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Studies on xenobiotic metabolism as a defense system in Carnivora

近藤, 充希 北海道大学

2022.12.26

概要

Chapter 1: General introduction
Chemicals, including pesticides, persistent organic pollutants, and pharmaceuticals, are sometimes released into the environment as environmental contaminants and have continuous impacts on wildlife and ecosystems. Xenobiotics metabolizing enzymes are known as "detoxifying" enzymes that alter the chemical properties of these chemicals. These are classified into Phase I through Phase III reactions. Phase I reactions include oxidation, reduction, and hydrolysis, phase II reactions include conjugation, and phase III reactions include post-metabolism efflux. Through these reactions, chemicals are often converted into less toxic forms that can be easily excreted from the body. The ability to metabolize chemical substances involving these metabolic enzymes varies greatly among animal species, and is a major factor that determines the sensitivity to chemical substances among animal species. Therefore, the evaluation of chemical-metabolizing enzymes in wild mammals, which are continuously exposed to environmental pollutants, is an urgent task. "Carnivorans" are a group of mammals that includes cats, dogs, bears, weasels, seals, and others. They are generally located at higher levels of the ecosystem and are susceptible to bioaccumulation of highly persistent environmental contaminants. Furthermore, as an umbrella species, they are a key species group for ecosystem conservation and are important to assess their effects on environmental pollutants. In this study, I attempted to comprehensively elucidate the evolutionary, genetic, and enzymatic properties of chemical metabolizing enzymes in these carnivores by using both in vitro and in silico analyses.

Chapter 2: Specific duplication and loss of Cytochrome P450 families 1-3
This chapter details the analysis of Cytochrome P450 (CYP), the most important xenobiotics metabolizing enzyme involved in phase I reactions. CYP metabolize variety of chemicals including exogenous substances such as drugs and environmental chemicals, and endogenous substances such as steroids, fatty acids, and cholesterol. Through duplication and loss events, CYPs have created their original feature of detoxification in each mammal. I performed a comprehensive genomic analysis to reveal the evolutionary features of the main xenobiotic metabolizing family: the CYP1-3 families in Carnivora. I found specific gene expansion of CYP2Cs and CYP3As in om-nivorous animals, such as brown bear, black bear, the dog, and the badger, revealing their daily phytochemical intake as the causes of their evolutionary adaptation. Further phylogenetic analysis of CYP2Cs revealed Carnivora CYP2Cs were divided into CYP2C21, 2C41, and 2C23 orthologs. Additionally, CYP3As phylogeny also revealed the 3As evolution was completely different to that of the Caniformia and Feliformia taxa. These studies provide us with fundamental genetic and evolutionary information on CYPs in Carnivora, which is essential for appropriate interpretation and extrapolation of pharmacokinetics or toxicokinetic data from experimental mammals to wild Carnivora.

Chapter 3: Duplication and loss of UDP-glucuronosyltransferase
This chapter focuses on glucuronosyltransferases (UGTs), the major detoxification enzymes, among the enzymes involved in phase II reactions that were not evaluated in chapter 1. UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of order include various carnivorous species yet there is huge variation of food habitat. Recently UGT1A and 2B lower activity were shown in Felidae and pinniped suggesting evolutional loss of these isoforms. However comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. I found specific gene expansion of UGT1As in Canidae, brown bear and black bear. I also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, I discovered contraction or complete loss of UGT1A7-12 in phocids, some otariids, felids and some Mustelids. These studies highly indicated even closely related species, they have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans.

Chapter 4: Enzymatic and genetic features of Sulfotransferases
This chapter focuses on sulfotransferases (SULTs), which, same as the UGTs evaluated in Chapter 3, are important for phase II reactions. Along with UGTs, it is involved in the metabolism of a variety of exogenous substances and endogenous substances such as steroid hormones, neurotransmitters such as dopamine, and thyroid hormones. In this chapter, I performed the same genetic analysis as in the previous chapters, as well as in vitro enzymatic evaluation using wild animal livers.

Genetic analysis revealed that SULT1E1, an important molecule for estrone metabolism, and SULT1D1 are genetically defective in pinnipeds. Furthermore, in vitro analysis of the metabolic activity of estradiol, the main molecule metabolized by SULT1E1, revealed that the activity of SULT1E1 is significantly lower in pinnipeds. These results suggest that the sulfate conjugation activity may be weak against estrogen, various drugs, and environmental pollutants in pinnipeds. SULT1E1 is also involved in the metabolism of endogenous substances, suggesting that there may be significant species differences in estrogen metabolism among carnivores.

Chapter 5: Conclusion and Future investigation
These studies revealed a comprehensive characterization of the chemical metabolism enzymes important for "detoxification" in carnivores. The results indicate that a wide variety of Xenobiotics metabolism enzymes have evolved even within the evolutionarily close order Carnivora. In particular, I found characteristic duplication of CYPs in brown bears, American black bears, and badgers; genetic expansion of UGT in brown bears, black bears, and canids; genetic contraction of UGT in pinnipeds and cats; and loss of SULT1E1 in pinnipeds. Our findings will allow for more accurate characterization of xenobiotic metabolism enzymes and help to accurately extrapolate findings of xenobiotic metabolism enzymes obtained in laboratory animals. Furthermore, I believe that this research was also important in estimating animals that are sensitive to environmental chemicals by evaluating these enzymes, which are important as a defense mechanism against xenobiotics.

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