Exposure to atrazine affects the expression of key genes in metabolic pathways integral to energy homeostasis in Xenopus laevis tadpoles.

In our laboratory, Xenopus laevis tadpoles exposed throughout development to 200 or 400 µg/L atrazine, concentrations reported to periodically occur in puddles, vernal ponds and runoff soon after application, were smaller and had smaller fat bodies (the tadpole's lipid storage organ) than controls. It was hypothesized that these changes were due to atrazine-related perturbations of energy homeostasis. To investigate this hypothesis, selected metabolic responses to exposure at the transcriptional and biochemical levels in atrazine-exposed tadpoles were measured. DNA microarray technology was used to determine which metabolic pathways were affected after developmental exposure to 400 µg/L atrazine. From these data, genes representative of the affected pathways were selected for assay using quantitative real time polymerase chain reaction (qRT-PCR) to measure changes in expression during a 2-week exposure to 400 µg/L. Finally, ATP levels were measured from tadpoles both early in and at termination of exposure to 200 and 400 µg/L. Microarray analysis revealed significant differential gene expression in metabolic pathways involved with energy homeostasis. Pathways with increased transcription were associated with the conversion of lipids and proteins into energy. Pathways with decreased transcription were associated with carbohydrate metabolism, fat storage, and protein synthesis. Using qRT-PCR, changes in gene expression indicative of an early stress response to atrazine were noted. Exposed tadpoles had significant decreases in acyl-CoA dehydrogenase (AD) and glucocorticoid receptor protein (GR) mRNA after 24 h of exposure, and near-significant (p=0.07) increases in peroxisome proliferator-activated receptor ß (PPAR-ß) mRNA by 72 h. Decreases in AD suggested decreases in fatty acid ß-oxidation while decreases in GR may have been a receptor desensitization response to a glucocorticoid surge. Involvement of PPAR-ß, an energy homeostasis regulatory molecule, also suggested changes in energy status. Despite, or possibly because of, these early gene changes, there were no differences in either absolute ATP levels or ADP:ATP ratios early in the exposure. However, livers from animals exposed to 200 µg/L atrazine had near-significant (p=0.06) increases in ADP:ATP ratios at the end of exposure suggesting tadpoles may have had difficulty maintaining energy homeostasis. Perturbations in the expression of genes regulating energy metabolism by 24 h into exposure to 400 µg/L atrazine was noteworthy, especially since these tadpoles were significantly smaller than controls by 72 h of exposure.

Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles.

Six studies were performed regarding the effects of atrazine, the most frequently detected pesticide in fresh water in the US, on developing Xenopus laevis tadpoles exposed 5 days post-hatch through Nieuwkoop Faber Stage 62. The levels of atrazine tested included those potentially found in puddles, vernal ponds and runoff soon after application (200 and 400 µg/L) and a low level studied by a number of other investigators (25 µg/L). One study tested 0, 25 and 200 µg/L, another tested 0, 200 and 400 µg/L, while the remaining four studies tested 0 and 400 µg/L. During all exposures, mortality, growth, metamorphosis, sex ratio, fat body (a lipid storage organ) size and liver weights, both relative to body weight, were evaluated. In selected studies, feeding behavior was recorded, livers and fat bodies were histologically evaluated, liver glycogen and lipid content were determined by image analysis, and immunohistochemical detection of activated caspase-3 in hepatocytes was performed. The NOEC was 25 µg/L. None of these exposure levels changed sex ratios nor were intersex gonads noted, however, no definitive histological evaluation of the gonads was performed. Although a marginal increase in mortality at the 200 µg/L level was noted, this was not statistically significant. Nor was there an increase in mortality at 400 µg/L versus controls. At the 400 µg/L level, tadpoles were smaller than controls by 72 h of exposure and remained smaller throughout the entire exposure. Appetite was not decreased at any exposure level. Slowed metamorphosis was noted only at 400 µg/L in two of five studies. Livers were significantly smaller in the study that tested both 200 and 400 µg/L, yet no pathological changes or differences in glycogen or lipid stores were noted. However, livers from 400 µg/L exposed tadpoles had higher numbers of activated caspase-3 immunopositive cells suggesting increased rates of apoptosis. Fat body size decreased significantly after exposure to 200 and 400 µg/L although these organs still contained some lipid and lacked any pathology. Since this was noted across all studies, it was considered the most sensitive indicator of atrazine exposure measured. The changes noted in body and organ size at 200 and 400 µg/L atrazine indicated exposure throughout development compromised the tadpoles. Significant reductions in fat body size could potentially decrease their ability to survive the stresses of metamorphosis or reduce reproductive fitness as frogs rely on stored lipids for these processes.