Doi:10.1016/j.pestbp.2008.01.007

Available online at www.sciencedirect.com Biochemistry & Physiology Pesticide Biochemistry and Physiology 91 (2008) 90–95 Warfarin resistance in Rattus losea in Guangdong Province, China Jianshe Wang a, Zhiyong Feng b, Dandan Yao b, Jingjing Sui b, Wenqin Zhong a, Ming Li a, Jiayin Dai a,* a Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101, PR China b Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Wushan Road, Tianhe District, Guangzhou 510640, PR China Received 12 December 2007; accepted 28 January 2008 Available online 5 February 2008 Control of rodent populations is performed worldwide with coumarin derivatives, such as warfarin. After widespread use, their eﬀect has been diminished by the rapid spread of resistant rodents. Warfarin resistance in Rattus loseas in Jiangmen and Zhanjiang City,Guangdong Province, was investigated by lethal feeding tests. Twenty-three of 30 R. loseas trapped in Jiangmen City were assayed aswarfarin-resistant individuals, whereas only 1 of 30 rodents in Zhanjiang was resistant. These results emphasize the need for thoroughresistance monitoring as a basis for adequate control measures to prevent the use of ineﬀective rodenticides in Jiangmen City. The resis-tance mechanism mainly involves VKORC1, the molecular target for coumarin drugs. VKORC1 mRNA expression in wild-caught resis-tant animals showed no diﬀerence compared with that in susceptible individuals. Mutation screening of VKORC1 was carried out and anArg58Gly mutation was identiﬁed as the prevailing type in R. loseas from Jiangmen City, which may constitute the genetic basis of anti-coagulation resistance in R. losea in this resistance region.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Rattus losea; Warfarin resistance; VKOR; VKORC1; Coumarin; Hemostasis; Mutation; SNP; Gene expression; Alignment tor vitamin K hydroquinone into vitamin K 2,3-epoxide.
Availability of vitamin K for this reaction requires prior Control of rodent populations is maintained worldwide reduction of vitamin K 2,3-epoxide by the vitamin K epox- with coumarin derivatives nowadays, which are also widely ide reductase (VKOR) complex in the endoplasmic reticu- used for oral anticoagulant therapy in humans. Coumarin lum (ER) membrane This cyclic interconversion of derivatives act as antagonists of vitamin K, which func- vitamin K metabolites is known as the vitamin K cycle tions as a cofactor for the posttranslational carboxylation . Coumarin derivatives inhibit the vitamin K cycle, and of glutamate residues to gamma-carboxyglutamate (Gla) consequently inhibit the coagulation reaction.
in vitamin K-dependent proteins. Gla-containing proteins Many biochemical analyses have shown that the warfa- are mainly involved in hemostasis (coagulation factors II, rin resistance mechanism mainly involves the VKOR com- VII, IX, X; proteins C, S and Z), as well as in bone plex, though the components of the VKOR complex metabolism (osteocalcin), cell proliferation and apopto- have not been identiﬁed yet. In 2004, the gene encoding sis (growth-arrest-speciﬁc proprotein 6, Gas6) . The car- an essential protein of VKOR enzymatic activity (named boxylation modiﬁcation is accomplished by the enzyme vitamin K epoxide reductase subunit 1, VKORC1) was gamma glutamyl carboxylase (GGCX), which also func- cloned and sequenced in humans and rats . The tions as an epoxide synthase, because it converts the cofac- encoded protein was concomitantly identiﬁed as the molec-ular target for coumarin drugs. And the following studies showed that VKORC1 is a member of a large family of Corresponding author. Fax: +86 10 64807099.
E-mail address: (J. Dai).
homologs that are represented among vertebrates and 0048-3575/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.pestbp.2008.01.007 J. Wang et al. / Pesticide Biochemistry and Physiology 91 (2008) 90–95 arthropods, as well as protists, plants, and bacteria .
the test with the amount of active ingredient ingested Recent study showed that VKORC1 protein alone was able (>10 mg/kg bodyweight) were considered as evidence of to catalyze the reduction of the vitamin K epoxide to vita- resistance .
min K and vitamin K to vitamin KH2 Rattus losea, mainly distributed in southern China, Lao 2.2. Total RNA extraction and reverse transcription reaction People's Democratic Republic, Thailand, Viet Nam, andTaiwan Province of China, is one of the main pests in agri- Total RNA was isolated using Trizol reagent (Invitro- culture. After the widespread use of warfarin in some areas gen, Carlsbad, CA,). RNA concentrations were evaluated in Guangdong Province, China, eﬀective R. losea control spectrophotometrically at 260 nm. The 260/280 ratios were has become hampered by the rapid development of warfa- between 1.8 and 2. Reverse transcription was achieved rin resistance in this rodent pest. Warfarin has also been using Oligo (dT)15 primer (Promega, Madison, WI) and widely used in Jiangmen and Zhanjiang cities, especially M-MuLV reverse transcriptase (New England Biolabs, in Jiangmen City, where farmers have used warfarin six Hitchin, UK) according to the supplier's instructions. Con- times every year for more than two decades. In the present ditions for reverse transcription were as follows: 60 min at report, warfarin resistance was detected in R. losea in 42 °C, followed by 5 min at 98 °C.
Jiangmen City and Zhanjiang City, Guangdong Province,as well as mutations of VKORC1 that are probably 2.3. VKORC1 ampliﬁcation and sequencing responsible for the resistance.
The ORF sequence of VKORC1 was identiﬁed using R.
loseas trapped from Ruyuan Yaozu Autonomous County, 2. Materials and methods Shaoguan City, Guangdong Province ), where anti-coagulation rodenticides had never been used and rodents 2.1. Animals for warfarin feeding tests were all warfarin susceptible. R. loseas were killed underisoﬂurane anesthesia (n = 4). Livers were excised, rapidly The study population consisted of 60 R. loseas, 30 each frozen in liquid nitrogen, and stored at 80 °C. The total from farms in Jiangmen City (January 2006) and in Zhanji- RNA was isolated and reverse transcribed to cDNA.
ang City (March 2006), in Guangdong Province, China.
VKORC1 in R. losea were ampliﬁed using primers based The sampling sites are shown in . All animals were on corresponding sequences for Rattus norvegicus in Gen- healthy adults and females were not pregnant. They were Bank (Accession No. NM_203335). The primers amplify conﬁned in individual cages and received the same food a 511 bp fragment spanning the whole ORF of VKORC1 and water ad libitum. All animals received humane care mRNA. Nucleotide sequences for the sense primer and in compliance with the Guide for the Care and Use of Lab- oratory Animals published by the US National Institutes of Health. Resistance to warfarin was assayed by feeding respectively. PCR was performed using 1 U of VentR studies developed by the World Health Organization DNA Polymerase (New England Biolabs) in a ﬁnal concen- (WHO) with some modiﬁcations: an acclimatization per- tration of 1 PCR buﬀer as formulated by New England iod, followed by a pretest diet assessment of 10 days, then Biolabs, 250 lM of dNTP, and 0.5 lM of each primer set by a 9-day no-choice feeding schedule of 0.002% warfarin- in a total volume of 20 lL. PCR conditions consisted of containing corn (diet consumption was monitored daily), 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, and 21 days of posttreatment observation. Survival during 58 °C for 30 s, and 72 °C for 40 s, with a ﬁnal extensionperiod of 72 °C for 5 min. PCR products were run on a1.5% agarose gel containing ethidium bromide. The corre-sponding fragments were cut and puriﬁed using a QIA-quick Gel Extraction Kit (Qiagen, Valencia, CA), andsequenced on a commercial ABI 3730 capillary sequencer.
2.4. Database and sequence analyses The prediction of the amino acid sequence of R. losea VKORC1 was carried out using the ORF ﬁnder programin NCBI. Sequence alignments with its orthologs in otherspecies were performed with program clustalW in EBI.
2.5. VKORC1 mRNA expression and statistical analysis Fig. 1. The map of Guangdong Province, showing the wild R. loseacollection sites: Jiangmen City, Zhanjiang City, and Ruyuan Yaozu To compare the expression of VKORC1 mRNA in Autonomous County.
the two groups of animals, total RNA from susceptible J. Wang et al. / Pesticide Biochemistry and Physiology 91 (2008) 90–95 (n = 4) and resistant (n = 4) rats was extracted from their eﬀectiveness is jeopardized by the evolution of resistance.
livers and reverse transcribed. The resulting cDNA Many resistant rodent strains have been identiﬁed so far (1 lL) was ampliﬁed by PCR using speciﬁc primers for in the world (in Europe, USA, Canada, Japan, and Austra- R. losea VKORC1 and GAPDH. The sense and anti- sense primer sequences for the GAPDH gene were 50- Thirty R. loseas trapped in Jiangmen City were assayed by a lethal feeding test with warfarin. Twenty-three of the 30 individuals survived. The resistance rate of these 580 bp fragment. The cycle number within the exponen- animals was 76.7%. Only one female animal out of 30 tial phase of the ampliﬁcation curve was chosen. The R. loseas from Zhanjiang survived the feeding test after ampliﬁcation was performed at 94 °C for 2 min, 25 (for ingesting a total of 14.15 mg/kg warfarin. The doses of GAPDH) or 29 (for VKORC1) cycles of 94 °C for active ingredient and mortality are listed in Even 30 s, 58 °C for 30 s, 72 °C for 40 s, followed by a ﬁnal rats from Jiangmen City that died consumed more active extension at 72 °C for 5 min. PCR products were run ingredient (12.90 ± 4.24 mg/kg warfarin) compared with on a 1.5% agarose gel containing ethidium bromide those (7.60 ± 2.69 mg/kg warfarin) from Zhanjiang City and band densities were measured using Bandleader Soft- (P < 0.0001), indicating that the rodents in Jiangmen City ware. The transcripts of R. losea VKORC1 from suscep- had higher warfarin resistance. Inappropriate use of anti- tible and resistant rat hepatic tissue samples were coagulant at resistance foci can apply a selection pressure normalized to the abundance of GAPDH. All values that tends to increase both the spread and severity of resis- are expressed as means ± SE. Two-tailed Student's t test tance among rodent populations . Our results imply was used to determine the statistical diﬀerence between that the extensive resistance in Jiangmen City may enable the two groups. P < 0.05 was considered signiﬁcant.
resistant individuals to survive treatment with warfarin inthe ﬁelds. And the results emphasize the need for thorough 2.6. VKORC1 mutations and single nucleotide resistance monitoring as a basis for adequate control mea- polymorphisms (SNPs) in potential warfarin resistance sures to prevent the use of ineﬀective rodenticides in this In order to screen the mutations in the coding region of VKORC1 in resistance R. losea, hepatic RNA from six 3.2. Nucleotide and amino acid sequence of VKORC1 in resistant animals, as well as 51 additional animals caught in Jiangmen City (39 animals) and Zhanjiang City (12 ani-mals) on April 2007, was extracted and reverse transcribed Though there are many factors that aﬀect warfarin sen- to cDNA. Their nucleotide sequences were obtained by sitivity, e.g., genetic variants in CYP2C9 (Cytochrome cDNA sequencing as described in 2.3. Mutation and poly- P450, subfamily IIC, polypeptide 9) (the principal drug- morphism screens were then carried out by sequence metabolizing enzyme that catalyzes the hydroxylation of warfarin warfarin resistance mainly involves theVKOR complex. We therefore identiﬁed the coding region 3. Results and discussion sequence of VKORC1 mRNA in warfarin susceptibleR. loseas trapped in Ruyuan Yaozu Autonomous County.
3.1. Warfarin-resistant R. losea in Jiangmen City The nucleotide sequence of VKORC1 was deposited inGenBank as Accession No. EF028346. Amino acid Coumarin derivatives, e.g. warfarin, which can eﬀec- sequence alignment was carried out with VKORC1 from tively repress blood coagulation, remain one of the main R. norvegicus, Homo sapiens, and other eukaryotes tools available to control rodent populations worldwide.
(). The deduced product had 161 amino acid residues, They have been widely used since the 1950s in Europe which is only one residue diﬀerent from R. norvegicus and 1980s in China. However, after widespread use, their VKORC1. The overall amino acid sequence identity com- Table 1Results of warfarin feeding test on R. losea trapped in Jiangmen and Zhanjiang City Total consumption of active ingredient (mg/kg) J. Wang et al. / Pesticide Biochemistry and Physiology 91 (2008) 90–95 pared with H. sapiens VKORC1 (GenBank Accession No.
NP_076869) is 83%.
VKORC1 mRNA expression from warfarin-resistant R.
loseas was similar to that from susceptible individuals 3.3. Expression of VKORC1 mRNA by PCR via PCR (It seems that the transcriptional diﬀer-ence of VKORC1 mRNA can not be the reason for resis- It has been reported that a French strain of rat tance in the R. losea in Jiangmen City, Guangdong showed a lower expression of VKORC1 mRNA in resis- tant rat livers compared with susceptible ones . And asimilar phenomenon was also observed in a Danish strain 3.4. Mutations and SNPs in VKORC1 mRNA of rat The expression of VKORC1 mRNA was alsocompared in our resistant and susceptible animals; the Several studies have focused on mutations in VKORC1 transcripts of R. losea VKORC1 from the two groups in both human disorders and in laboratory strains and wild .: **. **:. :**: ** * :* Fig. 2. Alignment of VKORC1 orthologs using the algorithm ClustalW. The numbers on the left indicate the amino acid positions of each protein. The‘‘*" and ‘‘:" indicate constitutive and semiconstitutive amino acids. Dashes represent gaps introduced to optimize alignment. The ﬁve completely conservedresidues (Cys43, Cys51, Cys132, Cys135, and Ser57) are indicated in the hatched box. Rn: Rattus norvegicus (GenBank Accession No. NP_976080); Hs:Homo sapiens (GenBank accession no. NP_076869); Rl: Rattus losea (GenBank Accession No. ABK27271); Gg: Gallus gallus (GenBank Accession No.
NP_996530); Mm: Mus musculus (GenBank Accession No. NP_848715); Tr: Takifugu rubripes (GenBank Accession No. AAR82912); Dm: Drosophilamelanogaster (GenBank Accession No. NP_001014533).

J. Wang et al. / Pesticide Biochemistry and Physiology 91 (2008) 90–95 Gly mutation, and was probably the speciﬁc genetic basis for resistance in Jiangmen City.
Hydrophobicity plots and secondary structure predic- tions suggest a topology including 3–4 a-helical transmem-brane analyses of VKORC1 homologous protein sequences iden-tiﬁed Cys43, Cys51, Cys132, Cys135, and Ser57 as com- pletely conserved residues in all species surveyed, andthese residues should comprise catalytic site residues .
Especially, the C132–X–X–C135 motif, it is characteristic of the active site of many redox proteins And the mutation of either of the cysteine residues in this motifresulted in loss of VKORC1 activity Several residues, Fig. 3. VKORC1 mRNA expression in warfarin-resistant (R) andsusceptible (S) R. loseas by PCR. PCR products were subjected to such as L120, L128 and Y139, which located in the pre- electrophoresis on a 1.5% agarose gel. A representative result is shown (A) dicted transmembrane domains , though not the high- ‘‘S1–S4" and ‘‘R1–R4" represent diﬀerent individuals of susceptible and est conserved residues, were also important for warfarin resistant rodents, respectively. ‘‘M" represents the DNA marker. Band resistance Serines are generally known to be important densities were normalized for GAPDH levels, and no diﬀerence in the residues in active sites of reductases. The highly conserved expression in the two groups was obtained statistically (B). Error barsrepresent SEM.
Ser57 in VKORC1 is a potential candidate for the bindingsite of the substrate vitamin K epoxide. And the substitu-tion of serine by alanine at 57 resulted in an almost com- catches of R. norvegicus. Several mutations (Arg35Pro, plete loss of VKOR activity . The Arg58Gly mutation Ser56Pro, Trp59Gly, Leu120Gln, Leu128Gln, Tyr139Ser, in our resistant R. loseas lies next to the proposed substrate Tyr139Cys, and Tyr139Phe) have been identiﬁed in warfa- binding site Ser57, and probably leads to warfarin resis- rin-resistant rats and four mutations in human beings resis- tance by reducing the binding activity with anticoagulants.
tant to warfarin (Val29Leu, Val45Ala, Arg58Gly, and Continuous inappropriate use of anticoagulant in Jiang- Leu128Arg), suggesting that coding-region variants of men City increased the proportion of higher resistant R.
VKORC1 are extremely detrimental . However, no losea in the population. Our feeding test showed that even information is available on the genetic mechanism occur- the animals which died during the test in Jiangmen City rence of resistance in R. losea.
survived a longer time and consumed more warfarin than Mutation screening was carried out across the ORF those from Zhanjiang City. We speculated that this phe- region of VKORC1 cDNA from six of our warfarin-resis- nomenon can partly be due to the heterozygous 58th muta- tant R. losea samples. A unique single nucleotide mutation tion individuals. Heterozygous individuals carried this was observed in all the resistant individuals. The single mutation possess higher resistant ability than wild type, nucleotide diﬀerence leads to a mutation in the 58th residue though cannot survive the feeding test.
of the translation product: the replacement of arginine in There were other types of mutations observed in the susceptible individuals by glycine ). The Arg58Gly wild rodents, including two SNPs and an Arg35Cys muta- mutation has been reported in warfarin-resistant patients tion () showing a high degree of sequence diver- by Rost and his colleagues . Furthermore, they also gence in wild R. loseas. Though SNPs do not alter the detected the enzyme activity of this type of mutation, show- amino acid sequence of the protein, it was shown that non- ing that recombination expression of the Arg58Gly muta- coding polymorphism in VKORC1 can also contribute to tion led to a lower functional eﬃciency of the enzyme the variability in the maintenance dose of warfarin in complex. Our ﬁeld studies showed that the missense muta- patients The R35C substitution, which was only tion Arg58Gly was prevalent in resistance foci (Jiangmen obtained in one individual from Jiangmen City, may lead City), with 36 individuals in 39 animals carried the Arg58- to functional impairment of VKORC1 activity and/or Fig. 4. The Ary58Gly mutation of VKORC1 in warfarin-resistant R. losea from Jiangmen City, Guangdong Province, China. The 58th amino acid inwarfarin-susceptible animals is arginine (A), while in warfarin-resistant individuals is glycine (B).
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