Determining gene expression in the Drosophila melanogaster after multigenerational exposure to chlorophenoxy herbicides.(COMMUNICATIONS--UNDERGRADUATE)(Report)

Recent studies have shown an increase in both respiratory and circulatory birth defects in wheat producing areas of the U.S. This risk is increased when the child, especially in males, are conceived during the application of herbicides. It is suggested that the exposure to chlorophenoxy herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA), is responsible, but the mechanism remains unknown. Chlorophenoxy herbicides that have been used since the 1940's and multiple generations have been exposed. Therefore, it is our hypothesis that the risk of birth defects increases due to exposure of multiple generations. The goal of these experiments is to determine the effect of multigenerational exposure of 2,4-D and MCPA on development and identify and determine the changes in gene expression after exposure. The Drosophila melanogaster was chosen as a model organista to study the effect of multiple generations because of its short generation time and genetic homolog with human genes involved in development.

Drosophila embryos were collected for 20 minutes after a pre-lay period to ensure embryos of equal age. Embryos were added to a vials with food containing chlorophenoxy herbicides in the following concentrations (control, 1 [micro]M and 3mM 2,4-D or MCPA). Development was observed every 24 hrs for a period of 16 days. Concurrently, Drosophila adults (75 lema|es and 50 males) were put into 8 bottles with food containing the same concentrations as above to produce the next generation. After 7 days the parents were removed stored and stored at -20[degrees]C until further use. After 14 days embryos were collected for the next developmental study and adults were used to produce the next generation. Development and growth were followed for 4 generations.

Adults were used to determine gene expression. After isolation of mRNA using Trizol, mRNA from two sources (for example 4th generation 2,4-D and 4th generation control) was labeled with Cy5 and Cy3 using PCR and ...