Oil-Related Toxicology Research at the Gulf Coast Research Laboratory

Research in the Griffitt Lab focuses on the effects of anthropogenic substances (introduced by human activity) on aquatic or marine species.  Our research focuses on three key areas: nanotoxicology, toxicogenomics, and the effects of the Deepwater Horizon oil spill.  In each case, we use a broad approach that examines effects from a molecular level to the complete individual organism.  Often, this uses a combination of techniques, such as quantitative PCR (polymerase chain reaction and microarrays.  We look at changes in gene expression patterns, and see if these alterations show up at larger scales – in structural changes in organs or reproduction, for example.   

Background

The polymerase chain reaction (PCR) is a technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. (Wikipedia)

One nanometer (nm) is one billionth of a meter.  The largest particles in Figure 2 are smaller than 100 nm, about 1/1000 the diameter of a typical human hair.

Figure 1. A chronic flowthrough exposure being performed.

 

We also commonly expose larval, juvenile, and adult individual fish to understand effects on different life stages.  This must be done under specific environmental conditions and contaminant levels.  The Shoemaker Toxicology Laboratory at GCRL allows us to perform both acute (96 hours or less) and chronic (several weeks) exposures while ensuring that the fish are  maintained at controlled temperature, salinity and oxygen levels. Using these controlled exposures, we can rapidly and accurately assess the impact of a particular contaminant on a variety of fish species. 

Nanotoxicology

Nanotoxicology is the study of nanoparticles and the effect they have on living organisms.  It is the fastest growing sector of the high-tech economy.  The rapidly increasing use of nanoparticles in industry and consumer products is causing concern in regulatory agencies about their contaminant potential.  Our research focuses on the effects of nanoparticulate metals, particularly silver, on several species of fish and invertebrates.  We’ve shown that exposure can produce serious adverse effects.  Nanoparticulate silver is found in many consumer products including cosmetics and athletic wear, where they are advertised as being effective for sun screening or odor control.  Nanoparticles are a concern because they are not consumed in their original uses and can persist in the environment.  Their potential effects on organisms are not well understood.

One of our research projects examines the effect of particle surface charge on uptake and retention of nanoparticles by organisms.  To do this, we exposed a small crustacean called daphnia to quantum dots, tiny semiconductors which are highly fluorescent and can be modified to have different properties. We showed that charge is an important factor determining the rate of uptake and clearance in daphnia.

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Figure 2. Nanoparticulate silver suspended in water.

Figure 3. Daphnia exposed to quantum dots readily ingest the particles.  Here, you can see the QD outlining the digestive system of daphnia.

Other research projects are aimed at understanding the effects of nanoparticles on fish, particularly focusing on gills.  We know that nanoparticulate metals are capable of producing reactive oxygen species (chemically reactive molecules containing oxygen, which may damage cell structures), and this has been suspected to be one of the ways in which nanometals cause toxicity to fish.  If this is occurring, the ROS might be causing damage to DNA in the fish gill cells.  When we exposed zebrafish to nanosilver, and used an assay to examine the effects, we saw that it did appear that nanosilver was damaging DNA.

 

 

Figure 4.  Exposure to nanosilver particles causes DNA damage in exposed fish.  The assay is called a comet assay because the damaged cells look like a tiny comet under a microscope.  When DNA is broken, it forms a "tail" that moves away from the unbroken DNA (the "head").

 

Toxicogenomics

Toxicogenomics is the study of how exposure to contaminants affects organisms at the level of gene expression.  (Gene expression is the process by which genes tell cells which proteins to make and in what amounts.)  Gene expression profiles are developed by measuring the expression of thousands of genes simultaneously.  They are very useful in determining what contaminant an organism has been exposed to, as well as finding out which molecular response pathways have been altered by the exposure. 

A common tool used to do this is the microarray.  This is a small glass slide that holds short sections of DNA that are unique to a particular gene for a given species.   We isolate total RNA (which is the messenger that goes from DNA to the ribosome to make proteins), label them with a fluorescent dye, and hybridize them to the slide.  By comparing fluorescence intensities for each gene-specific spot (called a probe) between exposed and unexposed individuals we can tell exactly how much the expression of the parent gene was affected by the exposure.


Figure 5. Heatmap of altered gene expression following exposure to different nanometals.

Reference: Griffitt et al., 2009. Toxicological Sciences 107(2), 404–415.

The results are plotted as shown above to produce a “heatmap” (a two dimensional plot of data using color to show quantities or intensities, not related to heat)  Each individual block represents a gene, with each row of blocks showing all the genes for a particular fish specimen.  Red colors indicate increased expression, and blue colors indicate decreased expression.  The labels on the right show the contaminant to which the fish was exposed.  Three control samples (no exposure to nanometals) are shown at the bottom.

Treatments producing similar effects are clustered together by the bracket lines on the right side; this information allows us to make inferences about pathways affected.  In the above case, exposure to nanosilver (nanoAg) produced a response that is very different from the other exposures. 

Description: 48 nanosilver dn metal ion binding

These gene expression profiles can then be used to identify molecular pathways that are affected, so that we can understand the potential effect on the organism.  A molecular pathway is a series of actions among molecules in a cell that leads to a certain end point or cell function.  These pathways are how cells get their jobs done; for example, multiple pathways work together to provide an organism’s respiration function.

 

 

Figure 6.  Molecular pathways affected by
a particular exposure.
 

Deepwater Horizon Oil Spill

Dispersed oil in a separatory funnel prior

Our third area of research addresses the environmental effects of the Deepwater Horizon oil spill.  The 2010 explosion and resultant uncontrolled release of oil resulted in an estimated 4.9 million barrels of crude oil being released into the northern Gulf of Mexico.  In an attempt to control the spread of the oil, an unprecedented amount of Corexit dispersant was used, both at the surface and 5000 feet below the surface at the wellhead.  Understanding how this event affected the ecosystem of the Gulf is a tremendous undertaking, one that will take several decades. 

Our research is funded by several sources, including the National Science Foundation, the Northern Gulf Institute, the Gulf of Mexico Research Initiative, and the National Resources Damage Assessment (NRDA).  (We have received no direct funding from BP and have no obligation to review our findings with BP.)  We are examining the effects of both crude oil and oil dispersed by mixing with Corexit on economically and ecologically important species of fish and shrimp.  Our work goes beyond mortality effects and considers various sub-lethal effects of exposure.  Further, it focuses specifically on the type of crude oil released from the Deepwater Horizon well.

Figure 7. Dispersed oil in a separatory funnel prior to use.  

Our research has been ongoing since the summer of 2010, but this is a necessarily long and complicated process and we are determined to get it right.  We have several manuscripts in preparation, with two oil-related papers expected to be published in 2013.  We are eager to submit our work for peer review and eventual release to the public; we hope that it will help to inform the public about the aftereffects of the Deepwater Horizon event.