Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants.

Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorc1 gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.

Distribution of VKORC1 single nucleotide polymorphism in wild Rattus norvegicus in France.

BACKGROUND: Anticoagulant rodenticides are commonly used to control rodent pests all over the world. These pesticides inhibit one enzyme of the vitamin K cycle, Vkorc1, and thus prevent blood clotting and cause death by haemorrhage. Resistance to anticoagulants was first observed in Scotland in 1958, and more potent anticoagulants have been developed to overcome this obstacle. Unfortunately, these chemicals are very toxic and cannot be used everywhere. Some authors have shown that resistance to anticoagulants seems closely linked with single nucleotide polymorphism (SNP) in the Vkorc1 gene. RESULTS: This study draws a map of SNP and haplotypes found in Vkorc1 in rats from different areas of France. Some of them had never been described before. Moreover, the Y139F mutation, described previously in France and Belgium, is the most frequent in France. This mutation is known to be associated with a strong resistance to anticoagulants, and it was found in 28% of the samples. CONCLUSION: This biomolecular approach to studying and detecting resistance is easier to carry out than the phenotypic approach measuring blood coagulation time because it can be conducted on biological samples from dead animals, and it is less dangerous for the operator.

Consequences of the Y139F Vkorc1 mutation on resistance to AVKs: in-vivo investigation in a 7th generation of congenic Y139F strain of rats.

OBJECTIVES: In humans, warfarin is used as an anticoagulant to reduce the risk of thromboembolic clinical events. Warfarin derivatives are also used as rodenticides in pest control. The gene encoding the protein targeted by anticoagulants is the Vitamin K-2,3-epoxide reductase subunit 1 (VKORC1). Since its discovery in 2004, various amino acid and transcription-regulatory altering VKORC1 mutations have been identified in patients who required extreme antivitamin K dosages, or wild populations of rodents that were difficult to control with anticoagulant rodenticides. One unresolved question concerns the dependency of the VKORC1 on the genetic background in humans and rodents that respond weakly or not at all to anticoagulants. Moreover, an important question requiring further analyses concerns the role of the Vkorc1 gene in mediating resistance to more recently developed warfarin derivatives (superwarfarins). METHODS: In this study, we bred a quasicongenic rat strain by using a wild-caught anticoagulant resistant rat as a donor to introduce the Y>F amino acid change at position 139 in the Vkorc1 into the genetic background of an anticoagulant susceptible Spraque-Dawley recipient strain. RESULTS AND CONCLUSION: In this manuscript we report the prothrombin times measured in the F7 generation after exposure to chlorophacinone, bromadiolone, difenacoum and difethialone. We observed that the mutation Y139F mediates resistance in an otherwise susceptible genetic background when exposed to chlorophacinone and bromadiolone. However, the physiological response to the super-warfarins, difenacoum and difethialone, may be strongly dependent on other genes located outside the congenic interval (28.3 cM) bracketing the Vkorc1 in our F7 generation congenic strain.

Heterogeneity of the coumarin anticoagulant targeted vitamin K epoxide reduction system. Study of kinetic parameters in susceptible and resistant mice (Mus musculus domesticus).

Vitamin K epoxide reductase (VKOR) activity in liver microsomes from a susceptible and a genetically warfarin-resistant strain of mice (Mus Musculus domesticus) was analyzed to determine the mechanism of resistance to this 4-hydroxycoumarin derivative. Kinetic parameters for VKOR were calculated for each strain by incubating liver microsomes with vitamin K epoxide +/- warfarin. In susceptible mice, an Eadie-Hofstee plot of the data was not linear and suggested the involvement of at least two different components. Apparent kinetic parameters were obtained by nonlinear regression using a Michaelis--Menten model, which takes into account two enzymatic components. Component A presents a high Km and a high Vm, and as a consequence only an enzymatic efficiency Vm/Km was obtained (0.0024 mL/min/mg). Estimated warfarin Ki was 0.17 microM. Component B presented an apparent Km of 12.73 microM, an apparent Vm of 0.32 nmol/min/mg, and an apparent Ki for warfarin of 6.0 microM. In resistant mice, the enzymatic efficiency corresponding to component A was highly decreased (0.0003-0.00066 mL/min/mg) while the Ki for warfarin was not modified. The apparent Vm of component B was poorly modified between susceptible and resistant mice. The apparent Km of component B observed in resistant mice was similar to the Km observed in susceptible mice. These modifications of the catalytic properties are associated with a single nucleotide polymorphism (T175G) in the VKOR-C1 gene, which corresponds to a Trp59Gly mutation in the protein.

Warfarin resistance in a French strain of rats.

A warfarin-resistant strain and a warfarin-susceptible strain of wild rats (Rattus norvegicus) maintained in enclosures of the National Veterinary School of Lyon (France) were studied to determine the mechanism of the resistance to anticoagulant rodenticides. A low vitamin K epoxide reductase (VKOR) activity has been reported for many resistant rat strains. As recently suggested, mutations in the vitamin K epoxide reductase subunit 1 (VKORC1) gene are the genetic basis of anticoagulant resistance in wild populations of rats from various locations in Europe. Here we report, for our strain, one of the seven described mutations (Tyr139Phe) for VKORC1 in rats. In addition, a low expression of mRNA encoding VKORC1 gene is observed in resistant rats, which could explain their low VKOR activity. We calculated kinetic parameters of VKOR in the warfarin-resistant and warfarin-susceptible rats. The V(max) and the K(m) of the VKOR obtained in resistant rats were lowered by 57 and 77%, respectively, compared to those obtained in susceptible rats. As a consequence, the enzymatic efficiency (V(m)/K(m)) of the VKOR was similar between resistant and susceptible rats. This result could be a good explanation to the observation that no clinical signs of vitamin K deficiency was observed in the warfarin-resistant strain, while a low VKOR activity was found. VKOR activity in warfarin-resistant rats was poorly inhibited by warfarin (K(i) for warfarin is 29 microM and 0.72 microM for resistant and susceptible rats, respectively).

Identification and characterization of the FMO2 gene in Rattus norvegicus: a good model to study metabolic and toxicological consequences of the FMO2 polymorphism.

BACKGROUND: In lung of many animal species flavin-containing monooxygenase 2 (FMO2) is a 535-amino acid residues drug-metabolizing enzyme. In humans FMO2 exhibits a genetic polymorphism. The major allele encodes a truncated FMO2, the minor allele a full-length FMO2. In laboratory rats we previously reported a FMO2 gene encoding a truncated FMO2 (432-AA residues). In these strains, a double deletion leads to the appearance of a premature stop codon. All laboratory rat strains were derived from the same wild ancestor, Rattus norvegicus. METHODS: A PCR-based method able to specifically recognize either the wild-type or the mutant allele was developed to investigate a putative FMO2 polymorphism in a population of wild rats. The FMO2 gene was analyzed in 42 wild rats. RESULTS: A genetic FMO2 polymorphism similar to that described in humans was found in R. norvegicus. We observed three different genotypes: homozygotes for the wild-type FMO2 (33.3%), homozygotes for the mutant FMO2 (38.1%) and heterozygotes (28.6%). Comparative FMO2 mRNA and protein expressions in lungs were studied by reverse transcription-PCR and western blotting. FMO2 mRNA expression was identical between the three groups. In contrast, major differences in the expression of FMO2 protein were detected. FMO2 was strongly expressed in lungs of homozygotes for the wild-type FMO2, faintly expressed in lungs of heterozygotes and non-expressed in lungs of homozygotes for the mutant FMO2. Comparative catalytic properties of lung microsomes were studied by the determination of the oxygenation of methimazole. FMO2 genetic polymorphism was associated with major differences in the S-oxidative metabolism.

Cloning, sequencing and tissue distribution of rat flavin-containing monooxygenase 4: two different forms are produced by tissue-specific alternative splicing.

The nucleotide sequence of rat flavin-containing monooxygenase 4 (FMO4) mRNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and 5'/3' terminal extension. Complete cDNA was amplified, cloned, and sequenced from the mRNA obtained from rat kidney and brain. Two different transcripts (short and long) stemming from the splicing of an internal region of 189 bases pair, corresponding to exon 4 were identified. This alternative splicing seems to be specific of the brain. The long cDNA encodes a protein of 560 amino acids with a predicted molecular mass of 63,395 Da. The short cDNA encodes a protein of 497 amino acids with a predicted molecular mass of 55,871 Da. Both of these encoded sequences contain the NADPH- and FAD-binding sites and a hydrophilic carboxyl terminus. These sequences are 80 and 79% identical to the sequences of human and rabbit FMO4. By Northern blotting and/or RT-PCR, the long-form FMO4 mRNA was detected in the rat kidney, intestine, and liver and the short form particularly in the brain. For the first time, the expression of FMO4 protein was demonstrated. By Western blotting using the two different forms of FMO4 antibodies, a long FMO4 protein was detected in the rat kidney, whereas in the rat brain, only the short form of FMO4 was observed.

The FMO2 gene of laboratory rats, as in most humans, encodes a truncated protein.

We describe the isolation and characterization of cDNAs for FMO2 from the laboratory rat. In contrast to FMO2 in other animals, each of which contain 535 amino acid residues, analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the ORF of the laboratory rat FMO2 encodes a polypeptide of only 432 residues. This truncated protein is due to the presence of a double deletion corresponding to 1263 and 1264 nucleotides of the orthologous FMO2 cDNAs. This double deletion provokes a frame-shift, with the appearance of a premature stop codon in position 1297-1299. By Northern blotting, the probe for FMO2 hybridized a 2.5-kb transcript in lung and kidney samples only. Heterologous expression of the cDNA revealed that the truncated protein was catalytically inactive. By Western blotting, FMO2 was faintly detected at approximately 50 kDa in laboratory rat lung.

Cloning, sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1 and FMO3 in the dog.

The expression of flavin-containing monooxygenases (FMOs) in dog liver microsomes was suggested by a high methimazole S-oxidase activity. When the reaction was catalyzed by dog liver microsomes, apparent V(max) and K(m) values were 6.3 nmol/min/mg and 14 microM, respectively. This reaction was highly inhibited (73%) in the presence of imipramine, but it was also weakly affected by trimethylamine, suggesting the involvement of different isoforms. The sequences of dog FMO1 and FMO3 were obtained by reverse transcription-polymerase chain reaction and 5'/3' terminal extension. The cDNAs of dog FMO1 and dog FMO3 encode proteins of 532 amino acids, which contain the NADPH- and FAD-binding sites. The dog FMO1 amino acid sequence is 88, 86, and 89% identical to sequences of human, rabbit, and pig FMO1, respectively. The dog FMO3 amino acid sequence is 83, 84, and 82% identical to sequences of human, rabbit, and rat FMO3, respectively. Dog FMO1 and dog FMO3 exhibited only 56% identities. The FMO1 and FMO3 recombinant proteins and the FMO1 and FMO3 microsomal proteins migrated with the same mobility (56 kDa), as determined in SDS-polyacrylamide gel electrophoresis and immunoblotting. By Western blotting, dog FMO1 and dog FMO3 were detected in microsomes from liver and lung but not in kidney microsomes. By Northern blotting, the probe for FMO1 specifically hybridized a 2.6-kilobase (kb) transcript in liver and lung samples only. The probe for FMO3 hybridized two transcripts of approximately 3 and 4.2 kb in the liver and lung samples.