爬虫類の有鱗目と亀目における生体異物代謝経路－生物学的防御システムの機序と特徴

2014 Fiscal Year Annual Research Report

• Project/Area Number: 13J01324
• Research Institution: Hokkaido University
• Principal Investigator: オロスラーニュ バラージュ 北海道大学, 獣医学研究科, 特別研究員(DC1)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: pyrene / reptile / turtle / tortoise / yeast

Outline of Annual Research Achievements

This year my research focused on confirming the effect of pyrene on freshwater turtle species(1.), establishing an environment to turtles and tortoises as laboratory animals (2.), further investigating the molecular background of species differences (3.), and continue developing a yeast model for reptile metabolism (4.).
1. Previous results from freshwater turtle species indicated a dominance of pyrene-1-sulfate metabolite. To determine whether this is a general Testudines characteristic, or is due to the aquatic environment, the results were reconfirmed in Chinese soft-shell turtles, and exposure studies are under way with Greek and Russian tortoises.
2. Reptiles are non-standard laboratory animals, and just providing an adequate environment provide challenges. I have developed a feeding formula for Chinese soft-shell turtles, and established a tortoise-friendly environment.
3. Reptile genetic data is still missing for repositories, and my lack of experience with molecular techniques might be the reason that my attempt to clone the enzymes failed. Based on the new data from three seawater turtles, I have re-started the cloning efforts.
4. Current enzyme kinematic experiments using liver samples requires to sacrifice the target animals. To reduce the experimental animal use, I use Saccharomyces cerevisiae to express xenobiotic metabolism enzymes. I am developing a method adapted to our laboratory’s environment.
With this method, I can combine the yeast method with the molecular results, and use it as a non-lethal, minimally invasive screening method for turtle metabolism.

Current Status of Research Progress

4: Progress in research has been delayed.

Reason

Turtles (and reptiles in general) are non-standard laboratory animals.
In my initial research plans, calculation of the required time for each phase of the experiment was based on my (limited) experiment with standard laboratory species, such as rats and mice.
When I started working with reptiles, I realized that not only providing an adequate environment provide challenges, but (compared to mammals) even the experiments might need an elongated time. For example, with pyrene, in rats we are expect to see a peak of metabolites hours (or a few days) after exposure. Chinese softshell turtles, on the other hand, still produced metabolites 4 weeks after exposure.

I realize that with better time management (and with parallel experiments) the effect of these elongated experiment time would have been greatly reduced. I hope that this year I can catch up, and reach the initial goals.

Strategy for Future Research Activity

I would like to combine and thus conclude my research. I will continue the following three parallel methods, namely;
1. In vivo exposure and in vitro kinetics with pyrene. Polycyclic aromatic hydrocarbons (PAHs) are one of the most common persistent organic pollutants (POPs) in the environment. The metabolism of pyrene, a common PAH is well studied in various animal model systems. However, we have no data of pyrene metabolism in reptiles.Our investigation with freshwater turtles suggested that contrary to mammals, pyrene-1-sulfate is the most abundant metabolite. To determine whether this difference is due to environmental factors or a common characteristic of reptiles, further experiments with tortoises is conducted.
2. Genetic background of the reptile xenobiotic metabolism.To understand the reptilian metabolism, sequencing and characterizing of the involved enzymes is necessary.We are focusing on Cytochrome P450 1A5, a Phase I metabolic enzyme, to be isolated from 3 freshwater turtle and 2 tortoise species. The development of a sequencing pipeline will help to broaden this method with further reptile species.
3. Expressing the selected enzymes in genetically modified yeast. S. cerevisiae was successfully used to express a wide variety of mammalian xenobiotic enzymes. We hope that we will have similar success with reptilian proteins.
By the end of this fiscal year, I plan to interweave and summarize the results of these methods, and write a thesis that will be an important stepping stone for further reptilian research as well as the base of reptile conservation initiatives.