DEVELOPING NEW TECHNOLOGIES & METHODS

FOR ENVIRONMENTAL ASSESSMENT

increasing the power to evaluate health effects on wildlife

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Kelley [SICB SF 2012] MultMeas[v.2]  Col

Collaboration

Prior research by us and others repeatedly demonstrate that endocrine and physiological disruptions occur in wildlife exposed to contaminant chemicals in the environment.  From this research, it has become increasingly evident that different kinds of effects in fish can reflect the specific types of contaminants that are present and active. 

To date, most research has developed and used single or a limited set of biomarkers (e.g., measurement of a hormone level, or expression of an mRNA, etc.) to identify and assess an impact in an organism.  However, methods that measure multiple biomarkers in the same sample can provide for a more powerful and integrative diagnostic tool to evaluate physiological impairments and the underlying mechanisms causing them.  In addition, since different environmental stressors often act through distinct pathways (e.g., actions of environmental estrogens will be different from that of a given pesticide, etc.), a multiple-measures approach has strong potential to serve as a screening methodology to predict types or classes of active environmental stressors and risk to the ecosystem. 

One type of multiple-measures approach is called proteomics.  This technology allows a small sample of tissue to be evaluated for its profile of proteins that are being made.  This provides the opportunity to compare a sample from a wild species that is exposed to an environmental contaminant(s) versus one that is not exposed, and then evaluate which proteins are higher (up-regulated) and which are lower (down-regulated).  As example, when a male is exposed to environmental estrogen, we might see his liver producing vitellogenin, an egg yolk protein made by females - not expected in males of course, unless environmental endocrine disruption is occurring.  As a researcher identifies each new protein change, each tells its own physiological story, and with more and more, one learns increasing details about the physiology and health of the organism being measured!

In some of our collaborative research, wild English sole have been evaluated when sampled from a regional wastewater treatment plant location versus from reference locations.  Their liver proteome was compared (see pictures shown below) to find proteins whose expression was altered due to location, contaminant exposure, and functionality of the endocrine system (endocrine disruption).  All parameters were measured in the same individuals, a highly integrative analytical strategy.  Of 840 proteins evaluated, 110 showed significant alterations in association with the treatment plant location.  These proteins were significantly correlated with contaminant loads and/or endocrine status in the animals.  Using correlative and multivariate statistical analyses, it appears that both contaminants and endocrine factors are associated with specific protein differences. 

While the process of protein identification continues, over 80 English sole proteins have been identified to date, using MALDI TOF/TOF mass spectrometry techniques. 

Expression changes of proteins point to altered hepatic fuel metabolism, toxicological responses, oxidative effects, and altered signaling (endocrine, intracellular) in fish exposed to different kinds of environmental contaminants. 

 

The results of this study are “proof positive” that development of this approach can provide powerful diagnostic tools for evaluating impacts of water/environmental quality.

[Research funded in part through NOAA/USC Sea Grant]

See a talk by Scott Johnson and Jesus Reyes on the development of a proteomics evaluation methodology for insects, as a new way to evaluate health of insect populations in important watershed environments.

-click here-

A Picture Tour of the Proteomics Approach:

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This picture shows results of 2-dimensional gel electrophoresis, a method used to separate all of the proteins in a given tissue sample. 

 

Hepatic (liver) proteome of a fish from a wastewater treatment location is compared with that of a fish from a clean reference locations.  Computer software compares each gel and indicates which proteins show different levels - red circles identify proteins that are visibly different between left and right proteomes.

Proteins of interest are then removed from the gel and their identity is determined using mass spectrometry.

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This picture highlights the results of the proteomics approach discussed above.  

MALDI/ToF-ToF mass spectrometry was used to determine the identity of each protein of interest.  (The instrument shown in the photo is the MALDI/ToF-ToF used in this study)

A large number of proteins were identified, several of which indicated important differences in physiological condition and health of the fish.