ARS | Publication request: Atrazine Is a Competitive Inhibitor of Phosphodiesterase But Does Not Affect the Estrogen Receptor

Submitted to: Toxicology Letters
Publication Acceptance Date: July 2, 2004
Publication Date: August 25, 2004
Citation: Roberge, M.T., Hakk, H., Larsen, G.L. 2004. Atrazine Is A Competitive Inhibitor Of Phosphodiesterase But Does Not Affect The Estrogen Receptor. Toxicology Letters 154/1-2:61-68.

Interpretive Summary: Phosphodiesterase (PDE) is an enzyme that controls cellular signaling. It turns signals off that control the manufacture of certain proteins, some of which are involved in the onset of sexual maturation and the estrous cycle. Atrazine (ATR) is one of the most commonly used pesticides in the United States. ATR under environmental conditions can be degraded into a number of metabolites such as atrazine desethyl, atrazine desisopropyl, atrazine desethyl desisopropyl or terbuthylazine. Other researchers have indicated that ATR and its degradation products affect the proper development of different species and have suggested that ATR interferes with the estrogen receptor. However, we have shown that ATR and its breakdown products do not bind to the estrogen receptor. Instead, ATR inhibits PDE, which suggests that it can disrupt cellular signaling. The degradation products of ATR inhibited PDE with a potency 50 to 500-fold less than ATR, indicating as ATR is degraded in the environment it becomes a less potent inhibitor of PDE. These studies also demonstrated that ATR does not bind directly to the active site on the PDE enzyme, which means that ATR does not compete with the natural ligand. The present research was limited in that it was performed in a (simplified) laboratory setting and not on a (complex) whole organism. Nonetheless, this is an important first step in determining that ATR can interfere with natural enzymatic processes and that this interference occurs at the level of cell signaling.

Technical Abstract: Atrazine (ATR), 2-chloro-4-ethylamino-6-isopropylamino-s-triazine, has been implicated in numerous studies to act as an endocrine disruptor. ATR and its metabolites, ATR-desethyl (ADE), ATR-desisopropyl (ADI), ATR-desethyldesisopropyl (ADD) and terbuthylazine (TBZ) did not affect the equilibrium between estrogen receptor-alpha and estradiol. Therefore, ATR and its degradation products were studied to determine their ability to inhibit phosphodiesterase (PDE), the enzyme responsible for hydrolyzing the second messenger cAMP to 5'-AMP. Using fluorescence polarization (FP), it was found that ATR inhibited PDE with an IC50 value of 1.8 microM. This is lower than the known PDE inhibitor isobutyl methylxanthine (IBMX), which had an IC50 value of 4.6 microM. The ATR degradation products ADE, ADI, ADD and TBZ were less effective than ATR at inhibiting PDE when assayed using FP. Classical competitive binding assays, using radiolabeled 14C-cAMP in conjunction with thin layer chromatography (TLC), were used to determine that ATR is a non-competitive inhibitor of PDE.