Candidate: Ronald Celestine

Degree of: Doctor of Philosophy

Department: Biological Sciences

Title: Molecular Analysis of the Effects of Atrazine on Xenopus Laevis Frogs

Committee: Dr. Charles Ide, Chair
Dr. Anna Jelaso Langerveld
Dr. John Spitsbergen
Dr. Cindy Linn

Date: Friday, May 26, 2006 2:30 p.m. – 4:30 p.m.
1718 Wood Hall

Abstract: Atrazine is the most commonly used agricultural herbicide in the USA. Recent research has implicated atrazine as one of the factors which could be partially responsible for the reported worldwide decline of frogs and other amphibians. Atrazine was considered a relatively safe chemical because of its low bioaccumulation, low bioamplification and low sedimentation. In order to investigate atrazine's effects on amphibians DNA microarray and qRT-PCR technology were used to assay global gene expression patterns characteristic of frog adaptive response to atrazine exposure.

In this study I treated Xenopus laevis tadpoles from 5 days post fertilization to NF stage 62 with a 400 ppb concentration of technical grade atrazine (Sigma, St. Louise, MO). Endpoints tested were metamorphic rate, growth rate (weight and length), and fat body development. Results showed significantly decreased metamorphic rate (p=0.0165), growth rate (weight, p= 0.0001; length, p=0.0005), and significantly underdeveloped fat bodies (p=0.0001). Total RNA was then extracted from control (n=6) and treated (n=6) tadpoles and DNA microarray analysis was used to identify underlying gene expression contributing to observed effects of atrazine. The raw data were analyzed using a non-parametric t-test. Significantly different signal intensity values (p= 0.05) were further filtered using fold change cut off values: = 1.5 for upregulated genes and = -1.5 for downregulated genes. This resulted in a final dataset of 49 genes. These were grouped according to physiological functions. Results showed genes concentrated mainly in groups associated with digestive system (11 genes), blood and plasma function (8 genes), and cell adhesion (12 genes). Four genes from the final dataset (pancreatic trypsin, chitinase, chitobiase and CYP-P450) were chosen to validate microarray DNA analysis using highly quantitative real-time RT-PCR. Three of the 4 genes used coincided with results from the microarray analysis. Taken together these results suggest that atrazine may be having an effect on food intake or energy assimilation of tadpoles. Stored energy in the form of lipids and proteins is vitally important to tadpole survival because they do not feed during metamorphic climax and this is the only source of energy available for metabolic maintenance and development. DNA microarray technology is a novel and powerful method for determining specific changes in gene expression that control specific physiological processes. This technology gives molecularly based insight into how atrazine affects tadpoles, and would provide a starting point for the production of new molecular tools for risk assessment related to atrazine in the environment.

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