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Integrating In Silico And In Vitro Approaches to Understand Cross-Species Predictions of Chemical Susceptibility for Aromatase
Citation:
Vliet, S., J. Cavallin, S. Mayasich, AND C. Lalone. Integrating In Silico And In Vitro Approaches to Understand Cross-Species Predictions of Chemical Susceptibility for Aromatase. SOT, Nashville, TN, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.22277455
Impact/Purpose:
Scientific research suggests that environmental contaminants can disrupt the endocrine system by mimicking naturally produced hormones and binding to receptors in the body. This can lead to negative health outcomes in both humans and wildlife. Aromatase is a particularly important endocrine target because many environmental chemicals can interfere with enzyme activity and disrupt biological processes. Identifying chemicals that interfere with Aromatase essential to determine the risk of these chemicals to human health and the environment. Although it’s clear that some chemicals cause endocrine-disrupting effects, very few chemicals have been tested because of the many resources and animals needed to test each chemical. New screening methods with mammalian cells can quickly test chemicals and prioritize them for further testing. Although these screening methods are useful, it’s unclear if the results of these mammalian tests will predict toxicity in non-mammalian species. To address this question, the goal of this research is to understand how Aromatase is similar and different across groups of organisms and how these differences many change the toxicity of chemicals. Using computer-based experiments and conducting cell-based confirmation studies, this research will help determine whether current mammalian-based screening methods can predict Aromatase activity in other organisms, as well as help identify potential refinements to computer-based tools.
Description:
Endocrine active chemicals are of concern for chemical hazard evaluation, and several new approach methodologies (NAMs) have been developed to rapidly screen chemicals for biological activities leading to endocrine disruption. Aromatase (CYP19A1), for example, catalyzes the biosynthesis of estrogens from androgens and can be inhibited by chemicals such as the fungicide Prochloraz. Many NAMs that focus on aromatase currently rely on mammalian-based test systems; however, the applicability of these approaches to non-mammalian targets remains uncertain. This study employs in silico approaches and subsequent in vitro confirmation assays to investigate cross-species predictions of chemical susceptibility for aromatase inhibition. The U.S. EPA’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool identifies whether known critical amino acids involved in catalytic function are conserved across species and makes predictions of susceptibility based on amino acid molecular weight and side chain classification. For this evaluation, SeqAPASS was used to identify amino acid differences between vertebrate species. Predictions then guided the selection of amino acids for in vitro site-directed mutagenesis (SDM) studies. In vitro SDM of the wildtype (WT) human Cyp19A1 gene sequence was used to create six enzyme variants representing amino acid differences identified in other vertebrates. The variant proteins, expressed in cell culture, were subsequently screened for aromatase inhibition. Results of in vitro inhibition assays agreed with SeqAPASS predictions for five of the six mutants. One variant, P308Q, predicted as not a match in SeqAPASS, demonstrated similar enzyme inhibition relative to WT enzyme. Virtual docking of testosterone to aromatase structural models representing the WT and amino acid substituted enzymes support in vitro results, also indicating similar activity between the P308Q variant and WT enzyme. Overall, these results demonstrate the power of multi-tiered approaches for predicting and understanding chemical susceptibility across species, and suggest that targeted refinements to silico tools could be made to enhance current predictions. This abstract neither constitutes nor necessarily reflects U.S. EPA policy.
URLs/Downloads:
DOI: Integrating In Silico And In Vitro Approaches to Understand Cross-Species Predictions of Chemical Susceptibility for Aromatase
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