Investigation of the Function and Evolution of Vitamin K Epoxide Reductases

  • Zoe Hollis

Student thesis: Doctoral Thesis


Vitamin K epoxide reductase complex subunit 1 (VKORC1) is an important enzyme in the vitamin K cycle, and the target of anticoagulant rodenticides. Polymorphisms identified in VKORC1 in Rattus norvegicus(brown rat) can cause anticoagulant resistance. Previous studies have shown several anticoagulant resistance polymorphisms that are found in the UK in brown rats, but the results are mainly derived from laboratory rat samples or frozen tissue samples collected between 1990-2000. The current status of anticoagulant rodenticide resistance in wild brown rat populations in the UK is therefore largely unknown. At present there has been no study of genetic resistance in Swedish rodents. In this study DNA sequencing of the VKORC1 gene of brown rats was done to assess the frequency and distribution of anticoagulant resistance polymorphisms in wild brown rats in the UK and Sweden. DNA sequencing revealed that 69% of brown rats in the UK and 14% of Swedish brown rats contained a known anticoagulant resistance polymorphism (p.L120Q, p.L128Q, p.Y139C, p.Y139S) . As well as these known polymorphisms a novel p.R40G polymorphism was identified in UK and Swedish Rattus norvegicus samples. A baculovirus expression system was used to express the p.R40G VKORC1 variant and wild type rat VKORC1 to compare the effect of five anticoagulant rodenticides (warfarin, bromadiolone, brodifacoum, difenacoum and flocoumafen) using a high pressure liquid chromatography assay to produce dose-response curves, which IC50 values were derived. It was found that the novel p.R40G VKORC1 variant is resistant to warfarin but sensitive to the other rodenticides tested. This is also the first study in which the effects of flocoumafen were tested against wild type brown rat VKORC1 and variants in vitro.

With the diverse anticoagulant resistance polymorphisms found in the UK and Sweden in this study, it was of interest to observe if there was a relationship between resistance
polymorphisms and mitochondrial D loop haplotypes. DNA sequencing of the mitochondrial D-loop of brown rats were completed to identify the current population structure of UK and Swedish wild brown rats. This revealed that certain resistance polymorphisms are associated with mitochondrial D-loop haplotypes. As a result of anticoagulant resistance in Rattus norvegicus there has been an increased usage of more potent second generation anticoagulant rodenticides to control populations of these pests. This has in turn resulted in an increased reports of non-target poisoning in animals belonging to all vertebrate classes. At present there is little known about the effects of anticoagulant rodenticides on many of these animals, including humans. Multiple sequence alignments were used as a prediction tool to determine the potential structure, functions and susceptibility to anticoagulant rodenticides throughout vertebrate VKORC1 sequences by comparing them with known resistance polymorphisms in human and rat VKORC1s. Four representative VKORC1 sequences (Rattus norvegicus - brown rat, Homo sapiens - human, Bos taurus - cow and Python bivittatus - python) were chosen for heterologous expression in the baculovirus expression system. The human, cow and brown rat VKORC1 variant were compared to test the effect of five anticoagulant rodenticides (warfarin, bromadiolone, brodifacoum, difenacoum and flocoumafen) using a high pressure liquid chromatography assay to produce dose-response curves, which IC50 values were derived. The results showed that the brown rat VKORC1 was more sensitive to warfarin compared to the human and cow VKORC1, but against the other four anticoagulant rodenticides the opposite was shown. The results showed a difference in potency for the anticoagulant rodenticides depending on the species tested.

The general overall phylogeny of the VKOR family of enzymes has been well characterized in terms of phylogenetic trees and relationships of VKORs. With respect to amino acid sequence comparisons these studies have however only used a relatively small number of vertebrate VKORC1 and VKORC1L1 sequences to assess the structural, functional and evolutionary relationships between VKORs in different species and between the two enzymes. In this study multiple sequence alignments were used to determine the structural, functional and evolutionary relationships between the 135 vertebrate VKORC1 sequences and 164 VKORC1L1 sequences. It can be predicted that all vertebrate VKORC1 and VKORC1L1 enzymes have the ability to bind both vitamin K and vitamin K epoxide and also have the ability to bind warfarin. It can also be predicted that some vertebrate VKORC1 sequences may have a natural resistance or sensitivity to anticoagulant rodenticides.
Date of Award2023
Original languageEnglish
SupervisorJarek Bryk (Co-Supervisor) & Dougie Clarke (Co-Supervisor)

Cite this