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The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. 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). 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. We 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 IC 50 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 (V max /K m ). 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 Lys30, Phe55, 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. 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. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the V max of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450., (Copyright © 2018. Published by Elsevier Inc.)

Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 µM. The Vmax values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The Km values of resistant rats (47 ± 32 µM) were similar to those of sensitive rats (59 ± 18 µM). Warfarin-sensitive rats exhibited enzyme efficiencies (Vmax/Km) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

Warfarin is commonly used worldwide as a rodenticide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. It has been reported that repeated or long-term treatments with this drug cause resistance in wild rodents. However, the mechanism of warfarin resistance in rodents is still not known precisely. Recent studies reported and identified the function of the molecule, vitamin K epoxide reductase complex subunit 1 (VKORC1), which is the main unit of VKOR. An amino acid substitution in VKORC1 is one of the supposed mechanisms of warfarin resistance. An accelerated detoxification system involving cytochrome P450 (CYP) could also cause the rodenticide resistance. Administration of SKF-525A, a potent inhibitor for P450, increased the mortality due to reduction of warfarin metabolism in warfarin-resistant rats. Meanwhile, the appearance of warfarin-resistant rodents has led to the development of the more effective and toxic rodenticide superwarfarin, which is widely used in Europe and the USA. However, animals resistant to this second-generation rodenticide have already been reported in Europe. In this review, we focus on the mechanism and the pleiotropic effects of pesticide resistance in wild rodents.

Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases. Pest control is important for public health, and warfarin is a commonly used rodenticide worldwide. However, continual use of warfarin may cause drug resistance in rodents and lead to failure of their control, especially in urbanized areas. In warfarin-resistant rats, the warfarin level in plasma was significantly lower after oral administration than that in the control warfarin-sensitive rats. Warfarin is metabolized by cytochrome P450 (P450), and hydroxylation of warfarin by P450 isoforms was significantly higher in warfarin-resistant rats (2-fold). Western blot analysis indicated that the level of CYP3A2 expression in warfarin-resistant rats was significantly larger than in warfarin-sensitive rats. The NADPH-P450 reductase activities in resistant rats were 8-fold higher than those in sensitive rats. In vivo, the administration of the P450 potent inhibitor proadifen (SKF-525A) increased the mortality of warfarin in the warfarin-resistant roof rats. We concluded that the mechanism of warfarin resistance in Tokyo roof rats is caused by increased clearance of warfarin.

Abstract: Warfarin is commonly used worldwide as a rodenticide. It inhibits blood coagulation by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity leading to hemorrhage. However, it has been reported that repeated or long-term treatment with warfarin results in resistance emerging in wild rodents. Such resistance may explain why it is difficult to control rodents in many regions in Japan. In this report, we studied mutations in the VKOR gene (including the VKOR complex subunit 1 (VKORC1)), while also analyzing VKOR and clotting factor activity in black rats (Rattus rattus) in order to understand better the mechanism of warfarin resistance in this species. We sequenced the VKORC1 gene from 275 rats living in the wild in Japan. We found several types of novel base substitutions, some of which conferred warfarin resistance. There was no difference in coagulation times between warfarin-sensitive and resistant rats measured under physiological conditions. However, after warfarin administration, no effect was noted in warfarin-resistant rats, although a prolonged coagulation time was noted in warfarin-sensitive rats. We also determined the kinetic differences in hepatic microsomal VKOR-dependent activity between warfarin-resistant and sensitive rats. Warfarin-resistant rats showed 2–3-fold lower Vmax /Km values than did sensitive rats. In addition, we report that resistant rats found in the Tokyo area had a VKOR activity which was poorly inhibited by warfarin. Finally, we conclude that reduced VKOR activity and warfarin resistance in the Japanese black rat might be due to mutations in the VKORC1 gene. However, further study is needed to clarify how such rats can maintain adequate vitamin K-dependent clotting factor levels, while simultaneously exhibiting low VKOR activity and warfarin resistance. [Copyright &y& Elsevier]