Cells and Instruments, but no Folsom Prison Blues

Brian Wuertz, Warren Wilson College

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In my previous post, “Hiding in plain sight”, I introduced DOSS, a compound that has been recently identified as a probable obesogen. We are especially concerned about the potential of this compound to cause obesity symptoms in developing children through exposure from their mothers. While DOSS is in many products we use daily, such as homogenized milk and makeup products, it is commonly prescribed to pregnant women in the form of Colace stool softener. I am investigating both how much DOSS is in certain places in the body and how it may promote obesity.

One of the main concerns about obesity is that it elevates the risk of developing other diseases such as diabetes or cancer by causing a state of chronic inflammation (Bianchini 2002).  Chronic inflammation in  adipose tissue is regulated by immune cells, including macrophages. Macrophages are immune cells found throughout the body that help to fight against infection by recognizing invading bacteria and engulfing them in a process called phagocytosis, literally meaning to eat the other cells. In addition to phagocytosis macrophages are important regulators of the larger inflammatory response by secreting proteins that tell other cells to initiate or maintain a state of inflammation (Fujiwara 2005). This inflammatory reaction may be induced by DOSS. We have seen evidence of increased inflammation and obesity in mice treated with DOSS, so in order to figure out what causes that I am focusing on macrophages because of the way they regulate inflammation.

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I am isolating macrophages from breast milk samples under this hood in a sterile environment to make sure they are not contaminated with bacteria.

One way to study the inflammatory response of macrophages is to expose them to DOSS and then see if they produce the inflammatory proteins. Instead of trying to measure the secreted proteins, we can measure how much RNA is made in the cell. The RNA is the translator molecule that takes the plan for the protein from the DNA and makes it available for the cell to read and make the right protein. I identified genes for four different inflammatory proteins to measure the RNA so we can test if DOSS causes the macrophages to make more of any of them. I am testing macrophages that I am isolating from human placenta and breast milk tissue because the developing child is influenced by inflammation in the placenta and breast milk. Macrophages in these tissues could be the source of inflammation that influences how the child develops.

Okay so we have talked about cells, but what about the instruments? In my last post I introduced my instrument of choice, but did not call it that. It is not a guitar or a saxophone, but the HPLC, or high performance liquid chromatograph. This is simply a fancy instrument used to separate chemical compounds by forcing them through a tiny filter column filled with tiny beads. Some compounds stick more to the beads than others, so when you flow a liquid through the column the compounds come out of the column at different times. It is essential to separate the compounds in a sample because then you can measure the amounts of individual compounds.

We want to know where DOSS goes in the body, so we need to be able to measure how much of it is in a sample. I am working to get a system up and running to measure the amounts of DOSS in samples from different cells and tissues. We want to be able to measure DOSS in humans and in marine mammals such as dolphins. Dolphins are exposed to DOSS in the COREXIT oil spill dispersal agent that is applied to large and small scale oil spill issues along coastlines and in harbors. Dolphins are an important sentinel species, meaning that they can provide insight into human health issues.

I have to prepare a column and get the right mixture of solvents to make DOSS come off of the column in a timely fashion and in a way that we can measure it. The measurement is actually done with a mass spectrometer, which measures allows us to identify the compound based on how much it weighs. The number of atoms and types of atoms in the compound determine the mass of the compound. This mass is how the instrument measures the compound. The technique I am using is therefore called liquid chromatography mass spectrometry or LC-MS and the instrument is also referred to by LC-MS. Hopefully by the end of the summer I will be able to find beautiful data with this instrument that will make a coherent tune rather than a jumble of notes.

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This is the MS part. It measures the mass of the compound and then breaks it apart and measures the mass of the pieces of the compounds and the amount of the compounds.

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This is the LC or liquid chromatography part of the LC-MS instrument. Most of the work is figuring out the best solvent system to the sample through the small column with the red tag on it.

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

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References:

Bianchini, F., Kaaks, R., and Vainio, H. (2002). Overweight, obesity, and cancer risk. The Lancet Oncology 3, 565–574.
Fujiwara, N., and Kobayashi, K. (2005). Macrophages in Inflammation. Current Drug Target -Inflammation & Allergy 4, 281–286.

Our complicated relationship with chemicals

Nina Sarmiento, Binghamton University

Chemicals found all around us that have been altered, mimicked, and synthesized to be added to our products, are behind the success of our modern society. They have made our plastics strong, our crops prosperous, and our medicines effective. But I have always wondered about the toxicity of these chemicals.  When you look at their biological activity, a chemical might possess the potential to do harm, like interfere with biological processes. The safety of a potentially harmful chemical is based on exposure and dose. It is important to know if we are touching it, eating it, or breathing it in, and for what period of time. The study of evaluating the harmful effects of substances on exposed organisms is what toxicology is all about. They have such an important job because their findings influence what we know is safe and unsafe, for us and organisms all around us.

I learned early on from pursuing biology that we are exposed to many things we are unaware of. Not only are we exposed to potentially harmful chemicals, but we facilitate exposure to other living organisms that may more sensitive. Take dogs for simple example. The toxic dose of something like chocolate for humans is very high, whereas leaving a small amount of chocolate out for a dog to eat could easily kill it.  Rachel Carson is someone I greatly admire, whose work on the pesticide DDT also exemplifies this reality. Food crops were the target for DDT, but birds were indirectly ingesting it, explaining the decline in the Bald Eagle population.  She is one of the people that sparked my interest in ecotoxicology, looking at the effects of harmful substances on ecology, not just humans.

unnamedThis is an example of some of the questions ecotoxicologists ask when there is a potentially harmful substance found in the environment. Photo credit: globe.setac.org.

Here is a chemical product you may not suspect as a threat, sunscreen. In sunscreens, UV filters protect you from getting burned, but also can act as endocrine disruptors, altering hormones and growth (1). Sunscreens are only meant for human skin, however they end up in our lakes, rivers and oceans through swimming or through waste water treatment effluent (2). Unintentionally, many more organisms become exposed.

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Photo credit: thesleuthjournal.com

In my project I will be using sea urchins as a model organism to study the effects sunscreens might be having on coral reefs.  I am learning how to preform toxicity tests on sea urchin sperm and embryos which involve an exposure period with sunscreen formulations and then evaluation of effects. I hope to investigate if the chemicals from sunscreens in the water can have negative impacts on coral reproduction.  My work can potentially help create understanding of how humans are contributing to coral reef decline, and influence others to take action to protect them.

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This is a picture of sunscreen water accomodated fractions (WAFs) I am making. They are a mix of sunscreen and seawater and I will be exposing the sea urchin embryos to each solution!

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This is me in the lab with a microscope I use to look at sea urchin sperm and embryos! Photo by Bob Podolsky

My research is funded by the National Science Foundation and College of Charleston partnered with National Oceanic and Atmospheric Administration

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Works cited:

1 Krause M.,, Klit A., Jensen M., Soeborg T., Fredrickson H., Schlumpf M., Litchensteiger W., Skakkebaek N E., Drzewieck K T. 2012. Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters. International journal of andrology. 35 424-436.

2Kyungho C., Kim  S. 2014. Occurances, toxicities, and ecological risks of benzophenone-3, a common component of organic sunscreen products: a mini review. Environment International. 70 143-157.