Claremont Center for the Mathematical Sciences

Identifying biomarkers for exposure to environmental contaminants

EcoArray is an environmental testing company using microarrays to observe the changes in gene expression that occur in fish that are exposed to various chemicals.  Gene expression profiling is a more complex version of the idea of the canary down the mineshaft.  This testing is promising because current tests observe either the presence of a chemical in water or the death rate of a sentinel species exposed to a chemical.  Gene profiling speaks to biological impact (rather than just presence in the environment) and is considerably more sensitive (and quick) than counting dead fish.  

The microarray I will concentrate on for the statement of the problem is our 15,000-gene array in fathead minnows (“FHM”), the most common “sentinel species” for fresh water testing used in the United States.   We have exposed FHM to various levels of 14 chemicals representing chemical families of concern in environmental studies (PCB-126, several metals, herbicides, estrogen, androgen, pesticides).  The task is to determine patterns of expression in those 15,000 genes that distinguish each chemical.  We call the unique group of genes that is differentially expressed for each chemical its biomarker fingerprint.  Ideally, we would like to be able to test a small sample of fish from a water source and determine what chemicals are affecting that fish … identifying more than one chemical if such is the case. 

I have attached a brief overview of how a microarray is processed.  After applying RNA from the fish treated with specialized dyes, the microarray is scanned by a specialized scanner, which produces data in the form of intensity statistics.  When the data is produced, it goes through two stages of analysis.  The first is normalization, in which the signal read from the scanner is converted into usable data.  This is done in software associated with the scanner.  The second stage is analysis, or bioinformatics.  We use a fairly sophisticated software, GeneSpring GX version 10, that allows for a great deal of statistical manipulation and visualization.  GeneSpring is powerful for analyzing specific experiments and for filtering data sets. 

In the bioinformatics stage of analysis, intensity ratios for genes of fish exposed to known contaminants are then compared to ‘control’ (null) expression intensity. A gene can be up-regulated or down-regulated compared to the control.  A good biomarker is one which is significantly different from a control value for one single chemical and which responds only to that chemical.
We have done some preliminary work on 7 of the chemicals.  From this we have determined several things:

1. Good biomarkers are relatively rare and vary widely from chemical to chemical.  In a study of the first 7 of our chemicals, we found that 2 chemicals have only 6 usable biomarkers (of which only one apiece is unique to that chemical), whereas hormones show hundreds of marker genes.  

Final Report
Title: Identifying Bio-markers for EcoArray (pdf)