International E co G en Incorporated




Flow Cytometry Analysis

-a screening procedure for DNA damage

 

APPLICATIONS

  • Environmental effects monitoring
  • Baseline studies before development
  • Assessment of the nature and scope of the contaminant problem
  • Assessment of the effectiveness of remediation activities to detoxify the effluent, soil or water
  • Risk assessment analyses

 

THE PROBLEM

A mutagen can cause:

  • deletions of DNA 
  • chromosomal breakage 
  • translocations of DNA to another chromosome 
  • loss of a whole or part chromosome 

When this damage is not repaired, there is an unequal distribution of nuclear DNA passed on to the resultant daughter cells after division. This damage then becomes fixed in the respective daughter cell lines which includes all their subsequent cell descendents. If this damage occurs in the rapidly dividing stem cells or transit cells, then the variation can be greatly bio-magnified in tissues such as human and other mammalian lymphocytes, the nucleated red blood cells of non-mammalian vertebrates, the sperm cells of males, the liver cells of juvenile animals. This increased variation in nuclear DNA content of cells is diagnostic for DNA damage when compared to a control or reference group and can be detected by the flow cytometer as an increase in the coefficient of variation (CV) of the G0/G1 peak. Thus the flow cytometry method detects damage that has not been repaired and that may result in significant effects.

 

THE PROCEDURE

Flow cytometry has been used to detect environmental mutagenesis in field populations of fish, frogs, birds, turtles and mice where contaminated sites were compared with reference sites. Flow cytometry has also been used to detect genetic damage in chinook salmon under controlled field bioassay exposures to pulp mill effluent as well as in ocean sampled fish naturally exposed to sediments and prey species containing PAH’s. Our method detects DNA damage that 

  • persists in cell populations
  • and, as a result, can produce significant impacts in the animal such as cancer, immune dysfunction and the disruption of cell function
  • and, ultimately, affect the population through reduced reproductive potential of the individual and its offspring.

To determine the nature of the contaminant impact, we can examine a range of tissues from blood to liver, kidney and germ cells. We can examine any vertebrate animal (i.e. mice, fish, frogs, snakes, birds, humans, etc.) and most invertebrates. Our only limitation is the tissue freezing protocol for some invertebrates. The samples can be frozen in the field and shipped to us on dry ice from any location in the world. We can do the complete study design for maximum efficiency and cost effectiveness and then work with consultants located near the study site to implement the design.

 

We typically examine 10,000 nuclei per animal with each treatment group being represented by at least 20 individuals. 30 animals of the same sex per group enables maximum resolution for this method. In pre-reproductive animals, sexing may not be possible and so a random sample is necessary. The assay is sufficiently sensitive to distinguish between populations of cells whose nuclear DNA content differs by as little as 1 to 2% and can be performed on a wide array of tissue types, blood being the tissue of choice. Results of flow cytometry have been verified by classical cytogenetic methods. We use a weighted least squares statistical procedure for the analysis of the CV values. This new procedure which we derived expressly for CV value analysis is the only valid method presently available for analyzing data of this kind. An additional benefit of this statistical method is that the ability to detect differences between treatment groups is increased by an order of magnitude compared to historical (and incorrect) methods of statistical analysis.

 

The service includes advice on statistical design, sample collection, sample preparation for freezing, flow cytometry analysis, a highly sensitive statistical analysis of the Coefficient of Variation values generated by the flow cytometry and an interpretive report. If necessary, we can also assist in sample collection.

 


For further details, please contact
Dr. Michael Easton in Canada at (1) (604) 986-2400
by e-mail at michael_easton@intl-ecogen.com




Types of Mutation
Consequences of Mutation
Our DNA Damage Detection Method

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Updated by Michael Easton 2009.