Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Pesticide Biochemistry and Physiology 88 (2007) 203–208 www.elsevier.com/locate/ypest

Comparison of the inhibition eVect of diVerent anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat R. Lasseur, A. Grandemange, C. Longin-Sauvageon, P. Berny, E. Benoit ¤ UMR 1233 INRA/DGER, Métabolisme des Xénobiotiques et Mycotoxines, Lyon College of Veterinary Medicine, 1 av. Bourgelat, 69280 Marcy l’Etoile, France Received 3 August 2006; accepted 15 November 2006 Available online 22 November 2006

Abstract Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of Wrst generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory eVect of diVerent anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (Ki D 0.72 § 0.1 M; 29 § 4.1 M), chlorophacinone (Ki D 0.08 § 0.01 M; 1.6 § 0.1 M), diphacinone (Ki D 0.07 § 0.01 M; 5.0 § 0.8 M), coumachlor (Ki D 0.12 § 0.02 M; 1.9 § 0.2 M), coumatetralyl (Ki D 0.13 § 0.02 M; 3.1 § 0.4 M), difenacoum (Ki D 0.07 § 0.01 M; 0.26 § 0.02 M), bromadiolone (Ki D 0.13 § 0.02 M; 0.91 § 0.07 M), and brodifacoum (Ki D 0.04 § 0.01 M; 0.09 § 0.01 M) on VKOR activity. Analysis of the results leads us to highlight diVerent anticoagulant structural elements, which inXuence inhibition parameters in both susceptible and Y139F resistant rats. © 2006 Elsevier Inc. All rights reserved. Keywords: Vitamin K epoxide reductase; Liver microsomes; Warfarin resistance; Inhibition parameters; Chlorophacinone; Diphacinone; Bromadiolone; Difenacoum; Brodifacoum; Coumatetralyl; Coumachlor; Rattus norvegicus

1. Introduction Coumarin derivatives such as warfarin are used as anticoagulant drugs in human thromboembolic diseases. They have also been widely used since 1950s in the control of rodent populations. These drugs belong to the anti-vitamin K group (AVK). They inhibit the vitamin K epoxide (K>O) reduction activity (VKOR), which is part of the vitamin K cycle and as a consequence they block the vita-

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Corresponding author. E-mail address: [email protected] (E. Benoit).

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min K dependent -carboxylation of the coagulation factors. Finally, the suppression of VKOR activity by AVK compromises the coagulation process [1]. In humans and rodents, genetic warfarin resistances are observed [2–4]. In these patients or in these animals, anticoagulant activity of warfarin is lowered and the warfarin inhibiting activity towards liver microsomal VKOR is reduced [2,3,5,6]. In rats, a large-scale utilization for 50 years of these chemicals has created an intense selection of resistant individuals. Nowadays, some places count up to 75% of resistant animals [7]. Control of such populations leads to the utilization of much more potent chemicals, such as difenacoum, bromadiolone or brodifacoum [8]. Nevertheless,

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resistances to difenacoum and bromadiolone are now observed, which conWrms the evolution of resistance mechanisms and rodent adaptation. Unfortunately, because of their increased activity, these rodenticides are much more threatening for non-target species [9]. Many studies on resistance mechanisms have been conducted and recently a protein of the VKOR complex has been identiWed and named VKORC1 (Vitamin K epoxide reductase subunit 1) [2,10]. The heterologous expression of this protein in SF9 (insect cells) or HEK (Human Embryonic Kidney) cells was found to be able to catalyze vitamin K>O reduction, this reaction being sensitive to inhibition by warfarin. An important step was achieved in the understanding of resistance mechanisms, identifying diVerent SNPs (Single Nucleotide Polymorphism) in warfarin resistant human and rodent VKORC1 sequences. In rodents, mutated VKORC1 showed a reduced enzyme activity and a partial resistance towards warfarin inhibition [3]. The study by Pelz et al. [3] comparing diVerent resistant laboratory strains and wild-caught rats for mutations in the VKORC1 gene showed that the mutation Y139F confers the most potent warfarin resistance in the rat. This Y139F SNP has been identiWed in wild resistant rat strains from France and Belgium [3]. In our laboratory, we have studied catalytic properties of VKOR in liver microsomes from wild and mutated (Y139F) rats [11]. The genotypes of these rats were homozygous for the wild VKOR-C1 or homozygote for the Y139F mutation. The warfarin Ki was highly increased (from 0.7 to 29 M) in the Y139F rats. In view of rapid resistance evolution in rodents, it was interesting to understand the behavior if inhibitors on the pharmacological target VKOR. We screened diVerent anticoagulants (warfarin, chlorophacinone, diphacinone, coumachlor, coumatetralyl, bromadiolone, difenacoum and brodifacoum) on VKOR activity from wild-type and Y139F rat liver microsomes. The aim of this study was to observe the consequences of this mutation on the activity of the diVerent AVK available. 2. Materials and methods 2.1. Animals Warfarin-susceptible and warfarin-resistant rats bred in the Lyon College of Veterinary Medicine (ENVL) were initially trapped on French farms in the 1980s. Each strain received the same feed and water ad libitum. Animals were not supplemented in vitamin K and received a standard feed. The warfarin-resistant rats survived a 6-day no-choice feeding test of 0.025% warfarin-containing oats [12]. Susceptibility was determined at 100% for the susceptible rat strain. All experimental protocols were approved by the ethics committee of the Lyon College of Veterinary Medicine and followed the guidelines described in the National Health Institute for the Care and Use of Laboratory Animals. VKORC1 was genotyped according to [11]. Only rats homozygous for the VKORC1 Y139F SNP or VKORC1

wild type were used for the study of vitamin K epoxide reductase activity (VKOR). Rats were euthanatized by decapitation under isoXurane anaesthesia, 3–4 months after the termination of warfarin exposure. The liver from 10 warfarin resistant and 10 warfarin susceptible rats were removed and stored in liquid nitrogen until assay. 2.2. Tissue preparation from rat livers Microsomes were prepared from thawed livers by diVerential centrifugation as described by [13]. Protein concentrations were determined by the method of Bradford [14] using bovine serum albumin as a standard. Microsomes were frozen at ¡80 °C until analysis. 2.3. Kinetics studies on vitamin K epoxide reductase Microsomal vitamin K epoxide reductase (VKOR) activity was assayed as described by [6,15,16], except that the reaction solution contained 2 mM dithiothreitol (DTT) and buVer solution was 200 mM Tris–HCl/0, 15 M KCl buVer (pH 7.4); therefore 1.5 mg of protein was added for assay of liver microsomes in a Wnal volume of 1 ml. VKOR activity was assayed at 30 °C. The reaction was started by the addition of vitamin K>O solution in 1% Triton X-100 (10 l volume). The incubation time was 20 min and the reaction was stopped by adding of 4 ml of iced 1:1 isopropanol/hexane solution. After centrifugation (5000g, 5 min), the hexane layer was removed and was dried under nitrogen. The dry residue was immediately dissolved in 0.2 ml of isopropanol. Vitamin K was measured by HPLC (D7000 HS Hitachi AS 2000 automatic injector, injection volume of 50 l, L 7100 pump, L 4000 UV detector). Separation was achieved on a LiChrospher 100 RP18e, 150 £ 4 mm, 5 m analytical column run at 1.5 ml/min in 100% methanol HPLC grade. Internal standard (Vitamin D3) was added and quantiWed at 254 nm. In order to determine apparent Km values, eight diVerent concentrations of the substrate vitamin KO were used ranging from 12.5 to 100 M. Incubations were performed in triplicate. Inhibition parameters were Wrst determined with anticoagulant concentrations of 0.01; 1; 10 and 30 M and further more precisely characterized using anticoagulant concentrations from about 0.05 £ Ki to 20 £ Ki. A non-linear model using the RWt Program developed by Ihaka and Gentleman [17] was Wtted to the data (free access on internet). 2.4. Chemicals Vitamin K (Phylloquinone) was converted to vitamin K>O according to [18]. Vitamin K, DTT, Tris buVer, isopropanol, hexane and methanol were purchased from Sigma–Aldrich (France). Sodium warfarin, coumachlor, difenacoum and brodifacoum were purchased from Sigma– Aldrich (France). Coumatetralyl and diphacinone were from Laboratory Dr. Ehrenstorfer-Schäfers (Germany). Chlorophacinone and bromadiolone were supplied by

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rats and 29.0 § 4.1 M for resistant ones. Such results are closely similar to results obtained in [11].

LiphaTech (Pont du Casse, France) and of the highest purity available (>99.7%). Primers for VKORC1 genotyping were synthesized by Proligo (Paris, France). DNA genomic Isolation System was from Promega (Charbonnières les Bains, France).

3.3. Coumachlor inhibition Coumachlor is structurally very close to warfarin except for the chlorine atom. Ki values was 0.12 § 0.02 M for susceptible rats and 1.9 § 0.2 M for resistant rats. These data show that VKOR in microsomes from (genetic) resistant rats is more inhibited by a chlorinated molecule than by warfarin.

3. Results 3.1. Preliminary investigations Preliminary experiments showed that enzymatic activity had a linear relationship with protein content and time of incubation (data not shown). Kinetic parameters of VKOR found were similar to the ones published previously [11], Km was 79.7 § 20.6 M for susceptible rats and was 20.9 § 12.4 M for warfarin resistant rats. Chemical structure of the anticoagulants studied are presented in Fig. 1.

3.4. Bromadiolone inhibition Ki was lower than warfarin Ki, values was 0.13 § 0.02 M for susceptible rats and 0.91 § 0.07 M for resistant ones. These results show that the genetic resistance is also responsible for a bromadiolone resistance in a liver microsomal system.

3.2. Warfarin inhibition 3.5. Difenacoum and brodifacoum inhibition Warfarin inhibition of VKOR was studied using microsomes from susceptible rats and from resistant rats. Data were Wtted to the Michaelis-Menten non-competitive inhibitor model. Ki obtained was 0.72 § 0.1 M for susceptible O

Brodifacoum has the same type of inhibiting eVect as difenacoum, except that a higher Ki for difenacoum was observed in microsomes from resistant rats. Ki for

O

O

O

O O

O OH

OH

A

OH

C

B Br O

Cl

O O

OH

O

O

OH

D

OH

E

O

Br

O O

O

O O O

OH

G Cl

F

H

O

Fig. 1. Chemical structure of eight anticoagulants (six derivatives 4-hydroxy-coumarin and two derivatives indane-dione). (A) Warfarin; (B) Coumachlor; (C) Coumatetralyl; (D) Bromadiolone; (E) Difenacoum; (F) Brodifacoum; (G) Diphacinone; (H) Chlorophacinone. Inhibitory eVect on VKOR activity of these anticoagulants was compared in microsomes from susceptible and resistant Y139F rat.

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100

Susceptible rats Resitant rats

80

60

40

20

0 0

0,05

0,1

0,2

1

2

5

10

20

Chlorophacinone concentration ( μM)

Fig. 2. Vitamin K epoxide reductase activity (expressed in percent of the control activity) as a function of Chlorophacinone concentration (M). Inhibition eVect on VKOR activity was studied in microsomes from susceptible and resistant rat (VKORC1 Y139F polymorphism) for each anticoagulant and this representation is an example. Control activity (without anticoagulant) is represented by the Wrst bar and is considered as 100% of VKOR activity. Each bar represents means (§ SEM) of three measurements.

susceptible rats was 0.07 § 0.01 M for difenacoum and 0.04 § 0.01 M for brodifacoum. Ki for resistant rats was 0.26 § 0.02 M for difenacoum and 0.09 § 0.01 M for brodifacoum. 3.6. Diphacinone inhibition Diphacinone is a Wrst generation inhibitor (such as warfarin) but not from the same chemical group. Warfarin is a coumarin derivated product while diphacinone is an indane-dione one. We observed a resistance phenomenon to this product (like in vivo observations (internal data)). Ki was 0.07 § 0.01 M for susceptible rats and 5.0 § 0.8 M for resistant ones. 3.7. Chlorophacinone inhibition Chlorophacinone is structurally very close to diphacinone except for a chlorine atom. We observed Ki of 0.08 § 0.01 M for susceptible rats and 1.6 § 0.1 M for resistant ones (Fig. 2). 4. Discussion In human beings, resistances to anti-vitamin K molecules are observed. Some of these resistances are due to mutations in the VKORC1 gene [2–4,10]. At present, about 10 mutations are described and are responsible for an important part of the AVK dosages variability [4,19].

Mutations on the same gene are also observed in wild rat (Rattus norvegicus) and are responsible for resistances to Wrst generation chemicals [3]. Yet, a massive utilization of AVK since 1950 has created a strong selection of these resistant animals [20]. The control of these resistant strains is complicated by the loss of eYciency of available anticoagulants. So it’s of great importance to understand inhibition eVect of AVK on VKOR carrying diVerent mutations identiWed in the Weld. We hold two strains of wild rats, maintained in enclosures, one of which is a resistant strain homozygous for the mutation Y139F. This strain shows an in vivo high degree of resistance to warfarin [11]. Consequently, we tested various AVK and analyzed their eVect on VKOR activity from both susceptible and resistant rats. Among AVKs, we can distinguish two main families. The ones derived from 4-OH-coumarin, the ones from indanedione. Concerning Ki values, in susceptible rat, warfarin has a singular position. Indeed, its Ki is quite high (0.72 M). Coumachlor’s Ki is about 0.12 M while its only diVerence with warfarin is a chlorine atom on a lateral phenyl chain. The importance of this halogen is not observed with chlorophacinone/diphacinone Ki values. The only diVerence between these two chemicals (indanedione structure) is also a chlorine atom on the lateral chain. Nevertheless, Ki values for these active compounds are so low that such an eVect of the chlorine atom would be diYcult to observe. Hydrophobia of the lateral chain on the hydroxycoumarin structure seems to be in favor of a decrease in Ki values

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1000000 Ki for susceptible microsomes

100000

Ki for resistant microsomes 10000 P/1000 1000

100

10

1

0,1

um

um

ne

Br

od

ifa

co

co na ife D

om Br

ha op or C

hl

ad

ci

io

no

lo

ne

ne no ci ha ip D

C ou m at et ra ly l

m ou C

W

ar

ac

fa

hl

or

rin

0,01

Fig. 3. Comparison of inhibition eVect of six anticoagulants, in microsomes from susceptible and resistant rats, in relation with the hydophobicity of each anticoagulant represented by P/1000 (partition coeYcient/1000).

(Fig. 3). Indeed, coumatetralyl’s Ki (0.13 § 0.02 M) is actually higher than difenacoum’s one (0.07 § 0.01 M) (cf Fig. 1). Moreover, a bromide at the end of the lateral chain decreases Ki as shown by the couple difenacoum/brodifacoum. For Y139F (resistant) rats, Ki of hydroxycoumarin and indanedione derivatives systematically increase as compared to susceptible rats. Nevertheless, the observations are not in the same range of values depending on anticoagulants studied. Warfarin’s Ki is highly increased (from 0.72 to 29 M for resistant rats). For the structurally related molecules such as coumachlor or bromadiolone, Ki is also increased 1.9 (£16) and 0.91 M, (£7), respectively. In other derivatives, mutation eVect is limited (£3.9 and £2 for difenacoum and brodifacoum, respectively). For these chemicals, Ki observed remain very low, and should be compatible with an in vivo toxic eVect. Hydrophobicity and length of lateral chain increase the interaction of anticoagulant as much in susceptible animal as in resistant animal. With increasing hydrophobicity, there is a decreased destabilizing eVect of this mutation on the interaction with anticoagulant (Fig. 3). Moreover, the eVect of the chlorine atom on the lateral phenyl chain (coumachlor) is observed in resistant rats for both coumachlor and chlorophacinone in comparison with diphacinone and warfarin, respectively. Indanedione structure seems to interact with VKOR more eYciently than 4-OH coumarine. This study was conducted with the Y139F mutation, which is involved in anticoagulant resistance. Catalytic

consequences of this mutation are aYnity changes of vitamin KO and warfarin for VKOR [11]. This paper describes aYnity changes of VKOR activity for all AVK compounds used and highlights the importance of the OH-coumarine vs indanedione nucleus, the chlorine atom and the hydrophobicity of the lateral chain. Nevertheless, some other mutations in VKORC1 have no catalytic consequences on VKOR activity [21]. It would be interesting to study consequences of all identiWed VKORC1 mutations in rodents on the susceptibility of the mutated enzyme to anticoagulants. This work concerns the inhibitory potential of diVerent anticoagulants on VKOR. This study does not take into account metabolism and kinetics that have a real part in the in vivo toxicity of these chemicals, and in inter-species variability [22]. Nevertheless, knowing the resistance status of a given rat population would certainly be of great use, in order to use the most adapted anticoagulant compound, with a high eYcacy on VKOR. It would also allow us to select one with less dramatic consequences on non-target species. Acknowledgments This research was supported by the INRA (Institut National de la Recherche Agronomique) and the DGER (Direction Generale de l’Enseignement et de la Recherche). The authors thank A. Bourret and F. Peigneaux for their technical assistance.

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