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Partnerships for enhanced engagement in research (PEER) SCIENCE
Cycle 2 (2012 Deadline)

Biodiversity and adaptations of CYP enzymes in the Amazon Loricariidae fishes

PI: Thiago Parente (, Fundação Oswaldo Cruz (Fiocruz), (formerly at Universidade Federal do Rio de Janeiro)
U.S. Partner: Mark Hahn (Woods Hole Oceanographic Institution)
Project Dates: September 2013 to July 2015

CYP1 enzymes are responsible for the biotransformation of natural compounds and anthropogenic pollutants. Usually the reactions catalyzed by CYP1 enzymes lead to detoxification, when the compound is eliminated from the body without causing harm. CYP1 enzymes, however, are also known to catalyze bioactivation reactions, in which one of the reaction products is more toxic than its parent compound. The equilibrium between the detoxification (beneficial) and bioactivation (detrimental) roles of CYP1 enzymes has been fine tuned for each and every vertebrate species over the course of evolution. This PEER Science project is closely aligned with the National Science Foundation-supported work of the U.S. partner, Dr. Mark Hahn, as both involve the study of different naturally evolved and selected solutions for the same issue: the balance between detoxification and bioactivation by CYP1 enzymes using fish species as vertebrate models. The adaptation of Killifish (studied by Dr. Hahn) is a well documented event classified as dramatic, rapid, convergent, and triggered by anthropogenic pollutants that balance the dual role of CYP1 enzymes at the gene expression level. However, the adaptation of Loricariidae fish is poorly known and most probably can have the opposite classification: gradual, slow, divergent, and triggered by chemicals naturally present in the fish microhabitat. The goals of this project are to determine whether the adaptations of CYP1 enzymes in Loricariidae fishes are convergent or divergent and how they change the susceptibility of this species to the toxic effects of petrogenic compounds.

Dr. Parente and his research team will sequence the CYP1 genes of 100 Loricariidae species from the Amazon, and these gene sequences will be used to determine the enzyme sequences, which in turn will be aligned and compared for amino acids substitutions and interaction with classical CYP1 substrates. Selected Loricariidae species will be used for biological assays to evaluate the toxic effects of petrogenic derivates and their molecular mechanisms of action. Due to the current and future prospects for crude oil drilling activities in the Amazon region, it is imperative to understand the metabolism of petrogenic hydrocarbons by Amazonian biota. This is especially true in the case of Loricariidae fishes, as it is already known that those species have CYP1 enzymes with distinct affinity for substrates. It needs to be determined whether these changes will unbalance the evolved equilibrium of CYP1 dual roles to the beneficial or to the detrimental side. This knowledge will be crucial to better evaluate the risks of oil drilling activities for Amazonian biodiversity.

Brazil Partnership Photo 1Brazil Partnership Photo B
The research team at the Andorinhas waterfall on the Roncador river in the district of Santo Alexio. Pictured are Paolo Buckup (top), Emmanuel Neuhaus (center), Thiago Parente (left foreground), and Jose Gomes (right foreground). (Photo: Carla Quijada).Labwork to obtain sequencing of genes 
(Photo: Dr. Parente).


Summary of Recent Activities

During the first year of the project the research team successfully completed their planned fish sampling. In total, 24 Siluriformes species (mostly loricariids) from the Amazon area and 32 other Siluriformes species from the rain forest habitat were sampled. RNA was extracted from more than 200 individual fish for cDNA synthesis. Every cDNA sample was used for PCR amplification with specific primers for CYP1A. The rate of positive PCRs (e.g., a PCR reaction where a product with the expected molecular weight is detected) was much lower than expected, and of all the collected species, the team was able to amplify by PCR and sequence CYP1A by the Sanger method from only seven species. Although these unexpectedly low positive PCR reactions somewhat delayed progress, they also indicate that the molecular diversity of CYP1A genes of the analyzed species is higher than expected. By agreement with the U.S. partner, the research team decided to change their traditional Sanger method approach and sequence the desired genes using the Next Generation Sequencing (NGS) method. The team is now sequencing the transcriptomes, a new approach that will give the research team the sequence of not only the two desired genes, CYP1A and AHR, but also all genes that were expressed at the time of fish liver sampling. This method will yield more crude data that will be analyzed during the second year of the PEER project. In order to accomplish the project goals, the research team will focus on a bioinformatic target gene search.

After a successful month-long visit to Woods Hole, Massachusetts, in July 2014 to conduct bioinformatics analysis with the raw NGS data and participate in the Marine Biological Laboratory Workshop on Molecular Evolution, Dr. Parente is planning to take his two undergraduate students to the Woods Hole Oceanographic Institution to sequence individual genes of additional fish species during January and February 2015. He also plans to participate in the 18th International Symposium on Pollutant Responses in Marine Organisms (18 PRiMO) in Norway (May 24-27, 2015) and the 19th International Conference on Cytochrome P450 in Tokyo (June 12-15, 2015).

Link to Project Blog

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