PI: Thiago Parente (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.
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).
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.
Summary of Recent Activities
Labwork to obtain sequencing of genes (Photo courtesy Dr. Parente).
Project activities from the first quarter of 2014 chiefly centered on lab work. Dr. Parente reports obtaining the full CYP1A sequence for three out of the six fish species that had positive polymerase chain reactions (PCR) amplifications as described in the first quarterly report. The project team decided not to send the other three PCR amplicons because of the methodological change to the Next Generation Sequence (NGS) technology. After considering all the available possibilities, costs, expected results, and time-frame, it was decided to use the Illumina Hi-Seq 2500 platform to sequence the transcriptome of 40 Siluriform species. The sequencing will be done free of charge at the Brazilian National Cancer Institute (INCA) in Rio de Janeiro. The project team is only paying for the kits needed to prepare 40 messenger RNA libraries and for the reagents for a full Hi-Seq run. In addition to their sequencing efforts, the team also spent some weeks working on fish classification and identification with Prof. Paulo Buckup’s group at the Brazilian National Museum (MNRJ), where the sampled fish were deposited. This was important in order to speed up fish identification and to decide which species’ genes would be sequenced.
The team plans a fish sampling trip to the Amazon cities of Manaus and Santarem during the third and fourth weeks of May. From this expedition, about five to ten species will be included in the planned HiSeq transcriptome sequencing run, which should be initiated in July. Once the raw NGS data are obtained, the project team will face months of bioinformatics analysis. Dr. Parente is planning a month-long visit to Woods Hole, Massachusetts, beginning in late July to start the bioinformatics analysis with the raw NGS data and to participate in the Marine Biological Laboratory Workshop on Molecular Evolution.
Link to Project Blog