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Investigation of mechanisms of rodenticide-resistance using a closed colony of rodenticide-resistant black rats (Rattus rattus) from Tokyo
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| Title: | Investigation of mechanisms of rodenticide-resistance using a closed colony of rodenticide-resistant black rats (Rattus rattus) from Tokyo |
| Other Titles: | 東京由来殺鼠剤抵抗性クマネズミ(Rattus rattus）クローズドコロニーを用いた殺鼠剤抵抗性獲得機序の解明 |
| Authors: | 武田, 一貴 Browse this author |
| Issue Date: | 25-Mar-2020 |
| Publisher: | Hokkaido University |
| Abstract: | Chapter I - Introduction -
Anti-blood coagulation rodenticides, such as warfarin, have been used since the 1940s. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats all over the world. Thus, it is necessary for development of novel rodenticide to reveal mechanism of the resistance. I investigated the mechanism with a closed colony of warfarin-resistant black rats (Rattus rattus) from Tokyo.
Chapter II - Target enzyme: VKOR -
Mutations of the VKOR enzyme can lead to resistance to those compounds. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138) of VKOR. However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. Thus, I conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant.
On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC50 value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (Vmax/Km). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Phe55, Lys30, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.
Chapter III - Assessment of ADME -
Absorption, distribution, metabolism, and excretion (ADME) is known to have a significant effect on diversity of drug effect. Herein, pharmacokinetics and pharmacodynamics of warfarin in those rats were investigated. Prothrombin time of R-rats did not change markedly and there was no significant difference compared to the value at 0 h. R-rats had low levels of warfarin in serum and significantly higher clearance activity. Warfarin is metabolized to hydroxywarfarin by cytochrome P450 (P450). They can rapidly metabolize warfarin by hydroxylation shown as significantly earlier Tmax of hydroxywarfarin. The levels of accumulation in the organs of R-rats were lower than those of S-rats.
To investigate warfarin metabolism in R-rats, in situ liver perfusion of warfarin was performed with those rats. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. R-rats showed significantly higher amount of all of five metabolites (hydroxywarfarin) than those of S-rats. The results indicated R-rats showed higher hepatic warfarin hydroxylation which can lead to enhanced excretion of warfarin in in vivo.
Chapter IV - Investigation of the mechanism of enhanced metabolism -
In this chapter, I investigated a possible mechanism of enhanced hepatic warfarin hydroxylation activity. In vitro warfarin metabolism assay was performed to investigate kinetic parameters of cytochrome P450. I prepared two fractions from their liver. One is microsomes containing P450, the other is S9 fraction, including P450 and natural nicotinamide adenine dinucleotide phosphate (NADPH) secretion ability. NADPH plays a role in electron donation to P450. In the assay, NADPH was added to microsomes, while NADP+ was added to S9. The Vmax of microsomes of R-rats showed modest difference, however, that of S9 of R-rats was significantly higher than that of S-rats. These results indicated the enhanced metabolism shouldn’t be due to P450 but NADPH production ability.
I measured NADPH production activity by pentose phosphate pathway; the major source of NADPH. R-rats showed enhanced NADPH production. There were no significant differences in amino acid sequence and expression level of glucose 6 phosphate dehydrogenase (G6PD) playing role in NADPH production in pentose phosphate pathway. G6PD is known to have post-transcriptional regulation. These results suggested the mechanism of higher NADPH production may lie in post-transcriptional regulation of G6PD.
Chapter V - Conclusion -
In this study, I revealed warfarin-resistant black rats from Tokyo had VKOR mutation and enhanced hepatic warfarin metabolism due to high NADPH production activity. Notably, this is the first report that shows enhanced NADPH production activity can contribute to rodenticide-resistance. These results have some potential to contribute to further understanding of rodenticide-resistant rodents and to the development of novel rodenticide. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第14111号 |
| Degree Level: | 博士 |
| Degree Discipline: | 獣医学 |
| Examination Committee Members: | (主査) 教授 石塚 真由美, 教授 乙黒 兼一, 准教授 池中 良徳, 助教 川合 佑典（帯広畜産大学） |
| Degree Affiliation: | 獣医学研究科（獣医学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/80766 |
| Appears in Collections: | 課程博士 (Doctorate by way of Advanced Course) > 獣医学院(Graduate School of Veterinary Medicine)
学位論文 (Theses) > 博士 （獣医学）
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