DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 DMD FastThis Forward. article has notPublished been copyedited on Septemberand formatted. The 29, final 2006 version as maydoi:10.1124/dmd.106.011775 differ from this version.

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Elevated metabolism in warfarin-resitant roof rats (Rattus rattus) in Tokyo

Mayumi Ishizuka, Fumie Okajima, Tsutomu Tanikawa, Heewon Min, Kazuyuki D.

Tanaka, Kentaro Q Sakamoto, Shoichi Fujita

Downloaded from

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate

School of Veterinary Medicine, Hokkaido University; MI, FO, HM, KDT, KQS, SF dmd.aspetjournals.org

Technical Research Laboratory, IKARI Corporation; TT at ASPET Journals on September 27, 2021

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Running title: Elevation of P450-dependent metabolisms in warfarin resistant rats

To whom correspondence should be addressed

Shoichi Fujita, Ph.D.

Laboratory of Toxicology, Department of Environmental Veterinary Sciences Downloaded from

Graduate School of Veterinary Medicine, Hokkaido University

N18, W9, Kita-ku, Sapporo 060-0818, Japan dmd.aspetjournals.org

Tel: +81-11-706-6948, Fax: +81-11-706-5105

E-mail: [email protected] at ASPET Journals on September 27, 2021

Number of text page: 33

Number of table: 1

Number of figures: 6

Number of references: 24

Number of words in

Abstract: 173

Introduction: 520

Discussion: 746

1 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Abstract

Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases. The is important for public health, and warfarin is a commonly used worldwide. However, continual use of Downloaded from 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 dmd.aspetjournals.org significantly lower after oral administration than that in the control warfarin-sensitive rats.

Warfarin is metabolized by cytochrome P450 (CYP), and hydroxylations of warfarin by at ASPET Journals on September 27, 2021

CYP isoforms were significantly higher in warfarin-resistant rats (2-fold). Western blot analysis indicated that the level of CYP3A2 expression in warfarin-resistant rats were significantly larger than in warfarin-sensitive rats. The NADPH-P450 reductase activities in resistant rats were 8-fold higher than those in sensitive rat. In vivo, the administration of P450 potent inhibitor 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.

2 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Introduction

Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases, such as plague, hemorrhagic fever with renal syndrome (HFRS), Weil’s disease, Hepatitis E virus and others. Although the pest control Downloaded from is important for public health, continual use of -based may cause

drug resistance in rodents and lead to failure of their control, especially in urbanized dmd.aspetjournals.org areas.

Warfarin is a commonly used rodenticide worldwide. It inhibits coagulation of blood by at ASPET Journals on September 27, 2021 inhibiting vitamin K epoxide reductase (VKOR) activity (Thijssen et al., 2004). An inadequate supply of vitamin K blocks the production of prothrombin and cause hemorrhaging.

Warfarin-resistant rats have been already reported in urban areas such as Liverpool, UK

(Greaves et al., 1973), California and Texas, USA (Jackson et al., 1979) and Tokyo, Japan

(Ito et al., 1981). Many studies have been performed to elucidate the mechanism of warfarin-resistance. Rost et al. (2004) recently reported that warfarin resistance is attributable to a mutation (Tyr139Phe) in the vitamin K epoxide reductase complex subunit 1 (VKORC1) gene. Pelz et al. (2005) concluded that mutations in VKORC1 are

3 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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the genetic basis of resistance of rodents in the experiments using recombinant VKORC1 mutants. Recently, Lasseur et al. (2006) reported that the resistant rats with mutated at 139 in VKORC1 showed VKOR activity with reduced Vmax and very small Km, to the extent that Kcat (Vmax/Km) become larger than that of sensitive

(wild type) rats. This means that resistant rat has higher VKOR activity than sensitive Downloaded from rat at the low concentrations of VKO in the physiological conditions. However, mutations

alone cannot explain all aspects of resistance in rodents (Wajhi et al., 2004), and we dmd.aspetjournals.org actually could not detect the mutation at the position of 139 in VKORC1 gene of Japanese roof rat. Pelz et al. (2005) agree with that variations in anticoagulant uptake, metabolism, at ASPET Journals on September 27, 2021 and clearance may influence the efficacy of rodenticides in vivo.

Another possible mechanism for annuals to acquire warfarin resistance is the enhanced warfarin metabolism and elimination. Several forms of cytochrome P450 (P450 or

CYP) are known to metabolize warfarin (Daly and King et al., 2003). In rats, warfarin hydroxylations are mainly catalyzed by CYP2C, CYP2B, CYP1A, and CYP3A subfamilies (Figure 1). The five hydroxides are identified as 4’-, 6-, 7-, 8- or 10-OH warfarin (Guengerich et al., 1982), and these primary metabolites are made more water-soluble by UDP-glucuronyltransferase (UGT) activity, and excreted in urine.

In a recent study, Sugano et al. (2001) newly reported the potent resistance of wild roof

4 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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rats to warfarin in the Tokyo area of Japan. They focused on the low level of mRNA expression of the warfarin-metabolizing enzyme, CYP3A which catalyzes warfarin

10-hydroxylation in resistant rats, and concluded that reduced metabolic activation of warfarin might cause the resistance. However, it has been reported that CYP3A induced by St. John’s wort decreases the anticoagulant effect and increases the tolerance for Downloaded from higher doses of warfarin in human (Izzo et al., 2005).

In present study, we identified the clearance and metabolic profiles of warfarin in the dmd.aspetjournals.org

Japanese roof rat to establish the novel mechanism of warfarin resistance in wild rodents.

at ASPET Journals on September 27, 2021

5 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Materials & Methods

Animals

Warfarin-sensitive and -resistant roof rats were supplied by Ikari Corporation.

Warfarin-resistant (R) rats were caught in buildings in the Tokyo area, namely, Shinjuku Downloaded from

(Tokyo), Akabane (Tokyo), Ichikawa (Chiba), and Kamagaya (Chiba). They were given

food containing 0.025% warfarin for 1 month. Rats who survived more than 1 month dmd.aspetjournals.org were used as warfarin-resistant rats according to WHO regulations. Warfarin-sensitive rats were originally from the Ogasawara Islands (Tanikawa and Suzuki et al., 1993). Six at ASPET Journals on September 27, 2021 rats (3 males and 3 females) were caught in the islands in 1989, and a closed colony is maintained in the laboratory of Ikari Corporation. Rats from the closed colony were used as warfarin-sensitive (S) rats.

All rats were housed, one per cage, under standard laboratory conditions (12 h light: 12 h dark) with food and water available ad libitum. They were used for experiments after 2-4 weeks of acclimatization. Treatments of all animals were performed according to the policies of the Institutional Animal Care and Use Committee of Hokkaido University.

We determined the ages of roof rats by paired eye-lens weight (Tanikawa et al., 1993).

Both eyes were removed and placed in 10% formalin immediately after sacrifice. We

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fixed eyes in formalin at room temperature for 4 weeks. After fixation, the tissue around the lens was removed, and the lenses were washed with distrilled water. The lenses were dried at 60°C for 3 days in an oven, and were weighed to estimate the rat’s age according to the formula in previous report. Three- to six-month-old rats were used for all experiments. Downloaded from

Chemicals dmd.aspetjournals.org

Glucose-6-phosphate (G6P), Glucose-6-phosphate dehydrogense (G6PDH), and

NADPH were from Oriental Yeast Co. Ltd. (Tokyo, Japan). β-Glucuronidase was from at ASPET Journals on September 27, 2021

Roche Ltd (Indianapolis, IN, USA). Anti-rat CYP 1A1, 2B1, 2C6, and 2C11 antibodies from goat and anti-rat CYP 3A2 antibody from rabbit were purchased from Daiichi Pure

Chemicals (Tokyo, Japan). Anti-goat IgG and anti-rabbit IgG conjugated with HRP

(horseradish peroxidase) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma Aldrich Inc., respectively. Dicoumarol and warfarin were from Sigma Aldrich

Inc. (St. Louis, MO, USA). Warfarin metabolites (4’-, 6-, 7-, 8-, or 10-hydroxywarfarin) were from Ultrafine Chemicals (Manchester, UK).

Measurement of warfarin in plasma

7 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Rats were given 15 mg/kg warfarin orally, and blood was taken from the tail at 0 and 60 min. Warfarin in plasma was extracted according to the method of Takahashi et al. (1997).

We added 30 µl of 2 N HCl, 150 µl of diethylether, and 19.5 µl of 5 µM dicoumarol into

15 µl of plasma in the tube as the internal standard (500 nM, final conc.). The tubes were vigorously shaken for 10 min, and then centrifuged at 1200 g at 4°C for 10 min. The Downloaded from organic layer was obtained and evaporated to dryness by a centrifugal evaporator

(EYELA CVE-200D, Tokyorikakikai Co. Ltd., Tokyo Japan). The residue was dmd.aspetjournals.org reconstituted with 150 µl of mobile phase (1.5% acetic acid (pH 4.85):acetonitrile=70:30) for HPLC analyses. The solutions were injected into the HPLC system (ODS-2, at ASPET Journals on September 27, 2021

4.6x250mm, 5µm) and measured at 307 nm (Kaminsky, 1979).

Treatment of rats with Warfarin metabolism inhibitor, SKF-525A

We treated four warfarin resistant male rats with 0.05 % or 0.1 % of SKF-525A in feeding bait containing 0.025 % of warfarin. As controls, we administered 0.05 % or 0.1 % of

SKF-525A in bait to three male rats. The daily feeding amount and body weight were measured, and mortality was determined at day 12.

Preparation of liver microsomes

8 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Livers were taken from roof rats after the sacrifice, and microsomes were prepared according to the method of Omura and Sato (1964). The livers were homogenized with three times their volume of 1.15% KCl. The homogenates were centrifuged at x9000 g at

4°C for 20 min. The supernatant was decanted to an ultra-centrifugation tube and centrifuged at x105000 g at 4°C for 60 min. The pellet was homogenized in potassium Downloaded from phosphate buffer (0.1 M, pH 7.4, containing 1 mM DTT, 1 mM EDTA, 2 µg/ml aprotinin,

final concentration) in ice. The microsomal homogenates were transferred to 1.5 ml tubes dmd.aspetjournals.org and stored at -80°C until use.

at ASPET Journals on September 27, 2021

P450 protein expression

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done according to the method of Laemmli et al. (1970). The protein concentrations were determined by the method of Lowry et al. (1951). Microsomal protein was separated by

SDS-PAGE and transferred to a nitrocellulose membrane (Toyo Roshi Kaisha Ltd.,

Tokyo). Anti-rat CYP1A1, CYP2B1, CYP2C6, CYP2C11 and CYP3A2 antibodies were used to detect each CYP isoform. Anti-CYP1A1, CYP2B1 and CYP2C6 antibodies cross react with CYP1A2, CYP2B2 and CYP2C13, respectively. Anti-rat CYP2C11 and

CYP3A2 antibodies cross react with other CYP2C and CYP3A subfamilies, respectively.

9 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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The membrane was immunostained using diaminobenzidine. The results of staining were analyzed by using NIH Image v. 1.63 (Lennard, 1990).

Warfarin metabolism

Warfarin metabolism by liver microsomes was analyzed by Kaminsky et al., (1979). Downloaded from

MgCl2 (3 mM in final conc.), G6P (5 mM in final conc.), and warfarin (400 µM in final

conc.) were mixed and added to microsomes (1 mg protein/ml, final conc.) or potassium dmd.aspetjournals.org phosphate buffer for the negative control. The total volume of each sample was 1 ml.

Samples were pre-incubated at 37°C for 5 minutes. A mixture of G6PDH (2 IU/ml in final at ASPET Journals on September 27, 2021 conc.) and NADPH (0.5 mM in final conc.) was added to each sample to start the reaction.

The reaction was kept at 37°C for 10 minutes, and 20 µl of 60% perforic acid was added to stop the reaction. Samples were centrifuged at x3000 g, 4°C for 5 min, and the supernatants were filtered through nitrocellulose (0.22 µm, 13 mm, Millipore). Filtrated samples were injected into the HPLC system. We used Inertsil ODS-2 colomun (GL

Science Inc. 4.6x250 mm, 5µm) and detected at 306 or 307 nm. Mobil phase consisted of

1.5% acetic acid (pH 4.85): acetonitrile=70:30 or 15 mM KH2PO4 (pH 3.00, adjusted with 1N HCl):methanol:acetonitrile=52:32:16.

10 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Testosterone metabolism

The incubation mixture for the assay of testosterone metabolism consisted of an

NADPH generating system as warfarin assay. Substrate concentration was 0.25 mM and corticosterone was spiked as an internal standard after the reaction. Testosterone metabolites were measured according to the methods of Ishizuka et al. (1998) by HPLC Downloaded from using a SUPELCOSIL LC-318 (SPELCO, Bellefonte, PA, USA) column. Mobil phase

consisted of methanol: water: tetrahydrofuran = 35:55:10, and the detection wavelength dmd.aspetjournals.org was 245 nm.

at ASPET Journals on September 27, 2021

NADPH P450 reductase

NADPH-P450 reductase activity was measured according to Omura and Takesue (1970).

Cytochrome c reduction was monitored at 550 nm and 37 °C. The incubation mixture contained 0.1 M potassium phosphate buffer, pH 7.4, 50 µM cytochrome c, 50 µM

NADPH, and 25 µg/ml microsomal protein. One unit of activity was defined as the amount of microsomal protein catalysing the reduction of 1 nmol of cytochrome c/min under the above conditions.

Vitamin K1 epoxide reductase complex subunit 1 (VKORC1) fragment cDNA

11 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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amplification and isolation.

Total RNA was prepared from each liver sample of sensitive and resistant Rattus rattus

(male) using TriReagent (Sigma-Aldrich, CA, USA). The concentration and purity of the RNA fraction was determined spectrophotometrically at 260 and 280nm, respectively.

Oligo (dT) primed total RNA was reverse-transcribed to cDNA using reverse Downloaded from transcriptase. VKORC1 primers were designed to amplify partial cDNA fragment, based

on the conserved regions of rat, mouse and human VKORC1 sequences retrievable from dmd.aspetjournals.org

GenBank. The sense primer for VKORC1 was 5’-GTTTGCGGCTTGCACTATG–3’, and antisense primet was 5’- ACCTCAGGGCTTTTTGACCT-3’. The PCR product at ASPET Journals on September 27, 2021 was freshly cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), followed by the transforming of bacterium DH5α with that plasmid DNA. The bacterial cells were cultured for 14-hrs on average. Purified plasmids were directly sequenced by the method of dye terminator cycle sequencing. The cycle sequencing reactions were performed with the vector-specific M13 Primers (Invitrogen, Carlsbad, CA) using the ABI PRISM

Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied

Biosystems, Foster, CA). The nucleotide sequence of the amplified fragment was determined by an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer).

12 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Statistical Analysis

Student’s t-test and two-way ANOVA were performed using JMP IN® v. 5.1. P<0.05 was considered significant. Columns show the means, and error bars show the standard deviation (SD) in bar charts.

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

13 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Results

Warfarin concentration in plasma in roof rats

Warfarin concentrations were determined after oral administration to investigate warfarin concentration of warfarin sensitive and resistant rats (Figure 2). The warfarin Downloaded from concentration in the plasma of warfarin-sensitive rats was higher than that of

warfarin-resistant rats at 60 min after administration (8 fold). dmd.aspetjournals.org

Warfarin metabolizing enzymes in roof rats at ASPET Journals on September 27, 2021

We also measured the warfarin metabolic activities in liver microsomes of roof rats. The rates of formations of all hydroxywarfarins were significantly higher in warfarin-resistant rats than those in -sensitive rats after the reaction resulting in 2 fold difference in total warfarin metabolizing activities (Figure 3).

To determine the expression of P450s, we compared expressions of each CYP related to warfarin metabolism (Figure 4). Expression of CYP3A subfamily was significantly higher (4.1 fold) in warfarin-resistant rats than -sensitive rats. In male rats, expression of

CYP2C11 was also significantly (1.2 fold) higher in warfarin-resistant rats than that in

-sensitive rats. Expressions of the other CYPs (CYP1A, CYP2B and CYP2C6) showed

14 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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no significant difference between warfarin-resistant and -sensitive rats. It should be noted that in spite of the fact that all the pathway of warfarin metabolism showed higher activities in resistant rats but not all the CYP isozymes catalyzing those pathway showed higher expression levels.

We also measured P450 contents (Omura and Sato, 1964). Although the contents of P450 Downloaded from showed the tendency of induction in resistant male rats, the induction was not significant

(data not shown). dmd.aspetjournals.org

Testosterone metabolism at ASPET Journals on September 27, 2021

Since the expression of CYP3A was induced in the liver of resistant rat, we measured

CYP3A-dependent activity using testosterone as model substrate. The testosterone

6-beta hydroxylation activity was significantly elevated in the liver of resistant rat; metabolic activity was 4-fold higher in resistance than that in sensitive rats (Figure 5).

The 16-beta and 7-alpha hydroxylase activities were also higher in livers of resistant rat than those of sensitive animals (1.9 and 1.8 fold, respectively). There was no significant difference in the activity of 2-alpha hydroxylation, which was catalyzed by CYP2C11, between resistant and sensitive animals (data not shown).

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NADPH P450 reducatase activity

Since the rats of P450 reduction is a significant determinant of the rate of P450 dependent reactions, we measured the NADPH P450 reductase dependent activities in sensitive and resistant rats (Figure 6). The activity of NADPH P450 reductase dependent cytochrome c reductase in liver microsomes was 3.00 ± 1.77 nmol/min/mg in sensitive rat, and 23.42± Downloaded from

3.91 nmol/min/mg in resitant animals.

dmd.aspetjournals.org

In vivo inhibition of warfarin metabolism in resistant rat

We treated warfarin resistant rats with warfarin or P450 potent inhibitor, SKF-525A at ASPET Journals on September 27, 2021

(Table 1). All rats, which were administered with warfarin or SKF-525A alone, survived during the administration period (12 days) and we did not observe any hemorrhage symptom among them. However, in the co-treated group, which was administered with

0.05 % of SKS-525A and 0.025 % warfarin, number of dead was 1 of 4 rats at day 12, and yet another rat died at day of 14. We observed that 2 of 4 male rats died after co-treatments of 0.1 % SKF-525A and 0.025 % warfarin at day 12.

16 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Discussion

Recent study showed substituted VKORC1 (Tyr139Phe) was poorly inhibited warfarin, and the mutation in VKORC1 was one of the factor for warfarin resistance (Lasseur et al.

2006). Thus, we cloned the fragments of VKORC1 gene from lover of sensitive and Downloaded from resistant Rattus rattus, and sequenced (data not shown). However, in this study, we did not

detect the mutation at the position of 139 in VKORC1 in liver of resistant roof rat. Then, dmd.aspetjournals.org we focused on the possibility that the clearance of warfarin in resistant rat might be accelerated, since CYP metabolism has been suggested to cause warfarin resistance in at ASPET Journals on September 27, 2021 humans. Also, induction of P450 by other drugs decreases warfarin levels in the human

(Dingemanse et al. 2004).

We suggest that elevation of ability to detoxify foreign chemicals due to the induction of enzyme expression or genetic-polymorphism in wild roof rats may adapt them to a rodenticide-contaminated environment. Exposures to environmental pollutants such as might induce the expression of P450 isoforms in wild rodents to accelerate detoxification. Actually, Fujita et al. (2001) reported the elevation of CYP2B and CYP3A expressions in wild voles (Clethrionomys rufocanus) that inhabited at highly -contaminated areas. Alternatively, polymorphically higher expression of CYP

17 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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may enhance the ability of detoxification. Hoshi et al. (1995) also found a polymorphic

P450 isoform in wild rodents, and reported the extensive and poor metabolizers of xenobiotics.

We studied the warfarin residue level in plasma of sensitive and resistant roof rats after oral administration (Fig. 2). Warfarin levels in plasma were lower in warfarin resistant Downloaded from than in -sensitive animals. Warfarin metabolic activities in resistant rats were higher than

those of sensitive animals as we expected (Figure 3). dmd.aspetjournals.org

According to the immunoblotting results (Figure 4), high expression of CYP3A2 seems to be reasons for high warfarin metabolism. Sugano et al. (2001) showed suppression of at ASPET Journals on September 27, 2021

CYP3A2 mRNA expression in their warfarin-resistant rat. In current study, we measured the expression levels of CYP3A2 apoprotein in addition to enzymatic reaction. Moreover, the hydroxylation activity of testosterone 6-beta, which was catalyzed by CYP3A2 in male rat, was also higher in resistant rat compared to sensitive animals (data not shown).

Expression of the CYP3A subfamily has been demonstrated to be regulated by nuclear receptors, such as PXR (pregnane X receptor) and GR (glucocorticoid receptor), and neonatal hormonal imprinting (Pascussi et al. 2000). In rats, CYP3A2 has a gender difference in its expression profile, and the concentration of this isoform is usually low in immature males. Although Sugano et al. did not indicate the ages of their rats, an

18 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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estimation of age might be essential to consider the CYP expression in wild rats.

In our study, the formation of 10-hydroxywarfarin, formed by a CYP3A dependent reaction, was higher in warfarin-resistant rats than -sensitive rats. However, formations of other metabolites (4’-, 6- and 8-hydroxywarfarins) were also higher in resistant rats.

Assay of testosterone metabolism was also elucidated the acceleration of total Downloaded from

P450-dependent metabolisms in resistant rats. We found not only the elevation of

CYP3A-dependent 6-beta testosterone hydroxylation in resistance rats, but also high dmd.aspetjournals.org level of 7-alpha and 16-beta hydroxylase activites (CYP2A and CYP2B-dependent activities, respectively). Thus, high CYP3A might not be the only reason for higher at ASPET Journals on September 27, 2021 warfarin metabolism in resistant rats. NADPH P450 reductase activity is involved in every P450 activity and it might be the rate-determining step of reactions of P450. We found that NADPH cytochrome c reducatse activity dependent on NADPH cytochrome

P450 reductase was markedly higher in resistant rats than in sensitive rats. This may be the reason for high activities of every pathway of warfarin metabolism in resistant rats.

To establish the P450 contribution to warfarin resistance, we treated roof rats with feeding bait containing warfarin and P450 potent inhibitor SKF-525A (Table 1).

Although the number of rats which used for this examination was limited, we have already confirmed that warfarin injection was not fatal for the resistant rats and the

19 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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mortality rate was 0 %. Increase of mortality rate in co-treated rats with warfarin and

SKF-525A indicated the possibility that CYP-dependent warfarin metabolism might contribute to warfarin resistance in wild rodents.

In present study, we reported that warfarin-resistant rats had high metabolic activity of warfarin by P450, which might be due to the high expression of CYP3A and elevated Downloaded from activity of NADPH P450 reductase. We concluded that the elevation of P450-dependent

warafarin metabolism also gave rise to warfarin resistance in wild rats. This is the first dmd.aspetjournals.org report which showed that elevation of P450-dependent metabolisms caused the pesticide resistance in wild mammals. at ASPET Journals on September 27, 2021

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Footnotes

This study was partly supported by Grants-in-Aid for Scientific Research from the

Ministry of Education, Culture, Sports, Science and Technology of Japan awarded to S.

Fujita (No. 15201009). Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

27 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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Figure legends

Figure 1. Metabolic pathway of warfarin

Figure 2. Warfarin concentration in plasma at 60 min after the oral administration of Downloaded from warfarin. Warfarin was extracted from plasma by diethylether and injected into an HPLC

system. The mobile phase was 1.5% acetic acid (pH 4.85):acetonitrile=70:30. Warfarin dmd.aspetjournals.org concentrations in plasma of sensitive (n=4) and resistant roof rats (n=4) were measured.

at ASPET Journals on September 27, 2021

Figure 3. Hepatic microsomal warfarin hydroxylase activities in warfarin sensitive and resistant rats. Reaction was carried out for 10 min at 37°C, and samples were injected into an HPLC system to measure warfarin and its metabolites. Columns show means, and error bars show SD. : p<0.05. Warfarin hydroxylase activities (nmol/min/mg protein) in liver microsomes from warfarin sensitive (n=3) and resistant males (n=3) were measured.

Figure 4. Results of western blotting of P450. Microsomal protein was separated by

SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was

28 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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immunostained using diaminobenzidine (A). Lane 1 and 2 showed typical CYP expressions in individual warfarin sensitive rats, and lane 3 and 4 showed CYPs in two resistant rats, respectively. The relative value of the color density of immunoblotting was analyzed by NIH Image v. 1.63 (B). Liver microsomes from warfarin sensitive (n=4) and resistant males (n=4) were measured. Columns show means, and error bars show SD.*: Downloaded from p<0.05

dmd.aspetjournals.org

Figure 5. Hepatic microsomal testosterone hydroxylase activities in warfarin sensitive and resistant rats. Reaction was carried out for 10 min at 37°C, and samples were injected at ASPET Journals on September 27, 2021 into an HPLC system to measure warfarin and its metabolites. Columns show means, and error bars show SD. *: p<0.05. Testosterone hydroxylase activities (nmol/min/mg protein) in liver microsomes from warfarin sensitive (n=3) and resistant males (n=3) were measured.

Figure 6. Results of NADPH P450 reductase dependent cytochrome c reductase activities in sensitive and resistant roof rats. Reductase activities in liver microsomes from sensitive (n=4) and resistant males (n=6) were measured. The vertical axis shows activity

(µmol/min/mg protein). Reaction was carried out for 1 min at 37°C, and samples were

29 DMD Fast Forward. Published on September 29, 2006 as DOI: 10.1124/dmd.106.011775 This article has not been copyedited and formatted. The final version may differ from this version.

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measured using spectrophotometer. Columns show means, and error bars show SD. *: p<0.05. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

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Table 1 Effects of inhibition of warfarin metabolism on mortality of warfarin-resistant rats treatment Mortality at day 12 (number of dead)

SKF-525A (0.05 %) 0/3

SKF-525A (0.05%) + warfarin (0.025 %) 1/4 (2/4 at day 14) Downloaded from

SKF-525A (0.1 %) 0/3 dmd.aspetjournals.org SKF-525A (0.1%) + warfarin (0.025 %) 2/4

Four warfarin resistant male rats were treated with 0.05 % or 0.1 % of SKF-525A in

feeding bait containing 0.025 % of warfarin. As controls, three male rats were at ASPET Journals on September 27, 2021 administered 0.05 % or 0.1 % of SKF-525A in bait. The daily feeding amount and body weight were measured, and mortality was determined at day 12.

31 Figure 1

Warfarin This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMD FastForward.PublishedonSeptember29,2006asDOI:10.1124/dmd.106.011775 8-OH

CYP1A1 CYP2C11 CYP2C6 8 O O CYP2B1 7-OH 7 4’ 4’-OH

6 5 CYP2C11 OH CYP1A1 10 O 6-OH CYP3A2

10-OH

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2.4 DMD FastForward.PublishedonSeptember29,2006asDOI:10.1124/dmd.106.011775 2.0 1.6 1.2 0.8 warfarin (uM) 0.4 * 0 sensitive resistance

Rattus rattus

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2.5 * DMD FastForward.PublishedonSeptember29,2006asDOI:10.1124/dmd.106.011775

2

7&10OH 1.5 8-OH 6-OH 1 4'-OH nmol/min/mg protein nmol/min/mg 0.5

0

sensitive resistant

Downloaded from from Downloaded dmd.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at Figure 4 1432 CYP1A1

CYP2B2 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMD FastForward.PublishedonSeptember29,2006asDOI:10.1124/dmd.106.011775 CYP2C6

CYP2C11

CYP3A

5.0 1A1 * 4.0 2B1

2C11 3.0 3A

2.0 2C6 relative value * 1.0

0.0 sensitive resistance

Rattus rattus

Downloaded from from Downloaded dmd.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at Figure 5 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

8 7α * DMD FastForward.PublishedonSeptember29,2006asDOI:10.1124/dmd.106.011775 7 6β 6 16β 5 4 3 * 2 *

nmol/min/mg protein nmol/min/mg 1 0

sensitive resistant

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0.040

0.032 * 0.024

0.016

µmol/min/mg ) ( 0.008

0.000

Sensitive Resistant

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