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.
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Hiding in plain sight

Brian Wuertz, Warren Wilson College

Headshot

How much do we really know about all the chemicals that we are exposed to every day? Do we even know when we come into contact with them? How much do we know about what is in homogenized milk, soda, stool softeners, baby formula, and personal care products such as eyeliner? The answer may be “not enough” for one compound found in all of those products, dioctyl sodium sulfosuccinate, or DOSS. DOSS has recently been identified by my mentor, Dr. Spyropoulos and his Ph.D. student, Alexis Temkin, as a probable obesogen. Obesogens are a class of compounds that promote obesity by interfering with the body’s hormone signaling pathways related to energy use, fat cell regulation, and inflammation. These pathways are especially important in the developing fetus, where hormone signals influence development and may have long lasting effects on the health of the child after birth (Holder 2016).

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I am working on a High Performance Liquid Chromatography  (HPLC) system, in the early stages of developing a method to measure the amount of DOSS in cell extracts. (More to come in future posts!)

We are especially concerned with regards to the developing fetus and child because stool softeners containing DOSS are are commonly taken by pregnant women. Approximately 35% of over 20,000 women who gave birth at MUSC in recent years reported taking a stool softener containing DOSS during their pregnancy. I am working to help understand the biochemical pathways DOSS may follow to affect changes in the  developing fetus through a mother’s exposure to DOSS. I am also working on a method to measure the amount of DOSS in cells so that we can learn where in the body DOSS goes and how much of it there actually is.

You might be wondering how this fits into the theme of marine organism health at this point since all I have talked about is human health and a compound found in products we put in our bodies, DOSS. A red flag was raised about DOSS through research on COREXIT, one of the agents used to clean up the Deepwater Horizon oil spill. Over 40 million gallons of COREXIT was dumped into the ocean as a part of the cleanup effort and DOSS is one of the major components (Temkin 2016).  DOSS was flagged as a potential human health hazard because of the research done on marine environmental degradation. It amazes me how a perhaps seemingly unrelated topic can end up having human health implications. I am excited to keep working on this puzzle to learn more about DOSS and how it interacts with the systems in our bodies!

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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Sources:

Holder, B., Jones, T., Sancho Shimizu, V., Rice, T.F., Donaldson, B., Bouqueau, M., Forbes, K., and Kampmann, B. (2016). Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal–Placental Messaging. Traffic 17, 168–178.
Temkin, A.M., Bowers, R.R., Magaletta, M.E., Holshouser, S., Maggi, A., Ciana, P., Guillette, L.J., Bowden, J.A., Kucklick, J.R., Baatz, J.E., et al. (2016). Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen. Environ Health Perspect 124, 112–119.