! Nevertheless, I learned a lot and had another great day.

The nitrogen tanks and minus 80 degrees Celsius freezers.

Day 5: Cornea’s and Cancer

Today I began my second week at the National Toxicology Program with Dr. Malarkey, or the pathology group. Grace Goetz also ventured to the National Institute of Environmental Health Sciences to join us. Early in the day, Dr. Malarkey showed Grace and I his shelf of interesting medical items that he has collected over his life. His collected contained memorabilia ranging from the first microscope he was given as a child to a 19^th century copy of a veterinary manual title Diseases in Large Animals. It was mostly about cows.

Later, Dr. Malarkey obtained one of the microscopes previously by a postdoctoral fellow for me to use so I could review histopathology slides on my own. Dr. Malarkey handed me a couple of organ slices from the tissue of an eye and asked me to identify the different components of the eye, the different lesions, and the location and type of tumors. After a while of switching between the microscope and the internet, I was able to identify the iris, lens, cornea optic nerve, retina, cornea, vitreous humor of the eye. With the help of Dr. Malarkey, I located melanoma, a tumor commonly found in the skin, near the near the lens of the eye. Because of the melanoma, the iris and retina of the eye were folded and rendered inoperable. I also located a separate tumor that resulted in unbounded muscle growth besides the optic nerve and cataracts under the lens epithelium. I then headed to the ten-headed with microscopes to look at some brain slices with a variety of cancers, known as neuroglial neoplasms. Glioma is name for a brain cell that falls into a miscellaneous category, other cells are ependymal cells, astrocytes, oligodendrocytes, microglia, and Schwann cells. Each cell can also cause a unique cancer where that cell has unbounded reproduction, for example astrocytes can cause astrocytoma and oligodendrocytes can cause oligodendroglioma. Dr. Malarkey also explained the immunohistochemistry used to visualize different tumors. Once a tumor cell type has been identified, antibodies with dye are splashed over the cell that attach to a certain protein prevalent in the cancerous cell. The loose dye is then washed away, leaving only the proteins in the tumor with dye, thus visualizing the cancerous areas. We specifically looked at an example of a brain histopathology slide with a malignant glioma, and the protein S100. If the cell contained S100, the brown dye would remain, and if it didn’t the dye would wash away. Grace and I also got a chance to explore the necropsy lab, where the dissections take place.

An electron microscope!

Day 4: Histopathology and Chemical Testing

For my fourth day at the National Toxicology Program, I continued to work with Dr. DeVito in the Predictive Toxicology and Screening Group. Early in the day, I attended a peer review session as an observer at the ten-headed microscope. The review consisted of a group of pathologists who gathered to make suggestions regarding the research of another pathologist. Reviews like this allow for research to be vetted and checked for accuracy before it is published, to avoid any controversial or misleading conclusions. The review session I attended was meant to confirm or reject diagnoses of cytoplasmic alterations, fibrosis, and bile duct hyperplasia in histopathology slides of rat livers exposed to different doses of dibutyl phthalate. Dibutyl phthalate is commonly found as a plasticizer in nail polish.

After looking at slides through the ten-headed microscope, without exactly knowing what I was looking for, I returned to my cubicle to begin reading about how to identify different lesions under a microscope. I learned that fibrosis appears as an increase in cell density at random areas, and hyperplasia appears an enlargement of the cells. Through my time at the ten-headed microscope, I have become accustomed to using the microscope and identifying key parts of the organ slices, such as the nucleus or cytoplasm of the cell.

Later, I joined Dr. DeVito in his office to discuss the numerous projects the Predictive Toxicology and Screening Group pursues. Dr. DeVito told me about the central challenge the National Toxicology Program Laboratory faces: testing thousands of chemicals efficiently and effectively. To combat this challenge, the group employs a variety of techniques. To group chemicals, Dr. DeVito and his lab use a logistical prediction model to determine the possible adverse effects a chemical may have on the body, so they know which areas they should focus their testing on. Their tests range from using donated human liver cells to stimulating cortex cells with electric charges. I also learned about some of the different animals used in the lab, such as infant zebrafish, to test for prenatal effects of the chemicals. Lastly, Dr. DeVito explained some of the research conducted in the Biomolecular Screening Branch, which uses robots and automation to screen thousands of chemicals at a time.

Finally, I observed an NIEHS fellow working with the spheroids Dr. DeVito and his lab created. She was testing the effects of six different chemicals, and three times per weeks she changes the growth medium and chemical doses in her wells using an automated pipette machine that can fill and mix the solutions in all 384 wells at once. I also learned about the constant threat of contamination in a lab, and the different safeguards used by the lab staff to prevent it.

Day 3: Testing Toxins

Today I took a break from my reading and research and traveled to one of the neighboring labs here in the National Toxicology Program. I ventured out of the pathology group and instead joined Dr. Michael DeVito, leader of the Predictive Toxicology and Screening Group, for the day. For a few trying hours in the morning, we attended a mandatory NTP meeting focused on statistics, citations, and publication format; I may have been the only high school student in the room, but I was not the only one that struggled to remain attentive. Later, though, I had the much more exciting opportunity to join Dr. DeVito and his lab staff. One of their current projects is focused on creating a predictive model of the liver that can be used to efficiently test various chemicals for toxicity. While traditional in vitro, or outside of an organism, hepatocyte (liver cells) cultures are grown in two dimensions, Dr. DeVito has found a method to grow these cells in sphere culture systems, similar to stem cells. This three-dimensional innovation has allowed in vitro toxicology and carcinogenic testing to more accurately model the biological effects on human organs than the effects measured in cells grown in two dimensions. More accurate in vitro testing also allows for a decreased reliance on in vivo, or inside of an organism, testing. I also learned about some of the equipment used in Dr. DeVito’s lab, such as a mass spectrometer (MS) and liquid chromatographer. The MS is used to finely quantitate the mass of molecules down to a couple of decimal places, and the liquid chromatographer is used to identify the different chemicals in a mixture.

While in Dr. DeVito’s lab, I also learned about how toxicology testing works in a real-world context. When a company finds a new chemical compound to use in their products, they must first gain approval from the FDA (Food and Drug Administration) or the EPA (Environmental Protection Agency), which nominates the chemical to the NIEHS or NTP. The National Toxicology Program is then responsible for finding if the chemical has negative effects, and if so, at what dosage. Here is a very elementary description of this process:

Dr. DeVito and his lab grow cell cultures in wells, then splash them with a fluorescent dye that sticks to the nucleus of each cell; next, using a handy motorized pipette, they dissolve the dye with dimethyl sulfoxide (DMSO), a universal solvent; next, they wash the cells with a phosphate-buffered saline solution a couple of times (I was allowed to complete this); finally, they splash the cells with whichever chemical they are testing for toxicology.

After a period of incubation, they take the cells out and insert them into a fluorescence microplate reader, which reads the fluorescence signature from each well and compiles the measurements into a document, so at the end of reading there are 384 measurements (one for each well) compiled. The premise of the test is that the less fluorescence emitted by a well, the fewer nuclei in the well, which means fewer cells, which means more cell death. So, Dr. DeVito and his lab can predict at what dosage cell death begins to take place by measuring the dosage at which the fluorescence begins to decrease. This test, however, is simply to find out whether or not chemicals are harmful at any reasonable dosage. The real challenge, which is far beyond my understanding, is identifying how the chemicals harm the cells and what these damages translate to in terms of humans.

If there was one thing that I took away from my time in Dr. DeVito’s lab, though, it was not their intriguing projects nor their complicated machines: it was that collaboration is an essential part of their research. No one person is an expert in everything or even more than one thing, and everyone relies on each other to complete the work.

Day 2: Beginning to Understand

Upon arriving for my second day working with the National Toxicology Program (NTP) Pathology Group I was excited to learn more about the projects I was introduced to yesterday. I returned to my cubicle, now broken-in and sufficiently cozy, and I began to read about some of the proteins I looked at yesterday with a post-doctoral fellow at the ten-headed microscope. Three of the proteins were CD45, S129 (alpha-synuclein), and GP91 (Nox2). CD45 is a type of protein tyrosine phosphatase involved with antigen receptor signaling for two different cell types. As of now, it is understood that CD45, and protein tyrosine phosphatases in general, can either act as inhibitors or activators in tumor growth. S129 is thought to be associated with neurodegenerative diseases, such as Parkinson’s Disease, and GP91 is a key binding protein in the NADPH-oxidase, which is involved with phagocytosis in neutrophils, or the process of killing harmful pathogens that enter the body. After researching for a couple of hours, I joined Dr. Cynthia Wilson for a discussion about veterinary pathology, and about an upcoming project in the NTP pathology group regarding the potential negative effects of a popular chemical used in food production. After lunch, I traveled with Dr. Malarkey to an adjunct NIEHS and NTP building for a meeting to discuss and present the findings of one the first cell phone radiation studies in the broader project. The study focused on the changes of gene expression in rats and mice after exposure to various amounts of CDMA or GSM microwaves in three different organs. Returning back to the main NIEHS building with new topics of research, I ended my day with a deeper understanding of histology, or the study of microscopic tissues, which I was introduced to yesterday. I also developed a highly amateur ability to identify different tumors, such as carcinoma or schwannoma, under in organ slices. Additionally, I have begun to grasp some of the complexities of the research conducted by the NTP pathology group and even discuss my own conclusions with the scientists.

Day 1: Meeting the Lab

After making it through the various security check-points on my way to the National Institute of Health Sciences (NIEHS), and the security in the main lobby, I met with some of the staff of the National Toxicology Program (NTP) Pathology Group. The NTP Pathology Group designs, reviews, and analyzes long-term rodent studies in the field of toxicology and carcinogenesis. I will be working with Dr. Dave Malarkey, the head of the Pathology Group. Specifically, I will be contributing to one of the NTP’s more recent studies: GSM and CDMA Modulated Cell Phone Radio Frequency Radiation. Essentially, the goal of the study, conducted over a couple of years and by dozens of scientists, is to determine whether or not the radiation emitted by cellular devices is carcinogenic. After a quick briefing by Dr. Malarkey about the study and pathology in general, I made my way to the ten-headed microscope, where I joined a couple of veterinary students in identifying a variety of cancers in rodents using histopathology cases, or stained slices of organs. Then I reviewed some slices of a human bronchus and categorized the cells into neutrophils, eosinophils, macrophages, or lymphocytes. Finally, I traveled with Dr. Malarkey to review the research of one of the post-doctoral fellows working at the NIEHS, and also to an NTP Pathology Group birthday party for one of the scientists.