Molecular Analysis of the Effects of Atrazine on Xenopus Laevis Frogs

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Celestine, Ronald Edward
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Atrazine, the most commonly used herbicide in the U.S., has been implicated as one of the factors that may be causing the worldwide decline of amphibians. First, I examined effects of atrazine on laboratory raised Xenopus laevis tadpoles, treated from 5 days post fertilization to NF stage 65/66 with a concentrationof 25 ppb of atrazine. This study showed no significant difference in body weight, length and metamorphic rate.Second, I used DNA microarrays to examine mRNA expression levels of genes in atrazine treated and control Xenopus laevis tadpoles. I found significant changes in gene expression levels of 18 genes. These 18 genes in the final dataset were categorized into biologically functional groups. These were genes associated with immunity and defense (6 genes), protein degradation (2 genes), DNA binding and repair (3 genes), and signal transduction (2 genes). I used quantitative RT-PCR to validate 2 candidate genes: Proteasome beta and Calbindin D28K. One was not significant, while Calbindin D28K was near significant (p=0.072).Third, I spawned and reared Xenopus laevis tadpoles in atrazine at the sub-lethal concentration of 400 ppb after 5 days post fertilization to NF stage 62. I found significant difference in metamorphic rate, growth rate (weight and length), and fat body development of the atrazine treated tadpoles compared to untreated tadpoles.Next, I utilized DNA microarrays to identify gene expression that may relate to phenotypic changes observed. The results showed altered expression of 49 genes. These were mainly concentrated in the digestive system (10 genes), blood and plasma function (8 genes), and cell adhesion (12 genes). In order to validate the DNA microarray expression data, qRT-PCR was used on 4 candidate genes: pancreatic trypsin (upregulated), chitinase (upregulated), chitobiase (downregulated) and CYP-P450 (downregulated). Results showed 3 out of 4 of the transcripts were significant. This research indicates that atrazine may have a metabolic effect on treated tadpoles.